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Sorting out moss frogs: mtDNA data on taxonomic diversity and phylogenetic relationships of the Indochinese species of the genus Theloderma (Anura, Rhacophoridae)

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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.
Distribution of Theloderma stellatum complex in southern Indochina and Thailand. A dot in the center of an icon indicates the type locality. Filled icon indicates that the population has been assessed molecularly and its identification was confirmed by mtDNA sequencing. Locality information. Theloderma stellatum: Thailand: 1, Hauy Kha Khaeng Wildlife Sanctuary, Uthai Thani Province (this paper); 2, Wangnamkhiew District, Nakor Ratchasrima Province (this paper); 3, Khao Yai National Park, Nakhon Nayok Province (this paper); 4, Nang Rong waterfall, Nakhon Nayok Province (this paper); 5, Pangsida National Park, Sa Kaew Province (this paper); 6, Khao Soi Dao Wildlife Sanctuary, Chanthaburi Province (this paper); 7, Khao Ang Rue Nai Wildlife Sanctuary, Chonburi Province (this paper); 8, Khao Kitchakut National Park, Chanthaburi Province (this paper); 9, Phliu waterfall envrions, Chanthaburi Province (this paper); 10, Khao Se Bab, Nam Tok Plew (Phliu) National Park, Chanthaburi province (type locality of Theloderma stellatum Taylor 1962); 11, Koh Chang National Park, Trad Province (this paper); 12, Koh Kut, Trad Province (this paper); Cambodia: 13, Koh Kong Province (Neang and Holden, 2008); 14, Central Cardamom Protected Forest, Pursat Province (Neang and Holden, 2008); Theloderma vietnamense sp. nov.: Cambodia: 15, Khao Seima National Park, Mondulkiri Province (Neang and Holden, 2008; Rowley et al., 2011); 16, Steng Chhral, Kampot Province (this paper); Vietnam: 17, Phu Quoc National Park, Phu Quoc, Kien Giang Province (Nguyen and Nguyen, 2008; Nguyen et al., 2009; this paper); 18, Nui Dai Mt., An Nong, Tinh Bien Commune, An Giang Provine (Ngyen Ngoc Hung, communication; this paper); 19, Con Dao National Park, Con Son Island, Ba Ria-Vung Tau Province (Poyarkov and Vassilieva, 2011; this paper); 20, Lo Go-Xa Mat National Park, Tay Ninh Province (this paper); 21, Binh Chau-Phuok Buu National Park, Ba Ria-Vung Tau Province (this paper); 22, Binh Thuan Province (Rowley et al., 2011); 23, Bac Binh District, Binh Thuan Province (NCSM 80384; Dever et al., 2015); 24, Ma Da Forestry, Dong Nai Biosphere Reserve, Dong Nai Province (Nguyen et al., 2009); 25, Cat Tien National Park, Dong Nai Biosphere Reserve, Dong Nai Province (type locality of Theloderma vietnamense sp. nov., this paper); 26, Loc Bac (Loc Bao) Forestry, Lam Dong Province (this paper); 27, Bu Gia Map National Park, Binh Phuok Province (this paper); 28, Yok Don Mt., Yok Don National Park, Dak Lak Province (Nguyen et al., 2009; this paper); 29, Krong Pa, Gia Lai Province (Nguyen et al., 2009); 30, Kon Ka Kinh Nature Reserve, Gia Lai Province (Nguyen et al., 2009); 31, Phu Yen Province (Nguyen et al., 2014); 32, K'Bang; Kon Cha Rang Nature Reserve, Gia Lai Province (Nguyen et al., 2014); 33, Mang Canh environs, Kon Plong, Kon Tum Province (Orlov and Ananjeva, 2007; Nguyen et al., 2014; this paper); 34, Nui Son Tra Mt., Danang City (this paper); 35, Nam Sagi River Drainage Basin, Vilabouli District, Savannakhet Province, Laos (NCSM 76485-8; Dever et al., 2015); 36, Tham Bing Cave, Xepon Mines, Vilabouli District, Savannakhet Province, Laos (NCSM 76490; Dever et al., 2015); 37, Phong Nha-Ke Bang National Park, Quang Binh Province (Luu et al., 2013).
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SORTING OUT MOSS FROGS: mtDNA DATA ON TAXONOMIC DIVERSITY
AND PHYLOGENETIC RELATIONSHIPS OF THE INDOCHINESE SPECIES
OF THE GENUS Theloderma (ANURA, RHACOPHORIDAE)
Nikolay A. Poyarkov, Jr.,1,2 Nikolai L. Orlov,3Anna V. Moiseeva,1
Parinya Pawangkhanant,4Thiti Ruangsuwan,5Anna B. Vassilieva,1,2
Eduard A. Galoyan,2,6 Tao Thien Nguyen,7Svetlana S. Gogoleva2,6
Submitted September 3, 2015.
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 indi-
cate 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 tax-
onomic 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.
Keywords: mitochondrial DNA; molecular phylogeny; sequence divergence; morphology; morphometrics; adver-
tisement call; cryptic species; Vietnam.
INTRODUCTION
The family Rhacophoridae is one of the most diverse
and speciose amphibian groups in the world, consisting
of 388 currently recognized species in 17 genera and rep-
resenting almost 6% of all known anurans (Amphibia-
Web, 2015; Frost, 2015). This largely arboreal group of
frogs is distributed throughout sub-Saharan Africa,
China, Southern and Southeast Asia, Japan, Taiwan, the
Philippines, and the Greater Sunda Islands (Frost, 2015).
Among the rhacophorid genera, the taxonomy of
Theloderma Tschudi, 1838 has been revised several
times and has been a subject of recent intensive phylo-
genetic studies (e.g., Rowley et al., 2011; Dever et al.,
2015; Nguyen et al., 2015). To date, 23 recognized spe-
cies of Theloderma are distributed throughout Southeast
1026-2296/2015/2204-0241 © 2015 Folium Publishing Company
Russian Journal of Herpetology Vol. 22, No. 4, 2015, pp. 241 – 280
1Department of Vertebrate Zoology, Biological Faculty, M. V. Lomonosov Moscow State University, Leninskiye Gory, GSP-1, Moscow 119991,
Russia; e-mail: n.poyarkov@gmail.com.
2Joint Russian-Vietnamese Tropical Research and Technological Center under the A. N. Severtsov Institute of Ecology and Evolution RAS, South
Branch, 3, Street 3/2, 10 District, Ho Chi Minh City, Vietnam.
3Zoological Institute, Russian Academy of Sciences, Universitetskaya nab., 1, St. Petersburg 199034, Russia.
4Bansomdejchaopraya Rajabhat University, 1061 Soi 15, Itsaraphab Rd., Hiran Ruchi, Thon Buri, Bangkok 10600, Thailand.
5Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
6Zoological Museum, M. V. Lomonosov Moscow State University, Bolshaya Nikitskaya str. 6, Moscow 125009, Russia.
7Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam.
Asia from Assam in northeastern India to Myanmar,
southern China and Indochina, to Malay peninsula, Su-
matra and Borneo of the Greater Sundas, but new species
in the genus continue to be discovered with 11 species
have been described in the last 15 years (Frost, 2015).
The frogs of the genus Theloderma (see Fig. 1) are
small (Th. andersoni SVL up to 20 mm; Th. bambusicola
SVL up to 26 mm) to medium (Th. horridum SVL up to
45 mm, Fig. 1a) and large sized (Th. ryabovi SVL up to
65 mm, Fig. 1i,j;Th. corticale SVL up to 75 mm,
Fig. 1l) strictly arboreal species, which are supposed to
be distinguished from other Rhacophoridae by the fol-
lowing combination of morphological attributes: (1) hav-
ing some degree of tuberculate skin with sometimes cal-
cified warts on the dorsum [though skin is totally smooth
in Th. bambusicola (Fig. 1t), and almost smooth in
Th. laeve (Fig. 1v), Th. lateriticum (Fig. 1o) and Th. li-
cin (Fig. 1e)], (2) two slips of M. extensor digitorum
communis longus (but only shown for Th. gordoni and
Th. stellatum by Liem, 1970), (3) Y-shaped terminal pha-
langes (vs. simple or bifurcate in Philautus ), (4) a dis-
tinct tympanum, (5) rounded canthus rostralis (vs. sharp
in Nyctixalus Boulenger, 1882; Fig. 1d), (6) absence of
bony ridges from canthus rostralis to occiput (vs. present
in Nyctixalus), (7) skin on head not co-ossified with skull
(vs. co-ossified in Nyctixalus), and (8) reproductive be-
havior of depositing eggs in water-filled tree hollows,
karst crevices or man-made ponds (this feature shared
with closely-related genus Nyctixalus and more remote
Kurixalus ) (Taylor, 1962; Liem, 1970; Manthey and
Grossmann, 1997; Orlov et al., 2006, 2010, 2012;
McLeod and Norhayati, 2007; Rowley et al., 2011; Dever
et al., 2015; Nguyen et al., 2015).
However, none of these characteristics forms a mor-
phological synapomorphy for Theloderma with all of
them being convergent characters found in other Rhaco-
phoridae members, though their combination appears to
be unique for Theloderma (Liem, 1970; Dever et al.,
2015). Therefore, because no morphological synapomor-
phy is known for the genus Theloderma, the monophyly
of the group has been put into question (Wilkinson and
Drewes, 2000; Wilkinson et al., 2002; McLeod and
Norhayati, 2007; Yu et al., 2007, 2008; Bain et al., 2009;
Li et al., 2008, 2009, 2013; Rowley et al., 2011; Nguyen
et al., 2015; Dever et al., 2015). Furthermore, from the re-
sults of recent extensive fieldwork and molecular analy-
ses, some species with smooth skin or unwebbed fingers,
which were once placed in other Rhacophoridae genera
such as Philautus,Chirixalus, and Aquixalus, have been
transferred to this genus (e.g., Yu et al., 2007; Rowley
et al., 2011; Orlov et al., 2006, 2012; Nguyen et al., 2014,
2015).
Traditional works on phylogenetic relationships of
Rhacophoridae are scarce and include few members of
Theloderma. The most extensive work by Liem (1970)
suggested close affinities between Theloderma and Nyc-
tixalus based mostly on similarities in reproductive biol-
ogy; though the phylogenetic analysis implemented in
this work placed these genera distantly from each other.
Later, Channing (1989) suggested sister relationships
between Theloderma and Nyctixalus based on morpho-
logical data. Recently, Dubois (2005) assigned both The-
loderma and Nyctixalus to the tribe Philautini (including
also Aquixalus,Kurixalus, and Philautus), whereas more
recently Grosjean et al. (2008) proposed separation of
Theloderma and Nyctixalus in the new tribe Nyctixalini.
This assignment and distant phylogenetic position of
Theloderma and Nyctixalus in respect to other members
of the subfamily Rhacophorinae was also supported by
subsequent phylogenetic analyses (Li et al., 2008, 2009,
2013; Pyron and Wiens, 2011). Based on the analysis of
mtDNA genetic markers, Li et al. (2008) showed sister
clade relationships between Nyctixalus (N. pictus and N.
spinosus ) and Theloderma (Th. corticale,Th. rhododis-
cum, and Th. asperum) and were first to report the distant
phylogenetic position of an enigmatic rhacophorid speci-
men identified as Th. moloch, not forming a monophyly
with Theloderma +Nyctixalus clade.
Yu et al. (2008) based on the analysis of mtDNA ge-
netic markers, suggested sister clade relationships of
Theloderma and Nyctixalus and showed that Philautus
albopunctatus from China should be assigned to the ge-
nus Theloderma and appears to be a junior synonym of
Th. asperum. This result was also confirmed by Rowley
et al. (2011) who performed a DNA-barcoding study
based on short-length fragments of the 16S rRNA
mtDNA gene and obtained a phylogenetic tree, suggest-
ing paraphyly of Theloderma with respect to the genus
Nyctixalus, but poorly resolving phylogenetic relation-
ships among nine species of Theloderma (Th. rhododis-
cum,Th. palliatum,Th. bicolor,Th. corticale,Th. gordo-
ni,Th. nebulosum,Th. truongsonense,Th. asperum,
Th. stellatum) and one species of Nyctixalus (N. pictus)
studied. The study by Rowley et al. (2011) also illustrated
that Philautus truongsonensis should be shifted to the ge-
nus Theloderma, based on mtDNA genetic data.
One of the most extensive studies performed recently
is the work of Nguyen et al. (2015) who included 17 out
of 23 Theloderma species and all the three recognized
Nyctixalus species in their analysis, based on 2412 bp se-
quences of the mitochondrial DNA genes of 12S rRNA,
tRNAval, and 16S rRNA. Their study clarified prelimi-
nary phylogenetic relationships within the genus and as-
sumed sister clade relationships between Theloderma
and Nyctixalus. According to their data Theloderma mo-
242 Nikolay A. Poyarkov, Jr., et al.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 243
kl
mn
o
p
ij
cd
ef
gh
ab
st
uv
wx
qr
Fig. 1. Theloderma species of Indochina and Thailand. a,Th. horri-
dum (Thailand, Satun Province, Tha Le Ban N. P., photo N. A. Poyar-
kov); b,Th. stellatum (Thailand, Uthai Thani Province, Hauy Kha
Khaeng W. S.; photo P. Pawangkhanant); c,Th. cf. stellatum [de-
scribed below as Th. vietnamense sp. nov.] (Vietnam, Kon Tum Prov-
ince, Kon Plong); d,Th. pictum (Thailand, Satun Province, Tha Le
Ban N. P.; photo P. Pawangkhanant); e,Th. licin (Thailand, Satun
Province, Tha Le Ban N. P., photo P. Pawangkhanant); f,Th. cf. aspe-
rum, northern population (Vietnam, Vinh Phuc Province, Tam Dao;
photo N. L. Orlov); g,Th. cf. asperum, southern population (Viet-
nam, Kon Tum Province, Kon Plong; photo N. L. Orlov); h,Th. aspe-
rum (Thailand, Kamphaeng Phet Province, photo P. Pawangkhanant);
i,Th. ryabovi, male (Vietnam, Kon Tum Province, Kon Plong; photo
N. L. Orlov); j,Th. ryabovi, female (Vietnam, Kon Tum Province,
Kon Plong; photo N. L. Orlov); k,Th. bicolor (Vietnam, Lao Cai, Fan
Si Pan Mt.; photo N. L. Orlov); l,Th. corticale (Vietnam, Quang Tri
Province, Ban Cup; photo N. L. Orlov); m,Th. gordoni, northern
population (Vietnam, Lao Cai, Sa Pa; photo N. L. Orlov); n,Th. gor-
doni, southern population (Vietnam, Kon Tum Province, Kon Plong;
photo N. L. Orlov); o,Th. lateriticum (Vietnam, Bac Giang; photo
Nguyen Thien Tao); p,Th. rhododiscum (China, Yunnan; photo
N. L. Orlov); q,Th. palliatum (Vietnam, Lam Dong Province, Bi
Doup; photo E. A. Galoyan); r,Th. chuyangsinense (Vietnam, Dak
Lak Province, Chu Yang Sin; photo N. A. Poyarkov); s,Th. truong-
sonense (Vietnam, Quang Tri Province, Ban Cup; photo N. L. Orlov);
t,Th. bambusicola (Vietnam, Lam Dong Province, Cat Loc; photo
N. A. Poyarkov); u,Th. cf. nebulosum (Vietnam, Kon Tum Province;
photo N. L. Orlov); v,Th. cf. truongsonense (Vietnam, Kon Tum
Province, Kon Plong; photo N. L. Orlov); w, (Vietnam, Khanh Hoa
Province, Hon Ba; photo A. B. Vassilieva); x,Th. petilum (Vietnam,
Dien Bien Province, Muong Nhe; photo Le Trung Dung).
loch is phylogenetically outside Theloderma and Nycti-
xalus clade and closer to Chiromantis,Feihyla,Gracixa-
lus,Kurixalus,Philautus,Polypedates,Raorchestes, and
Rhacophorus.
However, these results contradict the recent results
by Li et al. (2013) based on the large dataset of 2041 bp
of mtDNA and 3225 bp of five nuclear DNA genes for all
rhacophorid genera. Although this study included a more
limited sampling of Theloderma (Th. rhododiscum,
Th. corticale,Th. asperum,Th. cf. horridum, and Th. mo-
loch) and Nyctixalus (N. pictus and N. spinosus), it con-
firmed Nyctixalini as the most basal split within Rhaco-
phorinae with exception for Liuixalus, indicated para-
phyly of Theloderma in respect with Nyctixalus, with
Th. horridum forming the most basal split within Nycti-
xalini, and nested Th. moloch within Theloderma +
Nyctixalus clade. Li et al. (2013) estimated the basal split
in Nyctixalini as ca. 29 mya, whereas the basal split of
Theloderma sensu stricto (excluding Nyctixalus and
Th. horridum) was estimated as 26.1 – 16.4 mya.
Within Southeast Asia, the Indochinese peninsula is
renowned for its high diversity of amphibian species
(Orlov and Ananjeva, 2007; Bain and Hurley, 2012 and
references therein). The southern part of Indochina is
comparatively poorly investigated area with many new
species of amphibians and reptiles having been discov-
ered from this region during the last several years, both
from highland areas of the southern Annamite, or Truong
Son Mountains (Nazarov et al., 2008, 2012; Rowley et
al., 2010, 2011; Ngo and Chan, 2010; Stuart et al., 2011;
Orlov et al., 2012; Vassilieva et al., 2014; Poyarkov et al.,
2014, 2015), and from lowland areas of southern Viet-
nam (Stuart et al., 2006; Ngo and Bauer, 2008; Geissler et
al., 2009, 2014; Orlov et al., 2009, 2012; Rowley et al.,
2012; Chan et al., 2013; Hartmann et al., 2013; Ngo,
2013; Vassilieva et al., 2013; Poyarkov et al., 2014).
