Corresponding authors: L. Lee Grismer (
Academic editor Raffael Ernst
The integrated results of morphological and molecular phylogenetic analyses confirmed the new species status of a recently discovered population of
Stream toads of the genus
The general life history of their tadpoles—adhering themselves to the surfaces of rocks beneath fast-flowing water or in the spray zones of cascades—restricts the distribution of
This is the case for a Thai-Burmese clade of nine species of
Distribution of the species of the Thai-Burmese clade of
Specimens were collected in Suan Phueng District, Ratchaburi Province, Thailand by Parinya Pawangkhanant, Platon V. Yushchenko, Mali Naiduangchan, Chatmongkon Suwannapoom, and Nikolay A. Poyarkov during several field surveys from 2016 to 2019. The location of the surveyed locality and the distribution of the Thai-Burmese clade of
Specimen collection and animal use protocols were approved by the Institutional Ethical Committee of Animal Experimentation of the University of Phayao, Phayao, Thailand (certificate number UP-AE61-01-04-022, issued to Chatmongkon Suwannapoom) and were strictly complacent with the ethical conditions of the Thailand Animal Welfare Act. Field work, including collection of animals in the field and specimen exportation, was authorized by the Institute of Animals for Scientific Purpose Development (IAD), Bangkok, Thailand (permit numbers U1-01205-2558 and UP-AE59-01-04-0022, issued to Chatmongkon Suwannapoom).
Observations on color pattern were based on the examination of specimens in life as well as digital images of living and euthanized specimens prior to preservation. Measurements were recorded with a Mitutoyo dial caliper under a Nikon SMZ 1500 dissecting microscope to the nearest 0.01 mm. Measurements of adult specimens generally following
Raw mensural data from the type series of
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ZMMU A-7605* | 24.9 | 8.3 | 7.5 | 3.8 | 2.9 | 1.9 | 1.8 | 2.5 | 2.7 | 1.9 | 1.4 | 1.1 | 0.3 | 13.1 | 7.4 | 1.3 | 11.4 | 11.8 | 9.9 | 1.8 | 1.8 | 1.3 | 0.7 | 35.6 | 17.3 |
ZMMU A-7606 | 25.6 | 8.4 | 7.7 | 3.9 | 2.7 | 1.5 | 1.9 | 2.6 | 2.7 | 1.8 | 1.3 | 0.9 | 0.4 | 13.1 | 6.6 | 1.3 | 10.2 | 11.4 | 9.8 | 1.5 | 1.6 | 1.2 | 0.5 | 34.9 | 16.8 |
ZMMU A-7607 | 23.9 | 6.9 | 7.2 | 3.7 | 2.9 | 1.8 | 1.7 | 2.9 | 2.8 | 2.1 | 1.4 | 1.3 | 0.0 | 12.5 | 6.6 | 1.6 | 10.3 | 11.1 | 8.9 | 1.4 | 1.0 | 0.8 | 0.4 | 33.9 | 16.2 |
ZMMU A-7608 | 23.2 | 7.6 | 7.8 | 4.4 | 3.0 | 1.7 | 1.8 | 2.5 | 3.3 | 2.5 | 1.5 | 1.2 | 0.4 | 12.7 | 6.7 | 1.4 | 10.0 | 10.8 | 8.9 | 1.5 | 1.6 | 1.2 | 0.7 | 33.1 | 16.7 |
ZMMU A-7609 | 23.5 | 7.4 | 7.7 | 4.4 | 3.2 | 2.2 | 2.0 | 2.9 | 2.8 | 2.1 | 1.4 | 1.4 | 0.6 | 12.6 | 6.6 | 1.4 | 11.7 | 11.2 | 9.4 | 2.0 | 1.6 | 1.0 | 0.6 | 37.7 | 19.5 |
ZMMU A-7610 | 23.7 | 7.4 | 7.6 | 4.1 | 3.2 | 2.1 | 2.1 | 2.8 | 3.0 | 2.0 | 1.2 | 1.6 | 0.3 | 12.6 | 6.4 | 1.4 | 10.4 | 11.4 | 9.6 | 1.7 | 1.4 | 1.2 | 0.6 | 35.9 | 19.1 |
ZMMU A-7611 | 24.7 | 7.4 | 7.7 | 4.3 | 3.5 | 2.0 | 2.2 | 2.9 | 3.2 | 2.1 | 1.4 | 1.4 | 0.4 | 12.6 | 6.7 | 1.3 | 10.8 | 12.0 | 9.8 | 1.7 | 1.7 | 1.2 | 0.6 | 36.8 | 18.1 |
