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Accepted by J. Rowley: 16 Jan. 2018; published: 2 Mar. 2018
ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN
1175-5334
(online edition)
Copyright © 2018 Magnolia Press
Zootaxa 4388 (2): 191
–
206
http://www.mapress.com/j/zt/
Article
191
https://doi.org/10.11646/zootaxa.4388.2.3
http://zoobank.org/urn:lsid:zoobank.org:pub:C95E6C8A-21FD-41C1-B018-7893EAE6C493
A new phytotelm-breeding treefrog of the genus Nasutixalus (Rhacophoridae)
from western Yunnan of China
JIAN-HUAN YANG
1
& BOSCO PUI-LOK CHAN
Kadoorie Conservation China, Kadoorie Farm and Botanic Garden, Lam Kam Road, Tai Po, Hong Kong, China.
1
Corresponding author. E-mail: jhyang@kfbg.org
Abstract
A new treefrog of the genus Nasutixalus is described from montane evergreen forest in Yingjiang County, in western
Yunnan, China. The new species, Nasutixalus yingjiangensis sp. nov., can be distinguished from its congeners by a
combination of the following characters: medium body size (SVL 39.5–40.0 mm in adult males, 47.5 mm in a single fe-
male); tympanum indistinct and covered with tubercles; disc diameter of third finger greater than tympanum diameter;
dorsal skin relatively smooth, scattered with small tubercles, those on head and anterior dorsum of body more dense and
more prominent; light brown above with a dark brown marking between eyes and two broad dark brown lateral strips on
the dorsum; iris with a weak “X”-shaped, light colored marking; interorbital distance shorter than the upper eyelid width;
comparatively short foot (mean TFL/SVL ratio 67.0% and 62.9% in males and female respectively). The new species
appears to be forest-dependent and was found in well-preserved montane rainforest; it inhabits the canopy and breeds in
tree holes during the rainy season. The type locality of the new species is an isolated forest fragment surrounded by
degraded landscape and efforts are already underway to protect the site. Nasutixalus yingjiangensis sp. nov. represents the
third known species of the newly established genus Nasutixalus.
Key words: new species, taxonomy, molecular, Nasutixalus yingjiangensis sp. nov., Yingjiang County
Introduction
The old world treefrogs of the family Rhacophoridae are currently one of the largest families of amphibians,
containing over 400 recognized species (Frost 2017). To date, 18 genera are recognized within the family, with
seven of them only recognized in the past decade (Frost 2017). The newly described rhacophorid genus
Nasutixalus was established by Jiang et al. (2016) on the basis of evidence from morphological analysis and the
phylogenetic position of its type species, N. medogensis Jiang, Wang, Yan & Che from Medog (=Motuo) in
southeast Tibet. Phylogenetically, the genus Nasutixalus was reconstructed as a member of the clade containing
Kurixalus, Philautus, Beddomixalus, Mercurana, Pseudophilautus and Rarochestes (Jiang et al. 2016). Almost
concurrently, Biju et al. (2016) also described a new genus of Rhacophoridae, Frankixalus, based on phylogenetic,
morphological, osteological and ecological studies, and assigned F. jerdonii (Günther) from north-eastern India as
the type species of the new genus. The phylogenetic data presented in Biju et al. (2016) indicated that Frankixalus
was sister to a clade encompassing Kurixalus, Beddomixalus, Mercurana, Raorchestes and Pseudophilautus; their
analysis also indicated the presence of an unnamed species, referred to as Frankixalus sp., from southern Tibet of
China (= described as N. medogensis in Jiang et al. 2016). Apparently, these two new genera belong to the same
phylogenetic lineage, and therefore Frankixalus is objectively invalid and regarded as a junior synonym of
Nasutixalus based on publication priority (see Sivongxay et al. 2016). As a result, two species of the genus
Nasutixalus are hitherto recognized and recorded from the eastern Himalayas, i.e., N. jerdonii from northeast India,
and N. medogensis from southeast Tibet of China. Both species are reported to inhabit the canopy in montane
evergreen forest at elevations above 1300 m a.s.l. (Biju et al. 2016; Jiang et al. 2016); N. jerdonii was reported to
be a tree hole breeder with peculiar oophagous tadpoles (Biju et al. 2016).
During field surveys between July 2016 and June 2017, we collected specimens of an unknown treefrog from
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Yingjiang County, Yunnan Province, China. The treefrog inhabits the upper tree layer and breeds in tree hole,
sharing the same niche and breeding habits as reported for N. jerdonii (Biju et al. 2016). Subsequent morphological
and molecular studies further revealed that the species is assigned to the genus Nasutixalus. However, this species
can be distinguished from the two known congeners and other rhacophorids by a combination of morphological
characters and molecular divergence; we therefore describe it herein as a new species.