In recent years, the Indochinese peninsula was the
center of discovery of new Theloderma species, with 14
species of Theloderma and 1 species of Nyctixalus being
reported from its territory (see Fig. 1); 9 of these species
were discovered and described within a decade (Orlov
and Ananjeva, 2007; Nguyen et al., 2009; Orlov et al.,
2010, 2012; Rowley et al., 2011; Frost, 2015). Some
newly discovered small-sized species are morphologi-
cally conserved, mainly differentiated by coloration and
their taxonomic relationships remain unclear, whereas
the others were transferred to Theloderma from other
rhacophorid genera (Rowley et al., 2011; Orlov et al.,
2012; Nguyen et al., 2014). Though the work of Nguyen
et al. (2015) had elucidated the phylogenetic relation-
ships within the genus, some of them remain controver-
sial and contradictory to the results by Li et al. (2013),
and the taxon sampling for Indochina remains limited.
Therefore, the present paper is focused on scrutiniz-
ing diversity of Theloderma frogs of Indochina by molec-
ular phylogenetic analysis of 1987 bp 12S rRNA – 16S
rRNA mtDNA fragment for 90 specimens of 21 nominal
species of Theloderma and 3 species of Nyctixalus.We
also discuss genus-level taxonomy of Nyctixalini and
update data on distribution and systematics of Indochi-
nese taxa. Finally, we describe a new species of Thelo-
derma frogs from southern and central Vietnam based on
genetic, morphological and acoustic lines of evidence.
MATERIAL AND METHODS
Sample collection. All specimens were collected
during fieldwork in Vietnam and Thailand between 2007
and 2014. The geographic position of the surveyed local-
ities and samples included in the molecular analyses is
given in Table 1. The distribution of Theloderma stella-
tum Taylor, 1962 in southern Indochina (eastern Thai-
land, southern Cambodia and Vietnam) is shown in
Fig. 5, surveyed localities are marked with filled icons.
Geographic coordinates and altitude were obtained using
a Garmin GPSMAP 60CSx GPS receiver and recorded in
datum WGS 84. The newly collected specimens were de-
posited in the herpetological collection of the Zoological
Museum of Moscow State University (ZMMU) in Mos-
cow, Russia, the Zoological Institute RAS (ZISP) in
St. Petersburg, Russia, and the Vietnam National Mu-
seum of Nature (VNMN) in Hanoi, Vietnam. Compara-
tive materials examined are stored in herpetological col-
lections of ZMMU, ZISP, and National Science Museum
of Thailand (NHMT) in Bangkok, Thailand.
Morphological analysis. Examined specimens are
listed in Appendix 1. Specimens were photographed in
life, and tissue samples for genetic analysis were taken
prior to preservation and stored in 96% ethanol. Speci-
mens were preserved in 72 – 75% ethanol. Sex was de-
termined by direct observation of calling in life, and by
examining for the presence of nuptial pads in males
and/or gonadal inspection by dissection.
Adults. We recorded morphological data from adult
specimens fixed and stored in 75% ethanol. All measure-
ments were taken using digital caliper under the light dis-
secting microscope to the nearest 0.1 mm; morpho-
metrics follow Orlov et al. (2012) and Milto et al. (2013)
and includes the following 27 measurements: SVL,
snout-vent length; A-G, axilla to groin, measured as dis-
tance from posterior base of forelimb at its emergence
from body to anterior base of hind limb at its emergence
from body; HW, head width at the greatest cranial width;
HL, head length measured from the rear of the lower jaw
to the tip of the snout; HD, head depth, measured as the
244 Nikolay A. Poyarkov, Jr., et al.
greatest transverse depth of head, taken beyond inter-
orbital region; UEW, upper eyelid width measured as the
greatest width of upper eyelids; IOD, interorbital dis-
tance; IND, internarial distance measured as distance
between nostrils; ED, horizontal diameter of eye; TD,
horizontal diameter of tympanum; ESL, tip of snout
eye distance; TED, tympanum eye distance measured
from anterior edge of tympanum to posterior corner of
the eye; END, eye to nostril distance measured as dis-
tance from anterior corner of eye to nostril; FLL, length
of forelimb measured from the tip of disk of finger III to
axilla; FFL, first finger length; TFL, third finger length;
FTD, maximal diameter of disc of finger III; NPL, nup-
tial pad length, measured for males only; MKTe, length
of external metacarpal tubercle; HLL, length of hindlimb
measured from the tip of disk of toe IV to groin; FL, fe-
mur length; TL, tibia length; FOT, length of hindlimb
measured from the tip of disk of toe IV to posterior edge
of tibia; FTL, first toe length; FFTL, fourth toe length;
HTD, diameter of the fourth toe tip measured as the
greatest diameter of disc on fourth toe; MTTi, length of
internal metatarsal tubercle. Skin texture, dorsal colora-
tion, ventral coloration, and presence of supratympanic
folds, circummarginal grooves, dorsolateral folds, vo-
merine teeth, hind limb and forelimb webbing, dorsal,
lateral and ventral coloration were recorded. The web-
bing formula followed Savage and Heyer (1997).
Larval stages. Morphological description of larval
stages included the following 16 measurements: TL, total
length; BL, body length; TaL, tail length; BW, maximal
body width; BH, maximal body height; TH, maximal tail
height; SVL, snout-vent length; SSp, snout-spiracle
length; UF, maximal upper tail fin height; LF, maximal
lower fin height; IN, internarial distance; IP, interpupilar
distance; RN, rostro-narial distance; NP, naro-pupilar
distance; ED, eye diameter; ODW, oral disk width. LTRF
(labial tooth row formula) was recorded following
Wassersug et al. (1981). Tadpoles were staged after Gos-
ner (1960); morphometrics followed Grosjean (2001).
The diagnosis of the genus Theloderma and morpho-
logical characters chosen for comparison were taken
from Wolf (1936); Taylor (1962); Inger (1966); Liem
(1970); Zhao and Adler (1993); Orlov (1997, 2007);
Orlov et al. (2006, 2010, 2012); McLeod and Norhayati
(2007); Rowley et al. (2011).
We obtained comparative morphological data from
museum specimens of Theloderma and allied taxa and
from photographs of these specimens in life and from the
following literature: Theloderma andersoni (Anderson,
1879; Ahl, 1927, 1931; Bourret, 1942; Fei et al., 1990,
2009, 2010; Mathew and Sen, 2010); Th. asperum
(Fig. 1f–h; Boulenger, 1886; Smith, 1930; Taylor, 1962;
Liu and Hu, 1962; Fei et al., 1990, 2009, 2010; Li et al.,
2011); Th. baibengense (Jiang et al. in Fei et al., 2009;
Fei et al., 2010); Th. bambusicolum (Fig. 1t; Orlov et al.,
2012); Th. bicolor (Fig. 1k; Bourret, 1937; Orlov et al.,
2010); Th. chuyangsinense (Fig. 1r; Orlov et al., 2012);
Th. corticale (Fig. 1l; Boulenger, 1903; Orlov et al.,
2010); Th. gordoni (Fig. 1m,n; Taylor, 1962; Chan-ard,
2003; Orlov et al., 2010); Th. horridum (Fig. 1a; Boulen-
ger, 1903; Smith, 1926, 1930; Taylor, 1962; Manthey and
Grossmann, 1997; Das, 2007); Th. kwangsiense (Liu and
Hu, 1962; Fei et al., 1990, 2009, 2010; Orlov et al., 2010;
Li et al., 2011); Th. laeve (Fig. 1t, V; Smith, 1924; Orlov
and Ananjeva, 2007; Orlov et al., 2012); Th. lateriticum
(Fig. 1o; Bain et al., 2009); Th. leporosum (Tschudi,
1838; Manthey and Grossmann, 1997; Chan and Norha-
yati, 2009); Th. licin (Fig. 1e; McLeod and Norhayati,
2007; Das et al., 2013); Th. moloch (Annandale, 1912;
Liem, 1970; Fei et al., 1990, 2009, 2010; Mathew and
Sen, 2010; Orlov et al., 2010); Th. nagalandense (Orlov
et al., 2006); Th. nebulosum (Fig. 1u; Rowley et al.,
2011); Th. palliatum (Fig. 1q; Rowley et al., 2011; Orlov
et al., 2012); Th. petilum (Fig. 1x; Stuart and Heatwole,
2004; Nguyen et al., 2014); Th. phrynoderma (Boulen-
ger, 1893; Ahl, 1927; Manthey and Grossmann, 1997;
Dever et al., 2015); Th. rhododiscum (Fig. 1p; Liu and
Hu, 1962; Fei et al., 1990, 2009, 2010; Orlov et al., 2010;
Li et al., 2011); Th. ryabovi (Fig. 1i,j; Orlov et al., 2006,
2010; Orlov, 2007); Th. stellatum (Fig. 1b,c; Taylor,
1962; Chan-ard, 2003; Orlov and Ananjeva, 2007; Orlov
et al., 2010); Th. truongsonense (Fig. 1s,w; Orlov and
Ho, 2005; Rowley et al., 2011; Orlov et al., 2012);
Nyctixalus margaritifer (Boulenger, 1882; Smith, 1931;
Inger, 1966; Manthey and Grossmann, 1997); N. pictus
(Fig. 1d; Peters, 1871; Taylor, 1962; Inger, 1966; Liem,
1970; Manthey and Grossmann, 1997; Das, 2007); and
N. spinosus (Taylor, 1920; Inger, 1954). Due to the high
undiagnosed diversity within the genus, where available,
we relied on examination of type specimens, topotypic
material and/or original species descriptions.
Statistical analyses. The morphological data of
Theloderma sp. nov. and Theloderma stellatum was ana-
lyzed by using software STATISTICA 10 (StatSoft,
2010). Subadult and juvenile specimens were excluded
from the statistical morphological analyses. We per-
formed an analysis of variance (ANOVA) of morphologi-
cal characters to identify characters showing significant
differences between the two OTUs (sexes were analyzed
together). Those characters that showed no significant
differences ( p> 0.05), were excluded from the conclud-
ing principal component analysis (PCA).
DNA isolation, PCR and sequencing. The full list
of samples included in the molecular analyses is given in
Table 1. For molecular analysis, total genomic DNA was
extracted from ethanol-preserved muscle or liver tissues
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 245
246 Nikolay A. Poyarkov, Jr., et al.
TABLE 1. Specimens and Genbank Sequences of Theloderma used in Molecular Analyses and the Newly Proposed Taxonomy
No. Taxon Specimen ID Genbank AN Country Locality Reference Proposed taxonomy
1Rana kukunoris KIZ 0152 KC465786 China Qinghai Li et al., 2013
2Mantella aurantiaca UMMZ 201411 DQ283035 Madagascar Toamasina: Moramanga Frost et al., 2006
3Buergeria buergeri TTU-R-11759 AF458122 Japan Wilkinson
et al., 2002
4Buergeria japonica UMFS 5821 DQ283055 China Taiwan: Yilan Frost et al., 2006
5Buergeria oxycephala SCUM 050267YJ EU215524 China Hainan Li et al., 2008
6Buergeria robusta TTU-R-11761 AF458125 China Taiwan Wilkinson
et al., 2002
7Chiromantis doriae SN 030051 EU215527 China Hainan Li et al., 2008
8Chiromantis xerampelina CAS;
no number AF458132 — Wilkinson
et al., 2002
9Feihyla vittata KIZ 0001rao GQ285684 China Yunnan Li et al., 2009
10 Feihyla kajau FMNH 269090 KC465789 Malaysia Sarawak: Bintulu Div. Li et al., 2013
11 Feihyla palpebralis Vietnam 712 GQ285681 Vietnam Lam Dong Li et al., 2009
12 Gracixalus gracilipes KIZ 060821196Rao GQ285668 China Yunnan: Daweishan Li et al., 2009
13 Kurixalus banaensis ROM 32986 GQ285667 Vietnam Gia Lai: Krong Pa Li et al., 2009
14 Kurixalus bisacculus 200602010 KC465801 China Yunnan: Wenshan Li et al., 2013
15 Kurixalus idiootocus SCUM 061107L EU215547 China Taiwan: Lianhuachi Li et al., 2008
16 Kurixalus verrucosus CAS HERP224381 KC465822 Myanmar Kachin: Putao Li et al., 2013
17 Liuixalus hainanus LJT V15 KC465826 China Hainan: Diaoluoshan Li et al., 2013
18 Philautus abditus ROM 33145 GQ285673 Vietnam Gia Lai: Krong Pa Li et al., 2009
19 Polypedates cf. leucomystax ROM 29908 KC465833 Vietnam Gia Lai: Tram Lap Li et al., 2013
20 Polypedates cf. megacephalus HN 0806103 KC465830 China Hainan: Jianfengling Li et al., 2013
21 Polypedates megacephalus 6212RAO KC465834 China Xizang: Motuo Li et al., 2013
22 Raorchestes sp. ROM 38828 KC465838 Vietnam Tuyen Quang: Pac Ban Li et al., 2013
23 Rhacophorus annamensis KIZ 64 JX219448 Vietnam Binh Phuoc: Bu Gia Map Li et al., 2012
24 Rhacophorus dennysi Li06 JX219433 China Hunan Li et al., 2012
25 Rhacophorus kio SCUM 37941C EU215532 China Yunnan: Xishuangbanna Li et al., 2008
26 Rhacophorus maximus Rao6241 JX219411 China Xizang: Motuo Li et al., 2012
27 Rhacophorus schlegelii NC007178 Japan Hiroshima Sano et al., 2005
28 Nyctixalus margaritifer TNHCJAM 3030 EU178087 Indonesia Java Biju et al., 2008 Th. margaritifer
29 Nyctixalus pictus FMNH 231095 DQ283133 Malaysia Sabah: Lahad Datu Frost et al., 2006 Th. pictum
30 Nyctixalus pictus FMNH 231094 GQ204777; GQ204726 Malaysia Meegaskumbura et al., 2010 Th. pictum
31 Nyctixalus pictus AF215349 Malaysia Vences 2000, unpubl. Th. pictum
32 Nyctixalus pictus NMBE 1056413 JN705355; JN377342 Malaysia Sarawak: Batang Ai Hertwig et al., 2013 Th. pictum
33 Nyctixalus pictus MVZ 239460 GQ204783; GQ204732 Indonesia Meegaskumbura et al., 2010 Th. pictum
34 Nyctixalus pictus FMNH 231094 – 2 AF458135 Malaysia Wilkinson et al., 2002 Th. pictum
35 Nyctixalus pictus AF268255 Malaysia Richards 2000, unpubl. Th. pictum
36 Nyctixalus pictus AH07001 GU154888 Malaysia Sarawak: Gunung Mulu Das and Haas 2010 Th. pictum
37 Nyctixalus spinosus pet trade KT461916 Philippines Mindanao this study Th. spinosum
38 Nyctixalus spinosus ACD 1043 DQ283114 Philippines Mindanao Frost et al., 2006 Th. spinosum
39 “Thelodermamoloch 6255Rao GQ285679 China Xizang: Motuo Li et al., 2009 Th. moloch
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 247
No. Taxon Specimen ID Genbank AN Country Locality Reference Proposed taxonomy
40 Theloderma asperum KIZ 060821217 EF564522 China Guangxi: Jinxiu Yu et al., 2008 Th. albopunctatum
41 Theloderma asperum KIZ 060821201 EF564521 China Yunnan: Jinping Yu et al., 2008 Th. albopunctatum
42 Theloderma asperum VNMN J2916 KJ802913 Vietnam Vinh Phuc Nguyen et al., 2014 Th. albopunctatum
43 Theloderma asperum VNMN 3540 KJ802914 Vietnam Lao Cai Nguyen et al., 2014 Th. albopunctatum
44 Theloderma asperum 060821203Rao GQ285677 China Yunnan: Jinping Li et al., 2009 Th. albopunctatum
45 Theloderma asperum ZRC 1.1. 9321 GQ204725; GQ204776 Malaysia Meegaskumbura et al., 2010 Th. asperum
46 Theloderma asperum pet trade KT461929 Malaysia Perak this study Th. asperum
47 Theloderma asperum asperum-1 KT461884 Vietnam Kon Tum: Kon Plong this study Th. albopunctatum
48 Theloderma asperum asperum-2 KT461908 Vietnam Kon Tum: Kon Plong this study Th. albopunctatum
49 Theloderma asperum asperum-3 KT461909 Vietnam Kon Tum: Kon Plong this study Th. albopunctatum
50 Theloderma asperum ZMMU NAP-03557 KT461910 Vietnam Hai Phong: Cat Ba this study Th. albopunctatum
51 Theloderma asperum ZMMU NAP-03566 KT461911 Vietnam Hai Phong: Cat Ba this study Th. albopunctatum
52 Theloderma asperum ZMMU NAP-03575 KT461912 Vietnam Hai Phong: Cat Ba this study Th. albopunctatum
53 Theloderma asperum HN0806100 GQ285678 China Hainan: Yinggeling Li et al., 2009 Th. albopunctatum
54 Theloderma bambusicolum ZMMU NAP-01640 KT461928 Vietnam Lam Dong: Cat Loc this study Th. laeve
55 Theloderma bambusicolum ZMMU NAP-01644 KT461907 Vietnam Lam Dong: Cat Loc this study Th. laeve
56 Theloderma bambusicolum ZMMU NAP-01645 KT461913 Vietnam Lam Dong: Cat Loc this study Th. laeve
57 Theloderma bambusicolum ZMMU NAP-02906 KT461883 Vietnam Binh Phuoc: Bu Gia Map this study Th. laeve
58 Theloderma bambusicolum ZMMU NAP-02907 KT461905 Vietnam Binh Phuoc: Bu Gia Map this study Th. laeve
59 Theloderma bambusicolum ZMMU NAP-02908 KT461906 Vietnam Binh Phuoc: Bu Gia Map this study Th. laeve
60 Theloderma bambusicolum ZMMU NAP-03383 KT461892; KT461900 Vietnam Lam Dong: Bao Loc this study Th. laeve
61 Theloderma bambusicolum ZMMU NAP-03408 KT461897; KT461898 Vietnam Lam Dong: Bao Loc this study Th. laeve
62 Theloderma bambusicolum ZMMU NAP-03409 KT461920 Vietnam Lam Dong: Bao Loc this study Th. laeve
63 Theloderma bicolor VNMN 1394 JX046475 Vietnam Lao Cai: Sa Pa Gawor et al., 2012 Th. bicolor
64 Theloderma bicolor bicolor-2 KT461923 Vietnam Ninh Binh: Cuc Phuong this study Th. bicolor
65 Theloderma bicolor bicolor-3 KT461891; KT461899 Vietnam Ninh Binh: Cuc Phuong this study Th. bicolor
66 Theloderma bicolor IEBR A. 2011.4 JX046474 Vietnam Lao Cai: Sa Pa Gawor et al., 2012 Th. bicolor
67 Theloderma bicolor VNMN 3536 KJ802915 Vietnam Lao Cai Nguyen et al., 2014 Th. bicolor
68 Theloderma chuyangsinense ZMMU NAP-02757 KT461896; KT461904 Vietnam Dak Lak: Chu Yang Sin this study Th. palliatum
69 Theloderma chuyangsinense ZMMU NAP-02756 KT461930 Vietnam Dak Lak: Chu Yang Sin this study Th. palliatum
70 Theloderma chuyangsinense ZMMU NAP-02735 KT461926 Vietnam Dak Lak: Chu Yang Sin this study Th. palliatum
71 Theloderma chuyangsinense ZMMU NAP-02736 KT461927 Vietnam Dak Lak: Chu Yang Sin this study Th. palliatum
72 Theloderma corticale AMNH A161499 DQ283050 Vietnam Vinh Phuc: Tam Dao Frost et al., 2006 Th. corticale
73 Theloderma corticale IEBR 3267 JX046477 Vietnam Vinh Phuc: Tam Dao Gawor et al., 2012 Th. corticale
74 Theloderma corticale corticale-1 KT461885 Vietnam Ninh Binh: Cuc Phuong this study Th. corticale
75 Theloderma corticale corticale-2 KT461886 Vietnam Ninh Binh: Cuc Phuong this study Th. corticale
76 Theloderma corticale IEBR E193.15 JX046476 Vietnam Vinh Phuc: Tam Dao Gawor et al., 2012 Th. corticale
77 Theloderma corticale VNMN J2892 KJ802916 Vietnam Tuyen Quang Nguyen et al., 2014 Th. corticale
78 Theloderma corticale VNMN J2932 KJ802917 Vietnam Vinh Phuc: Tam Dao Nguyen et al., 2014 Th. corticale
79 Theloderma gordoni VNMN 03013 JN688167 Vietnam Nghe An Rowley et al., 2011 Th. gordoni
80 Theloderma gordoni VNMN PAE217 KJ802918 Vietnam Son La Nguyen et al., 2014 Th. gordoni
TABLE 1 (continued)
