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A new species of the genus Megophrys Gunther, 1864 (Amphibia: Anura: Megophryidae) from Mount Wuyi, China

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A new species of horned toad, Megophrys ombrophila sp. nov., is described based on specimens found from Guadun village from Mount Wuyi in northwestern Fujian Province, China. The species is distinguished from other described Megophrys by morphology, bioacoustics, and molecular data of the 12S and 16S mitochondrial loci. The species is characterized by its rotund stature, head length approximately equal to head width, canthus rostralis well developed, tympanum large and distinct, vomerine teeth absent, margin of tongue smooth, not notched from behind, heels of the feet not meeting when femurs are held at 90° to the axis of the body and tibias are depressed against the femur, toes weakly webbed at base, dorsal skin mostly smooth with scattered granules and ridges, usually the presence of two discontinuous dorsolateral ridges, supratympanic fold distinct and well-developed, and with females ranging from 32.8–35 mm snout-to-vent length, and males ranging from 27.4–34.5 mm SVL. In its type locality, the species is sympatric with M. boettgeri and M. kuatunensis; and phylogenetically, the most closely related described species is M. obesa from southwest Guangdong province. As more cryptic species are described within the subfamily Megophryinae, the more apparent becomes the need for fine-scale molecular and phenotypic assessment to capture the many forms of this hyper-diverse group.
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Accepted by M. Vences: 21 Dec. 2018; published: 11 Feb. 2019
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https://doi.org/10.11646/zootaxa.4554.2.9
http://zoobank.org/urn:lsid:zoobank.org:pub:66A87D4F-6323-4D1D-ADCE-28487EC263E4
A new species of the genus Megophrys Gunther, 1864
(Amphibia: Anura: Megophryidae) from Mount Wuyi, China
KEVIN R. MESSENGER
1,2,6
, HOLLIS A. DAHN
3,4
, YUANRUI LIANG
5
, PENG XIE
5
,
YONG WANG
2
& CHANGHU LU
1
1
Nanjing Forestry University, Department of Zoology, Nanjing, Jiangsu, China
2
Alabama A & M University, Department of Biological and Environmental Sciences, Normal, Alabama, USA
3
University of Central Florida, Department of Biology, Orlando, Florida, USA
4
Current address: University of Toronto, Department of Ecology and Evolutionary Biology, Toronto, Ontario, Canada
5
Wuyishan National Nature Reserve, Wuyi City, Fujian, China
6
Corresponding author. E-mail: kevinrmessenger@gmail.com
Abstract
A new species of horned toad, Megophrys ombrophila sp. nov., is described based on specimens found from Guadun vil-
lage from Mount Wuyi in northwestern Fujian Province, China. The species is distinguished from other described Mego-
phrys by morphology, bioacoustics, and molecular data of the 12S and 16S mitochondrial loci. The species is characterized
by its rotund stature, head length approximately equal to head width, canthus rostralis well developed, tympanum large
and distinct, vomerine teeth absent, margin of tongue smooth, not notched from behind, heels of the feet not meeting when
femurs are held at 90° to the axis of the body and tibias are depressed against the femur, toes weakly webbed at base, dorsal
skin mostly smooth with scattered granules and ridges, usually the presence of two discontinuous dorsolateral ridges, su-
pratympanic fold distinct and well-developed, and with females ranging from 32.8–35 mm snout-to-vent length, and
males ranging from 27.4–34.5 mm SVL. In its type locality, the species is sympatric with M. boettgeri and M. kuatunensis;
and phylogenetically, the most closely related described species is M. obesa from southwest Guangdong province. As
more cryptic species are described within the subfamily Megophryinae, the more apparent becomes the need for fine-scale
molecular and phenotypic assessment to capture the many forms of this hyper-diverse group.
Key words: Amphibia, Anura, Megophryidae
Introduction
The horned toads of the genera Megophrys sensu lato represent a highly diverse and cryptic group of frogs found
throughout central and southern Asia, from as far west as northeast India, to as far east as Zhejiang Province,
China, and as far south as Malaysia. Interestingly, the genera are lacking from both Taiwan (adjacent to Zhejiang,
125 km gap) and Hainan Island (adjacent to Guangdong, 20 km gap). Megophrys s.l. has undergone several
taxonomic changes in the last decade, bouncing between genera and subgenera of Atympanophrys, Megophrys
sensu stricto, and Xenophrys (Chen et al. 2017; Li & Wang 2008; Liu et al. 2018; Mahony et al. 2017; Pyron &
Wiens 2011). As a whole, Megophrys s.l. contains approximately 73 species, with that number changing every year
from a combination of taxonomic changes and novel animals being found (Chen et al. 2017; Liu et al. 2018;
Mahony et al. 2017; Zhang et al. 2017). The most recent analysis of the assemblage by Mahony et al. (2017)
concludes no distinction and designates the following former taxa as subgenera of Megophrys: Atympanophrys,
Brachytarsophrys, Ophryophryne, Panophrys, Pelobatrachus, and Xenophrys, in contrast to work by Chen et al.
(2017) which found distinction between the groups. It is due to their secretive nature and cryptic diversity that up
until recently (2012), only three species were known from Eastern China, i.e., M. boettgeri Boulenger, M.
kuatunensis Pope and M. huangshanensis Fei & Ye, and it was proposed that the diversity of eastern China is likely
underestimated (Wang et al. 2012). From 2012 to 2017, an additional seven new species of Megophrys were found
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in southeastern China (Li et al. 2014; Wang et al. 2012, 2014, 2017). Among the subgenera, Xenophrys and
Panophrys are primarily found within China, with Panophrys being found almost exclusive to China, and more
specifically, eastern China (Rao & Yang 1997), while Xenophrys being found within China and neighboring
countries (Chen et al. 2017; Liu et al. 2018).
During herpetological surveys from 2013 to 2015, from May to July, at Wuyishan National Nature Reserve
(27.74ºN 117.69ºE; Fig. 1), in Fujian Province, China, we found an unknown frog that could be allocated to Family
Megophryidae, subfamily Megophryinae, genus Megophrys by having the following characters according to Fei et
al. (2009): head broad and depressed, tympanum distinct, tubercles above the eye, tubercles on the chest, vertical
pupil, supratympanic fold, no mid-dorsal fold, and a lack of black spines on the dorsum, but did not match either of
the two-known species of the genus to the area, i.e., M. boettgeri and M. kuatunensis (Fig. 2), as well as all other
recognized congeners of the genus. In this paper, we describe this frog as a new species based on morphological,
bioacoustic, and molecular data.
FIGURE 1. Location of Wuyishan, the type locality, represented by black star in southeastern China, as well as the localities of
the closest related species to M. ombrophila sp. nov.: M. obesa represented by the black circle, and M. sp. nov. 8 and 9 per Liu
et al. (2018) represented by the black square.
Materials and methods
Taxon sampling. From May to July 2013, May to June 2014, and May to June 2015, for a total of 55 days
(approximately 1,404 man-hours), we sampled the Wuyishan National Nature Reserve on the Fujian side of the
reserve. Permits did not allow us to sample the northern Jiangxi side of the reserve. Surveys were conducted
regardless of weather conditions and ranged in elevation from 400 m to 1540 m. Initial locations in 2013 were
surveyed for a minimum of 5 times, each time consisting of three surveys (morning, afternoon, evening), and each
survey lasting for a minimum of 3 hours, for the months of May, June, and July. In 2014 (15 days), search efforts
were concentrated on those areas that had previously yielded the most specimens of the unknown megophryid. In
2015, surveyed locations ranged from 700 m to 1400 m. During all surveys, environmental data was taken with a
Kestrel 4000 hand-held weather meter at shoulder height for each amphibian observation as well as GPS
coordinates (< 3 m).
Tissue samples collected include the undocumented megophryid as well as M. boettgeri, M. kuatunensis, and
Leptobrachella liui from Mount Wuyi, Guadun (= Kuatun) Village, Fujian Province and M. wushanensis from
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Shennongjia National Nature Reserve, Hubei Province. Tissues from M. obesa in Heishiding Nature Reserve,
Guangdong Province were provided by The Museum of Biology, Sun Yat-Sen University (SYS), Guangzhou,
Guangdong Province, China. From GenBank, sequences were incorporated from a subset of available
consubgeners and outgroups chosen based on the phylogenies of Pyron & Wiens (2011), Chen et al. (2017), and
Mahony et al. (2017). This included all available sequences from the Megophrys subgenera Panophrys and
Xenophrys, a limited number of representatives from other Megophrys subgenera (Mahony et al., 2017), the
species Megophrys dringi because it lacks a current subgenera assignment, and available sequences from
provisionally identified samples and putative species previously published under Megophrys sensu lato
(Supplementary Materials Table S1). Three species of the genus Leptobrachella were also incorporated as a more
distant outgroup.
Four of the collected specimens (WUYI2014101–WUYI2014104) were fixed in 10% buffered formalin after
preserving liver and muscle tissue in 95% ethanol. The whole specimens were later transferred to 75% ethanol, the
rest were placed in 95% ethanol and then later transferred to 75% ethanol. Permitting stipulated a set number of
specimens for each species (M. boettgeri, M. kuatunensis, and the unknown megophryid) for preservation (n = 10).
