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Letter to the editor Open Access
Mountainjade:Anewhigh-elevationmicroendemicspecies
ofthegenusZhangixalus(Amphibia:Anura:Rhacophoridae)
fromLaos
DEAR EDITOR,
We report on a new species, Zhangixalus melanoleucussp.
nov.,from PhouSamsoum Mountain(PSM)inXiengkhouang
Province, northeastern Laos, based on an integrative
taxonomicapproach,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
bodysize(SVL 34.4–36.3mmin males,53.7mm inasingle
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
irregularblackpattern;fingerwebbingformulaI2½–3II2–3III
2¼–2IV; toewebbingformulaI2–2½II1–2 III1–2 IV2–1 V;
toewebbing creamwith smallblack blotches;outermarginof
forearmsandfeet withweakdermal ridges;supracloacalfold
andpointedprojectionattibiotarsalarticulationabsent;andiris
reddish-orange. The new species is divergent from all other
members of Zhangixalus based on 16S rRNA gene
sequences(P=3.4%–8.8%) andisreconstructed asthe sister
species of Z. nigropunctatus. The advertisement call of the
newspeciesconsistsofclickingsoundsandincludesaseries
ofnotes,each0.28sindurationand consistingof2–3 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 200ma.s.l.Wepreliminary suggestthenewspecies
should be considered as Data Deficient (DD) following the
IUCN’sRedListcategories.
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
southwesternChina,Japan,throughoutIndochinasouthwards
toMalaysiaandIndonesia(Jiangetal.,2019;Poyarkovetal.,
2021).RecentlyregardedasapartofthegenusRhacophorus
Kuhl & Van Hasselt, 1822, members of Zhangixalus can be
morphologically diagnosed by: medium to large body size
(SVL30–120mm);snoutrounded;projectionsonsnout,upper
eyelids,and tarsaljoint absent;dermal foldsalongforearmor
tarsusabsentor weak;terminalphalangesofdigitsY-shaped;
dorsalskinsurfacessmoothor scatteredwithsmalltubercles;
fingers webbed; dorsal coloration generally green; and iris
lackingX-shapedpattern (Jiangetal., 2019).Currently,there
are40recognized speciesofZhangixalus, one-thirdofwhich
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 etal., 2022). The herpetofauna of Laos remains
oneoftheleaststudied andpoorlyknownincontinental Asia.
Presently, only three Zhangixalus species are recorded from
Laos, i.e., Z. dennysi (Blanford), Z. feae (Boulenger), and Z.
pachyproctusYu, Hui,Hou, Wu,Rao, &Rang(Nguyenetal.,
2020;Poyarkovetal.,2021).
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 819ma.s.l.),PhouXaiLaiLeng (2 714m a.s.l.),andPhou
Sao(2 597ma.s.l.).PSMandthesurroundingmontaneareas
of Xiengkhouang Province are recognized as an amphibian
diversity hotspot in Indochina, exhibiting a high level of local
endemism(seePoyarkovetal.,2021).
During our recent fieldwork to PSM in Xiengkhouang
Province,Laos,we encounteredseveral 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
molecularphylogenetic analysesrevealed thatthis 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
hereinasanewspecies.
Field surveys were conducted in July 2020 in PSM,
Xiengkhouang Province, northeast Laos (Figure 1A). Details
onspecimen collectionandpreservation arepresented inthe
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
2023
Foundationitems:Thisworkwassupportedby theUnitof Excellence2023
onBiodiversity andNaturalResources Management,University ofPhayao
(FF66-UoE003, specimen collection) to C.S., and partially by the Russian
Science Foundation (22-14-00037, molecular phylogenetic analyses) to
N.A.P.
This is an open-access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
providedtheoriginalworkisproperlycited.
Copyright ©2023 Editorial Office of Zoological Research, Kunming
InstituteofZoology,ChineseAcademyofSciences
Received:16 January 2023; Accepted: 09 March 2023; Online: 14 March
2023
Foundationitems:Thisworkwassupportedby theUnitof Excellence2023
onBiodiversity andNaturalResources Management,University ofPhayao
(FF66-UoE003, specimen collection) to C.S., and partially by the Russian
Science Foundation (22-14-00037, molecular phylogenetic analyses) to
N.A.P.
This is an open-access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
providedtheoriginalworkisproperlycited.