Materials and methods
Sampling. Specimens were collected during fieldwork in Yingjiang County, Yunnan Province of China, fixed and
preserved in 80% ethanol, and deposited at the Museum of Biology, Sun Yat-sen University (SYS). The geographic
position of the surveyed locality and the distribution records of the two other known Nasutixalus species are shown
in Fig. 1.
FIGURE 1. Map showing the known distribution records of the three recognized species of Nasutixalus (refers to Biju et al.
2016; Jiang et al. 2016). Red: Nasutixalus yingjiangensis sp. nov. in Yingjiang County of Yunnan Province, China; blue:
Nasutixalus jerdonii in India; purple: Nasutixalus medogensis in Tibet of China. Stars indicate the type localities of each
species.
DNA extraction and sequencing. Genomic DNA was extracted from the muscle tissue using TIANamp
Genomic DNA Kit. We amplified fragments of the mitochondrial 12S rRNA and 16S rRNA from our Nasutixalus
sp. specimens using the primer pairs L1091/H1478 and L3975/H4551 (Simon et al. 1994; Chen et al. 2013)
respectively. PCR amplifications were performed in a reaction volume of 25 µl containing 100 ng of template
DNA, 0.3 mM of each PCR primer and 10 µl Premix EX TaqTM (Takara). The PCR conditions were an initial
denaturing step at 95 °C for 4 mins, followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 52 °C for 30
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NEW TREEFROG OF NASUTIXALUS FROM CHINA
s an extension step at 72 °C for 1 min, and a final extension of 72 °C for 7 mins. PCR products were purified with
spin columns. The purified products were sequenced with both forward and reverse primers using BigDye
Terminator Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Waltham, Mass., USA) according to the
guidelines of the manufacturer. The products were sequenced on an ABI Prism 3730 automated DNA sequencer at
the ShangHai Majorbio Bio-pharm Technology Co.,Ltd.
TABLE 1. Samples and sequences used in the phylogenetic analysis in this study.
Phylogenetic analyses. In addition to the newly collected specimens, sequences of other two species of the
genus Nasutixalus, as well as representatives of each currently recognized genera of the family Rhacophoridae,
were included in the genetic analysis; Limnonectes poilani was used as an outgroup after Biju et al. (2016) and
Jiang et al. (2016) (Table 1). The resulting sequences were aligned by Muscle 3.6 (Edgar, 2004) under default
Species Locality Voucher No. GenBank No. (12S, 16S)
(1) Nasutixalus sp. Yingjiang, Yunnan, China SYS a005802 MG603585, MG603588
(2) Nasutixalus sp. Yingjiang, Yunnan, China SYS a005803 MG603586, MG603589
(3) Nasutixalus sp. Yingjiang, Yunnan, China SYS a005804 MG603587, MG603590
(4) Beddomixalus bijui Kadalar, Kerala, India SDBDU 2011.1006 KU170017, KU169995
(5) Buergeria oxycephala Hainan, China SCUM 050267YJ EU215524
(6) Chiromantis nongkhorensis Laos FMNH 255378 Q204774, GQ204723
(7) Feihyla palpebralis Lam Dong, Vietnam KIZ 712 GQ285681
(8) Ghatixalus variablilis Mukkurthi NP, Tamil Nadu, India SDBDU 2008.4409 KU170006, KU169981
(9) Gracixalus gracilipes Mt. Dawei, Yunnan, China 060821196RAO GQ285668
(10) Kurixalus odontotarsus Mengyang, Jinghong, China SCUM 060688L EU215549
(11) Liuixalus ocellatus Mt. Wuzhi, Hainan, China HN0806045 GQ285672
(12) Mercurana myristicapalustris Chathankod, Kerala, India SDBDU 2011.849 KU170016, KU169994
(13) Nasutixalus jerdonii Raenghzaeng, Manipur, India SDBDU 2009.46 KU170018, KU169996
(14) Nasutixalus jerdonii Cherrapunjee, Meghalaya, India SDBDU 2009.271 KU230453, KU169997
(15) Nasutixalus jerdonii Mawphlang, Meghalaya, India BNHS 5977 KU230454, KU169998
(16) Nasutixalus jerdonii Mawphlang, Meghalaya, India SDBDU 2009.1166 KU230455, KU169999
(17) Nasutixalus jerdonii Tseminyu, Nagaland, India SDBDU 2009.362 KU230456, KU170000
(18) Nasutixalus jerdonii Sechüma, Nagaland, India SDBDU 2007.054 KU230457, KU170001
(19) Nasutixalus jerdonii Sechüma, Nagaland, India SDBDU 2007.055 KU230458, KU170002
(20) Nasutixalus jerdonii Meriema, Nagaland, India SDBDU 2007.060 KU230459, KU170003
(21) Nasutixalus medogensis Sessa, Arunachal Pradesh, India SDBDU 2009.1295 KU230460, KU170004