ZMMU A-7612 | 24.8 | 7.5 | 7.4 | 4.6 | 3.3 | 2.0 | 2.4 | 2.9 | 3.1 | 2.3 | 1.3 | 1.3 | 0.3 | 12.8 | 6.8 | 1.3 | 11.7 | 11.8 | 10.2 | 1.8 | 1.7 | 1.0 | 0.5 | 37.8 | 18.0 |
ZMMU A-7613 | 25.4 | 7.9 | 10.0 | 4.5 | 3.7 | 2.2 | 2.3 | 2.9 | 3.1 | 2.3 | 1.1 | 1.4 | 0.7 | 13.3 | 7.0 | 1.4 | 11.2 | 11.8 | 9.9 | 1.7 | 1.6 | 0.9 | 0.6 | 35.8 | 19.8 |
AUP-00661 | 24.4 | 8.0 | 7.5 | 3.8 | 3.1 | 1.5 | 1.9 | 2.4 | 2.6 | 1.9 | 1.4 | 1.0 | 0.3 | 13.2 | 6.5 | 1.3 | 11.4 | 12.1 | 9.8 | 1.8 | 1.8 | 1.2 | 0.5 | 34.8 | 17.4 |
AUP-00662 | 24.6 | 8.1 | 7.7 | 3.7 | 2.9 | 1.6 | 1.9 | 2.4 | 2.6 | 1.8 | 1.3 | 1.0 | 0.3 | 13.1 | 6.5 | 1.3 | 11.1 | 12.0 | 9.7 | 1.7 | 1.8 | 1.2 | 0.5 | 34.2 | 17.4 |
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AUP-00663 | 28.9 | 9.2 | 8.7 | 4.4 | 3.5 | 2.3 | 2.2 | 2.8 | 2.9 | 2.4 | 1.5 | 1.4 | 0.4 | 14.3 | 8.0 | 2.1 | 13.4 | 14.1 | 11.6 | 1.9 | 2.4 | 1.4 | 0.9 | 43.6 | 21.8 |
AUP-00664 | 29.2 | 9.1 | 8.2 | 4.5 | 3.6 | 2.1 | 2.5 | 2.8 | 2.9 | 2.4 | 1,51 | 1.4 | 0.5 | 14.4 | 7.5 | 2.1 | 14.2 | 13.7 | 11.5 | 1.9 | 2.4 | 1.3 | 0.9 | 44.7 | 21.2 |
ZMMU A-7614 | 26.2 | 8.3 | 7.9 | 4.0 | 3.3 | 2.0 | 2.1 | 3.0 | 3.2 | 2.4 | 1.4 | 1.4 | 0.7 | 13.7 | 7.3 | 1.3 | 12.0 | 12.2 | 10.2 | 1.7 | 2.1 | 1.4 | 0.7 | 39.2 | 19.1 |
ZMMU A-7615 | 27.9 | 8.5 | 8.3 | 4.7 | 3.1 | 2.1 | 2.1 | 3.2 | 3.2 | 2.8 | 1.6 | 1.4 | 0.5 | 14.3 | 7.1 | 2.9 | 12.7 | 13.0 | 11.6 | 1.9 | 2.0 | 1.3 | 0.8 | 43.5 | 21.8 |
AUP-00665 | 28.8 | 8.9 | 8.1 | 4.2 | 3.5 | 2.3 | 2.1 | 2.6 | 2.8 | 2.3 | 1.5 | 1.3 | 0.4 | 14.0 | 7.0 | 2.1 | 13.1 | 13.1 | 11.5 | 1.8 | 2.4 | 1.4 | 0.9 | 44.1 | 20.5 |
Measurements on a single tadpole specimen AUP-02091 followed
The morphospatial clustering among sampled individuals from a selected subset of species and characters for which there was full coverage for each species were visualized using principal component analysis (
Two-sample
For the molecular phylogenetic analyses, we extracted the total genomic DNA from ethanol-preserved femoral muscle tissue of six specimens of the new Thai population using standard phenol-chloroform-proteinase K extraction procedures with consequent isopropanol precipitation, for a final concentration of about 1 mg/ml (protocols followed
We amplified mtDNA fragments covering partial 16S rRNA gene sequences to obtain a 560 bp-length continuous fragment per specimen. The 16S rRNA gene has widely been applied in biodiversity surveys in amphibians (
PCR products were loaded onto 1.5% agarose gels in the presence of ethidium bromide and visualized in agarose electrophoresis. When distinct bands were produced, we purified the PCR products using 2 μl of a 1:4 dilution of ExoSapIt (Amersham) per 5 μl of PCR product prior to cycle sequencing. A 10 μl sequencing reaction included 2 μL of template, 2.5 μl of sequencing buffer, 0.8 μl of 10 pMol primer, 0.4 μl of BigDye Terminator version 3.1 Sequencing Standard (Applied Biosystems) and 4.2 μl of water. The cyclesequencing used 35 cycles of 10 sec at 96°C, 10 sec at 50°C and 4 min at 60°C. We purified the cyclesequencing products by ethanol precipitation. We carried out sequence data collection and visualization on an ABI 3730xl Automated Sequencer (Applied Biosystems).