Vouchers of all focal specimens examined from Wuyishan were deposited at the Biology Museum at Nanjing
Forestry University, Nanjing, Jiangsu Province, China (NJFU), and the Wuyishan National Nature Reserve
headquarters, Wuyi, Fujian Province, China (WUYI). Photographic vouchers of non-captured specimens were
accessioned to HerpMapper.org (HerpMapper 2017), http://www.herpmapper.org/, for public retrieval.
DNA extraction, amplification, and sequencing. Total genomic DNA was extracted from tissue using
TIANamp Genomic DNA Kit (TIANGEN). The quality of the DNA extraction was assessed via gel
electrophoresis on a 2% agarose gel. Amplification of the mitochondrial loci 16S rRNA (16S) and 12S rRNA (12S)
was attempted for all tissue samples using the primers and standard PCR conditions listed (Table 1). Amplified
fragments were again visibly assessed via gel electrophoresis on a 2% agarose gel before sequencing in both
directions at Shanghai Invitrogen Biotechnology Co., Ltd.
TABLE 1. Locus and primer information for loci utilized in phylogenetic analyses.
Sequence alignment and phylogenetic analyses. Sequences were checked for quality and edited for
sequencing errors based on chromatograms in Sequencher 5.1 (Gene Codes Corp.). Standard IUPAC ambiguity
codes were inputted at ambiguous sites. Sequence alignments were assembled and aligned using the MUSCLE
alignment algorithm (Edgar 2004) on default settings in the program MEGA 7.0.18 (Kumar et al. 2016). The
section of 16S from position 202 to 273 could not be adequately aligned and was excluded from the phylogenetic
analysis. Mean genetic distances between species were also calculated in MEGA using both uncorrected p-
distances and the Tamura-Nei (TrN) model (Tamura & Nei 1993) (Tables 2, 3, and Supplementary Materials Tables
S2, S3, S4, S5). For this analysis, sequences with <200bp of overlap with our unidentified individuals were
excluded. Ambiguous sites were removed from each sequence pair using the pairwise deletion option in MEGA.
The Bayesian Information Criterion (BIC) and the greedy search scheme in PartitionFinder (Lanfear et al. 2012)
were used to determine the appropriate model of evolution for use in BI analysis, and ModelFinder
(Kalyaanamoorthy et al. 2017) for ML analysis (Table 1). Phylogenetic estimates were recovered via the programs
BEAST 2.5.0 (Drummond et al. 2012; Rambaut & Drummond 2012) and IQ-TREE 1.6.7 (Nguyen et al. 2015),
using Bayesian inference (BI) and maximum likelihood (ML) analyses respectively. In addition to the concatenated
analysis, independent gene trees were also recovered for both loci to assess possible gene tree discordance.
In BEAST, each analysis (12S gene tree, 16S gene tree, and concatenated analysis) was run for 100 million
generations, sampling every 10000th trees. Burn-in was set to discard the first 10% of trees in each run. We used
Locus Forward Primer Reverse Primers Temp
(°C)
MgCl
(mM)
Size
(bp)
BI Model ML Model Reference
16S FPhe40L 5'-AAA
GCA CAG CAC TGA
AGA YGC-3'
12S600Ha 5'-TTA TCG
ATT ATA GAA CAG
GCT CCT CT-3'
48 1.0 546 HKY + I + G GTR+F+R8 Zhang et al.
(2013)
12S L3975 5'-CGC CTG
TTT ACC AAA AAC
AT- 3 '
H4551 5'-CCG GTC TGA
ACT CAG ATC ACG T-3'
50 1.0 576 HKY + I + G GTR+F+R8 Simon et al.
(1994)
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Tracer 1.6.0 (Rambaut et al. 2013) to assess convergence of model parameters by confirming that all effective
sample size (ESS) values exceeded 300. In this way, we observed that the gene trees and concatenated BEAST
analysis failed to converge under the model of evolution suggested by PartitionFinder (GTR+I+G) and over-
parameterization was indicated given the data set. The BI analyses were then re-run modeling both loci under the
simpler model HKY+I+G, and all BI trees reached stationarity under this method (ESS>300). ML analyses were
conducted in IQ-TREE using ultrafast bootstrapping (Hoang et al. 2017) and run until they converged with a
correlation coefficient of at least 0.99. Topologies were visualized using the program FigTree 1.4.0 (Rambaut &
Drummond 2012) (Fig. 3). Nodes are considered supported when a posterior probability greater than or equal to
0.95 and bootstrap support greater than or equal to 95% are recovered under BI and ML respectively (Huelsenbeck
et al. 2001; Minh et al. 2013).
FIGURE 2. Dorsolateral view of sympatric Megophrys species with M. kuatunensis in upper left, M. boettgeri in upper right,
and the holotype of M. ombrophila sp. nov. (lower right).
Morphological characters. Character measurements followed that of Li et al. (2014). Sex of specimens were
determined by dissection and gonadal inspection. Measurements of the three sympatric species were made with
digital calipers to the nearest 0.1 mm. We measured snout-to-vent (SVL), head length (HDL), from tip of the snout
to articulation of the jaw, head width (HDW) via distance between left and right articulations of jaw, snout length
(SNT) from tip of snout to the anterior corner of the eye, eye diameter (EYE) from the anterior to the posterior
corner of the eye, internasal distance (IND), narrowest interorbital distance (IOD), greatest tympanum diameter
(TMP), tympanum-eye distance (TEY) from anterior edge of tympanum to posterior corner of eye, hand length
(HND) from distal end of radioulna to tip of finger III, radioulna length (RAD), foot length (FTL) from distal end
of tibia to tip of finger III, and tibia length (TIB). All specimens were measured by a single author (KM) to
minimize sampling error. Additional morphological data of other species was obtained from literature records
(Boulenger 1908; Fei 1999; Fei et al. 1992, 2009; Huang & Fei 1981; Inger & Iskandar 2005; Inger & Romer 1961;
Jiang et al. 2008; Le et al. 2015; Li et al. 2014; Liu 1950; Liu et al. 2018; Mahony 2011; Mahony et al. 2011, 2013;
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Malkmus et al. 2002; Mathew & Sen 2010; Mo et al. 2010; Munir et al. 2018; Neang et al. 2013; Nguyen et al.
2014; Ohler 2003; Ohler et al. 2002; Orlov et al. 2015; Poyarkov et al. 2017; Rao & Yang 1997; Smith 1921; Stuart
et al. 2006a, b; Tapley et al. 2017; Taylor 1920; Tran 2013; Tran et al. 2010; Wang et al. 2012, 2014, 2017; Ye &
Fei 1995; Zhang et al. 2017; Zhao et al. 2014).
Acoustic data. We recorded male advertisement calls from 20 individuals, 10 each, of the two sympatric
species of Megophrys at an average ambient air temperature of 20.4ºC + 0.6 (range: 19.2–22.1), and recorded calls
from two individuals of the putative new species, one recording from the field on 18 May 2012 and one recording
from a captive animal responding to playback on 28 May 2015, both at an ambient air temperature of ~20ºC. The
recording from the field was with a Sony ICD-MX20 Digital Voice Recorder, held within ca. 1–2 m of the calling
individual next to a mountain stream. The recording from the individual in captivity was with an Olympus Digital
Voice Recorder WS-822, held within ca. 1–2 m of the calling individual. For the recording from the field, due to
stream noise, we imported the audio (44.1 kHz sampling rate, 16-bit encoding) into the program Audacity 2.2.1
(Audacity, http://web.audacityteam.org/) to remove as much of the background noise as possible. To remove low
frequency stream noise, below that of the frog call, we ran a high pass filter and used a conservative cutoff
frequency of 1 kHz (determined through visual inspection of the spectrogram), such that only sounds below 1 kHz
would be affected. The call itself is above 2 kHz, so this removal should have no effect on the attributes of the call
itself. Lastly, to eliminate more stream noise and further isolate the call, we highlighted the noise profile of the
stream sound between calls. Once a portion was highlighted and sampled by the program, we ran a noise removal
filter with default presets: noise reduction 24 dB, sensitivity 0 dB, frequency smoothing 150 Hz, attack/ decay time
0.15 seconds. Once stream noise was reduced, audio files were analyzed in Raven Pro 1.5 software (Bioacoustics
Research Program: http://www.birds.cornell.edu/raven). Audiospectrograms in figures were created using a
Hanning window with a fast-Fourier transformation of 1024 points, 50% overlap, and 43.1 Hz grid-spacing.
Measured parameters followed Rowley et al. (2016). We defined a continuous vocalization with a pause less than 1
second as a call and defined the smallest non-split syllable as a note. For each recording, we measured the call
duration (s), intercall interval (s), number of notes per call, number of notes per second, note duration (s), internote
interval (s), high and low frequency (Hz), dominant frequency (Hz), frequency band-width (Hz), and percent of
call composed of the first note. Audio recordings used were accessioned to AmphibiaWeb.org (AmphibiaWeb
2017), http://amphibiaweb.org/, and HerpMapper.org (HerpMapper 2017), http://www.herpmapper.org/, for public
retrieval. For the specimen recorded in the wild that evaded capture, both the original audio and the cleaned-up
audio are available along with a photograph of the specimen (HM 171540), as well as photographs and audio of the
captive specimen that responded to playback (HM 198774/ NJFU2015502).
