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We report on a new species, Zhangixalus melanoleucus sp. nov from the Phou Samsoum Mountain in Xiengkhouang Province, northeastern Laos, based on an integrative taxonomic approach including morphological, molecular and bioacoustic lines of evidence. Morphologically, the new species is distinguished from its congeners by a combination of the following diagnostic characters: medium body size (SVL 34.4–36.3 mm in males, 53.7 mm in a single female); smooth and green dorsum; chest and belly lacking spots; flanks, axillae, ventral surfaces of forearms, inguinal, anterior and posterior surfaces of thighs white covered with irregular black pattern; finger webbing formula I 2½–3 II 2–3 III 2¼–2 IV; toe webbing formula I 2–2½ II 1–2 III 1–2 IV 2–1 V; toe webbing cream with small black blotches; outer margin of forearms and feet with weak dermal ridges; supracloacal fold and pointed projection at tibiotarsal articulation absent; iris reddish-orange. The new species is divergent from all other congeners in 16S rRNA gene sequences (P=3.4–8.8%) and is reconstructed as the sister species of Z. nigropunctatus. The advertisement call of new species represents clicking sounds and includes of a series of notes each 0.28 s in duration and consisting of 2–3 pulses with the dominant frequency ca. 3.14 kHz. To date, Zhangixalus melanoleucus sp. nov. is known only from montane evergreen forest of Phou Samsoum Mt. at elevations of 2000–2200 m asl. We preliminary suggest the new species should be considered as Data Deficient (DD) following the IUCN’s Red List categories.
Letter to the editor Open Access
We report on a new species, Zhangixalus melanoleucussp.
nov.,from PhouSamsoum Mountain(PSM)inXiengkhouang
Province, northeastern Laos, based on an integrative
taxonomicapproach,including morphological, molecular, and
bioacoustic lines of evidence. Morphologically, the new
species can be distinguished from its congeners by a
combination of the following diagnostic characters: medium
bodysize(SVL 34.4–36.3mmin males,53.7mm inasingle
female); dorsum smooth and green; chest and belly lacking
spots; flanks, axillae, ventral surfaces of forearms, inguinal,
anterior and posterior surfaces of thighs white, covered with
2¼–2IV; toewebbingformulaI2–2½II1–2 III1–2 IV2–1 V;
toewebbing creamwith smallblack blotches;outermarginof
forearmsandfeet withweakdermal ridges;supracloacalfold
reddish-orange. The new species is divergent from all other
members of Zhangixalus based on 16S rRNA gene
sequences(P=3.4%–8.8%) andisreconstructed asthe sister
species of Z. nigropunctatus. The advertisement call of the
ofnotes,each0.28sindurationand consistingof2–3 pulses
with a dominant frequency of ca. 3.14 kHz. To date,
Zhangixalus melanoleucussp. nov. is known only from the
montane evergreen forests of PSM at elevations of
2 000–2  200ma.s.l.Wepreliminary suggestthenewspecies
should be considered as Data Deficient (DD) following the
The genus Zhangixalus Li, Jiang, Ren & Jiang, 2019 is
widely distributed in East and Southeast Asia, ranging from
northeastern India, Nepal, Bhutan, Myanmar, southern and
Kuhl & Van Hasselt, 1822, members of Zhangixalus can be
morphologically diagnosed by: medium to large body size
eyelids,and tarsaljoint absent;dermal foldsalongforearmor
tarsusabsentor weak;terminalphalangesofdigitsY-shaped;
dorsalskinsurfacessmoothor scatteredwithsmalltubercles;
fingers webbed; dorsal coloration generally green; and iris
lackingX-shapedpattern (Jiangetal., 2019).Currently,there
are40recognized speciesofZhangixalus, one-thirdofwhich
have been described in the last two decades (Frost, 2023).
However, the actual diversity of Zhangixalus remains
underestimated due to the common occurrence of elevated
endemism and narrow-ranged species within the genus
(Dufresnes etal., 2022). The herpetofauna of Laos remains
oneoftheleaststudied andpoorlyknownincontinental Asia.