(22) Nasutixalus medogensis Sessa, Arunachal Pradesh, India SDBDU 2009.1261 KU230461, KU170005
(23) Nasutixalus medogensis Motuo, Xizang, China 6255Rao GQ285679
(24) Nasutixalus medogensis Motuo, Xizang, China KIZ016395 KU243082
(25) Nyctixalus pictus Sarawak, Malaysia, Borneo MVZ 239460 GQ204783, GQ204732
(26) Philautus aurifasciatus Java, Indonesia AY141805, GQ204702
(27) Polypedates megacephalus Mt. Daiyun, Fujian, China SCUM 050508C EU215552
(28) Pseudophilautus microtympanum Central Hills, Sri Lanka WHT5065 AF249030, GQ204678
(29) Raorchestes glandulosus Coorg, Karnataka, India SDBDU 2011.817 KU170012, KU16998
(30) Rhacophorus reinwardtii Java ZRC 1.1.5273 GQ204771, GQ204720
(31) Taruga fastigo Sri Lanka WHT2783 Y141802, GQ204690
(32) Theloderma asperum Malaysia ZRC 1.1.9321 GQ204776, GQ204725
(33) Limnonectes poilani Vietnam AMNH A163717 DQ283378
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settings and then checked by eye and adjusted if necessary. Phylogenetic trees were constructed using maximum
likelihood (ML) and Bayesian inference (BI). Maximum likelihood analysis was conducted in RAxML v8.2.4
(Stamatakis 2014). Confidence intervals were determined with 1000 bootstrap replicates utilizing the rapid
bootstrap option under the GTRGAMMA substitution model. Bayesian analyses were performed in MrBayes 3.2.6
(Ronquist et al. 2012) under the GTR+I+G model as suggested by the Akaike Information Criterion implemented
in jModelTest 2.1.2 (Darriba et al. 2012). We employed two separate MCMC runs, each with four Metropolis-
coupled chains. The analyses were run for 2.5 million generations, with parameters and topology sampling every
1000 generations. The stationary phase was detected using Tracer 1.6 (Rambaut et al. 2014). The first 1000 trees
were discarded as burn-in and posterior probabilities were determined from the remaining trees. We also calculated
the uncorrected pairwise genetic distances using MEGA 6.06 (Tamura et al. 2013).
Morphological characters. Measurements followed Fei et al. (2009) and Biju et al. (2016), and were taken
with digital callipers to the nearest 0.1 mm: SVL = snout-vent length; HL = head length from tip of snout to the
articulation of the jaw; HW = head width at the greatest cranial width; SL = snout length, from tip of snout to the
anterior corner of the eye; IND = internasal distance; IOD = interorbital distance, closest distance between the
upper eyelids; UEW = maximum upper eyelid width; ED = eye diameter, from the anterior corner of the eye to the
posterior corner of the eye; TD = tympanum diameter; TED = tympanum-eye distance, from the anterior edge of
tympanum to the posterior corner of the eye; LAHL = length of lower arm and hand; HAL = hand length, from the
carpal-metacarpal articulations to the tip of the longest finger; HLL = hindlimb length; FML = femur (thigh)
length; TIB = tibia (shank) length; LFT = length of foot and tarsus; FL = foot length; FDW = finger disc width; FW
= finger width, measured at the base of the disc; TDW = toe disc width; TW = toe width, measured at the base of
the disc; digit number is represented by roman numerals I–V. Webbing formulae are given following Savage (1975)
and Biju et al. (2016). Sex was determined by the presence of nuptial pads. Comparative morphological data of two
known species of Nasutixalus were obtained from the literature: Biju et al. (2016) and Jiang et al. (2016).
Acoustic analyses. The call of an unvouchered individual of the new species was recorded using a TASCAM
DR-40 digital sound recorder (44.1 kHz sampling rate and 24-bit encoding), held approximately 3 m from the
calling individual at an ambient temperature of 15 °C at 21:30 h on 21 April 2017. Calls were analysed with Raven
Pro © v.1.5 software (The Cornell Lab of Ornithology, available from http://www.birds.cornell.edu/raven). The
sonograms and waveforms were generated by Raven Pro 1.5 software with Fast Fourier samples 512 points and
overlap 50%, from which all parameters and characters were measured. For each call recording, we measured the
call duration (s), intercall-interval (s), call repetition rate (calls/min), and dominant frequency (kHz).