Results
Molecular analyses.Where molecular data were available, we found that the undescribed species differs from all
other currently described congeners. Comparisons of between-group genetic distances are reported in Tables 2 and
3, and Supplementary Materials Tables S2, S3, S4, and S5. For interpretations of these values, we followed the
conventions outlined in Vences et al. (2005) and Fouquet et al. (2007). Malone and Fontenot (2008), using data
from interspecific crosses, also found that 3% genetic distance is usually associated with low to moderate post-
zygotic isolation in their studied group of toads. We consider 3–5% genetic distance in 16S to be sufficient for
identifying putative divergent lineages to then be further investigated with other analyses and other forms of
evidence. Notably, one of the smallest between-group Tamura-Nei genetic 16S distances we recovered between
species of Megophrys was 0.006 (both TN and p-distance) between M. boettgeri and M. huangshanensis. This is
consistent with both Li et al. (2014) and Chen et al. (2017) who concluded M. boettgeri and M. huangshanensis
could not be adequately distinguished using 16S sequence and further assessment of the validity of these species
was warranted. This low value is more consistent with expected genetic distance between populations of the same
species than between different species. With respect to the undescribed species, the smallest genetic distances were
recovered for the putative species 8 and 9 identified by Liu et al. (2018). Both the Tamura-Nei and p-distances
were 16S: 0.002/12S: 0.012 for Liu et al. (2018) sp. 8 and 16S: 0.005/12S: 0.005 for Liu et al. (2018) sp. 9. It
appears likely based on these measures that these individuals are other populations within the undescribed species.
Further comparison of other lines of evidence are necessary to distinguish Liu et al. (2018) sp. 8 and sp. 9 from the
undescribed species. The two currently described species with the lowest 16S genetic distances are M. cheni (TN,
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16S: 0.026/12S: 0.041; p-distance 16S: 0.025/12S: 0.039), and M. obesa (TN, 16S: 0.022/12S: 0.035; p-distance,
16S: 0.021/12S: 0.034), though this measure is insufficient to determine sister taxa. This level of sequence
divergence suggests a close phylogenetic relationship but remains consistent with accepted levels of mitochondrial
sequence divergence between other species in the genus.
Our BI and ML analyses yielded different topologies with low support at the subgenus level, but robust support
at shallower nodes. The phylogenetic hypothesis most strongly supported by our analyses indicates the undescribed
species to be a distinct, monophyletic group within the subgenus Panophrys and sister to a clade containing
putative species number 8 and 9 indicated by Liu et al. (2018) (Fig. 3). The most closely related current species is
M. obesa. The reciprocal monophyly of the undescribed species and the clade comprised of Liu et al. (2018)
putative species 8 and 9 is well supported by our BI and ML analyses. Additionally, the relationship to the most
closely related described species, M. obesa, is also supported under both analyses. The Tamura-Nei genetic
distances between the undescribed species and M. obesa were found to be 0.022 in 16S and 0.035 in 12S (p-
distance, 16S: 0.021/12S: 0.034), consistent with species-level sequence divergence. Gene tree discordance was
observed between the 16S and 12S phylogenies relating to the arrangement of deeper nodes of the phylogeny than
are relevant to the conclusions of this study. These results are corroborated by our morphological and bioacoustics
analyses in delimiting the undescribed species as an evolutionary lineage distinct from other currently described
species.
Morphological comparisons. Here we compare the undescribed species with all known 73 congeners known
as of July 2018 as well as two putative species from Liu et al. (2018) (Supplementary Materials Table S6).
The undescribed species is a comparatively small species, with 1) the larger sex (females), having a maximum
SVL of 35.0 mm, and can be distinguished from 48 congeners where the smaller sex has a minimum SVL >35.0
mm: M. aceras Boulenger (Inger & Iskandar 2005; Li et al. 2014; Wang et al. 2012), M. ancrae Mahony, Teeling,
& Biju (Li et al. 2014; Mahony et al. 2013), M. auralensis Ohler, Swan, & Daltry (Li et al. 2014; Ohler et al. 2002;
Wang et al. 2012), M. baluensis Boulenger (Malkmus et al. 2002; Wang et al. 2012), M. baolongensis Ye, Fei, &
Xie (Li et al. 2014; Wang et al. 2012), M. binlingensis Jiang, Fei, & Ye (Li et al. 2014; Liu et al. 2018; Wang et al.
2012), M. carinense Boulenger (Fei 1999; Smith 1921), M. caudoprocta Shen 1994 (Li et al. 2014; Liu et al. 2018;
Wang et al. 2012), M. chuannensis Fei, Ye & Huang (Liu et al. 2018; Zhao et al. 2014), M. damrei Mahony (Li et
al. 2014; Mahony 2011; Neang et al. 2013; Wang et al. 2012), M. dringi Inger, Stuebing, & Tan (Inger & Iskandar
2005; Wang et al. 2012), M. edwardinae Inger (Inger & Iskandar 2005; Malkmus et al. 2002), M. feae Boulenger
(Fei 1999; Liu et al. 2018; Smith 1921; Zhao et al. 2014), M. gigantica Liu, Hu, & Yang (Li et al. 2014; Wang et al.
2012), M. glandulosa Fei, Ye, & Huang (Fei et al. 1992; Li et al. 2014; Mathew & Sen 2010; Wang et al. 2012), M.
hansi Ohler (Ohler 2003; Orlov et al. 2015; Poyarkov et al. 2017; Tran et al. 2010), M. huangshanensis Fei & Ye
(Li et al. 2014; Wang et al. 2012, 2017), M. intermedia Smith (Smith 1921; Tran 2013), M. jingdongensis Fei & Ye
(Li et al. 2014; Wang et al. 2012), M. jinggangensis Wang (Li et al. 2014; Wang et al. 2012, 2014), M. kobayashi
Malkmus & Matsui (Malkmus et al. 2002), M. lancip Munir, Hamidy, Farajallah & Smith 2018 (Munir et al. 2018),
M. latidactyla Orlov, Poyarkov, & Nguyen (Orlov et al. 2015), M. lekaguli Stuart, Chuaynkern, Chan-ard, & Inger
(Li et al. 2014; Stuart et al. 2006a; Wang et al. 2012), M. ligayae Tay lor (Taylor 1920), M. longipes Boulenger
(Inger & Iskandar 2005; Wang et al. 2012), M. major Boulenger (Hecht et al. 2013; Li et al. 2014; Tran 2013; Tran
et al. 2010; Wang et al. 2012), M. mangshanensis Fei & Ye (Fei et al. 1992; Wang et al. 2012), M. medogensis Fei,
Ye, & Huang (Li et al. 2014; Wang et al. 2012), M. montana Kuhl & Van Hasselt (Inger & Iskandar 2005; Taylor
1920), M. nankiangensis Liu & Hu (Li et al. 2014; Wang et al. 2012), M. nasuta Schlegel (Malkmus et al. 2002),
M. obesa Wang, Li, & Zhao (Li et al. 2014), M. omeimontis Liu (Li et al. 2014; Liu 1950; Wang et al. 2012), M.
pachyproctus Huang (Huang & Fei 1981; Li et al. 2014; Ohler 2003; Wang et al. 2012), M. palpebralespinosa
Bourret (Inger & Iskandar 2005; Li et al. 2014; Wang et al. 2012), M. parallela Inger & Iskandar (Inger & Iskandar
2005; Wang et al. 2012), M. parva Boulenger (Inger & Iskandar 2005; Wang et al. 2012), M. popei Zhao, Yang,
Chen, Chen & Wang (Liu et al. 2018; Zhao et al. 2014), M. robusta Boulenger (Li et al. 2014; Mathew & Sen
2010; Wang et al. 2012), M. sangzhiensis Jiang, Ye & Fei (Jiang et al. 2008; Li et al. 2014; Wang et al. 2012), M.
serchhipii Mathew & Sen (Li et al. 2014; Mathew & Sen 2010; Wang et al. 2014), M. shapingensis Liu (Li et al.
2014; Liu 1950; Wang et al. 2012), M. shuichengensis Tian & Sun (Li et al. 2014; Wang et al. 2012), M. spinata
Liu & Hu (Li et al. 2014; Wang et al. 2012), M. stejnegeri Taylor (Taylor 1920), M. synoria Stuart, Sok & Neang
(Stuart et al. 2006b; Poyarkov et al. 2017), and M. takensis Mahony (Li et al. 2014; Mahony 2011; Wang et al.
2012).
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TABLE 2. Calculated Tajima-Nei between-group mean genetic distances using the 12S locus between Megophrys ombrophila sp. nov., putative species #8 and #9 from Liu
et al. (2018), eleven most closely related congeners based on the BI phylogeny, and the outgroup species Leptobrachella liui. The number of base substitutions per site from
averaging over all sequence pairs between groups are shown. For distances between all included taxa and uncorrected p-distances, see Supplementary Materials Tables S2, S3.
- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Leptobrachella liui 1 -
Megophrys acuta 2 0.316 -
Megophrys boettgeri 3 0.323 0.052 -
Megophrys brachykolos 4 0.333 0.072 0.071 -
Megophrys caudoprocta 5 0.312 0.043 0.037 0.060 -
Megophrys cheni 6 0.311 0.051 0.048 0.061 0.025 -
Megophrys jinggangensis 7 0.311 0.037 0.045 0.054 0.029 0.025 -
Megophrys kuatunensis 8 0.334 0.047 0.048 0.055 0.022 0.029 0.030 -
Megophrys lini 9 0.323 0.062 0.064 0.073 0.041 0.045 0.050 0.050 -
Megophrys obesa 10 0.330 0.049 0.045 0.063 0.029 0.030 0.034 0.036 0.049 -
Megophrys ombrophila
sp. nov.