Presently, only three Zhangixalus species are recorded from
Laos, i.e., Z. dennysi (Blanford), Z. feae (Boulenger), and Z.
pachyproctusYu, Hui,Hou, Wu,Rao, &Rang(Nguyenetal.,
As the third highest summit in Laos, PSM (2  632 m a.s.l.)
forms part of the Central Highlands, along with Phou Bia
(2 819ma.s.l.),PhouXaiLaiLeng (2 714m a.s.l.),andPhou
Sao(2 597ma.s.l.).PSMandthesurroundingmontaneareas
of Xiengkhouang Province are recognized as an amphibian
diversity hotspot in Indochina, exhibiting a high level of local
During our recent fieldwork to PSM in Xiengkhouang
Province,Laos,we encounteredseveral unusual rhacophorid
specimens, which were assigned to the genus Zhangixalus
due to their superficial morphological similarity to Z.
dorsoviridis(Bourret),Z. nigropunctatus(Liu, Hu, &Yang), Z.
pinglongensis (Mo, Chen, Liao, & Zhou), and Z. jodiae
Nguyen, Ninh, Orlov, Nguyen, & Ziegler. Subsequent
molecularphylogenetic analysesrevealed thatthis population
represents a distinct evolutionary lineage, nested within a
single clade with Z. nigropunctatus. Based on a combination
of morphological and bioacoustic characters, as well as
molecular divergence analysis, this population is described
Field surveys were conducted in July 2020 in PSM,
Xiengkhouang Province, northeast Laos (Figure 1A). Details
onspecimen collectionandpreservation arepresented inthe
Supplementary Materials. Specimens were deposited in the
herpetological collections of the Biotechnology and Ecology
Institute, Ministry of Science and Technology of Laos (BEI,
Veintiane, Laos), School of Agriculture and Natural
Resources, University of Phayao (AUP, Phayao, Thailand),
Received:16 January 2023; Accepted: 09 March 2023; Online: 14 March
Foundationitems:Thisworkwassupportedby theUnitof Excellence2023
onBiodiversity andNaturalResources Management,University ofPhayao
(FF66-UoE003, specimen collection) to C.S., and partially by the Russian
Science Foundation (22-14-00037, molecular phylogenetic analyses) to
This is an open-access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://, which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
Copyright ©2023 Editorial Office of Zoological Research, Kunming
Received:16 January 2023; Accepted: 09 March 2023; Online: 14 March
Foundationitems:Thisworkwassupportedby theUnitof Excellence2023
onBiodiversity andNaturalResources Management,University ofPhayao
(FF66-UoE003, specimen collection) to C.S., and partially by the Russian
Science Foundation (22-14-00037, molecular phylogenetic analyses) to
This is an open-access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://, which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
Copyright ©2023 Editorial Office of Zoological Research, Kunming
Brakels et al. Zool. Res. 2023, 44(X): 1−6
Figure 1 Phylogenetic relationships, distribution, acoustics, and external morphology of
Zhangixalus melanoleucus
sp. nov.
A: Distribution of Zhangixalus melanoleucus sp. nov. in Phou Samsoum Mountain, Xiengkhouang Province, northeast Laos. B: Holotype of
Zhangixalus melanoleucus sp.nov. (AUP02505),adultmale,in situin dorsolateralview.C:Sonogramof maleadvertisement callofZhangixalus
melanoleucus sp. nov., with two pulses (C1) and three pulses (C2) per note, recorded at 16.5 °C (at type locality). D: Dorsal life coloration of
Zhangixalus melanoleucussp.nov.holotype(AUP02505).E:VentrallifecolorationofZhangixalus melanoleucussp.nov.holotype(AUP02505).F:
Maximum-likelihood topology of Zhangixalus based on 4  432 bp of concatenated 16S rRNA and COI gene sequences. Ancestral nodes for all
speciesshow strong UFBand BIPP support(100 and 1.00,respectively). Valuesat nodescorrespondto UFB/BIPP, respectively;black circles
support of >90% only; nodes lacking circles are not supported. For specimen and sequence data see Supplementary Table S1. Photos by P.
and Zoological Museum of Lomonosov Moscow State
University (ZMMU, Moscow, Russia). Descriptions of
morphological characteristics of adults and larvae followed
Poyarkov etal. (2015, 2018). Comparative data on
morphological and bioacoustic characteristics of other
(seeSupplementaryMaterials fordetails).Advertisementcalls
of the new species were recorded at the breeding site.