248 Nikolay A. Poyarkov, Jr., et al.
No. Taxon Specimen ID Genbank AN Country Locality Reference Proposed taxonomy
81 Theloderma gordoni KUHE 32447 KJ802919 Laos Houaphan Nguyen et al., 2014 Th. gordoni
82 Theloderma horridum LJT W44 KC465843 Malaysia Li et al., 2013 Th. horridum
83 Theloderma horridum LJT W45 KC465842 Malaysia Li et al., 2013 Th. horridum
84 Theloderma horridum ZMMU NAP-04015 KT461890 Thailand Satun this study Th. horridum
85 Theloderma leporosum LJT W46 KC465841 Malaysia Li et al., 2013 Th. leporosum
86 Theloderma leporosum leporosum-1 KT461922 Malaysia Selangor this study Th. leporosum
87 Theloderma leporosum KUHE 52581 AB847128 Malaysia Negeri Sembilan Nguyen et al., 2014 Th. leporosum
88 Theloderma licin KUHE 52599 KJ802920 Malaysia Selangor Nguyen et al., 2014 Th. licin
89 Theloderma nebulosum ROM 39588 KT461887 Vietnam Kon Tum: Ngoc Linh this study Th. nebulosum
90 Theloderma nebulosum AMS R 173409 JN688168 Vietnam Kon Tum: Ngoc Linh Rowley et al., 2011 Th. nebulosum
91 Theloderma nebulosum AMS R 173877/UNS00141 JN688169 Vietnam Kon Tum: Ngoc Linh Rowley et al., 2011 Th. nebulosum
92 Theloderma palliatum AMS R 173130 JN688172 Vietnam Lam Dong: Bi Doup — Nui Ba Rowley et al., 2011 Th. palliatum
93 Theloderma palliatum ZMMU NAP-01846 KT461893; KT461901 Vietnam Lam Dong: Bi Doup — Nui Ba this study Th. palliatum
94 Theloderma palliatum ZMMU NAP-02511 KT461894; KT461902 Vietnam Lam Dong: Bi Doup — Nui Ba this study Th. palliatum
95 Theloderma palliatum ZMMU NAP-02516 KT461895; KT461903 Vietnam Lam Dong: Bi Doup — Nui Ba this study Th. palliatum
96 Theloderma petilum HNUE MNA.2012.0001 KJ802925 Vietnam Dien Bien: Muong Nhe Nguyen et al., 2014 Th. petilum
97 Theloderma rhododiscum AMNH A163892; A163893 DQ283392; DQ283393 Vietnam Ha Giang: Tay Con Linh Frost et al., 2006 Th. rhododiscum
98 Theloderma rhododiscum KIZ060821063 EF564533 China Guangxi: Jinxiu Yu et al., 2008 Th. rhododiscum
99 Theloderma rhododiscum KIZ060821170 EF564534 China Guangxi: Jinxiu Yu et al., 2008 Th. rhododiscum
100 Theloderma rhododiscum SCUM 061102L EU215530 China Guangxi: Dayaoshan Li et al., 2008 Th. rhododiscum
101 Theloderma rhododiscum CIB GX200807048 KJ802921 China Guangxi Nguyen et al., 2014 Th. rhododiscum
102 Theloderma ryabovi ryabovi-1 KT461914 Vietnam Kon Tum: Kon Plong:
Mang Canh this study Th. ryabovi
103 Theloderma ryabovi ryabovi-2 KT461915 Vietnam Kon Tum: Kon Plong:
Mang Canh this study Th. ryabovi
104 Theloderma stellatum stellatum-1 KT461918 Thailand Chanthaburi: Phliu this study Th. stellatum
105 Theloderma stellatum ZMMU NAP-03961 KT461917 Thailand Nakhon Nayok: Nang Rong this study Th. stellatum
106 Theloderma stellatum VNMN 3686 KJ802922 Vietnam Phu Yen Nguyen et al., 2014 Th. vietnamense sp. nov.
107 Theloderma stellatum VNMN 3687 KJ802923 Vietnam Phu Yen Nguyen et al., 2014 Th. vietnamense sp. nov.
108 Theloderma stellatum ZMMU NAP-00707 KT461889 Vietnam Dong Nai: Nam Cat Tien this study Th. vietnamense sp. nov.
109 Theloderma stellatum ZMMU NAP-03680 KT461921 Vietnam Tay Ninh: Lo Go – Xa Mat this study Th. vietnamense sp. nov.
110 Theloderma stellatum ZMMU NAP-03723 KT461919 Vietnam Kien Giang: Phu Quoc this study Th. vietnamense sp. nov.
111 Theloderma stellatum ZMMU NAP-03724 KT461888 Vietnam Kien Giang: Phu Quoc this study Th. vietnamense sp. nov.
112 Theloderma stellatum AMS R 173283 JN688170 Vietnam Binh Thuan Rowley et al., 2011 Th. vietnamense sp. nov.
113 Theloderma stellatum AMS R 174047 JN688171 Cambodia Mondol Kiri Rowley et al., 2011 Th. vietnamense sp. nov.
114 Theloderma truongsonense ROM 39363 KT461925 Vietnam Quang Binh: Phong Nha —
— Ke Bang this study Th. truongsonense
115 Theloderma sp. ZMMU ABV-00301 KT461882 Vietnam Khanh Hoa: Hon Ba this study Theloderma sp.
116 Theloderma sp. ZMMU ABV-00319 KT461924 Vietnam Khanh Hoa: Hon Ba this study Theloderma sp.
117 Theloderma cf. truongsonense AMS R 171510 JN688174 Vietnam Quang Nam Rowley et al., 2011 Th. cf. truongsonense
Notes. AN, Accession number. Numbers of specimens (No. 1 – 117) correspond to those in Figs. 2 and 3.
TABLE 1 (continued)
using standard phenol-chloroform extraction procedures
(Hillis et al., 1996) followed with isopropanol precipita-
tion. The isolated total genomic DNA was visualized in
agarose electrophoresis in presence of ethidium bromide.
Concentration of the total DNA was measured in 1 ìlus
-
ing NanoDrop 2000 (Thermo Scientific), and conse-
quently adjusted to ca. 100 ng DNA/ìl.
We amplified mtDNA fragments of 12S rRNA,
tRNAval, and 16S rRNA mtDNA genes, to obtain a frag-
ment with length up to 1987 bp. DNA barcoding tech-
niques (e.g., Vences et al., 2005a, 2005b; Smith et al.,
2008; Jiang et al., 2013; Murphy et al., 2013) have great-
ly facilitated the assessment of cryptic taxonomic diver-
sity in amphibians. 16S rRNA is a molecular marker
widely applied for biodiversity surveys in amphibians
(Vences et al., 2005a; 2005b; Vieites et al., 2009), and
was also proven to be useful in studies of Rhacophoridae
diversity (Wilkinson and Drewes, 2000; Wilkinson et al.,
2002; Bain et al., 2009; Li et al., 2008, 2009, 2013; Mee-
gaskumbura et al., 2015), including the genus Theloder-
ma (Yu et al., 2007, 2008; Rowley et al., 2011; Nguyen et
al., 2014, 2015; Dever et al., 2015; and references
therein). Amplification was performed in 25 ìl reactions
using ca. 50 ng genomic DNA, 10 nmol of each primer,
15 nmol of each dNTP, 50 nmol additional MgCl2,Taq
PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl,
1.1 mM MgCl2and 0.01% gelatin) and 1 U of Taq DNA
polymerase.
Primers used in PCR and sequencing were obtained
from the previous studies (Hedges, 1994; Wilkinson et
al., 2002; Li et al., 2008, 2009) and were as follows: for-
ward primers: F0001 (5’-AGATACCCCACTATGCCTA
CCC-3’); F0483 (5’-GAAGAGGCAAGTCGTAACATG
G-3’); F0937 (5’-TGGGATGATTTTCAAGTAG-3’);
F1624 (5’-GTATCAACGGCATCACGAGGG-3’); 16S-
L-1 (5’-CTGACCGTGCAAAGGTAGCGTAATCACT-
3’); 16S-L2021 (5’-CCTACCGAGCTTAGTAATAGCT
GGTT-3’); reverse primers: R0483 (5’-CCATGTTACG
ACTTGCCTCTTC-3’); R1169 (5’-GTGGCTGCTTTTA
GGCCCACT-3’); R1624 (5’-CCCTCGTGATGCCGTT
GATAC-3’); Rend (5’-GACCTGGATTACTCCGGTCT
GA-3’); 16S-H-1 (5’-CTCCGGTCTGAACTCAGATCA
CGTAGG-3’); 16S-H2715 (5’-AAGCTCCATAGGGTC
TTCTCGTC-3’). The PCR conditions for amplification
of 16S rRNA fragments included an initial denaturation
step of 5 min at 94°C and 33 cycles of denaturation for
30 sec at 94°C, primer annealing for 30 sec at 48°C, and
extension step for 1 min 30 sec at 72°C, followed with a
final extension step for 10 min at 72°C. The PCR condi-
tions for amplification of 12S rRNA fragments included
an initial denaturation step of 5 min at 94°C and 35 cy-
cles of denaturation for 1 min at 94°C, primer annealing
for 1 min at 55°C, and extension step for 1 min at 72°C,
followed with a final extension step for 10 min at 72°C.
PCR products were visualized in 1% agarose gels
stained with Ethidium bromide in a Dark reader trans-
illuminator. If distinct bands were produced, products
were purified using 2 ìl from a 1:4 dilution of ExoSapIt
(Amersham), per 5 ìl of PCR product prior to cycle se-
quencing. A 10 ìl sequencing reaction included 2 ìlof
template, 2.5 ìl of sequencing buffer, 0.8 ìl of 10 pmol
primer, 0.4 ìl of BigDye Terminator version 3.1 Se-
quencing Standard (Applied Biosystems) and 4.2 ìlof
water. The cycle-sequencing reaction was 35 cycles of
10 sec at 96°C, 10 sec at 50°C and 4 min at 60°C. Cycle
sequencing products were purified by ethanol precipi-
tation. Sequence data collection and visualization were
performed on an ABI 3730xl automated sequencer
(Applied Biosystems). The obtained sequences are de-
posited in GenBank under the accession numbers
KT461882-KT461930 (see Table 1).
Phylogenetic analysis. 12S rRNA – 16S rRNA frag-
ment sequences of 117 specimens (90 representatives of
Theloderma, 25 sequences of outgroup members of Rha-
cophoridae (genera Rhacophorus,Raorchestes,Polype-
dates,Philautus,Liuixalus,Kurixalus,Gracixalus,Fei-
hyla,Chiromantis,Buergeria) and 2 outgroup sequences
of Rana kukunoris (Ranidae, KC465786) and Mantella
aurantiaca (Mantellidae, DQ283035), the data on
voucher specimens and GenBank sequences used in
phylogenetic analyses are summarized in Table 1) with a
total length of up to 1987 bp were included in the final
alignment and subjected to phylogenetic analyses.
Sequences were also submitted to a BLAST search in
GenBank to confirm that the intended sequences had
been amplified. The forward and reverse sequences were
checked visually in Chromas Pro software (Techne-
lysium Pty Ltd., Tewntin, Australia) and a consensus se-
quence was compiled with BioEdit 5.0.9 (Hall, 1999).
Nucleotide sequences were initially aligned using
ClustalX 1.81 (Thompson et al., 1997) with default pa-
rameters, and then checked by eye in BioEdit 7.0.5.2
(Hall, 1999) and MEGA 6.0 (Tamura et al., 2013) and
slightly adjusted.
The dataset was first divided into three partitions,
12S rRNA, tRNAval, and 16S rRNA. MODELTEST
v. 3.06 (Posada and Crandall, 1998) was used to estimate
the optimal evolutionary models for partitions to be used
for the data set analysis. The best-fitting model as sug-
gested by the Akaike Information Criterion (AIC) for
12S rRNA and 16S rRNA partitions was the general
time-reversible (GTR) model with a gamma shape pa-
rameter (G); for tRNAval partition the Kimura 2-parame-
ter model (+G+I) was selected as the best fit.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 249
Phylogenetic analyses were conducted in MrBayes
3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, 2003) software. Transitions and trans-
versions were equally weighted, and gaps were treated as
missing data. Confidence in tree topology was tested by
posterior probability (PP) for Bayesian inference (BI) in
MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). Two
independent runs of four Markov Chains for 10,000,000
generations were summarized by the BI, with the tree
sampled every 100 generations with a consensus topol-
ogy calculated for 70,000 trees (burn-in = 3,000,000).
We a priori regarded the tree nodes with posterior
probabilities values over 0.95 as sufficiently resolved,
those between 0.95 and 0.90 were regarded as tenden-
cies, those below 0.90 were considered to be non-re-
solved (Huelsenbeck and Hillis, 1993). Mean uncor-
rected genetic distances ( p-distances) between sequences
were determined with MEGA 6.0 (Tamura et al., 2013).
Acoustic analysis. Advertisement calls of the
Theloderma cf. stellatum were recorded in situ in the
natural habitat in Cat Tien National Park, Dong Nai Prov-
ince, Vietnam, on 5th April, 2012, at 18:48 at temperature
25.5°C (the temperature was measured at the calling site
immediately after recording with a digital thermometer
KTJ TA218A Digital LCD Thermometer-Hydrometer),
using a Nikon D7000 digital SLR camera (Nikon Corpo-
ration, Japan) in video mode with default sound settings
(16 bit, 48 kHz, linear PCM audio format and auto sound
level control); in total, 16 advertisement calls from a sin-
gle male individual were recorded. The total duration of
the recording was 54.25 sec.
For comparison we also analyzed the advertisement
calls of the Theloderma stellatum recorded in captivity
from a male individual, collected from environs of Khao
Yai National Park, Nakhon Nayok Province, Thailand.
The calls were recorded at temperature 25.0°C; in total,
32 advertisement calls from a single male individual
were recorded. The total duration of the recording was
32.30 sec. The both audio tracks were removed from
original recording using Avisoft SASLab Pro software
v. 5.2.05 (Avisoft Bioacoustics, Germany) with a
22.05 kHz sampling frequency and 16-bit precision.
Calls were analyzed using Avisoft SASLab Pro soft-
ware v. 5.2.05; the temporal parameters were measured
with the standard marker cursor in the main window of
Avisoft and the frequency parameters were measured
using the reticule cursor in the spectrogram window of
Avisoft. Spectrograms for analysis were created using
Hamming window, FFT-length 1024 points, frame 75%,
and overlap 93.75%. Figure spectrograms were created
using Hamming window, FFT-length 512 points, frame
100%, and overlap 75%. We reduced the low-frequency
noise using the low-pass filter (up to 500 Hz). In total we
measured 16 calls of Theloderma cf. stellatum from Viet-
nam and 32 calls of Theloderma stellatum from Thailand.