Results
Phylogenetic analyses. Two partial sequences of 12S and 16S rRNA genes were combined as a 1266 bp fragment
for the phylogenetic analyses. Both the ML tree and BI analyses based on the fragment combined strongly support
the placement of the unknown rhacophorid specimens from Yingjiang County in the genus Nasutixalus (Fig. 2) and
substantially separate these samples from all currently recognized genera of Rhacophoridae by a large uncorrected
sequence divergence (Table 2). The Nasutixalus sp. from Yingjiang County is reconstructed as a sister species of N.
jerdonii, whereas N. medogensis is a sister lineage to them. For the uncorrected pairwise divergence in the 12S and
16S rRNA genes fragment, there are significantly high genetic distances between the Nasutixalus sp. from
Yingjiang and the other two known congeners, p = 7.1–7.5% in between with N. jerdonii, and p = 7.9% in between
with N. medogensis, while the intraspecific genetic distances between N. jerdonii and N. medogensis are 9.8–
10.2%. These levels of divergences are typically indicative of differentiation at the species level in amphibians
(Vences et al. 2005). As the observed molecular differences are corroborated by differences in morphology (see
below), we therefore consider the Nasutixalus sp. from Yingjiang County to be an undescribed species.
Nasutixalus yingjiangensis sp. nov.
Holotype. SYS a005802, adult male, calling from inside a small tree hole on a small tree (Tetradium glabrifolium,
ca. 13 cm in diameter at breast height) in a montane evergreen broadleaf forest at Tongbiguan Town, Yingjiang
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County, Yunnan Province, China (24°37'21.59'' N, 97°37'13.29'' E, 1610 m above sea level), collected on 20 April
2017 by J.H. Yang.
Paratypes. SYS a005803, adult male; SYS a005804, adult female, both from the same locality as holotype,
collected on 10 June 2017 by J.H. Yang, Shen-Pin Yang, Li-Yan Wang and Rong-Jia Li.
Etymology. The specific name “yingjiangensis”, is a Latinized toponymic adjective in reference to the type
locality of the new species, Yingjiang County of Yunnan Province, China. For the common names, we suggest
“Yingjiang Tree-hole Frog” (English) and “Yíng jiāng léng bí shù wā” (Chinese).
Diagnosis. The new species is assigned to the genus Nasutixalus by the possession of the following
morphological characters considered to be diagnostic for the genus: medium body size ; outline of snout truncate in
dorsal view, semi-circular in ventral view, slightly vertical in lateral view; snout not protruding; canthus rostralis
obtuse and raised prominently, forming a ridge from nostril to anterior corner of eyes; webbing rudimentary on
fingers and well developed on toes; dorsal skin relatively smooth, scattered with small tubercles; iris with a weak
“X”-shaped, light colored marking (Biju et al. 2016; Jiang et al. 2016), and on the basis of molecular analyses (see
above and Fig. 2).
FIGURE 2. Bayesian inference (BI) tree derived from 1266 bp sequences of the mitochondrial 12S and 16S rRNA genes.
Numbers above branches are Bayesian posterior probabilities (> 70% retained) and numbers below branches indicate bootstrap
support values for maximum likelihood analyses (>70% retained).
Nasutixalus yingjiangensis sp. nov. can be distinguished from its congeners by a combination of following
morphological characters: medium body size (SVL 39.5–40.0 mm in adult males, 47.5 mm in a single female);
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tympanum indistinct and covered with tubercles; disc diameter of third finger greater than tympanum diameter;
dorsal skin relatively smooth, scattered with small tubercles, those on head and anterior dorsum of body more
dense and more prominent; light brown above with a dark brown marking between eyes and two broad dark brown
lateral strips on the dorsum; iris with a weak “X”-shaped, light colored marking; interorbital distance shorter than
the upper eyelid width; comparatively short foot (mean TFL/SVL ratio 67.0% and 62.9% in males and a single
female respectively).