Analyses of acoustic data generally followed Poyarkov etal.
Toreconstructthe matrilinealgenealogyofZhangixalus,we
obtained partial 16S rRNA and COI mtDNA sequences from
the PSM population and compared them with the 16S rRNA
and COI sequences of all available Zhangixalus species
reportedinearlierphylogeneticstudies (e.g.,Dufresnesetal.,
2022).Information onGenBank accessionnumbers, museum
summarized in Supplementary Table S1. DNA extraction,
amplification, and sequencing protocols followed Poyarkov
(ML) and Bayesian inference (BI) approaches (see
SupplementaryMaterials fordetails).Pairwise uncorrectedP-
distances among 16S rRNA sequences of Zhangixalus
species were used to estimate genetic divergence among
species (see Supplementary Table S2). In total, the
concatenated alignment (total length 4  432 bp) included 45
16S rRNA and 32 COI gene sequences from 45
Rhacophoridae specimens, representing 38 nominal species
of Zhangixalus and three outgroup taxa (see Supplementary
Monophyly of the genus Zhangixalus obtained only
moderate nodal support (94/0.97, corresponding to ML ultra-
fast bootstrap (UFB)/BI posterior probability (PP) values,
respectively (same below); see Figure 1F), consistent with
earlier studies (Jiang etal., 2019). Two well-supported major
clades were revealed within Zhangixalus (as in Dufresnes
etal.,2022): onejoiningthreespeciesinhabitingtheMalayan
Peninsula, Sumatra, and Borneo (clade Z1, 100/1.0; see
Figure 1F), the other comprising all remaining species from
EastAsiaand continentalSoutheastAsia (cladeZ2,100/1.0;
see Figure 1F). Within the latter clade, analyses uncovered
several subclades: i.e., Z. feae+Z. pachyproctus+Z.
smaragdinus+Z. suffry (95/0.97; Figure 1F); Z. dennysi+Z.
arvalis (94/0.96; Figure 1F); majority of species from Japan,
China, and northern Indochina (Z. chenfui group, 100/1.0;
Figure1F);andsixspecies fromsouthernmainlandChina(Z.
chenfui, Z. nigropunctatus, Z. pinglongensis, and Z.
yaoshanensis), northernmost Vietnam (Z. jodiae), and the
newly discovered population of Zhangixalus sp. from PSM.
Thelatter wasstronglysuggested tobe asisterlineage ofZ.
nigropunctatus from Guizhou Province in China (100/1.0;
variedfromP=3.4% (with Z. nigropunctatus)to P=8.8% (with
Z. suffry). Values were generally higher than the formal
P=3.0% threshold for the 16S rRNA gene, which is widely
used as an indicator of species-level divergence in anurans
(Vieites etal., 2009). At the same time, genetic distances
weremuchlower, e.g., nodivergencein the 16SrRNA gene
was revealed between Z. burmanus, Z. taronensis, and Z.
gongshanensis (P=0.0%), Z. duboisi and Z. pingbianensis
Z. dugritei and Z. hui (P=0.2%), consistent with Dufresnes
etal.(2022).These results suggest thatthetaxonomy of the
taxa. The congruent molecular, morphological, and acoustic
differences of Zhangixalus sp. from PSM compared to other
congeners suggest evolutionary distinctiveness. As such, we
considerthePhouSamsoumpopulation asadistinctspecies,
Taxonomic account
Zhangixalus melanoleucussp. nov.
(Figure 1; Supplementary Figures S1–S4; Supplementary
Holotype: Adult male BEI 01010 (field ID NAP-09192),
collected 16 July 2020 by P. Brakels, T.V. Nguyen, P.
Pawangkhanant, S. Idiiatullina, and N.A. Poyarkov from the
montane evergreen forest on Phou Samsoum Mountain,
Xiengkhouang Province, northeast Laos (N19.13101°,
E103.78408°;atanelevationof2 066ma.s.l.).