We measured two temporal parameters: the duration
of each call, the interval between successive calls and
five frequency parameters: the initial and final funda-
mental frequency, the minimum and maximum of funda-
mental frequency and the frequency of maximum ampli-
tude. All numeral parameters are given as mean ± SE, the
minimum and maximum values are given in parentheses
(min – max).
RESULTS
Molecular differentiation
Sequence and statistics. Of 1987 nucleotide sites of
the studied mtDNA fragment, 909 sites were conserved
and 1067 sites were variable, of which 904 were found to
be parsimony-informative. The transition-transversion
bias (R) was estimated as 3.621 (all data given for
ingroup only). Nucleotide frequencies were 30.61% (A),
21.31% (T/U), 24.46% (C), and 23.62%.
Sequence divergence. The uncorrected p-distances
among and within the studied 1987 bp. mtDNA fragment
of the Theloderma sensu lato taxa are shown in Table 2
(data given for the ingroup only). Values of the uncor-
rected genetic p-distances in ingroup and outgroup com-
parisons notably overlapped.
Preliminary phylogenetic relationships of Indo-
chinese Theloderma.Results of the phylogenetic analy-
sis of the studied mtDNA fragment are presented in
Fig. 2 (position of the genus Theloderma within Rhaco-
phoridae) and Fig. 3 (relationships within the ingroup).
Phylogenetic relationships between the accessed taxa of
Theloderma and Nyctixalus are well resolved with the ex-
ception for several nodes within the group level having
low values of support (BPP < 0.95). At the same time,
monophyly of subgeneric and species-level groups and
species complexes is significantly supported (BPP £
£0.95). Monophyly of Rhacophoridae, Buergeriinae,
Rhacophorinae is strongly supported (BPP = 1.0) (see
Fig. 2). Monophyly of all Rhacophorinae with the excep-
tion for Liuixalus is also strongly supported, though the
relationships within the former clade are not resolved
(see Fig. 2). Monophyly of Nyctixalini — the clade,
comprising Theloderma sensu stricto, Nyctixalus and
Theloderma moloch is strongly supported (1.0) (Fig. 2)
well in concordance with the results obtained in a large-
scale phylogeny by Li et al. (2013). Hereafter we indicate
this clade as Theloderma sensu lato.
The Bayesian tree (Fig. 3) infers the following set of
phylogenetic relationships among studied Nyctixalini
250 Nikolay A. Poyarkov, Jr., et al.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 251
TABLE 2. Uncorrected p-Distance (%) between 16S rRNA Sequences of Theloderma Species Included in Phylogenetic Analyses (below the diagonal) and Standard Error Estimates (above the
diagonal)
Clade 123456789101112131415161718192021222324
1—Th. moloch 2.20 2.44 2.13 1.60 1.89 1.47 2.17 1.65 2.12 1.99 1.88 1.86 1.98 1.91 1.86 1.93 1.72 1.77 1.90 1.93 1.61 2.07 1.90
2—Th. horridum 19.26 0.45 1.22 1.49 1.81 1.77 2.07 2.00 1.93 1.88 1.87 1.88 1.57 1.49 1.79 1.74 1.71 1.82 1.67 1.95 1.77 1.63 1.64 1.56
3—Th. stellatum 19.59 8.45 0.00 1.65 2.01 2.03 2.06 2.25 2.10 2.27 1.93 2.03 1.75 1.91 1.87 1.91 2.13 2.21 1.92 2.06 2.06 1.99 1.99 1.83
4—Th. vietnamense sp. nov. 19.10 10.28 9.38 1.9 1.97 2.00 1.91 2.05 1.90 2.13 1.89 1.89 1.51 1.63 2.00 1.95 1.89 1.91 1.83 2.05 1.96 1.73 1.72 1.72
5—Th. pictum 14.64 19.00 19.91 18.17 5.66 1.46 1.53 1.85 1.60 1.76 1.89 1.69 1.76 1.80 1.64 1.62 1.82 1.59 1.75 1.76 1.82 1.70 1.77 1.67
6—Th. spinosum 14.86 15.77 15.88 13.65 11.36 0.00 1.44 1.85 1.59 1.83 1.85 1.63 1.73 1.73 1.71 1.67 1.94 1.84 1.75 2.09 1.96 1.99 1.96 2.04
7—Th. margaritifer 6.90 13.65 11.64 11.91 7.46 6.03 1.91 1.58 2.10 1.62 1.59 1.86 1.93 1.98 1.94 1.77 1.64 2.10 1.94 1.78 1.71 1.89 1.75
8—Th. ryabovi 18.64 19.89 22.37 19.63 15.04 15.59 12.07 0.00 1.77 1.93 2.04 1.91 1.76 1.93 1.96 1.98 2.06 1.84 1.91 2.05 2.09 2.02 2.15 2.07
9—Th. albopunctatum 15.39 17.52 17.86 17.58 14.20 12.79 7.43 14.66 3.16 1.66 1.69 1.54 1.87 1.68 1.59 1.57 1.69 1.76 1.85 1.68 1.82 1.59 1.73 1.58
10—Th. petilum 17.23 16.10 16.89 17.15 15.51 14.86 10.78 15.25 9.25 1.74 1.94 1.92 1.80 1.80 1.82 1.79 2.05 1.93 1.71 2.01 1.77 1.72 1.65
11 — Th. licin 16.89 17.79 18.58 17.87 15.28 16.22 8.19 15.59 11.00 12.16 1.86 2.01 1.63 1.85 1.83 1.97 2.02 1.86 1.82 1.83 1.99 2.00 1.95
12—Th. asperum 15.88 18.58 19.59 19.01 15.98 16.22 7.33 16.61 12.24 13.85 13.18 1.76 1.71 1.78 1.74 1.77 1.69 1.76 1.76 1.77 1.78 1.96 1.70
13—Th. corticale 16.64 15.34 15.63 12.90 15.39 12.19 8.68 14.85 15.00 16.01 15.04 14.17 1.62 1.30 1.70 1.67 1.70 1.72 1.65 2.08 1.60 1.95 1.80 1.85
14—Th. bicolor 14.53 13.31 14.32 12.64 14.54 11.62 8.36 15.05 12.43 12.97 12.30 12.97 6.09 0.27 1.49 1.41 1.57 1.76 1.56 1.96 1.58 1.85 1.68 1.79
15—Th. rhododiscum Vietnam 15.20 15.09 15.20 15.93 15.42 14.53 11.21 14.24 12.66 13.51 13.18 13.51 11.27 8.58 0.82 1.84 1.71 1.72 1.77 1.87 1.99 2.00 1.76
16—Th. rhododiscum China 14.57 14.12 14.92 14.75 14.83 13.98 10.78 13.52 13.24 13.30 13.81 13.47 11.63 8.61 2.29 0.51 1.81 1.76 1.70 1.78 1.80 1.91 1.88 1.64
17—Th. leporosum 13.98 15.30 17.25 15.10 14.58 13.98 8.19 14.82 13.34 13.64 15.33 14.21 10.20 8.03 10.94 10.43 0.68 1.67 1.82 2.02 1.67 1.80 1.79 1.82
18—Th. gordoni 13.49 15.58 18.29 15.49 13.31 13.94 8.43 14.22 14.37 14.86 15.88 14.06 10.59 8.93 10.86 9.83 8.53 2.33 1.74 2.01 1.94 1.91 2.09 1.95
19—Th. palliatum 16.19 16.71 17.36 16.74 15.96 14.90 9.78 17.09 14.14 15.42 15.76 15.21 11.55 8.34 10.92 11.75 11.53 12.60 0.65 2.00 1.90 1.99 2.09 1.88
20—Th. laeve 19.49 21.13 22.28 20.19 17.37 16.92 13.21 13.67 13.75 14.52 17.59 16.88 17.59 17.18 15.58 15.33 15.92 16.84 17.27 0.92 1.73 1.81 1.84 1.73
21—Th. nebulosum 15.88 17.57 17.91 16.27 15.35 13.85 7.76 14.92 14.95 15.20 14.19 13.51 10.16 9.12 13.85 12.88 10.26 12.00 14.79 13.14 0.00 1.75 1.84 1.72
22—Th. truongsonense 13.90 18.31 17.63 16.41 15.50 16.27 8.19 14.63 14.55 14.92 16.61 13.90 14.22 12.54 14.58 13.69 12.33 14.33 14.96 13.11 9.49 1.22 1.34
23—Th. cf. truongsonense 16.61 18.41 17.69 16.21 16.94 16.25 9.39 17.03 16.89 16.61 16.61 16.97 13.75 11.91 15.16 14.22 13.37 15.26 18.02 13.93 9.75 4.35 1.25
24—Theloderma sp. 16.22 17.23 17.23 15.42 15.82 16.55 9.05 16.61 15.15 15.54 16.55 14.19 12.72 11.82 13.51 12.62 12.51 15.08 15.30 12.83 9.46 6.10 5.78 0.00
Notes. The ingroup mean uncorrected p-distances are shown on the diagonal and shaded with gray. Clade numbers correspond to those at Fig. 3.
(Theloderma sensu lato) species. Theloderma moloch
(Fig. 3, clade 1) is recovered as a sister clade to all other
Nyctixalini with strong support (BPP = 1.0). Monophyly
of the clade comprising Theloderma other than Th. mo-
loch +Nyctixalus is also strongly supported (BPP = 1.0).
General topology of the Bayesian tree suggests that
within Nyctixalini other than Th. moloch, the most basal
split separates Th. horridum from Malayan Peninsula
(Fig. 3, clade 2), Th. stellatum sensu stricto from Thai-
land (clade 3) and Th. cf. stellatum from Vietnam (clade
4); monophyly of this clade receives strong support
(BPP = 1.0). This topology with Nyctixalus being nested
within Theloderma, and Th. horridum forming a more
basal split, was also recovered in the study by Li et al.
(2013). However, monophyly of the group, joining Nyc-
tixalus (Fig. 3, clades 5 – 7) and the rest of Theloderma
species (Fig. 3, clades 8 – 24) got poor support (BPP =
= 0.86). Within the Th. stellatum Th. horridum group,
Th. stellatum sensu lato is clearly paraphyletic with re-
spect to Th. horridum (Fig. 3, clade 2), the latter species
form a well-supported clade (BPP = 0.96) with Th. stel-
latum from Thailand (clade 3), whereas Th. cf. stellatum
from Vietnam forms a distantly related clade 4 (p-dis-
tance = 9.38 – 10.28%, see Table 2). Certain variation is
observed between populations of Th. cf. stellatum from
southern (samples 108 – 111, Table 1, Fig. 3) and central
(samples 106 – 107) Vietnam (p= 1.90%).
Monophyly of Nyctixalus got high support (BPP =
= 1.0) (Fig. 3, clades 5 – 7), but relationships within the
group are not sufficiently resolved; there is a tendency
for N. margaritifer (clade 7) from Java to form a basal
split. We report a high divergence between different lin-
eages of N. pictus (samples 29 – 36; p= 5.66%): 4 sub-
clades are revealed within this species; taxonomy of
N. pictus complex appears to be insufficiently studied.
Monophyly of the clade, comprising Nyctixalini
members with exception for Th. moloch,Th. horridum
group and Nyctixalus (= Theloderma sensu stricto;
clades 8 – 24, Fig. 3), is strongly supported (BPP = 1.0).
General topology of the tree correspond well to the re-
sults of Nguyen et al. (2015) and suggests that Theloder-
ma sensu stricto is divided into the two major groups: the
first group joins Th. ryabovi,Th. asperum sensu lato,
Th. petilum and Th. licin (these species we indicate as
members of Th. asperum group; see Fig. 3, clades 8 –
– 12), while the second group (clades 13 – 24) comprises
all other Theloderma taxa from both Sundaland and the
mainland Southeast Asia including Th. leporosum (the
type species of Theloderma Tschudi, 1838).
Within the Th. asperum group, Th. ryabovi from Kon
Tum Province in central Vietnam (clade 8) forms the
most distant lineage ( p-distance 14.66 – 16.61%;
Table 2), sister to other Th. asperum group taxa. The rest
252 Nikolay A. Poyarkov, Jr., et al.
genus sensu latoTheloderma
0.1
14
21
10
24
5
2
13
9
23
18
25
11
12
17
15
27
16
7
26
6
22
8
1
20
0.81
0.72
0.91
0.85
0.93
19
3
4
Polypedates leucomystaxcf.
Buergeria buergeri
Polypedates megacephalus
Feihyla kajau
Rhacophorus dennysi
Buergeria oxycephala
Mantella aurantiaca
Feihyla vittata
Rhacophorus annamensis
Buergeria japonica
Rhacophorus kio
Feihyla palpebralis
Liuixalus hainanus Rhacophorus schlegelii
Chiromantis doriae
Rhacophorus maximus
Buergeria robusta
Chiromantis xerampelina
Polypedates megacephaluscf.
Kurixalus bisacculus
Kurixalus banaensis
Philautus abditus
Gracixalus gracilipes
Kurixalus idiootocus
Kurixalus verrucosus
Raorchestessp.
Rana kukunoris
*
*
*
*
*
*
***
*
*
*
*
*
*
*
**
*
*
Raorchestes
Polypedates
Rhacophorus
MANTELLIDAE
Feihyla
Liuixalus
Chiromantis
Buergeria
Kurixalus
Philautus
Gracixalus
RANIDAE
Fig. 2. Bayesian inference dendrogram of Rhacophoridae derived from the analysis of 1987 bp of 12SrRNA – 16S rRNA mtDNA gene fragment, showing position of the genus Theloderma
sensu lato within the Rhacophoridae. Voucher specimen IDs and Genbank accession numbers are given in Table 1. Rana kukunoris used as an outgroup. Color of node correspond to the support
value (black — highly supported; gray — moderate support; white — no significant support). Star near the node indicate significant value of PP node support (PP > 0.95). Numbers near branches
represent posterior probability (PP) for poorly supported nodes. Numbers of specimens (No.1–117)correspond to those in Table 1.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 253
57
40
85
104
76
47
115
103
30
43
110
108
35
69
31
34
116
58
111
62
117
95
56
107
81
93
89
46
45
68
106
94
84
96
83
112
38
60
101
91
48
90
66
97
64
79
51
36
73
54
32
63
80
72
29
92
42
100
50
75
87
82
74
114
65
71
102
55
109
88
44
61
59
78
41
33
53
113
28
86
98
99
77
52
67
49
37
70
0.89
0.63
0.83
0.94
0.86
0.71
0.92
0.94
0.75
0.61
0.97
0.75
0.91
0.94
0.84
0.73
0.80
0.93
0.96
0.63
0.91
0.93
1
0.95
0.94
0.65
39
105
1—Th. moloch
2 — (Malaysia; S Thailand)Th. horridum
3 — (E Thailand)Th. stellatum
4— sp.nov.Theloderma vietnamense
5 — (Malaysia; Indonesia)Th. pictum
6 — (Philippines: Mindanao)Th. spinosum
7 — (Indonesia: Java)Th. margaritifer
8 — (Vietnam: Kon Tum)Th. ryabovi
9—
(N, C Vietnam; S China)
Th. albopunctatum
10 — (N Vietnam; N Laos)Th. petilum
11 — (Malaysia)Th. licin
12 — (Malaysia; S Thailand)Th. asperum
13 — (N Vietnam)Th. corticale
14 — (N Vietnam)Th. bicolor
15 — (N Vietnam)Th. rhododiscum
16 — (S China)Th. rhododiscum
18 — (Vietnam; Laos)Th. gordoni
17 — (Malaysia)Th. leporosum
19 —
including
(S Vietnam: Langbian Plateau)
Th. palliatum
Th. chuyangsinense
20 —
including
(S Vietnam)
Th. laeve
Th. bambusicola
21 — (C Vietnam: Kon Tum Plateau)Th. nebulosum
22 — (C Vietnam: Quang Binh)Th. truongsonense
23 — . cf. (C Vietnam: Quang Nam)Th truongsonense
24 — sp. (S Vietnam: Khanh Hoa)Theloderma
Th. asperum group
Nyctixalus Th. leporosum group
Theloderma s.str.
Th. laeve group
Stelladerma
subgen. nov.
SVietnam
CVietnam
OUTGROUPS
0.1
Genus
sensu lato
Theloderma
*
*
**
*
*
*
*
*
*
*
*
*
*
*
*
*
*
**
*
*
**
*
*
**
***
**
*
*
*
*
*
*
**
*
*
**
*
Fig. 3. Bayesian inference dendrogram of Theloderma and its relatives derived from the analysis of 1987 bp of 12SrRNA – 16S rRNA mtDNA gene fragment, showing differentiation within
Theloderma sensu lato. Voucher specimen IDs and Genbank accession numbers are given in Table 1. For outgroups see Fig. 2. Color of node correspond to the support value (black — highly
supported; gray — moderate support; white — no significant support). Star indicate significant values of PP node support (PP > 0.95). Numbers near branches represent posterior probability (PP)
for poorly supported nodes. Numbers of specimens (No.1–117)correspond to those in Table 1.
of the Th. asperum group is divided into the two well-
supported subgroups corresponding to Indochinese
(Th. petilum from Laos and Vietnam (clade 10) and
Th. asperum” populations from Vietnam and southern
China (clade 9); monophyly support BPP = 0.97) and
Malayan (Th. licin (clade 11) and Th. asperum sensu
stricto (clade 12), both from peninsular Malaysia; mono-
phyly support BPP = 0.92) populations. Thus, our results
clearly indicate the paraphyly of Th. asperum sensu lato
with respect to Th. licin and Th. petilum. Genetic differ-
entiation between Malayan and Indochinese Th. asperum
lineages is quite profound ( p= 12.24%, see Table 2).