Description of holotype (measurements in mm). SYS a005802 (Fig. 3 & 4), adult male, medium body size
(SVL 40.0), body habitus moderate; head width (HW 15.4) slightly greater that head length (HL 13.8); snout length
(SL 5.8) nearly equal to horizontal diameter of the eye (ED 5.4); snout truncated in dorsal view, semi-circular in
ventral view, and nearly slightly vertical in lateral view, not protruding; canthus rostralis rounded and raised
noticeably, forming a ridge from nostril to anterior corner of eye; loreal region slightly concave; interorbital
distance (IOD 3.7), slightly shorter than width of the upper eyelid (UEW 4.0), and internasal distance (IND 4.0);
nostrils notably anterolaterally protuberant, oval, slightly closer to tip of snout (NS 2.4) than to eye (EN 2.6);
tympanum barely distinct and circular (TD 2.2), 0.42 times of eye diameter, distance from eye (TED 0.7) by about
one third of its own diameter; vomerine ridge distinct, obtuse, closer to choanae than each other; vomerine teeth
absent; tongue deeply emarginated without median lingual process; supratympanic fold distinct and gently curved
ventrally, extending from behind the eye, over the tympanum to axilla.
Forelimbs moderately long, robust, lower arm and hand length (LAHL 20.6) about half of body length; relative
length of fingers I<II<IV<III; all finger tips dilated with well-developed disks with distinct circummarginal
grooves (FDWI 1.6, FWI 1.3; FDWII 2.3, FWII 1.3; FDWIII 2.5, FWIII 1.3; FDWIV 2.2, FWIV 1.4), third finger
disk width slightly larger than tympanum diameter; all fingers with distinct lateral fringes on the inner and outer
sides, webbing on fingers rudimentary (I2
+
–2
+
II2–3III2
2
/
3
–2
1
/
2
IV); subarticular tubercles prominently domed,
rounded, formula 1, 1, 2, 2; thenar tubercles well developed; inner and outer metacarpal tubercles distinct, oval and
long, inner one greater than outer one; distinct small supernumerary tubercles present on the base of all fingers;
distinct nuptial pad present, surface microgranular, covering the dorsal surface of the basal phalange of finger I.
Hindlimbs long and slender (HLL 61.0), 171% of SVL; tibiotarsal articulation reaching the posterior corner of
eye when adpressed along the body; shank (TIB 18.4) slightly longer than thigh (FML 18.0), and slightly shorter
than foot (FL 18.9, LFT 26.6); toes moderately long and thick, relative lengths I<II<III<V<IV; tips of toes with
well-developed disks with distinct circummarginal grooves, disks slightly smaller than those of fingers; relative
width of discs I<II=III<V<IV (TDWI 1.5, TWI 1.0, TDWII 2.0, TWII 1.5, TDWIII 2.0, TWIII 1.4, TDWIV 2.4,
TWIV 1.5, TDWV 2.1, TWV 1.3); all toes with distinct lateral fringes on the inner and outer sides, toe webbing
moderate (I1
1
/
3
–2II1–2
+
III1–2
+
IV2–1V); subarticular tubercles distinctly domed, and rounded formula 1, 1, 2, 3, 2;
inner metatarsal tubercle prominent, oval and long (2.1 in length); outer metatarsal tubercle absent; small
supernumerary tubercles presents on the base of all toes; tarsal glandular ridge absent.
Dorsal surfaces of head, body and limbs smooth and scattered with small tubercles, those on dorsal head,
eyelids and anterior body relatively prominent and dense whilst those on posterior body, lower flanks and limbs
relatively weak and scarce; lateral surfaces of head covered with small tubercles, those on temporal region and
supratympanic fold relatively prominent and dense; tympanum finely covered with tubercles; ventral surfaces of
throat, chest, belly and basal thighs densely covered with distinct flat tubercles, those on throat and chest relatively
smaller; ventral surfaces of the forelimbs, shanks and tarsus scattered with small tubercles.
Coloration of holotype in life. Dorsal surface pale brown with a moderate-sized dark inverted triangular-
shaped blotch between the eyes extending to the upper eyelids, and two relatively broad dark lateral stripes that
extend from above scapular region to groin; the two dark stripes on dorsum not in contact and anteriorly separated
from the dark interorbital blotch between eyes; dark brownish broad transverse bands present on dorsal surface of
fore and hind limbs: three on the lower arm, three on the thigh and tibia; a dark line extends from snout, through
nostril and along canthus rostralis to the anterior corner of eye; a short and narrow light yellow stripe present on tip
of snout; a dark blotch present on upper lip under the eye; a distinct small dark spot present on between anterior
corners of eyes; supratympanic fold and tympanic region slightly darker; a relatively large dark blotch present on
above axilla; lower flanks scattered with irregular dark marbling. Ventral surface immaculate, violet-grey in color.
Iris dark blackish, with light golden pigmentation relatively scarce on anterior, posterior, upper and lower corners,
forming an indistinct X-shaped golden marking (Fig.3D).