Paratypes: Three adult males BEI 01011 (field ID NAP-
(field ID NAP-09194), and one adult female ZMMU A-7782
(fieldIDNAP-09196), withthe samecollectioninformation as
Referred materials: Three tadpoles ZMMU A-7783 (field ID
NAP-09244), with the same collection information as the
Diagnosis: The new species is assigned to Zhangixalus
basedonthe followingmorphologicalcharacters: (1)medium
bodysize(SVL 34.4–36.3mm inmales,53.7 mmin female);
andeyelids absent;(3)dermal foldson limbsabsent;and(4)
dorsalcolorationgreen (Jiang etal., 2019).The new species
can be distinguished from all congeners by a combination of
the following morphological characters: dorsum smooth and
uniform green with several dark and light-green spots; chest
and belly immaculate white; flanks, axilla, inguinal region,
ventralsurface offorearms, anteriorand posteriorsurfaces of
thighs white, covered with irregular black pattern; finger
webbing formula I 2½–3 II 2–3 III 2¼–2 IV; toe webbing
small black blotches; outer margin of forearms and feet with
weak dermal ridges; supracloacal fold and pointed projection
at tibiotarsal articulation absent; iris reddish-orange; tadpole
Description of holotype: Medium-sized frog specimen in a
good state of preservation; body moderately robust
(Figure 1D–E), SVL 35.0 mm. Head much longer than wide
(HW/HL 0.98), quite deep (HD/HL 0.50), convex above;
dorsally smooth with skin not co-ossified to skull, calcified
warts lacking; snout long (ESL/HL 0.45) and tapering, snout
tip rounded in dorsal view (Supplementary Figure S1A),
roundedin profile(SupplementaryFigureS1C),snoutnotably
projectingbeyond marginoflowerjaw(SupplementaryFigure
S1C),oriented dorsolaterally,located closerto eyethan totip
of snout (END/ESL 0.39; END/NS 0.67); canthus rostralis
distinct, rounded; loreal region slightly concave; eyes large
(ED/HL 0.34), eye diameter less than snout length (ED/ESL
0.75), notably protuberant in dorsal view (Supplementary
Zoological Research44(X):1−6,20233
Figure S1A) and profile (Supplementary Figure S1C), pupil
horizontal, ovoid (Supplementary Figure S1C); tympanum
barely distinct, rounded, with vertical tympanum diameter
equal to horizontal; supratympanic fold distinct, glandular,
S1C),tympanumcomprising halfeyediameter (TD/ED0.49),
located close to eye (TED/ED 0.22); vomerine teeth present,
in two oblique series, closer to choanae than to each other,
separatedbydistanceaboutas longaseachseries;choanae
ovoid;tongueattached anteriorly, deeply notchedposteriorly;
Forelimbs relatively robust; relative finger lengths:
I<II<IV<III; tips of all fingers with well-developed disks with
distinctcircummarginal grooves(SupplementaryFigureS1D),
disksrounded,slightly expanded transversally, diskon finger
III slightly larger than tympanum (FTD/TD 1.07); dermal
fringing along fingers weak (Supplementary Figure S1D);
fingersmoderatelywebbed,webbingformula:I2½–3II 2–3III
2¼–2IV; subarticulartubercles ratherlarge, rounded,notably
protruding, distinct on all fingers, finger subarticular tubercle
formula:1,1, 2,2;twometacarpal(palmar)tuberclespresent,
inner metacarpal tubercle large, ovoid; outer metacarpal
tubercle smaller in size, flattened and heart-shaped; four
supernumerarymetacarpaltubercles atbases offingersII–IV
(Supplementary Figure S1D); nuptial pad present, ovoid,
Hindlimbs robust, moderately short, tibiotarsal articulation
notreachingbeyondtipof snout;tibiaslightlyoverhalf snout-
II1–2 III1–2IV2–1V; weakdermalfringesreachingdisksof
all toes (Supplementary Figure S1E). Tips of toes bearing
disks with distinct circummarginal and transverse grooves;
0.91); relative toe lengths: I<II<V<III<IV; round, distinct, and
protuberant subarticular tubercles present on all toes, toe
subarticular tubercle formula: 1, 1, 2, 3, 2; inner metatarsal
tubercle well-developed, bean-shaped, and notably