11 0.333 0.069 0.058 0.064 0.039 0.041 0.049 0.055 0.061 0.035 -
Megophrys sp. ZL2018-8 12 0.309 0.057 0.057 0.065 0.042 0.040 0.045 0.055 0.056 0.035 0.012 -
Megophrys sp. ZL2018-9 13 0.307 0.055 0.051 0.057 0.037 0.034 0.040 0.047 0.051 0.029 0.005 0.006 -
Megophrys spinata 14 0.318 0.062 0.064 0.091 0.064 0.079 0.071 0.079 0.080 0.077 0.091 0.077 0.074 -
Megophrys
tuberogranulatus
15 0.299 0.031 0.034 0.054 0.025 0.028 0.024 0.028 0.044 0.032 0.047 0.045 0.040 0.058 -
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TABLE 2. Calculated Tajima-Nei between-group mean genetic distances using the 12S locus between Megophrys ombrophila sp. nov., putative species #8 and #9 from Liu
et al. (2018), eleven most closely related congeners based on the BI phylogeny, and the outgroup species Leptobrachella liui. The number of base substitutions per site from
averaging over all sequence pairs between groups are shown. For distances between all included taxa and uncorrected p-distances, see Supplementary Materials Tables S2, S3.
- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Leptobrachella liui 1 -
Megophrys acuta 2 0.316 -
Megophrys boettgeri 3 0.323 0.052 -
Megophrys brachykolos 4 0.333 0.072 0.071 -
Megophrys caudoprocta 5 0.312 0.043 0.037 0.060 -
Megophrys cheni 6 0.311 0.051 0.048 0.061 0.025 -
Megophrys jinggangensis 7 0.311 0.037 0.045 0.054 0.029 0.025 -
Megophrys kuatunensis 8 0.334 0.047 0.048 0.055 0.022 0.029 0.030 -
Megophrys lini 9 0.323 0.062 0.064 0.073 0.041 0.045 0.050 0.050 -
Megophrys obesa 10 0.330 0.049 0.045 0.063 0.029 0.030 0.034 0.036 0.049 -
Megophrys ombrophila
sp. nov.
11 0.333 0.069 0.058 0.064 0.039 0.041 0.049 0.055 0.061 0.035 -
Megophrys sp. ZL2018-8 12 0.309 0.057 0.057 0.065 0.042 0.040 0.045 0.055 0.056 0.035 0.012 -
Megophrys sp. ZL2018-9 13 0.307 0.055 0.051 0.057 0.037 0.034 0.040 0.047 0.051 0.029 0.005 0.006 -
Megophrys spinata 14 0.318 0.062 0.064 0.091 0.064 0.079 0.071 0.079 0.080 0.077 0.091 0.077 0.074 -
Megophrys
tuberogranulatus
15 0.299 0.031 0.034 0.054 0.025 0.028 0.024 0.028 0.044 0.032 0.047 0.045 0.040 0.058 -
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FIGURE 3. Majority-rule consensus tree recovered through concatenated Bayesian inference and maximum likelihood
analyses of the 16S and 12S mitochondrial loci. For a full listing of samples in the analysis, including outgroup taxa in
collapsed nodes, see Supplementary Materials Table S1. Nodal support values are listed as posterior probabilities (BI) above
bootstrap support (ML). Well-supported nodes (<0.95 / <95) are denoted with a black dot. Support values on nodes with very
low support (>0.15 / >10) are omitted. Where BI and ML analyses did not recover the same topology, the figure defaults to the
BI topology with bootstrap value omitted.
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FIGURE 4. Advertisement call of Megophrys ombrophila sp. nov. from the field at ~20°C ambient air temperature (i) 10 s
waveform of relative amplitude (Rel. amp.) over time, (ii) waveform and (iii) corresponding 2 s spectrogram of call expanded
from first section shown in i.
FIGURE 5. Waveform and spectrogram of advertisement call of NJFU2015502 in captivity, in response to the original call
from the field.
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FIGURE 6. Representative advertisement call of Megophrys boettgeri from the study area at ~20°C ambient air temperature (i)
10 s waveform of relative amplitude (Rel. amp.) over time, (ii) waveform and (iii) corresponding 2 s spectrogram of call
expanded from first section shown in i.
Of the remaining 25 species of congeners and two putative species, the undescribed species can be
distinguished by the following combination of characteristics, 2) a lack of vomerine teeth vs present in M.
zunhebotoensis Mathew & Sen (Li et al. 2014; Mathew & Sen 2010; Wang et al. 2012), M. zhangi Ye & Fei (Li et
al. 2014; Wang et al. 2012), M. daweimontis Rao & Yang (Le et al. 2015; Li et al. 2014; Wang et al. 2012), M.
megacephala Mahony, Sengupta, Kamei & Biju (Li et al. 2014; Mahony et al. 2011), M. liboensis Zhang, Li, Xiao,
Li, Pan, Wang, Zhang & Zhou (Zhang et al. 2017), M. oropedion Mahony, Teeling & Biju (Li et al. 2014; Mahony
et al. 2013), M. rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley 2017 (Tapley et al.
2017), and in putative Megophrys species 8 and 9 (Liu et al. 2018);
3) presence of a horn-like supraocular tubercle vs absent in M. wuliangshanensis Ye & F ei (Li et al. 2014;
Mathew & Sen 2010; Wang et al. 2012), M. wushanensis Ye & F e i (Fei et al. 2009; Li et al. 2014; Wang et al.
2012), M. binchuanensis Ye & F e i (L i et al. 2014; Wang et al. 2012), M. wawuensis Fei, Jiang, & Zheng (Li et al.
2014; Wang et al. 2012), as well as previously mentioned species M. megacephala, M. oropedion, M. zhangi, and
M. zunhebotoensis;
4) having a tongue not notched from behind vs having a notched or deeply notched tongue in M. kuatunensis
(Li et al. 2014; Wang et al. 2012, 2017; this study), M. vegrandis Mahony, Teeling & Biju (Li et al. 2014; Mahony
et al. 2013), M. minor Stejneger (Inger & Iskandar 2005; Li et al. 2014; Liu 1950; Wang et al. 2012), M. boettgeri
(Li et al. 2014; Wang et al. 2012, 2017; this study), M. gerti Ohler (Nguyen et al. 2014; Ohler 2003; Orlov et al.
2015; Poyarkov et al. 2017), M. cheni Wang & Liu (Li et al. 2014; Wang et al. 2014), and M. microstoma
Boulenger (Hecht et al. 2013; Liu et al. 2018; Ohler 2003; Orlov et al. 2015; Poyarkov et al. 2017), as well as
previously mentioned species M. liboensis, M. oropedion, M. rubrimera (weakly notched in one specimen), M.
wawuensis, and M. zhangi;
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FIGURE 7. Representative advertisement call of Megophrys kuatunensis from the study area at ~20°C ambient air temperature
(i) 10 s waveform of relative amplitude (Rel. amp.) over time, (ii) waveform and (iii) corresponding 2 s spectrogram of call
expanded from first section shown in i.
5) having a head-width to snout-vent ratio greater than 0.36:1 in both males (0.36–0.4:1) and females (0.38–
0.39:1) vs having a head-width to snout-vent ratio less than 0.36:1 in both males and female in M. elfina Poyarkov,
Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che & Mahony (0.27–0.3:1 in males, 0.26–0.28:1
in females; Poyarkov et al. 2017), M. poilani Bourret (0.27–0.33:1 in females; Ohler 2003; Poyarkov et al. 2017),
M. lini Wang & Yang (0.33:1 in males, 0.31–0.35:1 in females; Li et al. 2014; Wang et al. 2014), M. lishuiensis
Wang, Liu & Jiang (0.33–0.34:1 in males, 0.31–0.33:1 in females; Wang et al. 2017), M. tuberogranulatus Shen,
Mo & Li (0.33–0.35:1 in males, 0.31:1 in females; Li et al. 2014; Mo et al. 2010; Wang et al. 2012), and M. acuta
Wang, Li & Jin (0.33–0.36:1 in males, 0.33–0.34:1 in females; Li et al. 2014), as well as previously mentioned
species M. gerti (0.28–0.29:1 in males, 0.25:1 in females), M. microstoma (0.28:1 in males), M. boettgeri (0.31–
0.32:1 in males, 0.28–0.35:1 in females), M. liboensis (0.31–0.34:1 in males, 0.31–0.33:1 in females), and M.
vegrandis (0.34–0.35:1 in males).