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FIGURE 3. Holotype of Nasutixalus yingjiangensis sp. nov. (SYS a005802) in life: (A) anterolateral view of right head, noted
the X-shaped iris pattern and raised canthus rostralis; (B) general view, dorsolateral aspect; (C) dorsal view; (D) ventral view;
(E) thenar view of hand; (F) volar view of foot; (G) schematic illustration of foot webbing. Photos by J.H. Yang.
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FIGURE 4. Type s of Nasutixalus yingjiangensis sp. nov. in preservative: (A–B) holotype SYS a005802; (C–D) male paratype
SYS a005803; (E–F) female paratype SYS a005804. Photos by J.H. Yang.
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7$%/(8QFRUUHFWHGpGLVWDQFHVEDVHGRQIUDJPHQWRIWKH6DQG6U51$JHQHV7REHFRQWLQXHG
±
±NasutixalusVS
Beddomixalus bijui ±
Buergeria oxycephala ±
Chiromantis nongkhorensis ±
Feihyla palpebralis ±
Ghatixalus variablilis ±
Gracixalus gracilipes ±
Kurixalus odontotarsus ±
Liuixalus ocellatus ±
Mercurana myristicapalustris
±Nasutixalus jerdonii ± ± ± ± ± ± ± ± ±
±Nasutixalus medogensis ± ± ± ± ± ± ± ±
Nyctixalus pictus
Philautus aurifasciatus
Polypedates megacephalus
Pseudophilautus microtympanum
Raorchestes glandulosus
Rhacophorus reinwardtii
Taruga fastigo
Theloderma asperum
Limnonectes poilani
……continued on the next page
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7$%/(&RQWLQXHG
± ±
±NasutixalusVS
Beddomixalus bijui
Buergeria oxycephala
Chiromantis nongkhorensis
Feihyla palpebralis
Ghatixalus variablilis
Gracixalus gracilipes
Kurixalus odontotarsus
Liuixalus ocellatus
Mercurana myristicapalustris ±
±Nasutixalus jerdonii ±±
±Nasutixalus medogensis ±± ±
Nyctixalus pictus ± ±±
Philautus aurifasciatus ± ±±
Polypedates megacephalus ± ±±
Pseudophilautus microtympanum ± ±
Raorchestes glandulosus ± ± ±
Rhacophorus reinwardtii ± ± ±
Taruga fastigo ± ± ±
Theloderma asperum ± ± ±
Limnonectes poilani ± ± ±
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NEW TREEFROG OF NASUTIXALUS FROM CHINA
Coloration of holotype in preservative. Greyish brown above, dark patterns on dorsum and bandings on
limbs still visible; venter uniform grey, without distinct dark spots or marking; throat greyish white (Fig. 4). Light
golden pigmentation of iris coloration fades to greyish white.
Va ri at io n . The male paratype SYS a005803 and female paratype SYS a005804 match the overall characters of
the holotype (for measurements of the type series see Table 3). The two broad dark stripes in contact with each
other anteriorly and separated from the dark marking on head in male paratype SYS a005803, while the female
paratype SYS a005804 has the two broad dark stripes extend anteriorly and connect with the dark marking on head
(see Fig. 5). A narrow, light yellowish stripe borders the anterior edge of the dark interorbital blotch in the female
paratype SYS a005804 (Fig. 5C). In preservative, the ventral surfaces of body, thigh and tibia of male paratype
SYS a005803 are dull white and scattered with distinct, small dark brown spots and marbling, while the female
paratype SYS a005804 has an immaculate, dark brown venter.
Comparisons. For the two known members of the genus Nasutixalus, N. yingjiangensis sp. nov. differs from
N. medogensis by having a relatively smaller body size (males SVL 39.5–40.0 mm vs. 45.0 mm), interorbital
distance shorter than the upper eyelid width (mean male IOD/UEW ratio 87.9% in the new species vs. 110.3% in N.
medogensis), a relatively shorter foot (mean male TFL/SVL ratio 67.5% in Nasutixalus yingjiangensis sp. nov. vs.
72.7% in N. medogensis), dorsal surfaces of head and the anterior part of body with dense and prominent tubercles
(vs. relatively smooth in N. medogensis), and a different coloration pattern in life (dorsum of body and limbs
without light green patterns in the new species vs. light green patterns present in N. medogensis). Nasutixalus
yingjiangensis sp. nov. can be distinguished from N. jerdonii by having interorbital distance shorter than the upper
eyelid width (mean male IOD/UEW ratio 87.9% in the new species vs. 121.2% in N. jerdonii), a relatively shorter
foot (mean TFL/SVL ratio 67.0% and 62.9% in males and female respectively in the new species. vs. 70.6%
(N=11) and 66.5% (N=1) in N. jerdonii), tympanum indistinct and covered with tubercles (vs. tympanum fully
exposed and smooth in N. jerdonii), and nuptial pads only present on dorsal surface of finger I (vs. present on
dorsal surface of finger I and finger II in male paratype of N. jerdonii, refers to Biju et al. 2016). Finally, the new
species is distinct from all currently recognized species of Nasutixalus in the fragments of the 12S and 16S rRNA
mt DNA genes examined (p> 7.1%).