protuberant; outer metatarsal tubercle or supernumerary
Skin texture and skin glands: Dorsal skin smooth; ventral
surfaceofchest, venter, and thighscoarselygranular; dorsal
and ventral surfaces of limbs smooth; cloaca and posterior
surface of thighs granular; supracloacal fold and pointed
projection at tibiotarsal articulation absent (Supplementary
Coloration in life:Dorsalsurfaceuniformgrass-green;flanks,
axilla, ventral surfaces of forearms, inguinal, anterior and
posteriorsurfacesof thighs,ventral surfaceofshanks, dorsal
surfacesof feet,and fingersI, II,IIIwhite-creamcoveredwith
irregular black pattern; larger black spot above insertion of
arm. Venter and chest immaculate cream; throat gray with
dark-graymargins,groinregionand ventralsurfacesofthighs
cream; dark-brown stripe from elbow to outer metacarpal
tubercle. Webbing grayish-pink with small black blotches; iris
reddish-orange, scleral ring grayish-blue; pupil horizontal,
Coloration in preservative:Aftertwoyears inethanol,green
colorationturnedbluish-gray; lower part offlanksand ventral
Variationand sexual dimorphism: Male individuals of the
and body coloration (Supplementary Figure S2); SVL varied
from 34.4–36.3 mm in males and 53.7 mm in single female;
measurements of type series are shown in Supplementary
Table S3. Male paratype BEI 01011 had several dark-brown
spots on mid-dorsum; male paratype ZMMU A-7781 had
numerous light-green to yellow spots on dorsum
(Supplementary Figure S2A, B). Female paratype ZMMU A-
7782 had duller dark bottle-green dorsal coloration
(Supplementary Figure S2D). Coloration of Zhangixalus
melanoleucus sp. nov.showedslightvariationinresponseto
day period and microhabitat conditions. In life, dorsum was
somewhatlighternocturnally than duringthe day, withdorsal
surfacesappearing lighttodarkgreen(SupplementaryFigure
S3). Males can be distinguished from the female based on
nuptial pads present; single external subgular vocal sac
Larval morphology: Tadpoles at Gosner developmental
stage 35 were assigned to the new species based on 16S
partialsequencesobtained for onespecimen ZMMU A-7783.
Measurements of the new species tadpoles are presented in
apparatus are shown in Supplementary Figure S4. Detailed
Advertisement call: Call description is based on six
advertisementcallsfrom two individuals(holotype BEI 01010
and paratype ZMMU A-7781). The advertisement call of the
new species represented a series of clicking sounds (notes).
Each note was 0.24–0.30 s in duration (mean 0.28±0.02 s,
n=50) and consisted of 2–3 pulses (mean 2.25±0.38, n=50).
The first pulse was always shorter in duration (0.05–0.11 s,
mean 0.09±0.03 s; n=90) than the second and third pulses
was0.48–0.85 s(mean 0.61±0.09s; n=40)and thedominant
frequency was uniformly 3.14 kHz (3  040–3  280 Hz, mean
3 140±47.06Hz).
Natural history notes: Our knowledge on Zhangixalus
melanoleucussp. nov. biology is scarce. Specimens were
found at night during the rain between 1900h and 2200h in
tropical montane evergreen forest on PSM in Xiengkhouang
Provinceatelevationsof2 000–2 200ma.s.l.Mostspecimens
werefoundon treeor grassleaves,ca. 1.5–3.0mabove the
multi-layeredcanopyand heavy undergrowth, suggestingthe
observed from April to July; in July, males called loudly from
thegrass orwhilesittingonthe edgeor withintemporaryrain
puddles. Tadpoles of the new species were recorded in the
same puddles. Other amphibian species were found at the
same site, including Duttaphrynus cf. melanostictus
(Schneider), Limnonectes taylori Matsui, Panha, Khonsue, &
Kuraishi, Nanorana aenea (Smith), Leptobrachium
masatakasatoi Matsui, Boulenophrys palpebralespinosa
(Bourret), Microhyla butleri Boulenger, Nidirana lini (Chou),
Polypedates impresus Yang, Zhangixalus feae, Gracixalus
yunnanensis Yu, Li, Wang, Rao, Wu, & Yang, and
Rhacophoruscf. rhodopusLiu&Hu.