6) having a tibia-to-SVL ratio of 0.33–0.41:1 encompassing both males and females vs either sex have a value
greater than 0.33–0.41:1 in M. daweimontis (0.54:1 in males, 0.53–0.59:1 in females), M. wawuensis (0.51–0.59:1
in males), M. wuliangshanensis (0.5–0.51:1 in males, 0.49–0.52:1 in females), M. cheni (0.5–0.56:1 in males, 0.5–
054:1 in females), M. zhangi (0.49:1 in males), M. minor (0.49:1 in males), M. vegrandis (0.49–0.54:1 in males),
M. liboensis (0.48–0.51:1 in males, 0.46–0.47:1 in females), M. wushanensis (0.47–0.48:1 in males, 0.47:1 in
females), M. binchuanensis (0.46–0.48:1 in males), M. zunhebotoensis (0.46–0.5:1 in males), M. lini (0.46–0.53:1
in males, 0.49–0.52:1 in females), M. lishuiensis (0.45–0.46:1 in males, 0.4–0.43 in females), M. boettgeri (0.45–
0.49:1 in males, 0.41–0.47:1 in females), M. tuberogranulatus (0.45–0.51:1 in males, 0.45:1 in females), M. gerti
(0.45–0.58:1 in males, 0.43–0.44:1 in females), M. oropedion (0.44–0.46:1 in males, 0.42–0.43:1 in females), M.
elfina (0.43–0.58:1 in males, 0.42–0.54:1 in females), and M. megacephala (0.42–0.47 in males, 0.42 in females).
7) and lastly, the species is distinguished by lacking toe webbing vs. present in M. brachykolos Inger & Romer
(Li et al. 2014; Wang et al. 2012), as well as previously mentioned species M. binchuanensis, M. boettgeri, M.
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cheni, M. elfina, M. gerti, M. liboensis, M. lini, M. megacephala, M. wawuensis, M. minor, M. tuberogranulatus, M.
vegrandis, and M. wushanensis.
The closest relatives of the new species according to the molecular phylogeny are M. cheni and M. obesa, but
based on data provided in the previous paragraphs, these are morphologically clearly divergent based on the
presence (vs absence) of toe webbing in both species; margin of tongue notched behind (vs not notched) in M.
cheni; lateral fringes on toes present (vs not present) in M. cheni; subarticular tubercles on toes present (vs not
present) in M. obesa; tibiotarsal articulation reaching anterior region of eye in M. cheni (vs reaching posterior
margin of eye); SNT: HDL ratio in males of 0.35:1 in M. obesa and 0.33–0.42:1 in M. cheni vs 0.25–0.31:1 in the
new species; a TEY:TMP ratio in females of 0.88–1.09:1 in M. obesa and 0.83–1.55:1 in M. cheni vs 0.72–0.83:1
in the new species; a FTL:SVL ratio in females of 0.59–0.66:1 in M. obesa and 0.68–0.76:1 in M. cheni vs 0.55–
0.58:1 in the new species; and a RAD:SVL in females of 0.25–0.28:1 in M. obesa and 0.23–0.26:1 in M. cheni vs
0.2:1 in the new species. Furthermore, of the species listed in the above paragraphs, 10 have no genetic data
available. Nine of these are quickly dismissed due to their large sizes: females being between 61 to 118 mm, and
males being between 35 and 116 mm (M. edwardinae, M. latidactyla, M. baluensis, M. damrei, M. ligayae, M.
popei, M. shuichengensis, and M. kobayashi). In addition, all but M. edwardinae and M. shuichengensis possess
vomerine teeth. These two are further distinguished by possessing a notched tongue in M. shuichengensis, and both
possessing toe webbing. For the remaining species which lacks genetic data: M. [Ophryophryne] elfina is readily
distinguished by possessing lateral fringes on toes, toe webbing, and lacking subarticular tubercles on toes.
Bioacoustic analysis of advertisement call. Call descriptions are based on two recording sessions of the
putative new species, recorded at 20 ºC ambient air temperature (Figs. 4–5). Calls were an average of 1.3 s in
duration and consisted of 1–5 notes. Most of the calls were single noted (58%) followed by 25% double-noted and
8% consisted of 5 notes. The call sounds like a harsh, abrupt “renk,” similar to that of a referee whistle.
Compared to the two sympatric species, the calls of the undescribed species differ in many regards from the
calls of M. boettgeri and M. kuatunensis (Table 4; Figs. 6–7). In call duration, the undescribed species was shorter
(M=1.3s, SD=1.4) than M. boettgeri (M=6.8s, SD=0.04) or M. kuatunensis (M=22.2s, SD=10.5). The undescribed
species was shorter in intercall interval (M=1.8s, SD=1.1) than M. boettgeri (M=12.4, SD=10.9) or M. kuatunensis
(M=6.1, SD=3.5). Percent of call composed of note 1 was much higher in the undescribed species (M=75.3%,
SD=31.7) than M. boettgeri (M=3.9%, SD=0.7) or M. kuatunensis (M=3.7%, SD=2.6). Number of notes per call
was much lower in the undescribed species (M=1.8, SD=1.2) than M. boettgeri (M=26.6, SD=5.2) or M.
kuatunensis (M=36.2, SD=16.6). Note duration was longer in the undescribed species (M=0.46s, SD=0.1) than M.
boettgeri (M=0.1s, SD=0.02) or M. kuatunensis (M=0.2s, SD=0.03). Internote interval was longer in the
undescribed species (M=1.3s, SD=1.0) than M. boettgeri (M=0.2s, SD=0.04) or M. kuatunensis (M=0.5s, SD=0.1).
The undescribed species had a higher low frequency (M=2,702 Hz, SD=80) than either M. boettgeri (M=2,383 Hz,
SD=722) or M. kuatunensis (M=2,184 Hz, SD=536). The undescribed species had a lower high frequency
(M=4,857 Hz, SD=246) than either M. boettgeri (M=5,334 Hz, SD=642) or M. kuatunensis (M=5,851 Hz,
SD=759). Therefore, the frequency range was narrower in the undescribed species (M=2,155 Hz, SD=308) than
either M. boettgeri (M=2,951 Hz, SD=1,221) or M. kuatunensis (M=3,677 Hz, SD=1,284). The dominant
frequency of the undescribed species (M=3,536 Hz, SD=38) was between that of M. boettgeri (M=3,408 Hz,
SD=223) and M. kuatunensis (M=3,634 Hz, SD=155).
Due to genetic similarities we also compared the available data of the advertisement calls of the closest related
congeners to the undescribed species, namely M. obesa, M. cheni, and the putative species Megophrys sp. #9 from
Liu et al. (2018) and incorporated the data into Table 4. Call data is not available for Megophrys sp. #8 from Liu et
al. (2018), which is also closely related according to genetic data. However, both Megophrys sp. #8 and sp. #9 are
distinguished in morphology from the undescribed species (Liu et al. 2018). Among the closely related species, the
new species differs from M. obesa, M. cheni, and M. sp. 9 in the following ways: 1) having an average call duration
of 1.3s vs 9.1s in M. cheni (unpublished in M. obesa and M. sp. 9); 2) an average number of 1.75 notes per call vs
9.2 in M. cheni (unpublished in M. obesa and M. sp. 9); 3) in having an average note duration of 0.46s vs 0.24s in
M. obesa, 0.14s in M. cheni, and 0.07 in M. sp. 9; 4) having an average internote interval of 1.3s vs 0.3s in M.
obesa, 0.95s in M. cheni, and 0.19s in M. sp. 9; 5) having an average low frequency of 2.7 kHz vs 1.6 kHz in M.
obesa, and 1.4 kHz in M. sp. 9 (M. cheni having a similarly low frequency of 2.8 kHz); 6) having an average high
frequency of 4.9 kHz vs 6.6 kHz in M. obesa and M. sp. 9, and 4.0 kHz in M. cheni; and 7) having an average
frequency range of 2.2 kHz vs 5.1 kHz in M. obesa, 1.2 kHz in M. cheni, and 5.1 kHz in M. sp. 9.
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TABLE 4. Measurements of advertisement call parameters for sympatric Megophrys species in the Guadun village area and closely related species from Wang et al. (2014)
and Liu et al. (2018). Parameter values are given as means. Sample sizes refer to individuals and not calls.
Species Call
duration (s)
Intercall
interval (s)
% of call
composed of
note 1
Number of
notes per call
Note
duration (s)
Internote
interval (s)
Low
frequenc y
(Hz)
High
frequenc y
(Hz)
Frequency
range (Hz)
Dominate
frequency (Hz)
M. ombrophila (n=2)
Ave 1.33 1.82 75.3 1.75 0.46 1.29 2702 4857 2155 3536
SD 1.43 1.12 31.7 1.22 0.1 0.99 80 246 308 38
Range 0.7–1.97 1.3–2.4 61.7–88.8 1.3–2.2 0.34–0.53 1.1–1.4 2702 4835–4893 2133–2191 3505–3555
M. boettgeri (n=10)
Ave 6.75 12.43 3.9 26.6 0.1 0.16 2383 5334 2951 3408
SD 2.11 10.89 0.7 5.17 0.02 0.04 722 642 1221 223
Range 4.9–11.8 5.6–42.9 2.6–5.3 19–38 0.08–0.13 0.1–0.21 1248–3008 4363–6127 1418–4740 3280–3593
M. kuatunensis
(n=10)
Ave 22.19 6.06 3.7 36.2 0.21 0.47 2184 5851 3677 3634
SD 10.49 3.47 2.6 16.6 0.03 0.12 536 759 1284 155
Range 7.1–39.1 1.5–11.7 1.4–10 10–69 0.17–0.25 0.36–0.7 1653–2960 4651–6740 1734–5187 3388–3804
M. sp. 9 n/a n/a n/a n/a 0.073 0.193 1375.0 6148.1 4773.1 n/a
M. obesa n/a n/a n/a n/a 0.244 0.323 1557.3 6632.4 5075.1 n/a
M. cheni 9.1+2.5 n/a n/a 9.2+2.4 0.14+0.0 0.95+0.05 2799+22.2 4041+38.7 1242+48.6 n/a
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TABLE 5. Measurements (in mm) of the type series of Megophrys ombrophila sp. nov. (H = holotype, P = paratype,
other abbreviations defined in text).