Copyright ©2023 Editorial Office of Zoological Research, Kunming
InstituteofZoology,ChineseAcademyofSciences
Brakels et al. Zool. Res. 2023, 44(X): 1−6
https://doi.org/10.24272/j.issn.2095-8137.2022.382
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. (AUP02505),adultmale,in situin dorsolateralview.C:Sonogramof maleadvertisement callofZhangixalus
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 melanoleucussp.nov.holotype(AUP02505).E:VentrallifecolorationofZhangixalus melanoleucussp.nov.holotype(AUP02505).F:
Maximum-likelihood topology of Zhangixalus based on 4 432 bp of concatenated 16S rRNA and COI gene sequences. Ancestral nodes for all
speciesshow strong UFBand BIPP support(100 and 1.00,respectively). Valuesat nodescorrespondto UFB/BIPP, respectively;black circles
representnodeswithBIPPandUFBsupportof>0.95and95%,respectively;whitecirclesrepresentnodeswithBIPPsupportof>0.90onlyorUFB
support of >90% only; nodes lacking circles are not supported. For specimen and sequence data see Supplementary Table S1. Photos by P.
PawangkhanantandN.A.Poyarkov.
2www.zoores.ac.cn
and Zoological Museum of Lomonosov Moscow State
University (ZMMU, Moscow, Russia). Descriptions of
morphological characteristics of adults and larvae followed
Poyarkov etal. (2015, 2018). Comparative data on
morphological and bioacoustic characteristics of other
Zhangixalusspecieswereobtainedfrompreviouspublications
(seeSupplementaryMaterials fordetails).Advertisementcalls
of the new species were recorded at the breeding site.
Analyses of acoustic data generally followed Poyarkov etal.
(2018)andaredetailedintheSupplementaryMaterials.
Toreconstructthe matrilinealgenealogyofZhangixalus,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
reportedinearlierphylogeneticstudies (e.g.,Dufresnesetal.,
2022).Information onGenBank accessionnumbers, museum
vouchers,andoriginlocalityofsequencesusedinthisstudyis
summarized in Supplementary Table S1. DNA extraction,
amplification, and sequencing protocols followed Poyarkov
etal.(2018)andaredetailedintheSupplementaryMaterials.
Weinferredmatrilinealgenealogyusingmaximum-likelihood
(ML) and Bayesian inference (BI) approaches (see
SupplementaryMaterials fordetails).Pairwise uncorrectedP-
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
TableS1).
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 etal., 2019). Two well-supported major
clades were revealed within Zhangixalus (as in Dufresnes
etal.,2022): onejoiningthreespeciesinhabitingtheMalayan
Peninsula, Sumatra, and Borneo (clade Z1, 100/1.0; see
Figure 1F), the other comprising all remaining species from
EastAsiaand continentalSoutheastAsia (cladeZ2,100/1.0;
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;
Figure1F);andsixspecies fromsouthernmainlandChina(Z.
chenfui, Z. nigropunctatus, Z. pinglongensis, and Z.
yaoshanensis), northernmost Vietnam (Z. jodiae), and the
newly discovered population of Zhangixalus sp. from PSM.
Thelatter wasstronglysuggested tobe asisterlineage ofZ.
nigropunctatus from Guizhou Province in China (100/1.0;
Figure1F).
Uncorrectedgeneticdistancesbetweenthe16SrRNAgene
sequencesofZhangixalussp.fromPSMandothercongeners
variedfromP=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 etal., 2009). At the same time, genetic distances
betweenseveralrecognizedspeciesofthegenusZhangixalus
weremuchlower, e.g., nodivergencein the 16SrRNA gene
was revealed between Z. burmanus, Z. taronensis, and Z.
gongshanensis (P=0.0%), Z. duboisi and Z. pingbianensis
(P=0.0%),andonlyminimaldivergencewasrevealedbetween
Z. dugritei and Z. hui (P=0.2%), consistent with Dufresnes
etal.(2022).These results suggest thatthetaxonomy of the
genusZhangixalusisfarfromcompleteandfurtherintegrative
studiesareneededtoclarifythestatusoftheabovementioned
taxa. The congruent molecular, morphological, and acoustic
differences of Zhangixalus sp. from PSM compared to other
congeners suggest evolutionary distinctiveness. As such, we
considerthePhouSamsoumpopulation asadistinctspecies,
whichweformallydescribebelow.