Distributionand biogeography: The new species is
currently known only from the type locality in high-elevation
montane evergreen forest of PSM, Xiengkhouang Province,
Laos(Figure1A). Further recordsfromother mountainareas
intheHouaphan,Xaisomboun,and Bolikhamxayprovincesof
Laos and adjacent Nghe An Province in Vietnam are
Conservation status:Todate,Zhangixalus melanoleucussp.
nov. is known only from a narrow area within PSM in
XiengkhouangProvince,northeast Laos,whichbelongstothe
newly gazetted 98  873 ha Yod Nam Mo–Phou Samsoum
conservationstatus.We preliminarysuggest thenewspecies
Etymology: The specific epithet melanoleucus” is an
adjective in the nominative case derived from the Ancient
Greek μέλανος” for “black” and λευκός” for “white” and is
giveninreference to thecharacteristicwhite andblackspots
on the flank of the new species. We recommend Phou
Samsoum Treefrog”asthecommonEnglishname.
Morphological comparisons: The new species can be
distinguished from the 25 nominal Zhangixalus species
distributed in Indochina, China, India, and Myanmar by
inguinal, anterior and posterior surfaces of thighs white,
covered with irregular black pattern; and iris reddish-orange
(detailed comparisons are provided in Supplementary Tables
Morphological comparisons of Zhangixalus melanoleucus
sp. nov.withitssisterspeciesZ. nigropunctatusappeartobe
of Z. nigropunctatus and Supplementary Results for
measurementsofthe holotypeofthis species(CIB590405)).
The new species can be readily distinguished from Z.
nigropunctatus by coloration in life, in particular presence of
large irregular black blotches on axilla, flanks, anterior and
posterior surfaces of thighs forming continuous pattern (vs.
small separated indistinct black spots), small back spots on
absent),andiris brightreddish-orange (vs.yellowish-gold).In
morphometrics, males of the new species can be easily
differentiated from Z. nigropunctatus by comparatively larger
head (HL/SVL 36.7% (n=4) vs. 34.5% (n=20, data from
Sciences,2009) inZ. nigropunctatus,33.4% inholotypeofZ.
nigropunctatus, see Supplementary Results); larger
tympanum (TD/SVL 5.9% (n=4) vs. 4.9% (n=20), data from
Sciences, 2009) in Z. nigropunctatus, 4.1% in holotype of Z.
nigropunctatus); comparatively larger eyes (ED/SVL 16.7%
(n=4) vs. 14.0% in holotype of Z. nigropunctatus); larger
internarial distance (IND/SVL 12.2% (n=4) vs. 9.3% in
holotype of Z. nigropunctatus); and comparatively longer
hindlimbs (HLL/SVL 141.7% (n=4) vs. 131.5% in holotype of
Z. nigropunctatus). Furthermore, the new species is clearly
different from Z. nigropunctatus in keratodont row formula
(KRF)of tadpolemouth discs(1:5+5/1+1:2 vs.1:3+3/1+1:1 in
Z. nigropunctatus,datafromEditorialCommitteeofZoologyof
China, Chinese Academy of Sciences, 2009). Moreover, the
closest known population of Z. nigropunctatus in Guizhou
Province (China) is separated from the range of Zhangixalus
melanoleucussp. nov. by over 800 km, providing further
support for our hypothesis that the differentiation between
melanoleucussp. nov. with other congeners are detailed in
the Supplementary Materials and summarized in
The advertisement call of Zhangixalusmelanoleucus sp.
nov. can be readily distinguished from the calls of six other
congeners for which call descriptions are available by higher
dominant frequency (3  140±47.06 Hz) and lower number of
pulses per note (2.25±0.38); comparative bioacoustic
Our phylogenetic study agrees with previous research of
Zhangixalus in recognizing two distinct well-supported major
clades within the genus (Dufresnes etal., 2022; Jiang etal.,
2019). Moreover, several currently recognized species of
Zhangixalus showed almost no or minimal differentiation in
mtDNAgenes (SupplementaryTableS2),andtheir diagnosis
from morphological data alone is also not possible
(summarized in Supplementary Tables S5–S7). Hence, our
data confirmed the synonymy of Rhacophorus taronensis
Smith and R. gongshanensis Yang & Su with Z. burmanus
(Andersson), as proposed by Ohler (2009); and of
Polypedates pingbianensis Kou, Hu, & Gao with Z. duboisi
(Ohler, Marquis, Swan, & Grosjean), as proposed by Orlov
etal. (2002). We also tentatively propose that Rhacophorus
hui Liu should be considered as a junior subjective synonym
of Z. dugritei (David), as our study demonstrated that these
taxadid notdifferin16SrRNA sequences(0.2% divergence,
see Supplementary Table S2) and were morphologically
indistinguishable from each other (Supplementary Table S5).