Ingroup samples consisted of 128 individuals representing 32 nominal species and included three of the six samples from the new Thai population. Outgroups used to root the tree were
Calculated t and p values from the t-tests.
t-value |
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–2.9397 | 0.02401 | Welch t-test |
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–5.0071 | 0.00187 | Welch t-test |
EL | –7.3776 | 0.00022 | Welch t-test |
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–3.9494 | 0.00363 | Welch t-test |
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–8.1595 | 7.84E-05 | Welch t-test |
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–3.244 | 0.003133 | Student t-test |
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–3.9681 | 0.00656 | Welch t-test |
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–10.845 | 1.49E-06 | Welch t-test |
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–7.8502 | 3.65E-05 | Welch t-test |
Maximum Likelihood (
Stationarity was checked with Tracer v1.6 (
The
A. Majority-rule consensus tree from 1000
The new Thai population from Suan Phueng and its sister species
Summary statistics of the adjusted mensural characters used in the statistical analyses of
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mean | 1.943 | 1.853 | 1.052 | 0.762 | 0.224 | 0.756 | 0.960 | 2.346 | 2.164 |
± sd | 0.034 | 0.041 | 0.055 | 0.072 | 0.081 | 0.104 | 0.063 | 0.028 | 0.038 |
range | 1.907–1.987 | 1.804–1.929 | 0.967–1.112 | 0.672–0.850 | 0.105–0.310 | 0.585–0.851 | 0.847–1.031 | 2.312–2.384 | 2.113–2.212 |
1.987 | 1.929 | 1.112 | 0.850 | 0.310 | 0.851 | 1.031 | 2.384 | 2.212 | |
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mean | 1.983 | 1.995 | 1.167 | 0.986 | 0.369 | 0.853 | 1.066 | 2.486 | 2.298 |
± sd | 0.029 | 0.053 | 0.049 | 0.063 | 0.093 | 0.052 | 0.047 | 0.050 | 0.049 |
range | 1.907–2.030 | 1.804–2.068 | 1.041–1.263 | 0.809–1.108 | 0.198–0.569 | 0.723–0.946 | 0.957–1.161 | 2.312–2.557 | 2.173–2.367 |
Summary statistics and principal component analysis scores for the mensural characters of
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Standard deviation | 2.39726 | 0.96600 | 0.89174 | 0.83157 | 0.57186 | 0.47365 | 0.37752 | 0.32949 | 0.17559 |
Proportion of variance | 0.63854 | 0.10368 | 0.08836 | 0.07683 | 0.03634 | 0.02493 | 0.01584 | 0.01206 | 0.00343 |
Cumulative proportion | 0.63854 | 0.74222 | 0.83058 | 0.90741 | 0.94375 | 0.96868 | 0.98451 | 0.99657 | 1 |
Eigenvalue | 5.74685 | 0.93316 | 0.79520 | 0.69150 | 0.32702 | 0.22434 | 0.14252 | 0.10856 | 0.03083 |
HIL | 0.27098 | 0.54668 | –0.48265 | 0.10878 | –0.53108 | 0.16594 | –0.09269 | 0.22918 | –0.11062 |
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0.33941 | –0.03475 | –0.41842 | –0.11058 | 0.72617 | 0.08739 | –0.11690 | 0.33045 | –0.19611 |
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0.34608 | –0.14423 | 0.01457 | 0.50058 | –0.03803 | –0.62922 | –0.45158 | –0.05594 | 0.06492 |
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0.29530 | 0.23246 | 0.58207 | –0.45619 | –0.08608 | –0.26407 | –0.05060 | 0.35025 | –0.33087 |
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0.38077 | 0.19814 | 0.02006 | 0.16603 | 0.12146 | –0.24659 | 0.79279 | –0.04052 | 0.28647 |
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0.24973 | –0.73749 | –0.24304 | –0.17910 | –0.39792 | –0.03709 | 0.25438 | 0.12371 | –0.25072 |
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0.34435 | 0.06183 | –0.18036 | –0.58885 | –0.02297 | –0.06361 | –0.20669 | –0.60330 | 0.29507 |
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0.37290 | 0.01362 | 0.26001 | 0.31988 | 0.05980 | 0.42742 | 0.02970 | –0.48613 | –0.51707 |
FL | 0.37339 | –0.19475 | 0.30554 | 0.09243 | –0.06762 | 0.50026 | –0.18555 | 0.30861 | 0.57988 |
ZMMU A-7605 (field number NAP-06631), an adult male collected on 8 November 2016 at a forest stream within the montane evergreen forest of Khao Laem Mt., Suan Phueng District, Ratchaburi Province, Thailand (