Taxonomic account
Zhangixalus melanoleucussp. nov.
(Figure 1; Supplementary Figures S1–S4; Supplementary
TablesS1–S5)
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°;atanelevationof2 066ma.s.l.).
Paratypes: Three adult males BEI 01011 (field ID NAP-
09193),AUP02507(fieldIDNAP-09195),andZMMUA-7781
(field ID NAP-09194), and one adult female ZMMU A-7782
(fieldIDNAP-09196), withthe samecollectioninformation as
theholotype.
Referred materials: Three tadpoles ZMMU A-7783 (field ID
NAP-09244), with the same collection information as the
holotype.
Diagnosis: The new species is assigned to Zhangixalus
basedonthe followingmorphologicalcharacters: (1)medium
bodysize(SVL 34.4–36.3mm inmales,53.7 mmin female);
(2)supracloacalfoldanddermalprojectionsontibiotarsaljoint
andeyelids absent;(3)dermal foldson limbsabsent;and(4)
dorsalcolorationgreen (Jiang etal., 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,
ventralsurface offorearms, anteriorand posteriorsurfaces of
thighs white, covered with irregular black pattern; finger
webbing formula I 2½–3 II 2–3 III 2¼–2 IV; toe webbing
formulaI2–2½II1–2III1–2IV2–1V;toewebbingcreamwith
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
mouthdisckeratodontrowformula(KRF)1:5+5/1+1:2.
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),
roundedin profile(SupplementaryFigureS1C),snoutnotably
projectingbeyond marginoflowerjaw(SupplementaryFigure
S1C);nostrilovoid,slightlyprotuberant(SupplementaryFigure
S1C),oriented dorsolaterally,located closerto eyethan totip
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 Research44(X):1−6,20233
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,
elevatedaboveskinoftemporalregion(SupplementaryFigure
S1C),tympanumcomprising halfeyediameter (TD/ED0.49),
located close to eye (TED/ED 0.22); vomerine teeth present,
in two oblique series, closer to choanae than to each other,
separatedbydistanceaboutas longaseachseries;choanae
ovoid;tongueattached anteriorly, deeply notchedposteriorly;
singleexternalvocalsac.
Forelimbs relatively robust; relative finger lengths:
I<II<IV<III; tips of all fingers with well-developed disks with
distinctcircummarginal grooves(SupplementaryFigureS1D),
disksrounded,slightly expanded transversally, diskon finger
III slightly larger than tympanum (FTD/TD 1.07); dermal
fringing along fingers weak (Supplementary Figure S1D);
fingersmoderatelywebbed,webbingformula:I2½–3II 2–3III
2¼–2IV; subarticulartubercles ratherlarge, rounded,notably
protruding, distinct on all fingers, finger subarticular tubercle
formula:1,1, 2,2;twometacarpal(palmar)tuberclespresent,
inner metacarpal tubercle large, ovoid; outer metacarpal
tubercle smaller in size, flattened and heart-shaped; four
supernumerarymetacarpaltubercles atbases offingersII–IV
(Supplementary Figure S1D); nuptial pad present, ovoid,
elongated,coveringprepollexarea(MCTi/1FLi0.42).
Hindlimbs robust, moderately short, tibiotarsal articulation
notreachingbeyondtipof snout;tibiaslightlyoverhalf snout-
ventlength(TL/SVL0.42);dermalridgealongoutersurfaceof
tibiaweak;toesmoderatelywebbed,webbingformula:I2–2½
II1–2 III1–2IV2–1V; weakdermalfringesreachingdisksof
all toes (Supplementary Figure S1E). Tips of toes bearing
disks with distinct circummarginal and transverse grooves;
disksrounded,slightlysmallerthanthoseoffingers(HTD/FTD
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
tuberclesabsent(SupplementaryFigureS1E).
Skin texture and skin glands: Dorsal skin smooth; ventral
surfaceofchest, venter, and thighscoarselygranular; dorsal
and ventral surfaces of limbs smooth; cloaca and posterior
surface of thighs granular; supracloacal fold and pointed
projection at tibiotarsal articulation absent (Supplementary
FigureS1).