Furthermore, several currently recognized Zhangixalus
species demonstrated only shallow 16S rRNA gene
divergence (see Supplementary Table S2), including Z.
lishuiensis (Liu, Wang, & Jiang) and Z. zhoukaiyae (Pan,
Zhang,&Zhang) withonly1.6%divergence,aswellas alack
of reliable diagnostic characters to differentiate them
(summarizedinSupplementary TableS6). Further integrative
studies are required to clarify the taxonomic status of Z.
yaoshanensis (Liu & Hu) and Z. pinglongensis (Mo, Chen,
Liao,& Zhou)duetoshallowdivergence(3.4%in 16SrRNA)
and unclear morphological differentiation between these
species (summarized in Supplementary Table S7). Although
we refrain herein from formally proposing synonymy of the
abovementioned taxa, we call for additional investigations to
Ourstudy highlightstheimportantroleof thenorthern Laos
mountains as a crucial center of amphibian diversity and
surveys and integrative taxonomic analyses are required to
expand our understanding of this region’s exceptional
herpetofaunaldiversityand effectivelydevelop science-based
The electronic version of this article in portable document
format represents a published work according to the
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
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
Publication LSID: 
Zoological Research44(X):1−6,20235
Zhangixalusmelanoleucus, LSID: 
Fieldwork in Laos was permitted by the Biotechnology and
Ecology Institute, Ministry of Science and Technology, Lao
PDR (permit No. 009 of 23 June 2020). Specimens were
collected under approval from the Institute of Animals for
Scientific Purposes Development (IAD), which issued
P.B., T.V.N., C.S., and N.A.P. designed the study. P.B.,
N.A.P., T.V.N., P.P, S.S.I., and S.L. collected specimens in
the field. N.A.P., S.S.I., and S.L. performed molecular
experiments. T.V.N, S.S.I., and N.A.P. performed data
C.S., T.V.N., and N.A.P. revised the manuscript. All authors
NAP thanks Andrei N. Kuznetsov (JVRTRTC, Vietnam),
Leonid P. Korzoun (MSU, Russia), Vyacheslav V. Rozhnov
(IPEE RAS, Russia), and Hoi Dang Nguyen (JVRTRTC,
Vietnam)for organizingand supportinghis workin Indochina.
TVN thanks Thai Van Nguyen (SVW, Vietnam) and Toan
Quoc Phan (DTU, Vietnam) for considerable support. We
thankthe anonymousreviewers forcommenting onan earlier
draft of the manuscript. We are grateful to Jian Wang (SYS,
China)for providingaphoto ofZhangixalus nigropunctatusin
1IUCN Laos PDR, Vientiane 01160, Lao PDR
2Institute for Research and Training in Medicine, Biology and
Pharmacy, Duy Tan University, Da Nang 550000, Vietnam
3Faculty of Medicine, Duy Tan University, Da Nang 550000,
4Division of Fishery, School of Agriculture and Natural Resources,
University of Phayao, Phayao, Thailand
5Department of Vertebrate Zoology, Biological Faculty,
Lomonosov Moscow State University, Moscow 119234, Russia
6Biotechnology and Ecology Institute, Ministry of Science and
Technology, Vientiane 01000, Lao PDR
7Joint Russian-Vietnamese Tropical Research and Technological
Center, Nghia Do, Cau Giay, Hanoi 122000, Vietnam
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The results of herpetological surveys conducted throughout Laos in 2016-2019 resulted in significant records at the country and provincial levels for several amphibian and reptile species, other than lizards. Three species, namely Quasipaa verrucospinosa, Gracixalus quangi, and Theloderma lateriticum, were recorded for Laos for the first time. The occurrences of Glyphoglossus molossus, Subsessor bocourti, and Siebenrockiella crassicollis in the country were also confirmed. Species with expanded distributions are represented by new records of Nanorana aenea, Ophryophryne pachyproctus, Xenophrys palpebralespinosa, Glyphoglossus guttulatus, Rana johnsi, Gracixalus quyeti, Theloderma petilum, Zhangixalus feae, Gonyosoma prasinum, Hebius leucomystax, Lycodon futsingensis, Bungarus candidus, Pareas hamptoni, and Trimeresurus gumprechti, which are reported for Laos for the second time. Furthermore, new distribution and natural history data are presented on 27 other poorly-known species from several provinces of Laos. These results suggest that the herpetofaunal diversity in Laos is still underestimated and highlight the importance of conducting further field surveys and elaborating the appropriate conservation actions.