ZMMU A-7606 (field number NAP-06630), an adult male with same collection information as holotype; ZMMU A-7607 (no field number) and ZMMU A-7608 (field number AUP-00349), two adult males collected on 15 June 2018 at same locality as holotype by P. Pawangkhanant, C. Suwannapoom and M. Naiduangchan; ZMMU A-7609–A-7614 (field numbers NAP-10193–NAP-10198), five adult males and an adult female collected on 18 June 2019 at a forest stream within the montane evergreen forest on the northern slope of Khao Laem Mt., Suan Phueng District, Ratchaburi Province, Thailand (N 13.54581, E 099.20368; 758 m a.s.l. in elevation), by P. Yushchenko and M. Naiduangchan; AUP-00661–00665, two adult males and three adult females collected on 15 June 2019 at same locality as holotype by P. Pawangkhanant and M. Naiduangchan; and ZMMU A-7615 (field number NAP-09901), an adult female collected on 4 October 2019 at same locality as holotype by P. Pawangkhanant.
Adult male,
Holotype of
Forelimbs and fingers long and slender (
Top of head black (Fig.
Holotype of
After five years of storage in ethanol, the warm reddish, yellowish and orange tints have significantly faded, the specimen looks dark greyish-brown; however all
Raw and adjusted mensural data of the type series are presented in Tables
Male paratypes of
Description based on AUP-02091 at
The tadpole of
In life (Fig.
The new species inhabits a polydominant montane tropical evergreen forest on Khao Laem Mountain at elevations from ca. 700 to 750 m a.s.l., where the adult specimens were observed at night perched on leaves or stones (Fig.
Natural history of
The species of amphibians and reptiles recorded in sympatry with the new species at the type locality include:
The specific name “
Morphological and color pattern characters of the species of the Thai-Burmese clade of
Species |
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26.2–29.2 |
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24.0–27.9 |
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Maximum |
23.2–25.4 |
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24 | 19.9–23.9 | 23.1–25.4 |
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22.1–28.1 |
Snout projecting beyond lower jaw (1) or not (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Tympanum visible (1) or not (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 or 1 | 1 |
Light coloured rictal tubercle(s) at the corner of the jaw present (1) or not (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Interorbital tubercle ridges present (1) or not (0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Opening of vocal sac on right (1) or left (0) | 1 | 1 | 1 | 0 | / | 0 | 1 | 0 or 1 | 0 or 1 |
Finger tips rounded or forming small discs (1) or expanded and spatulate (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Toe tips rounded or forming small discs (1) or expanded and spatulate (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
1st finger reaching the disc of the 2nd (1) or not (0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
No. of fingers with nuptial pads | 1 | 1 | 0 | 1 | 0 | 2 | 2 | 1 | 1 + 2 |
No. of free phalanges of V toe | 0.5 | 0.5–1 | 2 | 0.5 | 1.5 | 0.5 | 0.5 | 1 | 0.5 |
No. of free phalanges of IV toe | 2.5 | 2.75 | 3–3.5 | 0.5–2.0 | 3 | 2.75–3 | 3 | 2 | 2.75–3 |
No. of free phalanges of III toe | 0.5 | 0.5–2.66 | 2 | 0.5–2.33 | 1.5–3 | 0.5–2 | 1 | 1 | 0.5–2 |
No. of free phalanges of II toe | 0.5 | 0.5–2 | 1–1.5 | 0.5–2.0 | 0.5–1.5 | 0.5–1 | 0.5 | 1 | 0.5–1 |
No. of free phalanges of I toe | 0.5 | 0.5–1 | 0.5 | 0.5–1.0 | 0.5 | 0.5 | 0.5 | 1 | 0.5 |
Tarsal ridge present (1) or not (0) | 0 | 0 |
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0 | 0 | 0 | 0 | 0 | 0 |
Inner metatarsal tubercle present (1) or not (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Outer metatarsal tubercle present (1) or not (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Submandibular tubercles in males present (1) or not (0) | 1 | 1 |