Megophrys ombrophila sp. nov. Messenger & Dahn
Holotype. Adult male, WUYI2014101, collected by Kevin R. Messenger and Hollis A. Dahn (hereafter KRM and
HAD, respectively) on 16 June 2014, from a bamboo plantation (Fig. 8) outside Guadun village, Wuyishan
National Nature Reserve (27.735980ºN 117.640810ºE, 1242 m above sea level), Wuyi City, Fujian Province,
China.
Paratypes. Nine adults (4 males, 4 females, 1 unknown): Two adult males, WUYI2014102 and
WUYI2014103, collected from the same locality as the holotype at 1257 to 1302 m a.s.l. by KRM and HAD on 16–
17 June 2014; an adult male, NJFU2015201, collected by KRM on 27 May 2015 in tea plantation, about 50 m from
bamboo forest edge; an adult male, NJFU2015202, collected by KRM on 28 May 2015, found on rocky path/
ephemeral stream in secondary bamboo forest; an adult female, WUYI2014104, collected by KRM and HAD, from
an abandoned tea plantation near (0.6 km linear distance) the type locality at 1262 m a.s.l. on 17 June 2014,
27.7317 117.6366; an adult female, NJFU2015203, collected by KRM on 30 May 2015 on footpath between tea
plantation and bamboo forest; an adult female, NJFU2015204, collected by KRM on 1 June 2015 north and west of
Guadun village in bamboo plantation, representing the northern and western most find as well as the highest
elevational record at 1350 m a.s.l.; 1 adult female, WUYI2015101, collected by KRM and HAD on 23 June 2015,
found after a rainstorm; 1 specimen of unknown sex, NJFU2015205, collected by KRM on 27 May 2015, was
killed on the road that leads into Guadun village at 27.734 117.646, which also represents the easternmost and
lowest elevation find at 1125 m a.s.l.
Etymology. Megophrys ombrophila: Greek: ombros (rainstorm), Greek: philos (that loves, that is fond of), in
reference to the species’ propensity for being especially active during and after heavy rainstorms. For an English and
Chinese standard name, we are recommending the name Yu Shen Horned Toad/ Yǔ Shén jiǎo chán (䴞⾔䀈㸴), after
the Chinese god of rain.
Diagnosis. The specimen matched the genus Megophrys based on the following characters: head broad and
depressed, tympanum distinct, tubercles present on upper eyelid, tubercles present on the chest, vertical pupil,
supratympanic fold present and distinct, no mid-dorsal fold, and a lack of black spines on the dorsum (Fei et al.
2009). Megophrys ombrophila sp. nov. is characterized by the combination of the following characters: (1) a small-
sized species with 27.4.0–34.5.0 mm SVL in adult males and 32.8–35.0 mm SVL in adult females; (2) head length
slightly shorter than head width (HDL/HDW ratio ranges from 0.85–1.00:1); (3) snout rounded in dorsal view,
slightly protruding, sloping backward to mouth in lateral view, protruding beyond the mandible; (4) canthus
rostralis developed; (5) tympanum large and distinct, TMP/EYE ratio ranging from 0.53–0.69:1; (6) vomerine teeth
absent; (7) heels of the feet not meeting when femurs are held at 90° to the axis of the body and tibias are depressed
against the femur; (8) tibio-tarsal articulation reaching forward to the posterior corner of the eye; (9) tibia relatively
Specimen Type status sex SVL HDL HDW SNT EYE IND IOD TMP TEY HND RAD FTL TIB
WUYI2014101 H M 27.4 9.4 10.8 2.8 3.2 3.5 2.7 1.9 1.9 5.8 5.4 16.3 10.1
WUYI2014102 P M 28.8 10.0 11.1 3.1 3.2 3.7 3.1 2.2 1.8 6.5 5.5 16.5 10.4
WUYI2014103 P M 30.0 10.1 11.4 2.5 4.0 3.6 3.2 2.2 1.6 6.5 6.2 18.1 12.2
WUYI2014104 P F 32.8 11.0 12.5 3.0 4.2 4.0 3.5 2.5 1.8 6.6 6.7 17.9 12.9
NJFU2015201 P M 30.4 11.3 11.2 3.1 4.1 4.0 3.2 2.2 2.4 7.9 6.7 17.8 11.4
NJFU2015205 P ? 34.9 12.1 13.2 3.3 4.4 4.3 4.2 2.6 1.8 7.9 7.0 19.3 14.1
NJFU2015502 P M 34.5 11.6 12.5 3.2 4.5 4.1 3.8 2.8 2.7 8.0 6.4 18.1 11.3
NJFU2015204 P F 34.9 11.3 13.2 4.0 4.4 4.4 4.0 2.4 1.9 7.9 7.0 19.3 12.5
NJFU2015203 P F 34.2 11.2 13.2 3.4 4.2 4.3 4.1 2.2 1.8 8.0 6.9 19.5 11.9
WUYI2015101 P F 35.0 12.3 13.6 4.2 4.1 4.6 3.9 2.6 2.0 8.2 7.1 20.3 13.0
Average 32.3 11.0 12.3 3.3 4.0 4.1 3.6 2.3 2.0 7.3 6.5 18.3 12.0
SD 2.9 0.9 1.1 0.5 0.4 0.4 0.5 0.3 0.3 0.9 0.6 1.3 1.2
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short, TIB/SVL ratio ranging from 0.33–0.41:1; (10) relative finger length I < II < IV < III; (11) toes lacking
webbing; (12) distinct subarticular tubercles at the base of each finger; (13) dorsal skin mostly smooth with
scattered granules and ridges; (14) triangular ridge with small granules between the eyes; (15) a Y-shaped dorsal
ridge with granules; (16) horn-like tubercle on posterior edge of eyelid, distinct but small; (17) supratympanic fold
distinct and well-developed, extending to scapula; (18) throat possessing a single short streak down the midline,
darker in color than surrounding tissue.
FIGURE 8. Habitat of type locality, a bamboo plantation, 0.5 km east of Guadun village.
Description of holotype. An adult male (Fig. 9A-F), 27.4 mm SVL; head wider than long, with HDW/HDL
ratio being 1.2:1; snout rounded in dorsal view, slightly protruding in lateral view, snout protruding beyond the
mandible, sloping backward to mouth; top of the head flat; eye diameter 34% of HDL, pupil vertical; nostril
oblique ovoid; canthus rostralis well developed, forming the beginning of a fleshy, protruding ridge, that continues
over the upper eyelid, and transitions into a supratympanic fold that terminates in the scapular region; internasal
distance slightly longer than interorbital distance; tympanum distinct and large, EYE/TMP ratio 1.7:1; ovoid
choanal slit at base of maxilla; vomerine teeth and ridge absent; maxillary teeth present; tongue margin smooth, not
notched from behind.
Radioulna length is 19.8% of SVL; hand without webbing, hand length measuring 21.3% of SVL; fingers
without lateral fringes, relative finger length I < II < IV < III; tips of fingers slightly dilated, with rounded tips;
subarticular tubercle at base of each finger; inner metacarpal tubercle enlarged, outer metacarpal tubercle smaller,
both reddish in color. Hindlimbs slender, tibio-tarsal articulation reaching forward to the posterior corner of eye;
heels not overlapping when tibia depressed against femur; tibia and foot measuring 36.7% and 59.5% of SVL
respectively; relative toe length I < II < V < III < IV; toe tips rounded and slightly dilated, without webbing; toes
without lateral fringes or subarticular tubercle; inner metatarsal tubercle short and ovoid, reddish in color, outer
metatarsal tubercle absent.
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FIGURE 9. A. View of the adult male holotype WUY2014201 of Megophrys ombrophila sp. nov. in life, among leaf litter. B:
Dorsolateral view of the holotype in life. C: Hand of the holotype. D: Dorsal view of holotype in preservation. E: Ventral view
of holotype in preservation. F: Foot of the holotype. G: Close up view of supraocular tubercles and supratympanic fold (HM
208556). H. Phenotypic variation within the species (WUYI2014102). I. Specimen found by JHY (HM171540) in May 2012.
Dorsal skin rough; several granules throughout the body, from the top of the head, the dorsum, the flanks, and
the dorsal side of limbs. Granules on the flanks are larger than granules elsewhere. Large, distinct tubercles along
the edge of the upper eyelid; series of small granules on dorsum running parallel to body axis. Ventral surface
smoother than dorsal surface but still has presence of small granules; ventral thigh mostly smooth, with several
small femoral glands towards the posterior aspect of thigh; pair of small, white pectoral glands on chest.
Measurements of holotype (in mm). SVL 27.38, HDL 9.35, HDW 10.84, SNT 2.8, IND 3.46, IOD 2.7, EYE
3.22, TMP 1.87, TEY 1.9, HND 5.84, RAD 5.42, FTL 16.29, TIB 10.05.