Coloration in life:Dorsalsurfaceuniformgrass-green;flanks,
axilla, ventral surfaces of forearms, inguinal, anterior and
posteriorsurfacesof thighs,ventral surfaceofshanks, dorsal
surfacesof feet,and fingersI, II,IIIwhite-creamcoveredwith
irregular black pattern; larger black spot above insertion of
arm. Venter and chest immaculate cream; throat gray with
dark-graymargins,groinregionand ventralsurfacesofthighs
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,
black(Figure1;SupplementaryFigureS1).
Coloration in preservative:Aftertwoyears inethanol,green
colorationturnedbluish-gray; lower part offlanksand ventral
surfaceofbodyandlimbsgraywhite,contrastingblackpattern
onflankswelldiscernable.
Variationand sexual dimorphism: Male individuals of the
typeserieswereverysimilarinmorphology,bodyproportions,
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.showedslightvariationinresponseto
day period and microhabitat conditions. In life, dorsum was
somewhatlighternocturnally than duringthe day, withdorsal
surfacesappearing lighttodarkgreen(SupplementaryFigure
S3). Males can be distinguished from the female based on
nuptial pads present; single external subgular vocal sac
present;andthroatsgrayish.
Larval morphology: Tadpoles at Gosner developmental
stage 35 were assigned to the new species based on 16S
partialsequencesobtained for onespecimen ZMMU A-7783.
Measurements of the new species tadpoles are presented in
SupplementaryTableS4.Tadpolecolorationinlifeandmouth
apparatus are shown in Supplementary Figure S4. Detailed
descriptionoflarvalmorphologyisgivenintheSupplementary
Materials.
Advertisement call: Call description is based on six
advertisementcallsfrom 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
(0.06–0.19s,mean0.15±0.04s;n=70).Theinter-noteinterval
was0.48–0.85 s(mean 0.61±0.09s; n=40)and thedominant
frequency was uniformly 3.14 kHz (3 040–3 280 Hz, mean
3 140±47.06Hz).
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
Provinceatelevationsof2 000–2 200ma.s.l.Mostspecimens
werefoundon treeor grassleaves,ca. 1.5–3.0mabove the
ground,inpatchesofprimaryundisturbedforestwithcomplete
multi-layeredcanopyand heavy undergrowth, suggestingthe
newspeciesisastrictforest-dwellingspecialist.Breedingwas
observed from April to July; in July, males called loudly from
thegrass orwhilesittingonthe edgeor withintemporaryrain
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
Rhacophoruscf. rhodopusLiu&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(Figure1A). Further recordsfromother mountainareas
intheHouaphan,Xaisomboun,and Bolikhamxayprovincesof
Laos and adjacent Nghe An Province in Vietnam are
4www.zoores.ac.cn
anticipated.
Conservation status:Todate,Zhangixalus melanoleucussp.
nov. is known only from a narrow area within PSM in
XiengkhouangProvince,northeast Laos,whichbelongstothe
newly gazetted 98 873 ha Yod Nam Mo–Phou Samsoum
NationalProtectedArea(NPA).Furtherresearchisrequiredto
clarifytheextentofitsdistribution,populationsize,trends,and
conservationstatus.We preliminarysuggest thenewspecies
beconsideredasDDfollowingtheIUCN’sRedListcategories
(IUCNStandardsandPetitionsCommittee,2022).
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
giveninreference to thecharacteristicwhite andblackspots
on the flank of the new species. We recommend “ Phou
Samsoum Treefrog”asthecommonEnglishname.
Morphological comparisons: The new species can be
distinguished from the 25 nominal Zhangixalus species
distributed in Indochina, China, India, and Myanmar by
dorsumgreen;belly,flanks,axilla,ventralsurfaceofforearms,
inguinal, anterior and posterior surfaces of thighs white,
covered with irregular black pattern; and iris reddish-orange
(detailed comparisons are provided in Supplementary Tables
S5–S7).
Morphological comparisons of Zhangixalus melanoleucus
sp. nov.withitssisterspeciesZ. nigropunctatusappeartobe
themostpertinent(seeSupplementaryFigureS5forlifephoto
of Z. nigropunctatus and Supplementary Results for
measurementsofthe holotypeofthis species(CIB590405)).