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1 | 1 | 1 | 1 | 0 | 1 |
Dorsal tubercles present (1) or not (0) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 |
Dorsolateral row of enlarged tubercles present (1) or not (0) | 1 |
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1 |
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1 |
Rows of tubercles on back (1) or not (0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Oblique flap of skin on each side of vent (1) or not (0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | / |
Abdomen coarsely granular (1), finely granular (2), or tuberculate (0) | 1 | 1 | 1 | 1 | 1 | 1 |
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1 |
Color or iris | Yellowish–gold | gold |
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Gold | Yellowish–gold | Gold | Gold | / | Yellow |
Gular spotting present (1) or not (0) | 0 |
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0 | 0 | 0 |
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0 |
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Wide, light patch below eye (1) or not (0) | 1 |
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0–1 |
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1 | 1 |
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1 |
White postorbital patch present (1) or not (0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Light spot between the scapulae present (1) or not (0) | 1 | 1 |
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1 | 1 | 1 | 1 |
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1 |
Light crossbar on hind limbs present (1) or not (0) | 1, irregular in shape | 1 |
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1 | 1 | 1 (faint) | 1 | 1 | 1 |
Vertebral stripe present (1) or not (0) | 0 | 0 | 0 | 0 | 0 |
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0–1 | 0 | 0 |
Discrete white or bright yellow spots along the underside of lower jaw large (2), small (1) or absent (0) | 2 | 2 |
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2 | 2 | 2 | 2 | 2 |
The genus
The northern Tenasserim Mountain region is notable for the recent discoveries of endemic amphibians and reptiles (
We would like to thank the Laboratory Animal Research Center, University of Phayao and The Institute of Animal for Scientific Purposes Development (IAD), Thailand for permission to do field work. We also want to thank the Rabbit in the Moon foundation for help during the field work, and especially to Charnchai Bindusen and Juthamas Wangaryattawanich for organizing and supporting our fieldwork; and to Krarok Wohde, Torn Wohde, Jo Wohde, Maiday Ta-Au for assistance in the field; we thank Kanokwan Yimyoo for constant support and Pattarawhich Dawwrueng, Thiti Ruengsuwan, Kawin Jiaranaisakul, Akkrachai Aksornneam for help during the field work. NAP also thanks Karen Sarkisian for his support and encouragement; NAP is grateful to Dr. Valentina F. Orlova and Roman A. Nazarov (ZMMU) for help during the work in collection under their care; NAP thanks the members of MSU HerpLab including Vladislav A. Gorin, Evgeniya N. Solovyeva, Andrei M. Bragin, Sabira S. Idiatullina, Alexey Trofimets, and Anna S. Dubrovskaya for support and assistance. We express our sincere gratitude to two anonymous reviewers for their useful suggestions on an earlier version of the manuscript. Fieldwork, specimen collection, morphological examination, molecular phylogenetic analyses and data analyses for this paper were conducted with the financial support of the Russian Science Foundation (RSF grant No. 19-14-00050 to Nikolay A. Poyarkov); specimen collection and data analysis were also partially supported by the grants of the Unit of Excellence 2022 on Biodiversity and Natural Resources Management, University of Phayao (No. FF65-UoE003, specimen collection) and Thailand Research Fund (DBG6180001; specimen collection, data analysis) to Chatmongkon Suwannapoom.