Coloration of holotype in life (Fig. 9A–B). Dorsal color light brown with dark brown “Y”-shape. The fork of
the “Y” comprised of a raised ridge. Lateral to the ridges of the “Y” are dorso-lateral ridges running lengthwise.
Dark brown interorbital triangle, two corners over each eye and third corner pointing posteriorly form the darkest
aspects of the triangle. Center of triangle is slightly lighter brown in color, more like the background coloration.
Dorso-laterally, the light brown of the dorsum transitions to orange on the venter. Venter is mostly light tan and
orange with dark brown stripes running parallel to the body axis. A prominent stripe running down the center of the
throat to the chest. Pectoral and femoral glands white. Iris of eye is a golden brown to auburn coloration. Dark
subocular stripe from the eye to lip. Another dark post-ocular stripe running from the eye to the tympanum. Dark
crossbars running perpendicular to the radioulna, thigh, tibia, and feet. The granules that are found throughout the
body can vary from red, brown, black, and white in color. Tubercles at the base of each finger are mottled white and
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brown (Fig. 9C) and tubercles at base of hand are red. The subarticular tubercle on the feet are small, ovoid, and
reddish in color (Fig. 9F). The rest of the sole of the foot is brown.
Coloration of holotype in preservation. Preservation caused increased contrast in specimen compared to life.
Light tan fades to lighter tan on dorso-lateral surface. The dark brown patterning turns darker. A reticulate pattern
emerges. The dark corners of the interorbital triangle are the most prominent and the inner aspect of the triangle is
closer to the background coloration. On the ventral surface, the groin is white, chest is mostly grey with black
stripes running parallel to body axis. Presence of crossbars running parallel to the radioulna, thigh, tibia, and feet
still present, as they were in life. Subocular and post ocular stripe present in life are still present in preservation.
Pectoral and femoral glands white.
Va ri at io n . There is variation in morphometric measurements of the type series (Table 5). In nearly all
measured criteria, females are larger than males. The only measurement in which females had a value less than
males was in the average TEY distance. There is much variation in color. Majority of specimens were orange or
yellow-orange in color, but other colors include slate grey, light brown, and dark brown. The dark brown coloration
is usually temporary (likely temperature induced) as these dark brown individuals lighten in captivity within a few
hours. Under preservation, most specimens retained the lighter coloration, making dorsal patterns more visible.
Some specimens have dorsal granules that create a “V” shape, others simply have granules that run in parallel
series to the body. Dorsal pattern most commonly gives the impression of a “Y,” but can also appear as a square, a
“V,” or an “X.” Figure 10 displays several examples of the variation in dorsal patterning.
FIGURE 10. Examples of variation in the dorsal pattern, which usually resembles a “Y” or asymmetrical “X.” Clockwise from
upper left: specimen #: HM208970, NJFU2015201, WUYI2015101, HM208971, HM208972.
Secondary Sexual Characteristics: Males have a single subgular vocal sac. Males collected lacked nuptial
pads or spines, and lacked any fleshy projection above the cloaca, but since our specimens were presumably
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sampled outside of the breeding season, this may be the reason. Sexes of males and females were determined
through inspection of the gonads via dissection.
Distribution and Ecology. We observed 25 individuals total (6 male, 4 female, 15 unknown), all through
active search methods. Save for one presumably roadkilled individual, all specimens were active in habitat. No
eggs or tadpoles were observed despite search efforts. Currently M. ombrophila sp. nov. is only known from the
outskirts of Guadun village (within 1 km of the town). Based on the supposedly preferred habitat and elevational
range in which we found specimens, the species should have an approximate distribution of 173 km
2
within the
Wuyishan National Nature Reserve on the Fujian side of the reserve, but there is no reason for it to not be found on
the Jiangxi side of the reserve. Five of the specimens were found in a bamboo plantation near the village. During
surveys that yielded M. ombrophila sp. nov., the species was found in syntopy with Megophrys boettgeri,
Theloderma rhododiscus Liu & Hu, and Leptobrachium liui Pope. We never found the species syntopic with M.
kuatunensis, though Jianhuan Yang (JHY) did observe a calling male M. ombrophila syntopically with M.
kuatunensis in mid-May. Two individuals were found in an abandoned tea plantation surrounded by bamboo
plantation and secondary forest. Another specimen was found dead on the road (presumably ran over by a car) at
the edge of a bamboo plantation. In general, the species was most commonly found in edge habitat, between
bamboo forest (including bamboo plantations) and mixed broad-leafed evergreen forest. Common vegetation and
tree species include Moso bamboo (Phyllostachys edalis), Long-ear Cane bamboo (Indocalamus longiauritus),
Masson Pine (Pinus massoniana), Chinese fir (Cunninghamia lanceolate), Tea tree (Camellia sinensis) and
Chinese cedar (Cryptomeria fortunei).
Several individuals (n=4) were found in an active tea plantation, but in all cases, the tea plantation was small
(<1 ha) and surrounded by bamboo forest. Specimens were never more than 70 m from bamboo edge. One
specimen was found in a secondary forest. Three specimens were found in secondary bamboo forest (non-
plantation) that was comprised mostly of Long-eared Cane Bamboo, Moso Bamboo, and sparse hardwoods. Eight
specimens were found on the edge habitat between a Long-eared Bamboo Forest and tea plantation. No specimens
were found within the city limits of Guadun village.
Provenance of finds ranged from 1125 to 1350 m, with an average of 1242 m (SD + 39.7 m; n=25). JHY found
the one and only calling male (not collected) on 18 May 2012. The species is more active and detectable during
rain events. Of our 55 days of sampling effort, 28 (51%) were days with rain and 27 (49%) had no rain for at least
24 hours. In May 2015, during nights of rain events, each night yielded 5–7 specimens. One day post rain yielded
2–3 specimens, and any day beyond 2-days post rain did not yield any specimens. Nighttime temperatures of finds
averaged 20.5 (SD + 0.8, range 19.2–22.3°C, n=23), with an average humidity of 92.3 (SD + 4.7, range 86.3–98.9,
n=23), and barometric pressure averaged 25.8 (SD + 0.1, range 25.57–25.93, n=23).
There have not been surveys for this species outside of May, June, and July. We cannot confirm the mating
season. Initially we thought the specimen calling on 18 May 2012 was likely calling toward the beginning of the
breeding season, and efforts were made to find calling males during the second half of May in 2014 and 2015, but
as none were found, despite finding several individuals each night, we now suspect that before the 18
th
of May is
around the end of the breeding season for this species. It is our recommendation that for future surveys for this
species, especially for parties interested in the breeding ecology that surveys for breeding males begin around
March and conducted to mid-May. It would also be of interest to see if the species is present on the Jiangxi
province side of the reserve.
Regarding activity, one specimen (HM 208556) was found in the day time (15:47 on 14 June 2013, a rainy
day) and 24 specimens were found at night. Nocturnal animals were found as soon as 49 minutes after sunset, to as
late as 4 hours 2 minutes after sunset. A higher number of specimens were found immediately after heavy rains,
and only a few specimens were found during heavy rains.
Megophrys ombrophila sp. nov. does not currently face excessive threats. It is not of economic concern, so
there is no worry about the species being used for consumption, medicine, or a high demand for the pet trade (prior
to our distinction/ recognition, none of the locals recognized it as being different from all the other “small frogs”).
Within the Wuyishan nature reserve, the biggest threat is that of habitat destruction. The species is semi-tolerant of
disturbance, seeming to prefer mildly disturbed areas to less disturbed. Mild disturbance in this context is referring
to the creation of a trail through the woods, or a small (<1 ha) tea plantation.
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Discussion
The Wuyishan area, and more specifically, the village of Guadun (spelled “Kuatun” until recent times), has been a
significant area for new species to Chinese herpetology for over 100 years. Wuyishan and Guadun were first made
known to science by Armand David, who visited the site in 1873 (Pope 1931). The tiny, remote village has been the
type locality for at least a dozen species of reptiles or amphibians. Following David’s visit, a subsequent visit by
John D. La Touché in 1896 and 1898 (Boulenger 1899) revealed four new snakes, one lizard, and three frogs, one
of which being Megophrys boettgeri (Boulenger 1899). In the 1920’s and 1930’s, Clifford H. Pope visited Guadun
village, and described an additional two snakes and two frogs, one of which was the second Megophrys species
with the type locality coming from Guadun: M. kuatunensis (Pope 1928, 1929). It is an interesting note of history
and coincidence that now an additional species of Megophrys shares the same type locality as the two-sympatric
species after nearly 100 years since the last species was described from the site.
Research in recent years has illustrated a need for re-examining the amphibian species diversity of southeast
China. The last few years have seen an explosion of several novel species, especially within megophryid diversity
(Li et al. 2014; Liu et al. 2018; Wang et al. 2012, 2014, 2017; Zhang et al. 2017; Zhao et al. 2014). Several other
taxa in southeastern China share similar aspects to Megophryidae, and that fact suggests there may be many more
cryptic complexes that are actually far more diverse than we currently believe them to be. This explosion of
diversity will hopefully incite a re-examining of southeastern herpetofauna in China.
The recent discoveries in southeastern China have illustrated a high degree of endemism and the significance
of isolated mountain ranges, which is where many of these forms are appearing (Li et al. 2014; Liu et al. 2018;
Wang et al. 2012, 2014, 2017; Zhang et al. 2017; Zhao et al. 2014). Southeastern China requires significant work
to determine how widespread and connected these species and populations are between one another as there are
still several more isolated mountain ranges that could hold yet more new species.