According to the recently published paper by Dever et al.
(2015) the poorly known species Th. phrynoderma is also
likely a member of the Th. asperum group. We also report
a certain genetic variation within Th. asperum from Indo-
china ( p= 3.16%, see Table 2).
The second group, comprising the rest of Theloder-
ma taxa, is also divided into the two subgroups. The first
subgroup joins small-sized Theloderma taxa from south-
ern and central Vietnam (Fig. 3, clades 20 – 24: Th. bam-
busicola,Th. laeve,Th. nebulosum,Th. truongsonense,
and two unidentified lineages, allied to Th. truongso-
nense), which we indicate as Th. laeve group (see Fig. 3;
monophyly support BPP = 1.0). The second subgroup
joins all other Theloderma species (Th. leporosum group:
clades 13 – 19; Fig. 3) and has a moderate value of
monophyly support (BPP = 0.93, see Fig. 3). According
to the phylogeny by Nguyen et al. (2015), the Indo-
chinese species Th. lateriticum is also likely a member of
Th. leporosum group.
However, phylogenetic structuring within both the
Th. laeve and Th. leporosum groups received low support
and relationships between members of these groups re-
main sufficiently unresolved and require further study.
Within the Th. leporosum group, Th. corticale (clade
13) and Th. bicolor (clade 14) form a monophyly (BPP =
= 1.0, Fig. 3); these species appear to be closely related
(p= 6.09%). Certain differentiation is revealed between
Vietnamese (sample 97) and Chinese (samples 98 – 101)
populations of Th. rhododiscum (p= 2.29%). An Indo-
chinese species Th. gordoni (clade 18) appear to be a sis-
ter species to Sundanese and Malayan Th. leporosum
(clade 17) ( p= 8.53%, monophyly support BPP = 1.0).
Finally, we revealed minimal variation (p= 0.65%)
between Th. chuyangsinense from Chu Yang Sin Mt. in
Dak Lak Province of Vietnam (Fig. 3, clade 19, samples
68 – 71) and Th. palliatum from Bi Doup and Hon Giao
Mountains in Lam Dong Province of Vietnam (Fig. 3,
clade 19, samples 92 – 95); samples from the two geo-
graphic populations are mixed and form a single clade
which we indicate as Th. palliatum.
Within the Th. laeve group the species indicated as
Th. bambusicolum by Orlov et al. (2012) from southern
Vietnam forms a most basal split, quite distant from the
other small-sized Theloderma (p= 12.83 – 13.93%).
Populations of this species from the 4 localities in Binh
Phuok, Dong Nai and Lam Dong Provinces of Vietnam
demonstrate low interpopulational differentiation ( p=
= 0.92%; data for Dong Nai Province not included). Dif-
ferentiation between populations of Theloderma cf.
truongsonense from Quang Binh (see Fig. 3; type local-
ity of Th. truongsonense, clade 22), Quang Nam (origi-
nally indicated as Th. truongsonense by Rowley et al.,
2011; reindicated as Th. laeve by Orlov et al., 2012; clade
23) and Theloderma sp. from Khanh Hoa (clade 24)
provinces of Vietnam varies from p= 4.35 to p= 6.10%
(Table 2); taxonomic status of these populations requires
further studies.
TAXONOMY
Status of “Thelodermamoloch (Annandale, 1912)
After its discovery by Annandale (1912) in Abor
country, Arunachal Pradesh (India) and description as
Phrynoderma moloch, this enigmatic species has
changed its generic affiliation several times, being listed
as Rhacophorus,Nyctixalus, and Theloderma (see Ahl,
1931; Dubois, 1981; Inger in Frost, 1985; Nguyen et al.,
2015). Li et al. (2009) were the first to report the distant
phylogenetic relationships between the specimen
6255Rao collected in Motuo (southern Tibet, China),
which they identified as Th. moloch, and the rest of the
Theloderma; the specimen 6255Rao was recovered in
their tree as a sister clade with respect to Philautus. This
result was also confirmed by the following research (Li et
al., 2009; Pyron and Wiens, 2011). Therefore, inclusion
of Phrynoderma moloch Annandale, 1912 in Theloderma
was widely rejected, and the species is preliminary listed
as “Thelodermamoloch in the Amphibian Species of the
World database (Frost, 2015).
However, in a consequent paper, Li et al. (2013)
showed that in the multilocus-based phylogeny of
Rhacophoridae, the sample 6255Rao is nested in the
well-supported Nyctixalini clade, together with Thelo-
derma sensu stricto and Nyctixalus. In the recent paper,
Nguyen et al. (2015) raised doubts on the validity of the
phylogenetic analyses by Li et al. (2013), and assumed
that the figure presented by the latter is incorrect. Though
Nguyen et al. (2015) did not confirm monophyly of The-
loderma sensu lato, their phylogenetic tree does not re-
solve the phylogenetic position of Theloderma moloch;
monophyly of Nyctixalini and Rhacophorinae other than
254 Nikolay A. Poyarkov, Jr., et al.
Liuixalus is also not supported in the tree of Nguyen et al.
(2015).
According to our phylogenetic analyses, the se-
quences of the sample 6255Rao form a well-supported
clade with the rest of Nyctixalini (Theloderma +Nyctixa-
lus); monophyly of the latter clade is also strongly sup-
ported (see Fig. 3). Thus, our results are in agreement
with the data by Li et al. (2013), but they do not contra-
dict the tree of Nguyen et al. (2015), since in their phylo-
gram, the position of Th. moloch is unresolved as well as
some basal nodes of the tree.
Thus, our data suggests the possible monophyly of
Th. moloch with the rest of Nyctixalini. However, to date,
all the debates on phylogenetic position of Th. moloch are
based on the sequence data from a single specimen
6255Rao, identification and taxonomic status of which
require clarification; it is not clear whether it really repre-
sents the species described by Annandale (1912) or not.
Therefore, in our opinion, no taxonomic decision on
Th. moloch is possible until a more detailed phylogenetic
and taxonomic study on additional material of this taxon
is performed, including careful comparison with the type
specimens.
Relationships between Theloderma Tschudi, 1838
and Nyctixalus Boulenger, 1882
The classical works on morphology and taxonomy of
Rhacophoridae (Taylor, 1962; Liem, 1970; Channing,
1989) indicated close proximity of the genera Theloder-
ma and Nyctixalus, based on significant morphological
and ecological similarities, especially similar breeding
habits of these frogs, with no clear synapomorphies dis-
tinguishing Theloderma from Nyctixalus. The following
molecular phylogenetic studies also reported Nyctixalus
and Theloderma as sister genera, mostly due to the in-
complete taxon sampling for both genera (Wilkinson et
al., 2002; Frost et al., 2006; Li et al., 2008, 2009; Yu et
al., 2009; Pyron and Wiens, 2011; Meegaskumbura et al.,
2015). A number of studies (Rowley et al., 2011; Nguyen
et al., 2014), however, recovered Nyctixalus pictus to be
nested within Theloderma, which was later explained as
a result of an insufficient taxon sampling and short se-
quences by Nguyen et al. (2015). Though Nguyen et al.
(2015) state the opposite, this is not the case of the multi-
locus phylogenetic reconstruction by Li et al. (2013),
which imbeds Nyctixalus within Theloderma, with a
clade marked as “Unidentified species” [specimen
ID: LJT W44-W45 from Malaysia; GenBank AN:
KC465842-43, see Table 1] forming a basal split within
Nyctixalini, being a sister clade to the rest of Theloder-
ma +Nyctixalus. Examination of the sequences by Li et
al. (2013) has shown that they correspond to Theloderma
horridum, and therefore their study suggests paraphyly of
Theloderma with respect to Nyctixalus. According to the
divergence time estimates by Li et al. (2013), the diver-
gence between Th. horridum clade, Nyctixalus, and
Theloderma sensu stricto should have taken place within
the time interval of 29 – 25 mya.
The study by Nguyen et al. (2015) provided evidence
for monophyly of Theloderma other than Th. moloch, and
Nyctixalus is recovered as a sister genus to the clade join-
ing Th. horridum clade and Theloderma sensu stricto (the
monophyly of the latter has not an absolute, but still a
high level of support: MLBS/BPP 88/1.0). In our phy-
logeny, Th. horridum +Th. stellatum clade is a sister
group to Theloderma sensu stricto + Nyctixalus, though
the monophyly of the latter group has no significant sup-
port (BPP = 0.86) (Fig. 3). Therefore, our analysis shows
the presence of the three deeply divergent groups of
Nyctixalini (Th. moloch excluded) with practically unre-
solved phylogenetic relationships between them: Th. hor-
ridum +Th. stellatum clade, Nyctixalus and the rest spe-
cies of Theloderma sensu stricto.
Thus, discussing the generic taxonomy of Theloder-
ma Tschudi, 1838 and the related taxa, excluding Th. mo-
loch, on which data are too scarce, the following should
be considered:
1. The three Nyctixalini clades: Th. horridum +Th.
stellatum clade, Nyctixalus and the rest species of Thelo-
derma sensu stricto, are highly divergent from each other
and have likely arisen within a narrow time interval (pre-
sumably, late Oligocene, ca. 29 – 25 mya, based on Li
et al., 2013). The branching order between these three
groups still remains controversial (e.g., compare Li et al.,
2013 and Nguyen et al., 2015).
2. All the three groups share multiple morphological
and ecological characters (Liem, 1970; Channing, 1989),
including the peculiar mode of reproduction, which dis-
tinguish them from all other Rhacophoridae (Meegas-
kumbura et al., 2015).
3. Morphological differences between these groups
are unclear; no synapomorphies are known to date to dif-
ferentiate them from each other (Liem, 1970; Channing,
1989; Rowley et al., 2011; Dever et al., 2015).
Apparently, there are currently five alternative,
equally valid taxonomic options:
1. Recognize no subgenera and place all of the spe-
cies in Theloderma.
2 Recognize a genus Theloderma, with two subgen-
era, Theloderma and Nyctixalus.
3. Recognize three subgenera of Theloderma:Thelo-
derma,Nyctixalus and the unnamed taxon for Th. horri-
dum Th. stellatum clade.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 255
4. Recognize two genera, with two subgenera of
Theloderma,Theloderma sensu stricto and the unnamed
taxon for Th. horridum Th. stellatum clade.
5. Recognize three genera.
At the current state of phylogenetic and morphologi-
cal knowledge, we tend to support the option 3. As noted
by Wake (2013): an “effective way of attaining <...>
a goal [to derive phylogenetic information from taxo-
nomic ranks, without resorting to an alternative, non-
Linnaean taxonomic system] is through the use of sub-
genera <...> this is especially effective when there is
strong evidence of monophyly from diverse sets of bio-
logically significant traits.”
Hence, we propose a new subgenus to designate the
clade encompassing Th. horridum and Th. stellatum,
which we describe below as:
Stelladerma subgen. nov.
Type species. Theloderma stellatum Taylor, 1962.
Holotype. EHT 35441, by original designation; now
FMNH 172249 (according to Frost, 2015).
Included species. Theloderma (Stelladerma)horri-
dum (Boulenger, 1903); Theloderma (Stelladerma)stel-
latum Taylor, 1962; Theloderma (Stelladerma)vietna-
mense sp. nov. (described below).
Diagnosis. Frogs of the subgenus Stelladerma
subgen. nov. are arboreal frogs of medium to large body
size (adult SVL from 22 to 45 mm) and can be differenti-
ated from other congeners by the following combination
of morphological character states: (1) highly tuberculated
skin on the dorsum with calcified warts and large asperi-
ties; (2) two slips of M. extensor digitorum communis
longus (only shown for Th. stellatum by Liem, 1970);
(3) Y-shaped terminal phalanges; (4) a distinct tympa-
num; (5) elongated snout with canthus rostralis rather
sharpened than rounded, nostrils being slightly protuber-
ant; (6) absence of bony ridges from canthus rostralis
to occiput; (7) skin on head not co-ossified with skull;
(8) reproduction in water-filled tree hollows and some-
times man-made waterbodies; (9) bluish coloration of
sclera; (10) iris coloration uniform, brownish to dark
golden with dark reticulations; (11) dark-brown to black-
ish belly coloration with light (whitish to bluish) mark-
ings forming star-like patterns or reticulations; (12) hand-
webbing present, finger and toe discs wide and reddish in
life; (12) vomerine teeth absent; (13) vocal openings in
males present.
Phylogenetic definition. The most inclusive mono-
phyletic taxon including the species Theloderma stella-
tum Taylor, 1962 and excluding the species Th. asperum
(Boulenger, 1886).
Etymology. Derived from Latin “stella” for “star,”
what refers to the scientific name of the type species of
the new subgenus — Theloderma stellatum Taylor, 1962
(“stellated”) and also is a reference to the beautiful belly
coloration of the members of this taxon with star-shaped
or reticulated whitish to bluish pattern on dark back-
ground. Gender is neutral.
Distribution. To date known from southern Indo-
china (from central and southern Vietnam, Gia Lai and
Kon Tum Provinces southwards to Phu Quoc Island and
Con Dao Archipelago), eastern Cambodia, Cardamom
Mountains in southern Cambodia and eastern Thailand,
westwards to Uthai Thani Province of Thailand; extreme
southern peninsular Thailand and Malaya south of the
Isthmus of Kra, also including northern Borneo (Sabah,
Malaysia, and possibly Brunei); also found in Pulau
Tioman, West Malaysia.
The proposed preliminary taxonomy of Nyctixalini is
provided in Appendix 2.
Taxonomic rearrangements
within the Theloderma asperum group
Our data show monophyly and distinctiveness of the
Th. asperum species group (Fig. 3), joining a number of
small to large-sized Theloderma, all of which are charac-
terized by presence of uniform reddish-brown iris and
large white blotches on the rear part of the dorsum (with
the exception of for Th. petilum, which has no white
blotches). Phylogenetic relationships within this group
are well-resolved and in general correspond well to those
presented by Nguyen et al. (2015). The large-sized spe-
cies Th. ryabovi (Fig. 3, clade 8) from central Vietnam is
recovered as the most basal lineage in Th. asperum spe-
cies group; it shows distant relationships to all other taxa
of this group (see Table 2, p= 14.66 – 16.61%). The rest
of the taxa are divided in two subgroups, one of which
joins populations from Indochina (Fig. 3, clades 9 – 10)
while the other one encompasses populations of the Ma-
layan Peninsula and Thailand south of the Isthmus of Kra
(Fig. 3, clades 11 – 12). Our study confirms the assign-
ment of Th. petilum to this group, made by Nguyen et al.
(2014, 2015); though morphologically Th. petilum does
not resemble other members of Th. asperum group (com-
pare Fig. 1xto Fig. 1ej).
Our study clearly indicates paraphyly of Th. asperum
sensu lato with Indochinese populations, traditionally as-
signed to this species, forming a well-supported mono-
phyly with Th. petilum (BPP = 0.97), while populations
of Th. asperum from Perak (Malaysia) are closer to Th. li-
cin with significant levels of support (BPP = 0.92). This
species was described by Boulenger (1886) as Ixalus
asper Boulenger, 1886, from “Hill Garden, Larut, Perak,
at an altitude of 3300 feet.” Later this species was trans-
ferred to the genus Theloderma by Taylor (1962). Mean-
while, a morphologically similar rhacophorid frog was
256 Nikolay A. Poyarkov, Jr., et al.
described from “Yang-liu-chung, Yaoshan” in Guangxi
Province of China as Philautus albopunctatus Liu et Hu,
1962. Later this rhacophorid was assigned to the genera
Aquixalus and Liuixalus (Fei et al., 2009; Hertwig et al.,
2012), until it was not synonymized with Th. asperum
Boulenger (1886) by Yu et al. (2007; 2008). However,
Yu et al. (2007; 2008) did not include any material from
the vicinity of the type locality of Th. asperum or, at least,
from the Malayan Peninsula. Although Orlov et al.
(2010) reported a presence of distinct northern and south-
ern populations of Th. asperum in Vietnam, our data indi-
cate that genetic variation among these populations is
quite limited ( p< 3.16%).
According to our data, Th. asperum sensu lato ap-
pears to be paraphyletic, with the Indochinese popula-
tions forming a distant ( p= 12.24%, see Table 1) and
well-supported clade (Fig. 3, clade 9; BPP = 1.0), show-
ing closer affinities to Th. petilum (Fig. 3, clade 10) than
to morphologically more similar Th. asperum (Fig. 3,
clade 12) and Th. licin (Fig. 3, clade 11) from the Ma-
layan Peninsula. This paraphyly and genetic distinctive-
ness is a strong argument towards recognizing the Indo-
chinese populations of Th. asperum sensu lato as a sepa-
rate species. In this case, the name Theloderma albo-
punctatum (Liu et Hu, 1962) comb. et stat. nov. should be
applied at least to the populations from southern China,
central and northern Vietnam and Laos, though the extent
of distribution of this species, as well as its genetic and
morphological distinctiveness from other members of
Th. asperum species complex, has still to be investigated.
Taxonomic status of small-sized Theloderma
from southern Vietnam
Orlov and Ho (2005) described a small-sized rhaco-
phorid frog from Quang Binh Province in central Viet-
nam as Philautus truongsonensis (Fig. 1s). Rowley et al.
(2011) applied 16S rRNA sequences for molecular iden-
tification of the small-sized rhacophorid frogs from cen-
tral and southern Vietnam and described the two new
species — Th. palliatum from Bidoup and Hon Giao
mountains in the Langbian Plateau, Lam Dong Province
(Fig. 1q) and Th. nebulosum from highlands in Kon Tum
Province (Fig. 1u), which were assigned to the genus
Theloderma based on molecular and morphological data.