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
ventralsurfacesofthighsandtarsus(vs.yellowish,backspots
absent),andiris brightreddish-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
EditorialCommitteeofZoologyofChina,ChineseAcademyof
Sciences,2009) inZ. nigropunctatus,33.4% inholotypeofZ.
nigropunctatus, see Supplementary Results); larger
tympanum (TD/SVL 5.9% (n=4) vs. 4.9% (n=20), data from
EditorialCommitteeofZoologyofChina,ChineseAcademyof
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 tadpolemouth discs(1:5+5/1+1:2 vs.1:3+3/1+1:1 in
Z. nigropunctatus,datafromEditorialCommitteeofZoologyof
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
thesetaxareachesspecieslevel.ComparisonsofZhangixalus
melanoleucussp. nov. with other congeners are detailed in
the Supplementary Materials and summarized in
SupplementaryTableS5.
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
informationispresentedinSupplementaryTableS8.
Our phylogenetic study agrees with previous research of
Zhangixalus in recognizing two distinct well-supported major
clades within the genus (Dufresnes etal., 2022; Jiang etal.,
2019). Moreover, several currently recognized species of
Zhangixalus showed almost no or minimal differentiation in
mtDNAgenes (SupplementaryTableS2),andtheir 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
etal. (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
taxadid notdifferin16SrRNA 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) withonly1.6%divergence,aswellas alack
of reliable diagnostic characters to differentiate them
(summarizedinSupplementary TableS6). Further integrative
studies are required to clarify the taxonomic status of Z.
yaoshanensis (Liu & Hu) and Z. pinglongensis (Mo, Chen,
Liao,& Zhou)duetoshallowdivergence(3.4%in 16SrRNA)
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
clarifythetaxonomicstatusofthesespecies.
Ourstudy highlightstheimportantroleof thenorthern Laos
mountains as a crucial center of amphibian diversity and
endemisminIndochina(Poyarkovetal.,2021).Additionalfield
surveys and integrative taxonomic analyses are required to
expand our understanding of this region’s exceptional
herpetofaunaldiversityand effectivelydevelop science-based
measuresforitsconservation.
NOMENCLATURAL ACTS REGISTRATION
The electronic version of this article in portable document
format represents a published work according to the
InternationalCommissiononZoologicalNomenclature(ICZN),
andhence thenewnamescontainedin theelectronic 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
beenregisteredinZooBank,theonlineregistrationsystemfor
the ICZN. The ZooBank LSIDs (Life Science Identifiers) can
be resolved and the associated information can be viewed
throughany standardwebbrowserbyappending theLSID to
theprefixhttp://zoobank.org/.
Publication LSID: urn:lsid:zoobank.org:pub:601DF810-CCDE-
4D0A-A8AF-81837DDE02EA
Zoological Research44(X):1−6,20235
Zhangixalusmelanoleucus, LSID: urn:lsid:zoobank.org:act:
A987B391-AFFD-4A47-BEB2-F51C0468F977
SCIENTIFIC FIELD SURVEY PERMISSION INFORMATION
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
fieldworkpermission(No.610104022).
SUPPLEMENTARY DATA
Supplementarydatatothisarticlecanbefoundonline.
COMPETING INTERESTS
Theauthorsdeclarethattheyhavenocompetinginterests.
AUTHORS’ CONTRIBUTIONS
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
analyses.P.B.,C.S.,T.V.N.,andN.A.P.wrotethemanuscript.
C.S., T.V.N., and N.A.P. revised the manuscript. All authors
readandapprovedthefinalversionofthemanuscript.
ACKNOWLEDGEMENTS
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 organizingand supportinghis workin Indochina.
TVN thanks Thai Van Nguyen (SVW, Vietnam) and Toan
Quoc Phan (DTU, Vietnam) for considerable support. We
thankthe anonymousreviewers forcommenting onan earlier
draft of the manuscript. We are grateful to Jian Wang (SYS,
China)for providingaphoto ofZhangixalus nigropunctatusin
life,andtoChung-WeiYou(Taipei,China)forassistance.
PeterBrakels1,#,TanVanNguyen2,3,#,
ParinyaPawangkhanant4,SabiraS.Idiiatullina5,
SengvilayLorphengsy6,ChatmongkonSuwannapoom4,*,
NikolayA.Poyarkov5,7,*
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,
Vietnam
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
#Authorscontributedequallytothiswork
*Correspondingauthors,chatmongkonup@gmail.com;
n.poyarkov@gmail.com
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