Acknowledgements and Ethics Statement
We thank Dr. Yulong Ding of Nanjing Forestry University for helping to initiate the work in 2013 and assisting in
providing proper permits to work in the Wuyishan Nature Reserve, and Justin Waraniak for his help in the field,
and stumbling across the first specimen that generated curiosity in this animal. Immense appreciation to Jianhuan
Yang for his assistance and advice over the course of the project. Advice and edits were graciously given by Dr.
Tiffany Doan of University of Central Florida as well as the anonymous reviewers, and the editors at Zootaxa.
Funding was provided by the National Science Foundation’s REU program, Alabama A & M University, and
partially funded by the PAPD program at Nanjing Forestry University. Permission to visit the study sites were
issued by the management administration of the Wuyishan National Nature Reserve and the Shennongjia National
Nature Reserve. We obtained permissions for specimen and tissue collection from the Fujian Provincial Forestry
Bureau and the Shennongjia National Nature Reserve administration. This study did not involve protected species
in China. All the animal operations were approved by Institutional Animal Care and Use Committee of Alabama A
& M University (Protocols # 10061 and 10062), and strictly complied with the ethical conditions by the Chinese
Animal Welfare Act. Specimens were anaesthetized and subsequently euthanized with 20% benzocaine via
cutaneous absorption on the venter.
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NEW MEGOPHRYS FROM MOUNT WUYI, CHINA
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... Results of the BLAST analyses, including sequences from the four specimens we collected from the Hoang Lien Range, showed that all of the least divergent samples were from species within the subgenus Panophrys. For phylogenetic analyses, 12S and 16S sequences (hereafter the 12S-16S data set) belonging to 47 of the 48 known species of this subgenus were obtained from the following papers with published sequence data (Zheng et al. 2004;Li et al. 2014;Xiang et al. 2013;Oberhummer et al. 2014;Wang et al. 2014;Chen et al. 2017;Mahony et al. 2017;Poyarkov et al. 2017;Tapley et al. 2017;Wang et al. 2017;Zhang et al. 2017;Mahony et al. 2018;Tapley et al. 2018a;Liu et al. 2018;Li et al. 2019Li et al. ["2018; Messenger et al. 2019;Wang et al. 2019a;Wang et al. 2019b;Wu et al. 2019;Cutajar et al. 2020;Liu et al. 2020;Lyu et al. 2020;Nguyen et al. 2020;Wang et al. 2020;Xu et al. 2020) and downloaded from GenBank (Benson et al. 2017), along with the following outgroup taxa representing all other subgenera: Megophrys (Atympanophrys) shapingensis Liu, 1950, M. (Brachytarsophrys) carinense (Boulenger, 1889) Kuhl and Van Hasselt, 1822, M. (Pelobatrachus) nasuta (Schlegel, 1858) and M. (Xenophrys) monticola (Günther, 1864). Two additional taxa, Leptobrachella oshanensis (Liu, 1950) and Leptobrachium boringii (Liu, 1945), were included to root the tree, following Chen et al. (2017). ...
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The genus Megophrys is known to harbour morphological cryptic species diversity. During field work on Vietnam’s third highest peak, Mount Ky Quan San, north-west Vietnam, we collected specimens of a new species of Megophrys at two locations more than 2000 m above sea level (asl). Phylogenetic analyses using mitochondrial markers place the new species within the subgenus Panophrys, a primarily Chinese radiation within the genus Megophrys. We describe the species based on an all-male type series and distinguish it from all other Megophrys species from mainland Southeast Asia, north of the Isthmus of Kra and nearby provinces of China (Yunnan, Guangxi and Guizhou) based on morphological, molecular and bioacoustic data. The new species is inferred to form a clade along with M. hoanglienensis and M. fansipanensis; it is syntopic with the former but has a non-overlapping range with the latter. Uncorrected p distances for the 16S rDNA and CO1 genes between the new species and closest relatives exceed values observed between other closely related species in the Panophrys subgenus. The new species is most similar to M. fansipanensis in terms of morphology, and several call parameters also overlap with this species; however, these two species call at different times of year. This is the fourth likely range restricted and Endangered Megophrys species described from the Hoang Lien Range since 2017, and this discovery further highlights the significance of the Hoang Lien Range for Vietnam’s amphibian diversity.
... Yet, at least M. wawuensis, M. shapigensis and M. gigantica significantly differ at nuclear sequences, with coalescent events older than 5 Myr Liu et al., 2018). Therefore, although low 16S divergences in some Megophrys taxa could indicate conspecific lineages (Messenger et al., 2019), here the pattern probably arises from incomplete lineage sorting of this marker -M. wawuensis/shapigensis/gigantica differ by > 6% at COI [P(S) > 0.5] -and should not invalidate these species. ...
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Biodiversity analyses can greatly benefit from coherent species delimitation schemes and up-to-date distribution data. In this article, we have made the daring attempt to delimit and map described and undescribed lineages of anuran amphibians in the Eastern Palaearctic (EP) region in its broad sense. Through a literature review, we have evaluated the species status considering reproductive isolation and genetic divergence, combined with an extensive occurrence dataset (nearly 85k localities). Altogether 274 native species from 46 genera and ten families were retrieved, plus eight additional species introduced from other realms. Independent hotspots of species richness were concentrated in southern Tibet (Medog County), the circum-Sichuan Basin region, Taiwan, the Korean Peninsula and the main Japanese islands. Phylogeographic breaks responsible for recent in situ speciation events were shared around the Sichuan Mountains, across Honshu and between the Ryukyu Island groups, but not across shallow water bodies like the Yellow Sea and the Taiwan Strait. Anuran compositions suggested to restrict the zoogeographical limits of the EP to East Asia. In a rapidly evolving field, our study provides a checkpoint to appreciate patterns of species diversity in the EP under a single, spatially explicit, species delimitation framework that integrates phylogeographic data in taxonomic research.
... nov. (Fei et al. 2012;Wang et al. 2017b;Messenger et al. 2019;Wang et al. 2020). The new species can be distinguished from these species by a series of morphological characters as follows. ...
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A new species of the Asian horned toad genus Megophrys is described from Zhejiang Province, China, based on multiple data. Molecular phylogenetic analyses based on mitochondrial DNA indicated the new species as an independent clade deeply clustered into the Megophrys clade. The new species is identified from its congeners by a combination of the following characters: body size small (SVL 28.4–32.4 mm in males); vomerine teeth absent; tongue not notched behind; tympanum distinctly visible, oval; a small horn-like tubercle present at the edge of each upper eyelid; two metacarpal tubercles distinctly visible in hand; toes without webbing; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level to middle of eye when leg stretched forward; an internal single subgular vocal sac in male; in breeding male, the nuptial pads present on the dorsal base of the first two fingers.
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Here we reported a new species, Xenophrys lishuiensis Wang, Liu and Jiang, sp. nov. (Fig. 1 and 2). Holotype: WYF00164, adult male, collected by WANG Yu-Fan on 20 May 2016 from Fengyang Forest Station and adjacent area in Liandu, Lishui, Zhejiang, China (28°11′51.72″N, 119°49′2.28″E, altitude 1 100 m); allotype: WYF00169, adult female, collected by WANG Yu-Fan from the locality in the same date as holotype; paratypes: 12♂♂ (WYF00165﹣WYF00168, WYF00171﹣WYF00178), 2♀♀ (WYF00170 and WYF11011), collected in the same date and same locality as holotype, by WANG Yu-Fan. The holotype, allotype and eight of the paratypes (WYF00165﹣WYF00168, WYF00171﹣WYF00173, WYF11011) were deposited in Herpetological Museum of Chengdu Institute of Biology, Chinese Academy of Sciences; the remaining six paratypes (WYF00170, WYF00174﹣WYF00178) deposited in Zhejiang Museum of Natural History. The new species distinguished by a combination of the following characters: 1) small body size, snout vent length (SVL) 30.7﹣34.7 mm in males, 36.9﹣40.4 mm in females; 2) head length larger than head width; 3) vomerine ridge and vomerine teeth absent; 4) tongue not notched behind; 5) two metacarpal tubercles present; 6) tibia length less than half snout-vent length; 7) no web and lateral fringe in toes; 8) a “X” shaped botch on dorsal body, or divided medially, the botch thick, the margin distinct and edged with light color; 9) the triangle botch on dorsal head not touched with the botch on dorsal body; 10) males with indistinct semicircular light-colored botch on shoulder; 11) males with nuptial pad on the first and second fingers, covered by dense and small nuptial spines, relatively less on the second finger (Table 2 and 3, Fig. 1 and 2). In addition, based on 397 based pairs (bp) of mitochondrial 16S rRNA sequences, we constructed phylogenetic relationships using Bayesian inference among 14 species of the genus Xenophrys (Table 1 and Fig. 3). The new species formed a monophyletic group (Clade B) with high support value (Bayesian posterior probability 98%), three of which formed a distinct lineage with high support value (Bayesian posterior probability 100%), there possesses a 3.9% genetic distance between X. lishuiensis and its close relative X. boettgeri, which is greater than the one between some other species in the Genus of Xenophrys (Table 4).
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