In particular, the both new species demonstrated the
bicolored iris, a character which, among the Southeast-
Asian Rhacophoridae, they shared only with Ph. truong-
sonensis and Nyctixalus. Rowley et al. (2011) also
showed that based on their phylogenetic data, the frog
they identified as Ph. truongsonensis (from Quang Nam
Province, central Vietnam) is also nested in the genus
Theloderma, and assigned it as Th. truongsonense.
Later, Orlov et al. (2012) argued that the specimen
which was identified as Th. truongsonense by Rowley et
al. (2011) was identified incorrectly. Orlov et al. (2012)
claimed that it is different from the type Philautus
truongsonensis Orlov and Ho, 2005 due to the smooth
skin on the dorsum and almost lacking asperities, and
likely corresponds to the species, which was identified as
Philautus laevis Smith, 1924 in the previous works
(Inger et al., 1999; Orlov and Ananjeva, 2007). Orlov et
al. (2012) proposed a new combination Th. laeve for this
species (Fig. 1v). In the same paper, Orlov et al. (2012)
described the two new small-sized Theloderma species.
Th. chuyansinense was described from a single specimen
from Chu Yang Sin Mountain in the northern edges of the
Langbian Plateau (Fig. 1r), and is morphologically simi-
lar to Th. palliatum.Th. bambusicolum was described
from low altitudes in southern foothills of the Langbian
Plateau (Fig. 1t); this species was assigned to the genus
Theloderma mainly by presence of the bicolored iris and
based on its reproductive biology — it breeds in wa-
ter-filled bamboo internodes and is strictly associated
with bamboo forests. However, in contrast to the other
Theloderma species, Th. bambusicolum has a completely
smooth skin with no signs of warts or asperities. The sta-
tus and phylogenetic position of all these taxa remained
controversial.
Our phylogenetic data (Fig. 3) indicate the
following:
(1) Samples of Th. palliatum from the type locality
(Bidoup Mt.) and Th. chuyangsinense from the type lo-
cality (Chu Yang Sin Mt.) form a clade with no clear
structuring and low intergroup genetic distances, p<
< 0.65% (Fig. 3, clade 19). In our opinion, this clearly in-
dicates that the two taxa are conspecific and Th. chuyang-
sinense Orlov et al., 2012 should be considered as a sub-
jective junior synonym of Th. palliatum Rowley et al.,
2011. Possibly, taking into account conservation treats
due to fragmentation of Langbian forests and isolated po-
sition of Chu Yang Sin Mountain at the northern edge of
the plateau, a subspecific status would be more appropri-
ate for the Chu Yang Sin population of Th. palliatum.
(2) The small-sized Theloderma of southern and
central Vietnam form a clade (Fig. 3, Th. laeve group)
with the poorly resolved phylogenetic relationships. Pop-
ulations, indicated by Orlov et al. (2012) as Th. bambusi-
colum (Fig. 1t), form a lineage (Fig. 3, clade 20), which
is quite distant from the other members of the group
(p= 12.83 – 13.93%, see Table 2) (the correct Latin
name should be modified to “bambusicola” as a noun
in preposition from “bambusa” (bamboo) and “-cola”
(tiller, inhabitant); gender is masculine).
(3) Th. nebulosum from Kon Tum Province repre-
sents a divergent lineage (Fig. 3, clade 21); it appears that
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 257
this species was misidentified with Th. truongsonense (as
Philautus truongsonensis) in some of the previous works
(Orlov and Ananjeva, 2007; Orlov et al., 2012).
(4) The topotypic Th. truongsonense from Quang
Binh (Fig. 3, clade 22), showing presence of dermal as-
perities on the dorsum (Fig. 1s), forms a well-supported
clade with the smooth Theloderma, lacking dorsal asperi-
ties (Fig. 1v), previously indicated as Th. truongsonense
by Rowley et al. (2011) and as Th. laeve by Orlov et al.
(2012) (Fig. 3, clade 23) ( p= 4.35%). An unidentified
Theloderma sp. from Khanh Hoa province with a moder-
ate level of dorsal asperities development (Fig. 1w)isre
-
covered as a sister clade of Th. truongsonense (Fig. 3,
clade 24; p= 5.78 – 6.10%, Table 2). Thus, the clades 22,
23, and 24 from central and southern Vietnam are closely
related to each other and show certain variation in dorsal
skin structure from the well-developed tiny dermal aspe-
rities in Th. truongsonense to almost completely smooth
dorsum in Th. cf. truongsonense. Here we tentatively join
all these three lineages under the name Th. truongso-
nense (see below).
In order to clarify taxonomic relationships within the
Th. laeve group, we had a chance to examine the holotype
of Philautus laevis Smith, 1924, stored in BMNH
1947.2.5.94 (formerly 1924.1. 31.1; formerly M. Smith
2439) (Fig. 4a–c). Smith (1924) described this species
from “Sui Kat, alt. 1000 m., Langbian Plateau, S. An-
nam” (now in Lam Dong Province, Vietnam). In all ma-
jor diagnostic characters, the type of Smith (1924) is in-
distinguishable from the form, described by Orlov et al.
(2012) as Th. bambusicolum; photos of the Th. bambusi-
colum holotype ZMMU A-4569 (Field ID ZMMU
NAP-02908) are given in Fig. 4d,e. During the recent
field surveys in Lam Dong Province (Bao Lam District)
258 Nikolay A. Poyarkov, Jr., et al.
ab
cde
Fig. 4. Type specimens of Philautus laevis Smith, 1924 and Theloderma bambusicolum Orlov, Poyarkov, Vassilieva, Ananjeva, Nguyen, Sang et
Geissler, 2012. Philautus laevis holotype BMNH 1947.2.5.94 (formerly 1924.1.31.1) in preservative: a, ventral view; b, dorsal view; c, lateral view
(photos A. V. Abramov); Theloderma bambusicolum holotype ZMMU A-4569 (field ID ZMMU NAP-02908) in life: d, dorsal view; e, ventral view
(photos N. A. Poyarkov).
of Vietnam, we discovered several new populations of
Th. bambusicola from altitudes up to 800 – 900 m a.s.l.,
almost at the same altitudes and in geographic proximity
to the type locality of Philautus laevis Smith, 1924.
Therefore, we consider that Th. bambusicolum Orlov,
Poyarkov, Vassilieva, Ananjeva, Nguyen, Sang et Geiss-
ler, 2012 should be considered a subjective junior syn-
onym of Philautus laevis Smith, 1924.
Differentiation within Theloderma (Stelladerma)
stellatum Taylor, 1962 species complex
Our work also sheds some light on taxonomy of
Theloderma stellatum Taylor, 1962, a medium-sized spe-
cies inhabiting southern Indochina from eastern Thailand
to southern and central Vietnam.
Molecular differentiation. Results of our molecular
analyses clearly indicate paraphyly of Th. stellatum sensu
lato with respect to Th. horridum: it appears that the Thai
populations of Th. stellatum form a sister clade to Th.
horridum (Fig. 3) while Th. cf. stellatum from Vietnam is
a more distant lineage. Genetic differentiation of Th. cf.
stellatum from Vietnam to closest congeners is quite sig-
nificant ( p-distance = 9.38 and 10.28% to Th. stellatum
sensu stricto and Th. horridum, respectively; see
Table 2). This degree of pairwise divergence in the 12S
rRNA and 16S rRNA genes is greater than that usually
representing differentiation at the species level in Anura
(Vences et al., 2005a, 2005b; Vieites et al., 2009).
Together with non-monophyly of Th. stellatum sensu lato
this suggests a species status for the Vietnamese popula-
tions.
Known distribution of the Theloderma stellatum spe-
cies complex is shown in Fig. 5: filled icons correspond
to populations, taxonomic identification of which was
checked by mtDNA sequencing [NB: populations 17
(Con Dao) and 19 (Binh Chau) were identified using dif-
ferent genetic markers, therefore are not included in
Table 1]. As Fig. 5 shows, the range of the species com-
plex lasts from central to eastern Thailand, then east-
wards along the Cardamom Mountains to southern Cam-
bodia, southern and central Vietnam, including Phu Quoc
and Con Dao islands. The type locality of Th. stellatum
Taylor, 1962 is situated in Khao Se Bab, Nam Tok Plew
(Phliu) National Park, Chanthaburi Province (locality 10,
Fig. 5). Our molecular phylogenetic analysis assessed the
two populations of Th. stellatum Taylor, 1962 from Thai-
land, including the close vicinity of the type locality in
Chanthaburi Province (locality 9, Fig. 5); other popula-
tions from Thailand and neighboring areas of Cambodia
were tentatively assigned to Th. stellatum Taylor, 1962;
their taxonomic status requires further investigations.
Known distribution of the Vietnamese form of Th. cf.
stellatum lasts from Gia Lai province in central Vietnam
southwards to Con Dao and Phu Quoc Islands; this spe-
cies is also recorded from Mondulkiri Province of Cam-
bodia. The extent of the Vietnamese form of Th. cf. stel-
latum distribution in Cambodia is unknown. Similar dis-
tribution pattern was reported for Indochinese reptiles
(Hartmann et al., 2013) and amphibians (Geissler et al.,
2015); it is interpreted as a role of the Mekong River
basin as a zoogeographical barrier for herpetofauna (see
Bain and Hurley, 2011; Geissler et al., 2015 for discus-
sion).
Thus, based upon the phylogenetic analysis of the
12S rRNA — 16S rRNA mtDNA sequence fragments,
the Th. cf. stellatum from Vietnam represents a highly di-
vergent mtDNA lineage, clearly distinct from all other
Theloderma species for which comparable mtDNA se-
quences are available.
Morphological differentiation. Specimens of the
Theloderma cf. stellatum from southern Vietnam and
Theloderma stellatum from Thailand are morphologi-
cally obviously different from other congeners (see com-
parison). Hence, other taxa of Theloderma were not in-
cluded in the PCA. Total samples of 10 adults of Th. cf.
stellatum from central Vietnam (5 males; 5 females), 9
adults of Th. cf. stellatum from southern Vietnam (7
males; 2 females) and 6 adults (all males) of Th. stellatum
sensu stricto from eastern Thailand were included in the
PCA. The morphological characters used in the PCA are
listed in Table 3 and are also marked (* or **) in Table 4.
The performed PCA discriminated well between the two
species (see Fig. 6); it also clearly distinguished the sam-
ple sets from southern and central parts of Vietnam. As
the result of the PCA, the F1 had an eigenvalue explain-
ing 53.53% of variability, the F2 explained 23.44% of
variability and the F3 just 5.54% (see Fig. 6).
At the same time, from the external appearance,
specimens of Th. stellatum from Thailand (Fig. 1b;
Fig. 7) and Th. cf. stellatum from southern and central
Vietnam (Fig. 1c; Fig. 8) look fundamentally similar to
each other and their diagnostics is very complicated, par-
tially due to the high individual variation in coloration.
This variation is enhanced by the presence of the lighter
night (Fig. 7b,c; Fig. 8d) and darker day coloration
(Fig. 7a; Fig. 8ac), different degrees of the dorsal dark
markings development and a high variation in extend of
whitish or beige blotches on the rear of the dorsum. In the
original description Taylor (1962) reported presence of
trifoliate dark pattern on the dorsum; we also observed a
trifoliate-shaped marking in all the Thai specimens we
had a chance to examine, whereas in Vietnamese popula-
tions the most common dark pattern in shoulders area
was a chevron in a shape of an inverted U or sometimes
Ë. In agreement with description of Taylor (1962), in life
Thai specimens have pinkish coloration of finger and toe
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 259
260 Nikolay A. Poyarkov, Jr., et al.
Fig. 5. Distribution of Theloderma stellatum complex in southern Indochina and Thailand. A dot in the center of an icon indicates the type locality.
Filled icon indicates that the population has been assessed molecularly and its identification was confirmed by mtDNA sequencing. Locality infor-
mation. Theloderma stellatum: Thailand: 1, Hauy Kha Khaeng Wildlife Sanctuary, Uthai Thani Province (this paper); 2, Wangnamkhiew District,
Nakor Ratchasrima Province (this paper); 3, Khao Yai National Park, Nakhon Nayok Province (this paper); 4, Nang Rong waterfall, Nakhon Nayok
Province (this paper); 5, Pangsida National Park, Sa Kaew Province (this paper); 6, Khao Soi Dao Wildlife Sanctuary, Chanthaburi Province (this
paper); 7, Khao Ang Rue Nai Wildlife Sanctuary, Chonburi Province (this paper); 8, Khao Kitchakut National Park, Chanthaburi Province (this pa-
per); 9, Phliu waterfall envrions, Chanthaburi Province (this paper); 10, Khao Se Bab, Nam Tok Plew (Phliu) National Park, Chanthaburi province
(type locality of Theloderma stellatum Taylor 1962); 11, Koh Chang National Park, Trad Province (this paper); 12, Koh Kut, Trad Province (this pa-
per); Cambodia: 13, Koh Kong Province (Neang and Holden, 2008); 14, Central Cardamom Protected Forest, Pursat Province (Neang and Holden,
2008); Theloderma vietnamense sp. nov.: Cambodia: 15, Khao Seima National Park, Mondulkiri Province (Neang and Holden, 2008; Rowley et al.,
2011); 16, Steng Chhral, Kampot Province (this paper); Vietnam: 17, Phu Quoc National Park, Phu Quoc, Kien Giang Province (Nguyen and
Nguyen, 2008; Nguyen et al., 2009; this paper); 18, Nui Dai Mt., An Nong, Tinh Bien Commune, An Giang Provine (Ngyen Ngoc Hung, communi-
cation; this paper); 19, Con Dao National Park, Con Son Island, Ba Ria — Vung Tau Province (Poyarkov and Vassilieva, 2011; this paper); 20,Lo
Go – Xa Mat National Park, Tay Ninh Province (this paper); 21, Binh Chau — Phuok Buu National Park, Ba Ria — Vung Tau Province (this pa-
per); 22, Binh Thuan Province (Rowley et al., 2011); 23, Bac Binh District, Binh Thuan Province (NCSM 80384; Dever et al., 2015); 24,MaDa
Forestry, Dong Nai Biosphere Reserve, Dong Nai Province (Nguyen et al., 2009); 25, Cat Tien National Park, Dong Nai Biosphere Reserve, Dong
Nai Province (type locality of Theloderma vietnamense sp. nov., this paper); 26, Loc Bac (Loc Bao) Forestry, Lam Dong Province (this paper); 27,
Bu Gia Map National Park, Binh Phuok Province (this paper); 28, Yok Don Mt., Yok Don National Park, Dak Lak Province (Nguyen et al., 2009;
this paper); 29, Krong Pa, Gia Lai Province (Nguyen et al., 2009); 30, Kon Ka Kinh Nature Reserve, Gia Lai Province (Nguyen et al., 2009); 31,
Phu Yen Province (Nguyen et al., 2014); 32, K’Bang; Kon Cha Rang Nature Reserve, Gia Lai Province (Nguyen et al., 2014); 33, Mang Canh envi-
rons, Kon Plong, Kon Tum Province (Orlov and Ananjeva, 2007; Nguyen et al., 2014; this paper); 34, Nui Son Tra Mt., Danang City (this paper);
35, Nam Sagi River Drainage Basin, Vilabouli District, Savannakhet Province, Laos (NCSM 76485-8; Dever et al., 2015); 36, Tham Bing Cave,
Xepon Mines, Vilabouli District, Savannakhet Province, Laos (NCSM 76490; Dever et al., 2015); 37, Phong Nha – Ke Bang National Park, Quang
Binh Province (Luu et al., 2013).
disks (Fig. 7), whereas in Vietnamese populations digit
disks are usually brick-reddish or rusty (Fig. 8). Finally,
we have observed relatively stable differences in the
belly pattern (Fig. 9). The three species of the subgenus
Stelladerma subgen. nov. show an increasing degree of
development of the dark (blackish to dark-brown) spots
on the belly, with Th. cf. stellatum from southern Viet-
nam (Fig. 9a) having the darkest ventral pattern with
comparatively sparse light (whitish to bluish) thin
reticulations on the belly and rare minute whitish dots on
the throat. Th. stellatum sensu stricto from eastern Thai-
land (Fig. 9b) shows a lighter ventral pattern, formed by
the thick irregular white or bluish star-shaped spots and
reticulations, usually connected to each other, and more
pronounced white spotting on the throat. Finally, Th.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 261
TABLE 3. Measurements of the Examined Series of Theloderma stellatum and Theloderma vietnamense sp. nov.