FIGURE 5. Nasutixalus yingjiangensis sp. nov.: (A–B) adult male paratype SYS a005803 in life; (C–D) adult female paratype
SYS a005804 in life. Photos by J.H. Yang.
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FIGURE 6. Habitat of Nasutixalus yingjiangensis sp. nov.: (A) type locality—mature evergreen montane forest fragment
surrounded by farmland, cleared forest and Betula alnoides timber plantation; (B) microhabitat of the male holotype SYS
a005802, red arrow denotes location of the tree hole, where the calling male holotype was found; (C) close-up of the tree hole
opening located ca. 4.5 m above the forest floor. Photos by Joanne Li (A, B) and Zhi-Hua Zhang (C).
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FIGURE 7. Advertisement call of an unvouchered male individual of Nasutixalus yingjiangensis sp. nov.: (A) waveform and
spectrogram showing a two note portion of a call group; (B) waveform and spectrogram showing a single call.
Distribution and natural history. Nasutixalus yingjiangensis is currently only known from its type locality in
Yingjiang County, western Yunnan, China. The type locality is a small, isolated patch of mature montane evergreen
forest with an average canopy height of 30 m (Fig. 6); this forest remnant is surrounded by a mosaic of farmland,
orchards, Betula alnoides timber plantation, and recently-cleared forest land.
The male holotype SYS a005803 was found calling inside a small tree hole on a Tetradium glabrifolium tree at
21:30 h on 20 April 2017 (Fig, 6B); it has rained during the day. The tree hole was about 4.5 m above the ground;
the opening was small and oval in shape (ca. 18 mm in width and 25 mm in length), and the tree hole cavity had
conical shape and was about 100 mm in depth, 30 mm at the widest and 15 mm at its narrow end. The single female
paratype SYS a005804 was found and collected from inside a large tree hole which was about ca. 5 m above
ground during the day survey on 11 June 2017; the opening was about ca. 80 mm in diameter, while the tree hole
cavity was about ca. 120 mm in depth. The other male paratype SYS a005803 was also found and collected from
inside a small tree hole which was about ca. 2 m above ground during the day survey; the opening was about ca. 40
mm in diameter, while the tree hole cavity was about ca. 80 mm in depth. These observations suggest that the new
species is a phytotelm-breeder, similar to N. jerdonii (Biju et al. 2016).
We conducted four field surveys at the type locality between July 2016 and June 2017. Many males were heard
calling from the canopy (mostly ca. 4 m above ground) during summer surveys on 5 May 2016, 20 April and 10
June 2017; no male calls were detected during the winter survey on 8 December 2016. This indicates the new
species breeds during the rainy season starting from mid-April. Calling activity was highest from dusk until
midnight. Sympatric rhacophorid treefrogs recorded in the forest fragment during the surveys include Rhacophorus
maximus Günther, Rhacophorus rhodopus Liu & Hu, Raorchestes longchuanensis (Yang & Li), Kurixalus
odontotarsus (Ye & Fei) and Polypedates cf. braueri (Vogt). No eggs and tadpoles of Nasutixalus yingjiangensis
were found during the survey.
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Advertisement call. The call series contains a single, pulsed call of 0.297–0.391 s duration (mean 0.344 ±
0.035 s, N=12). The intercall-interval is of 3.527–6.150 s (mean 4.195 ± 0.770 s, N=11). Calls are repeated in series
at a rate of 13.3 times per minute on average. All calls contain a fundamental frequency and a dominant frequency.
The dominant frequency range is of 1.56–1.64 kHz, whereas the fundamental frequency is of 0.76 kHz. A third
harmonic is weakly present at 2.32–2.41 kHz (Fig. 7). To the human ear, the advertisement call of the new species
is a short and low-pitched “uh”.
TABLE 3. Measurements of types of Nasutixalus yingjiangensis sp. nov., and comparison with two congeners
Nasutixalus jerdonii and Nasutixalus medogensis (all measurements in mm).
Remarks: measurements of N. jerdonii and N. medogensis obtained from Biju et al. 2016 and Jiang et al. 2016
respectively.