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We discuss phylogenetic relationships and taxonomic diversity of the rhacophorid frogs of the genus Theloderma in sight of the novel phylogenetic data obtained from the Bayesian analysis of the up to 1987 bp length fragment of mtDNA (12S rRNA, tRNAval, and 16S rRNA) from the 90 specimens of 21 nominal species of Theloderma and 3 species of Nyctixalus. Our data suggest monophyly of the tribe Nyctixalini, including Th. moloch, and indicate deep divergence between the three major clades: Th. horridum + Th. stellatum group, Nyctixalus and the rest of the Theloderma species (Theloderma sensu stricto). We establish new subgenus Stelladerma subgen. nov. for Th. horridum + Th. stellatum group and discuss provisional taxonomy of Nyctixalini.We also indicate that the taxonomic status of the certain Indochinese Theloderma requires reassessment. In particular, our data suggest deep divergence between Malayan and Indochinese taxa of Th. asperum group and indicate non-monophyly of Th. asperum sensu lato; we resurrect the name Th. albopunctatum (Liu et Hu, 1962) for the Indochinese species. We provide molecular evidence for synonimization of Th. chuyangsinense Orlov et al., 2012 with Th. palliatum Rowley et al., 2011; as well as morphological and genetic evidence for syninomization of Th. bambusicola Orlov et al., 2012 with Th. laeve (Smith, 1924). We indicate a deep morphological and genetic differentiation within the Th. truongsonense (Orlov et Ho, 2005) complex. Finally, we report on the deep divergence within Th. stellatum Taylor, 1962 from eastern Thailand, southern Cambodia, and Vietnam, and describe a new species, Theloderma vietnamense sp. nov., based on morphological, acoustic and genetic lines of evidence.
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Amphibians are in decline worldwide. However, their patterns of diversity, especially in the tropics, are not well understood, mainly because of incomplete information on taxonomy and distribution. We assess morphological, bioacoustic, and genetic variation of Madagascar's amphibians, one of the first near-complete taxon samplings from a biodiversity hotspot. Based on DNA sequences of 2,850 specimens sampled from over 170 localities, our analyses reveal an extreme proportion of amphibian diversity, projecting an almost 2-fold increase in species numbers from the currently described 244 species to a minimum of 373 and up to 465. This diversity is widespread geographically and across most major phylogenetic lineages except in a few previously well-studied genera, and is not restricted to morphologically cryptic clades. We classify the genealogical lineages in confirmed and unconfirmed candidate species or deeply divergent conspecific lineages based on concordance of genetic divergences with other characters. This integrative approach may be widely applicable to improve estimates of organismal diversity. Our results suggest that in Madagascar the spatial pattern of amphibian richness and endemism must be revisited, and current habitat destruction may be affecting more species than previously thought, in amphibians as well as in other animal groups. This case study suggests that worldwide tropical amphibian diversity is probably underestimated at an unprecedented level and stresses the need for integrated taxonomic surveys as a basis for prioritizing conservation efforts within biodiversity hotspots.
Editorial Committee of Zoology of China
Editorial Committee of Zoology of China, Chinese Academy of Sciences.
Amphibian species of the world 6.1, an online reference
  • D Frost
Frost D. [2023-01-11]. Amphibian species of the world 6.1, an online reference. New York, USA: American Museum of Natural History.
Guidelines for using the IUCN red list categories and criteria. Ver. 14. Prepared by the standards and petitions committee
  • Iucn Standards
  • Petitions Committee
IUCN Standards and Petitions Committee. [2022-09-01]. Guidelines for using the IUCN red list categories and criteria. Ver. 14. Prepared by the standards and petitions committee. documents/RedList-Guidelines.pdf.