Charac-
ter
Th. vietnamense sp. nov. Th. stellatum
Central Vietnam Southern Vietnam Thailand
males (N= 5) females (N= 5) males (N= 7) females (N= 2) males (N=6)
aver. max min aver. max min aver. max min aver. max min aver. max min
SVL 32.60 35.10 30.20 32.80 35.80 30.20 29.85 31.94 28.02 29.96 30.14 29.78 30.69 36.90 22.70
A-G 17.18 17.90 16.40 17.96 20.40 16.50 15.48 17.16 13.74 17.53 17.64 17.41 14.02 15.90 12.50
HW 13.44 14.10 12.90 14.20 16.20 12.40 11.76 12.99 10.52 11.73 11.88 11.57 12.15 13.20 11.19
HL 14.92 16.20 13.50 14.28 15.80 13.70 11.30 12.53 10.15 11.86 12.36 11.36 11.42 12.70 10.70
HD 6.06 6.30 5.80 6.54 7.10 6.00 4.20 4.69 3.90 4.23 4.27 4.18 3.63 4.19 3.40
UEW 4.22 4.40 4.00 4.70 5.60 4.10 2.80 3.10 2.54 2.64 2.71 2.56 3.47 4.00 2.54
IOD 5.26 5.70 5.00 5.82 6.80 5.10 3.51 3.83 3.12 3.59 3.74 3.44 3.77 4.70 3.10
IND 3.56 3.80 3.30 3.88 4.70 3.40 2.17 2.44 1.81 2.09 2.18 1.99 2.56 3.10 2.13
ED 4.78 5.20 4.60 4.94 5.60 4.30 3.94 4.20 3.67 3.87 3.91 3.82 3.80 4.40 3.00
TD 4.14 4.70 3.70 4.68 5.30 3.60 2.17 2.39 1.89 2.37 2.37 2.37 3.05 3.90 2.40
ESL 6.00 6.80 5.10 6.30 7.30 5.30 4.92 5.39 4.38 4.58 4.72 4.44 6.35 8.90 4.42
TED 1.36 1.40 1.30 1.42 1.80 1.20 1.07 1.28 0.88 1.21 1.30 1.11 1.71 2.30 1.30
END 3.80 4.10 3.60 4.34 5.30 3.60 3.59 3.99 3.26 3.44 3.51 3.37 5.43 9.70 3.59
FLL 23.60 25.40 21.80 24.92 29.60 21.90 18.16 18.89 17.00 19.83 20.11 19.55 16.53 19.10 14.00
FFL 3.80 4.00 3.50 4.00 4.70 3.60 2.65 2.96 2.26 2.97 3.08 2.85 3.21 4.10 2.30
TFL 7.30 7.60 7.00 7.58 8.00 7.20 5.44 5.89 4.80 5.73 5.84 5.62 7.27 9.60 4.70
FTD 1.86 2.20 1.60 1.86 2.20 1.60 1.84 2.06 1.66 1.98 2.17 1.78 2.37 2.80 1.90
NPL 3.22 3.50 2.90 2.48 2.99 1.26 2.57 3.30 1.51
MKTe 0.96 1.26 0.66 0.65 0.80 0.50 1.36 2.50 1.00
HLL 55.40 59.70 52.30 58.98 69.10 51.40 44.97 48.44 41.40 45.70 47.56 43.84 50.81 61.50 42.30
FL 14.08 14.90 13.70 14.72 16.40 13.80 13.62 14.81 12.31 13.90 14.43 13.37 14.79 18.80 11.10
TL 17.92 19.10 16.90 19.68 22.90 15.80 15.30 16.92 13.79 16.01 16.08 15.94 16.34 16.80 15.36
FOT 23.40 25.70 21.40 24.58 29.80 21.50 20.06 22.21 18.20 19.78 20.16 19.40 23.81 32.70 19.96
FTL 3.38 3.60 3.20 3.72 4.30 3.30 2.75 3.23 2.31 2.89 2.98 2.79 3.72 4.70 3.30
FFTL 9.32 10.00 8.80 9.78 11.50 8.70 7.93 9.94 6.82 8.40 8.74 8.06 12.48 12.60 7.98
HTD 1.12 1.40 0.90 1.22 1.60 0.90 1.25 1.51 1.05 1.17 1.45 0.88 2.50 4.10 1.48
MTTi 1.33 1.55 1.12 1.31 1.39 1.23 1.66 2.40 1.30
For abbreviations see Material and Methods; “aver.” for “average.” All measurements are given in mm.
6
–8 –8–6 –6–4 –422002244668810 10
Factor 1: 53.53%
ab
Factor 1: 53.53%
7
6
5
4
3
2
1
0
–1
–2
–3
–4
4
2
0
–2
–4
–6
–8
–10
Factor 2: 23.44%
Factor 2: 5.54%
Fig. 6. Two-dimensional principal component plot showing the suc-
cess of multivariate morphometrics in discriminating the members of
Theloderma stellatum complex: Th. stellatum from Thailand (dia-
monds), Th. vietnamense sp. nov. from southern Vietnam (black cir-
cles) and Th. vietnamense sp. nov. from northern Vietnam (white
circles): a, the first two factors of PCA; b, the first and third factors
of PCA.
horridum shows the lightest ventral coloration (Fig. 9c),
with dark coloration on the belly being reduced to numer-
ous blotches of irregular shape and distinct light reticula-
tions on the throat. Based on the material we had a chance
to examine (see Appendix 1 ), we consider that these dif-
ferences in belly coloration are usually stable.
We also found stable differences between the three
Stelladerma subgen. nov. species in the body morpho-
metrics, degree of finger and toe webbing development
and hand morphology. These characters are discussed in
details in the Comparisons.
Acoustics. The advertisement calls of Theloderma
cf. stellatum from southern Vietnam and Theloderma
stellatum Taylor, 1962 from eastern Thailand differ both
by temporal and frequency characteristics (see Fig. 10,
Table 5). Moreover, the advertisement calls of Theloder-
ma cf. stellatum from southern Vietnam have longer call
and intercall durations and show lower frequency range
than calls of Theloderma stellatum sensu stricto. Though
differences in frequencies and temporal parameters may
be partially related to minor differences in temperature
conditions of the recordings or may reflect differences in
body size of calling males. The frequency of maximum
amplitude coincides with the fundamental frequency in
87.5% calls of Theloderma cf. stellatum from southern
Vietnam and only in 34.4% calls of Theloderma stella-
tum. Finally, these two species significantly differ by the
shape of the frequency modulation — arched in Thelo-
derma stellatum Taylor, 1962 (see Fig. 10a; Table 5) vs.
weak lift in Theloderma cf. stellatum from southern Viet-
nam (see Fig. 10b; Table 5).
Taxonomic part
Thus, our molecular, morphological and acoustic
analyses (see below) provide solid evidence that the Viet-
namese populations previously referred as Th. stellatum
represent a distinct yet undescribed species of Theloder-
ma, clearly distinguishable from Th. stellatum sensu
stricto from Thailand and other congeners, which we de-
scribe below as:
Theloderma vietnamense sp. nov.
Synonymy. Theloderma stellatum Taylor, 1962 (par-
tim) — Tarkhnishvili (1994, 1995); Kuzmin and Tarkh-
262 Nikolay A. Poyarkov, Jr., et al.
TABLE 4. Mean Values and Standard Deviation of Morphometric and
Characters of Theloderma vietnamense sp. nov. and Th. stellatum
Character Th. vietnamense
sp. nov. (n= 19) Th. stellatum (n=6) p-value
SVL 31.36 ± 2.21 30.69 ± 4.77
A-G/SVL 53.54 ± 2.69 46.27 ± 5.93 **
HW/SVL 40.86 ± 2.14 40.26 ± 5.62
HL/SVL 41.61 ± 3.81 37.87 ± 5.76
HD/SVL 16.82 ± 2.79 12.05 ± 2.03 **
UEW/SVL 11.56 ± 2.41 11.55 ± 2.57
IOD/SVL 14.51 ± 2.78 12.39 ± 1.59
IND/SVL 9.40 ± 2.27 8.49 ± 1.63
ED/SVL 14.06 ± 1.30 12.41 ± 1.23 *
TD/SVL 10.60 ± 3.26 9.96 ± 1.44
ESL/SVL 17.55 ± 1.80 20.94 ± 2.61 *
TED/SVL 3.98 ± 0.51 5.76 ± 2.22 **
END/SVL 12.18 ± 0.95 17.89 ± 2.64 **
FLL/SVL 68.42 ± 7.03 55.13 ± 11.13 **
FFL/SVL 10.59 ± 1.64 10.43 ± 1.82
TFL/SVL 20.72 ± 2.41 23.90 ± 5.06 *
FTD/SVL 5.96 ± 0.61 7.83 ± 1.58 **
NPL/SVL 2.69 ± 1.46 8.4 ± 1.86
MKTe/SVL 6.44 ± 1.53 4.44 ± 1.62
HLL/SVL 163.48 ± 13.91 167.31 ± 10.98
FL/SVL 44.89 ± 1.94 49.29 ± 3.93
TL/SVL 54.70 ± 4.26 54.50 ± 7.03
FOT/SVL 70.22 ± 4.78 78.54 ± 13.41 *
FTL/SVL 10.12 ± 1.15 12.35 ± 2.54 **
FFTL/SVL 28.09 ± 2.39 34.14 ± 11.09 *
HTD/SVL 3.816 ± 0.61 8.20 ± 2.87 **
MTTi/SVL 5.37 ± 2.30 5.45 ± 1.36
Notes. All morphometric characters are given as % of SVL. Character
abbreviations are explained in Material and Methods.
*p< 0.5, ** p< 0.05.
TABLE 5. Temporal and Frequency Characteristics of Advertisement
Call Parameters for Theloderma stellatum and Theloderma vietnamen-
se sp. nov. [means ± SE (range)]
Parameters Th. vietnamense
sp. nov. Th. stellatum
Number of calls 16 32
Call duration, msec 161 ± 3
(125 – 175) 85 ± 2 (65 – 108)
Intercall interval duration, sec 2 ± 0.02
(1.91 – 2.2) 0.88 ± 0.08
(0.38 – 2.39)
Calling rate, calls/sec 0.47 0.95
Number of harmonics 2.6 ± 0.1 (2 – 3) 2.1 ± 0.1 (2 – 3)
Fbeg,Hz 1230±10
(1180 – 1290) 1480 ± 10
(1350 – 1740)
Fend,Hz 1330±10
(1180 – 1290) 1400 ± 20
(1220 – 1550)
Fmin,Hz 1220±10
(1160 – 1290) 1380 ± 20
(1220 – 1520)
Fmax,Hz 1340±10
(1310 – 1370) 1580 ± 10
(1220 – 1740)
Frange,Hz 120±10
(60 – 170) 200±12
(80 – 370)
Frequency modulation weak lift arched
Fpeak 1,Hz 1300±10
(1270 – 1330) 1530 ± 30
(1370 – 1720)
Fpeak 2,Hz 2550±10
(2540 – 2560) 2910 ± 10
(2840 – 2970)
nishvili, 1997; Orlov (1997; 2005); Inger et al. (1999);
Orlov et al. (2002; 2006; 2010); Orlov and Ho (2005);
Nguyen et al. (2005; 2009); Stuart et al. (2006); Nguyen
and Nguyen (2008); Rowley et al. (2011); Nguyen et al.
(2014; 2015).
Holotype. ZMMU A-5387 (field number
NAP-05238) collected on February 4th, 2015, by Anna B.
Vassilieva and Anna V. Moiseeva in Vietnam, Dong Nai
Province, Dong Nai Biosphere Reserve (former Cat Tien
National Park), Nam Cat Tien Sector, environs of Cat
Tien National Park headquarters, in a water-filled hollow
inaLagerstroemia calyculata (Lythraceae) tree, approxi-
mate coordinates: 11°25¢43¢¢ N 107°25¢38¢¢ E; altitude
ca. 110 m a.s.l. (Figs. 11 – 13).
Paratypes. ZMMU A-4661 (NAP-00081): Cat Tien
National Park, Dong Nai Province, Vietnam (1 sp.,
paratype); ZMMU A-5456 (NAP-03406): Loc Bac (Loc
Bao) forestry, Bao Lam District, Lam Dong Province,
Vietnam (1 sp., paratype); ZMMU A-4525 (NAP-
02998): Con Son island, Road to So Ray, Con Dao Archi-
pelago, Ba Ria — Vung Tau Province, Vietnam (1 sp.,
paratype); ZMMU A-5385 (NAP-03723 – 03725; NAP-
03811; NAP-03824): Phu Quoc National Park, Phu Quoc
Island, Kien Giang Province, Vietnam (5 sp., paratypes);
ZISP 12212 (NAP-03825): Phu Quoc National Park, Phu
Quoc Island, Kien Giang Province, Vietnam (1 sp.,
paratype).
Referred materials. Adults: ZMMU A-5457: Bu
Gia Map National Park, Binh Phuok Province, Vietnam
(1 sp.); ZMMU A-3172-1-2: Ma Da Forestry, Dong Nai
Province, Vietnam (2 sp.); ZMMU A-3778: Lo Go – Xa
Mat National Park, Tay Ninh Province, Vietnam (2 sp.);
ZMMU A-3951: Phu Quoc National Park, Phu Quoc Is-
land, Kien Giang Province, Vietnam (1 sp.); ZMMU
A-5460: Binh Chau – Phuok Buu National Park, Ba Ria –
Vung Tau Province, Vietnam (2 sp.); ZMMU A-3758:
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 263
ab
c
Fig. 7. Theloderma stellatum sensu stricto from Thailand in life: a, adult male from Nakhon Nayok Province, Khao Yai Nature Reserve; b, juvenile
from Uthai Thani Province, Hauy Kha Khaeng Wildlife Sanctuary; c, adults from Uthai Thani Province, Hauy Kha Khaeng Wildlife Sanctuary
(photos P. Pawangkhanant).
Mondulkiri Province, Cambodia (1 sp.); ZMMU
A-3773: Steng Chhral, Kampot Province, Cambodia
(1 sp.); ZISP 7556a-7556b; FMNH 253617 – – 253626:
Tram Lap, 40 km northwest of the town of Kannack,
900 m a.s.l. (Tay Nguyen Plateau), Gia Li Province, Viet-
nam (10 sp.); Larvae: ZMMU NAP-02442: Cat Tien Na-
tional Park, Dong Nai Province, Vietnam (2 sp.); ZMMU
NAP-02827: Loc Bac Forestry, Lam Dong Province,
Vietnam (1 sp.); ZMMU NAP-03680: Lo Go – Xa Mat
National Park, Tay Ninh Province, Vietnam (1 sp.);
ZMMU ABV-00832: Cat Tien National Park, Dong Nai
Province, Vietnam (2 sp.); ZMMU A-3172-3: Ma Da
Forestry, Dong Nai Province, Vietnam (1 sp.).
Etymology. The specific name “vietnamense”isa
Latin toponymic adjective in the nominative singular
(neutral gender), referring to distribution of the new spe-
cies covering central and southern parts of Vietnam.
Recommended vernacular name. We recommend
the following trivial name in English: South-Vietnamese
Bug-Eyed Frog. Recommended vernacular name in Viet-
namese: Ech Cay San Viet Nam.
Diagnosis. The new species is assigned to the genus
Theloderma by (1) having some degree of calcified tu-
berculate skin, (2) presence of a distinct tympanum,
(3) having terminal phalanx with a Y-shaped distal end,
(4) presence of the intercalary cartilage between the ter-
minal and penultimate phalanges of digits, (5) tips of dig-
its expanded into large disks bearing circummarginal
grooves, (6) reproductive behavior of depositing eggs in
water-filled tree hollows, karst crevices etc. (Liem, 1970;
Rowley et al., 2011; Nguyen et al., 2015), and molecular
data (see Fig. 3). Theloderma vietnamense sp. nov. is dis-
tinguished from all other Theloderma by a combination
of the following morphological attributes: (1) absence of
co-ossification of the head skin to the skull; (2) absence
of bony ridges from canthus rostralis to occiput;
(3) highly tuberculated skin on the dorsum with calcified
warts and large asperities; (4) elongated snout with can-
thus rostralis rather sharpened than rounded, nostrils be-
264 Nikolay A. Poyarkov, Jr., et al.
ab
cd
Fig. 8. Th. vietnamense sp. nov. from Vietnam in life: southern Vietnam: a, Kien Giang Province, Phu Quoc Island, Phu Quoc N. P. (photo N. A.
Poyarkov); b, Lam Dong Province, Loc Bac (Loc Bao) (photo N. A. Poyarkov); central Vietnam: c, Gia Lai Province, Kannack, Tram Lap (photo
N. L. Orlov); d, Kon Tum Province, Kon Plong, Mang Canh (photo N. L. Orlov).
ing slightly protuberant; (5) vocal opening in males pres-
ent; (6) absence of vomerine teeth; (7) male of medium
body size (SVL 28.0 – 35.1 mm, mean SVL 31.2 mm),
female (SVL 29.8 – 35.8 mm, mean SVL 32.0 mm);
(8) small tympanum size, tympanum diameter to eye di-
ameter (57% in males, 61% in females), tympanum al-
most completely smooth; (9) dorsal surface with distinct
calcified, white-tipped warts and small asperities (size
ranging from 0.1 – 0.5 mm in diameter), regularly scat-
tered across the dorsum, but being larger and denser at
the head basis; (10) ventral surfaces, including throat,
covered with numerous minute asperities giving it a
slightly granular appearance; (11) rudimentary webbing
between fingers (basal between all fingers; Fig. 12b);
(12) finger and toe discs wide with dorsal surfaces red-
dish in life; (13) supernumerary tubercles on palmar sur-
face absent, inner metacarpal tubercle present, elongated,
single outer metacarpal tubercle; (14) toe webbing exten-
sive, webbing formula I1–2
1/4II 1–2III 1–2
1/4IV
21/4–1
1/2V; (15) inner metatarsal tubercle present;
outer metatarsal tubercle absent; (16) iris coloration uni-
form, brownish to dark golden with brown veins, dark ra-
diating streaks and dark vertical stripe in the lower part of
iris; (17) bluish coloration of sclera; (18) brown dorsum
with irregular brown or rusty with white, occasionally
lavender gray markings, irregular brownish black spots
on dorsum, darker brown inguinal (groin) spots, rear
back and heels usually white, dark transverse stripes on
limbs, and darker, inverted U-pattern between shoulders
on lighter background; (19) dark-brown to blackish belly
coloration with slight whitish to bluish interrupted
reticulations, belly looks dark; throat with minute light
dots; (20) head size to SVL ratio (16.82 ± 2.79); eye di-
ameter to SVL ratio (14.06 ± 1.30); eye to nostril dis-
tance to SVL ratio (12.18 ± 0.95). The new species is also
markedly distinct from all congeners for which compara-
ble sequences are available (12S rRNA to 16S rRNA mi-
tochondrial DNA fragment; uncorrected genetic distance
>9.38%). The advertisement call of the new species, con-
sisting of a tonal call with frequency modulation ex-
pressed in the weak lift of fundamental frequency during
the call, average call duration 160 ± 4 msec, and with an
average dominant frequency of 1.23 – 1.33 kHz, also dis-
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 265
123sec
1234sec
kHz
a
b
kHz
10
10
8
8
6
6
4
4
2
2
Fig. 10. Oscillograms (top) and sonograms (bottom) of male adver-
tisement calls of Th. stellatum complex, recorded at 25.5°C. a,Thelo-
derma stellatum (Thailand, Nakhon Nayok Province, Khao Yai N. P.);
b,Theloderma vietnamense sp. nov. (Vietnam, Dong Nai Province, Cat
Tien N. P.).
ab c
Fig. 9. Ventral patterns in three species of Theloderma (Stelladerma subgen. nov.) in life: a,Th. vietnamense sp. nov. (Vietnam, Dong Nai Prov-
ince, Cat Tien N. P.; photo E. A. Galoyan); b,Th. stellatum sensu stricto (Thailand, Nakhon Nayok Province, Khao Yai N. P.; photo P. Pawang-
khanant); c,Th. horridum (Thailand, Satun Province, Tha Le Ban N. P.; photo P. Pawangkhanant).
tinguishes the new species from Theloderma species for
which calls are known.