Discussion
The discovery of Nasutixalus yingjiangensis greatly extends the distribution of the genus Nasutixalus from west
Bengal, through northeast India and southeast Tibet, to western Yunnan of China (Fig. 1), and represents the third
known species of this newly established genus. Due to their cryptic and arboreal habits, Nasutixalus species are
difficult to detect and collect in the wild. Since the original description of Polypedates jerdonii Günther, 1876 (now
Nasutixalus jerdonii) from “Darjeeling” in West Bengal, India, no additional specimens of this mysterious frog
were reported for more than 130 years, until Indian herpetologists rediscovered the species from several locations
in northeast India in 2007 and recognized it as N. jerdonii (Biju et al. 2016). It is noteworthy, that despite intensive
field research efforts, further attempts to find this species in the environs of its type locality in Darjeeling have been
unsuccessful (Biju et al. 2016). The two remaining members of the genus, N. medogensis and N. yingjiangensis
were only discovered in the last two years (Jiang et al. 2016; this study).
The type locality of N. yingjiangensis is less than one kilometer from the boundary of Tongbiguan Provincial
Nature Reserve, and ca. 5 km from the international border with Myanmar’s Kachin State; we can expect that the
new species also occurs in the well-preserved natural forests of Tongbiguan Nature Reserve and Kachin State of
Myanmar. The extensive forests of northern Myanmar is biologically still insufficiently explored; it is likely that
further survey efforts in northern Myanmar could lead to discovery of other Nasutixalus species in future.
Nasutixalus yingjiangensis sp. nov. N. jerdonii N. medogensis
SYS
a005802
SYS
a005803
mean ±SD
(N=2)
SYS
a005804
(N=1) mean ±SD
(N=11)
(N=1)
Sex Male Male Female Female Male Male
SVL 40.0 39.5 39.8±0.35 47.5 46.8 39.9±1.9 45.0
HL 13.8 14.1 14.0±0.21 16 15.7 13.2±0.6 15.5
HW 15.4 15.1 15.3±0.21 17.3 16.9 14.1±0.6 16.1
SL 5.8 5.7 5.8±0.07 6.6 6.7 5.6±0.2 6.8
IOD 3.7 3.5 3.6±0.14 3.7 4.7 4.1±0.1 4.3
IND 4.0 4.2 4.1±0.14 4.6 -- -- 4.9
UEW 4.0 4.2 4.1±0.14 4.6 4.3 3.4±0.3 3.9
ED 5.4 5.8 5.6±0.28 6.2 5.3 4.8±0.4 6.0
TD 2.2 2.2 2.2±0.00 2.1 1.6 1.7±0.2 2.4
LAHL 20.6 20.8 20.7±0.14 23.6 -- -- 24.4
FAL------ --9.87.9±0.6--
HAL 13.1 13.3 13.2±0.14 14.7 15 13.1±0.7 14.9
FML 18.0 17.7 17.9±0.21 19.7 19.7 17.1±0.7 20.8
TIB 18.4 18.2 18.3±0.14 21.3 20.6 18.2±0.5 21.2
TFL 26.6 26.7 26.7±0.07 29.9 31.1 28.2±1.5 32.7
FL 18.9 19.1 19.00.14 21.2 22 19.0±0.9 22.1
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During our survey, male calls of N. yingjiangensis were only detected in mature natural forest with complex,
multi-layered vegetation, and no calls were heard in adjoining old-growth monoculture plantation forest, despite
the fact that Betula alnoides is native to the region, suggesting that the new species is a forest-dependent species
and only live in well-preserved natural forest. In Yingjiang County of Yunnan Province, the major threat to the
local biodiversity is the clearance and degradation of natural forests for agricultural expansion, which severely
fragments the natural forests and threatens forest-dependent species including N. yingjiangensis. The forest
fragment of the type locality of N. yingjiangensis is now leased by a local conservationist who is determined to
protect the site from further encroachment. The Hong Kong-based NGO Kadoorie Farm and Botanic Garden
(KFBG), together with the Critical Ecosystem Partnership Fund (CEPF), are helping the landowner to preserve the
site as a community-protected forest.
Acknowledgements
This study was supported by Kadoorie Farm and Botanic Garden of Hong Kong. We thank Forestry Bureau of
Dehong Dai and Jingpo Autonomous Prefecture, Tongbiguan Nature Reserve, and Forestry Department of
Yingjiang County for their help and support for the survey. We are grateful to Li-Yan Wang and Rong-Jia Li for
their hospitality and kind help during the surveys; Guo-Liang Yang, Shen-Ping Yang, Zhi-Hua Zhang, Xi Zheng
and Joanne Li for field assistance.
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