Description of holotype. Medium-sized specimen in
good state of preservation; body habitus relatively robust
(Figs. 11 and 13). The holotype has no dissections; the
distal phalanx of the right third finger is absent; the right
fourth finger of the holotype is slightly damaged at the
basis (Fig. 11c,d; Fig. 13). Head. Head slightly longer
than wide (HW/HL 0.91), flattened; top of head sculp-
tured with calcified warts; snout comparatively short
(ESL/HL 0.37), obtusely truncate in dorsal view
(Fig. 11c; Fig. 13a) and gently rounded in profile
(Fig. 12a), snout slightly projecting beyond margin of the
lower jaw; nostril slightly oval-shaped, surrounded by a
slightly raised notably protuberant dermal rim (Fig. 11a),
with a distinct skin flap laterally, located much closer
to the tip of the snout than to the eye (Fig. 12a); loreal
region distinctly concave; canthus rostralis relatively dis-
tinct, rounded; eyes rather large (ED/HL 0.30), eye di-
ameter slightly less than snout length (ED/ESL 0.82),
notably protuberant in dorsal view and in profile
(Fig. 13a,c), pupil horizontal, diamond-shaped; tympa-
num distinct, round with vertical diameter being equal to
the horizontal diameter (vertical diameter to horizontal
diameter (TD) ratio 1.0), tympanic rim elevated relative
to skin of temporal region, tympanum relatively small
comprising 67% of eye diameter (TD/ED 0.67); tym-
panic rim notably elevated relative to skin of temporal
region; vomerine teeth absent; pineal ocellus absent; skin
not co-ossified to skull; choanae oval, at margins of
mouth roof; vocal sac openings present; tongue wide, at-
tached anteriorly with the free posterior end, notched at
posterior tip; supratympanic fold indistinct, being only
notable in the posterior edge of tympanum, running down
266 Nikolay A. Poyarkov, Jr., et al.
a
b
cd
Fig. 11. Holotype Theloderma vietnamense sp. nov. (ZMMU A-5387, male; filed number NAP-05238) in life: a, diurnal coloration; b, nocturnal
coloration; c, dorsal view; d, ventral view. Scale bar 10 mm. Photos by N. A. Poyarkov.
towards an area posteriorly to the corner of the mouth,
not reaching the level of axilla, supratympanic ridge in-
distinct (Fig. 12a). Forelimbs. Forelimbs slender, thin;
relative length of fingers I<II <<IV <III; tips of all
fingers with the well-developed disks with distinct cir-
cummarginal grooves (Fig. 12b), disks moderately wide
compared to finger width (third finger disk width 161%
third finger width), disks slightly wider than long, the
third finger disk width 136% of third finger disk length;
third finger disk width 73% of tympanum diameter; der-
mal fringing developed at all fingers (Fig. 12b); finger
webbing rudimentary, basal between all fingers, reaching
the level of the most proximal articular tubercles at the
inner sides of the first and second fingers, not reaching
the level of the most proximal articular tubercles on
the outer surfaces of all fingers, finger-webbing formula
I2–3
1/4II 3–3III 23/4–2
3/4IV; subarticular tuber-
cles small, slightly protruding, rounded, distinct on all
fingers, finger subarticular formula I(1), II (1), III (1),
IV (1) [subarticular tubercles indistinct at distal articula-
tions of fingers III and IV]; on the ventral surface of the
proximal parts of the third and fourth fingers a low der-
mal midventral ridge present; supranumary tubercles ab-
sent; nuptial pad present, elongated, oval-shaped, cover-
ing prepollex area; a single indistinctly paired outer
metacarpal (palmar) tubercle is barely distinct [the
holotype was collected out of the breeding season]
(Fig. 12b). Hindlimbs. Hindlimbs slender, relatively
long, heels overlap when legs are at the right angles to the
body, but tibiotarsal articulations reaches the level of the
middle of the eye, but not exceeding beyond the front of
the eye; tibia half of the snout-vent length (TL/SVL ra-
tion 0.51); toes fully webbed, toe-webbing formula I
1–2
1/4II 1–2III 1–2
1/4IV 21/4–1
1/2Vwith dermal
fringes reaching to disks at all toes. Tips of toes bearing
disks with distinct circummarginal and transverse
grooves; disks slightly smaller than those of the fingers;
relative toe length I<II <III <V<IV; round yet indis-
tinct subarticular tubercles on all toes, toe subarticular
formula I(1), II (1), III (2), IV (3), V(2); oval-shaped
inner metatarsal tubercle well pronounced and slightly
protuberant, 2.2 times longer than wide, outer metatarsal
tubercle absent (Fig. 12c). Skin texture and skin glands.
Dorsal skin covered with calcified, white-tipped small
pearly asperities, quite evenly distributed as single warts
on dorsum, body flanks and around the vent, but tending
to form several clumps on forehead and on the area poste-
rior to tympanum (Fig. 13a), upper eyelid with small tu-
bercles (Fig. 12a); tympanum almost completely smooth
with 3 tiny asperities discernable (Fig. 12a); ventral sur-
face of thighs and posterior surface of belly coarsely
granular, chest and throat smooth (Fig. 11d; Fig. 13b).
Dermal fringes and pointed projection at tibiotarsal artic-
ulation absent.
Color by Coloration? Dorsal surface diurnally dark
brownish-gray, with distinct dark blackish-brown
blotches and warm brown markings (Fig. 11a,c).
At night coloration more contrasting with dark-gray
ground color turning to warmer and lighter brown tint,
with dark dorsal markings being well-distinct (Fig. 11b).
In the center of the dorsum large blackish-brown blotch
of irregular shape lasts towards the scapular area, form-
ing a distinct dark-brown chevron of an inverted U-shape
(Fig. 11ac), anteriorly edged with light warm beige
(nocturnally, Fig. 11b) to rusty brown (diurnally,
Fig. 11a,c). Irregular diamond-shaped interorbital blotch
located between the posterior edges of the inner margins
of the orbital bulge, is also edged with light-brown
(Fig. 11b). A single dark brown pelvic band runs across
the body between the dark-blackish inguinal spots. Rear
of the dorsum, body flanks, dorsal surface at the basis of
hindlimbs and around the tibiotarsal articulation has
much lighter ground color, varying from dull gray-brown
(diurnally, Fig. 11a) to beige or creamy white (noctur-
nally, Fig. 11b). Three small (ca. 1 mm in diameter)
creamy spots in the middle of the dorsum between the
scapular chevron and the transverse pelvic dark band
(Fig. 11b). Dorsum is covered by numerous whitish or
bluish-white asperities, being brighter and more distinct
at night (Fig. 11b); they are also well-discernable on dor-
sal and lateral surfaces of the head (Fig. 12a). Tympanum
uniformly colored, purplish-brown (Fig. 12a). Lateral
surfaces of the body lighter than the dorsum, gray-brown,
with two distinct uniformly black large inguinal spots
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 267
a
b
c
Fig. 12. Holotype Theloderma vietnamense sp. nov. (ZMMU A-5387,
male; filed number NAP-05238) in life: a, head, lateral view; b, volar
view of the left hand; c, plantar view of the left foot. Scale bar 10 mm.
Photos by N. A. Poyarkov.
(Fig. 11a,b). Small pale bluish speckles on flanks and in
the groin region (Fig. 11a,b; Fig. 12a). Three dark
blackish-brown bands run across the dorsal side of fore-
arm, smaller transverse dark bands continue on fingers.
Three dark blackish-brown bands running across the dor-
sal surfaces of tibiotarsus and thigh; when leg is ad-
pressed, the medial dark band forms a continuous line
with the dark inguinal spot, which, in its turn, continues
to the dorsal pelvic band. Thigh has an extra-band proxi-
mally than the knee; knee with a dark-brownish patch,
forming a dark knee cap. Dorsal surfaces of finger and
toe disks dark brick-reddish with lighter gray margins;
finger disks are brighter than the disks on toes. Dorsal
and ventral surfaces of arms, hands and feet grayish
brown with faint bluish speckling (Fig. 11a,b;
Fig. 12b,c). Ventral surfaces dark blackish-brown with a
purple tint being especially prominent in the throat and
chin area, covered with faint pale bluish white thin
reticulations forming a network-like pattern (Fig. 11d).
Throat with faint white to bluish speckling (Fig. 11d), it
extends laterally onto the upper lip and flanks (Fig. 12a);
minute bluish speckles also present on ventral surfaces of
limbs (Fig. 12b,c).
Pupil horizontally oval; iris coloration uniform,
brownish to dark golden with brown veins, dark horizon-
tal radiating streaks and a more distinct dark vertical
stripe in the lower part of iris (Fig. 12a). Sclera light blu-
ish (Fig. 11b; Fig. 12a).
Color of holotype in preservative. The coloration of
specimens in ethanol depends on its color before eutha-
nasia. In preservative, the reticulated ventral pattern is
distinct, and the inguinal dark spot remains blackish. In
general, coloration remains much resembling the one ob-
served in life, but the dark brown base color of the dorsal
surface looses warm tints in tone and look darker, brown-
ish-gray; light ochre spots on dorsum fade to grayish
creamy-white, ventral coloration is also less warm in
tone (Fig. 13a). The white reticulations on the ventral
surface look more distinct and contrasting, though the
bluish tint fades completely (Fig. 13b).
Measurements of the holotype (all in mm). SVL
33.53; A-G 16.76; HW 12.98; HL 14.32; HD 5.84; UEW
2.68; IOD 3.39; IND 2.22; ED 4.34; TD 2.90; ESL 5.28;
TED 0.87; END 4.13; FLL 20.58; FFL 3.19; TFL 5.69;
FTD 2.12; NPL 2.09; MKTe 1.06; HLL 48.86; FL 14.97;
TL 16.99; FOT 22.07; FTL 3.05; FFTL 8.05; HTD 1.40;
MTTi 1.34.
Variation. All individuals in the type series are gen-
erally similar in morphology and body proportions; vari-
ation of the studied specimens (including the type series)
in morphometric characters is shown in Table 3. We
found no clear differences in body size between the sexes
268 Nikolay A. Poyarkov, Jr., et al.
ab
Fig. 13. Holotype Theloderma vietnamense sp. nov. (ZMMU A-5387, male; filed number NAP-05238) in preservative: a, dorsal view; b, ventral
view. Scale bar 10 mm. Photos by N. A. Poyarkov.
based upon the series examined from both the southern
and central Vietnam (see Table 3).
Specimen coloration may vary greatly in the number
and size of black spots on dorsum, in shape of dark ingui-
nal spots, dark dorsal pattern (may vary from an inverted
V or U-shaped chevron to almost trifoliate figure or sepa-
rate dark blotches), in presence and size of the white
blotches (in some specimens they are completely indis-
tinguishable, whereas in others they may occupy the
larger part of the dorsum) (Fig. 8c,d).
Like other rhacophorids (including most species of
Theloderma, see McLeod and Norhayati, 2007; Rowley
et al., 2011; Orlov et al., 2012), Theloderma vietnamense
sp. nov. can vary the pigmentation of portions of its skin
in response to diel period, stress and microhabitat condi-
tions. Similar to other Theloderma (McLeod and Norha-
yati, 2007; Rowley et al., 2011), in life, both sexes of the
new species show much lighter coloration of dorsum
(to grayish-brown or dark beige), belly (dark brown with
purple tint) and throat nocturnally (Fig. 8d; Fig. 11b)
than during the day time (Fig. 8ac; Fig. 11a). Diur-
nally coloration is much darker, dorsum turns dark warm
chocolate-brown, belly also turns much darker, colored
blackish-brown with violet tint and clear thin bluish-
white reticulations. White blotches on the rear of dorsum
are quite clear during night, whereas diurnally or when
handled and stressed; they turn grayish-beige and may be
almost indistinguishable. Similarly, beige or ochre edg-
ing of dorsal blotches and the dark pattern in scapular
area become much less distinct during the day.
Though we revealed certain differences in body mor-
phometrics between central and southern Vietnamese
populations of Theloderma vietnamense sp. nov. (see
Table 3 for details), in general morphology and body
coloration, population from Kontum Province corre-
sponds well to the type series (see Fig. 14).
Larval stage. Morphological description of larval
stages is based on eight tadpoles (Gosner stages 25 – 35)
collected in tree hollows filled with water: ZMMU
NAP-02442 (2 specimens, Cat Tien National Park, Dong
Nai Province), ZMMU NAP-02827 (Loc Bac Forestry,
Lam Dong Province), ZMMU NAP-03680 (Lo Go – Xa
Mat National Park, Tay Ninh Province) (see Appen-
dix 2 ).
The main morphometric parameters of the tadpoles
are given in Table 6. Body proportions are given as
mean ± SD. We also included data on two tadpoles at
hatching (Gosner stage 25) ZMMU ABV-00832 (2 speci-
mens, Cat Tien National Park, Dong Nai Province; not
included in Table 6).
External morphology. Body broadly oval
(BW/BL = 0.73 ± 0.04), depressed dorsoventrally
(Fig. 15); snout broad, rounded; in fixed specimens lat-
eral lymphatic sacs visible on each side of the body as
thickened horizontal ridges extending along the posterior
half of the body. Eyes small (ED/BL = 0.06 ± 0.02),
smaller at early developmental stages (mean ED/BL =
= 0.05) and slightly larger at more advanced stages
(mean ED/BL = 0.09), dorsal, pupils oriented dorso-
laterally. Tail relatively short (TaL/BL = 1.20 ± 0.1),
slightly longer at more advanced stages, with rounded tip
and well developed muscular part. Upper and lower fins
reach their maximum height in the medium tail part, with
the upper fin being slightly higher or approximately
equal to the lower fin in height (UF/LF = 1.1 ± 0.17), not
extending on the trunk. Spiracle sinistral, lateroventral,
representing short tube fused to the body wall without
free distal portion, with aperture directed posterodor-
sally; spiracle opening margins even, without serration.
Vent tube medial, attached to the lower fin, short, with
oblique aperture oriented ventrocaudally. Narial aper-
tures small, rounded, oriented rostrolaterally, surrounded
by a slightly raised rim; positioned closer to the snout tip
than to the eyes (NP/RN = 1.85 ± 0.50). Mouth antero-
ventral.
Sorting out Moss Frogs: Indochinese Species of the Genus Theloderma 269
a
c
b
d
Fig. 14. Theloderma vietnamense sp. nov. (FMNH 253623, male) in
preservative: a, dorsal view; b, ventral view; c, volar view of the left
hand; d, plantar view of the right foot. Scale bar 10 mm. Photos by
N. L. Orlov.
Tadpoles hatch at stage 25 with the fully developed
opercular fold. At hatching, mouth sheaths are well
formed, labial teeth are still developing. Digestive tract
filled with yolk. Two tadpoles at hatching had TL 10.8,
SVL 3.7 and TL 9.6, SVL 3.4 mm, respectively.
Oral disc. Oral disc (Fig. 16, based on NAP-02827,
Gosner stage 35) moderately wide (ODW/BW = 0.36 ±
± 0.04), elliptical; mouth corners and lower labium
fringed with continuous double (occasionally triple) row
of soft conical papillae. Mouth sheaths with densely ser-
rated cutting edges; upper sheath wide arch-shaped,
lower sheath smoothly U-shaped. Spike-like labial
denticles arranged along the upper labium edge and flat
unpigmented ridges having horizontal orientation: a sin-
gle undivided and several (2 – 4) divided rows on the up-
per labium and invariably 3 rows on the lower labium.
LTRF 1:3+3/3 (variants 1:2+2/3 or 1:4+4/3 are also
observed).
Coloration. Coloration in life (Fig. 15) and in pre-
servative are similar, slightly variable from dark brown to
yellowish brown on the dorsal and lateral surfaces of the
body and tail; ventral surface is paler, semi-transparent,
grayish or yellowish. Eyes and keratinized mouthparts
are black. Lines of neuromasts are faintly visible on the
dorsal surfaces of the head, trunk and tail.
270 Nikolay A. Poyarkov, Jr., et al.
TABLE 6. Main Morphometric Parameters of Theloderma vietnamense sp. nov. Tadpoles
Character NAP-02827 NAP-02827 NAP-02442 NAP-03680 NAP-03680 NAP-02442 NAP-02827 NAP-02827
Stage 25 25 25 27 28 31 31 35
TL 15.5 16.6 20.2 23.9 26.7 26.1 30.8 34.1
BL 7.0 7.6 9.2 11.5 12.8 11.6 13.5 14.1
TaL 8.5 9.0 11.0 12.4 13.9 14.5 17.3 19.7