Research Journal of Biotechnology
Vol. 18 (8) August (2023)
Res. J. Biotech.
Tadpoles of Papurana milleti (Amphibia: Anura):
molecular identification and morphological description
Le Thi Thuy Duong1,2*, Tran Gia Thinh1,2, Le Tran Tuyen1,2 and Pham Manh Hung1,2
1. Department of Ecology and Evolutionary Biology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City,
227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, VIETNAM
2. Vietnam National University, Ho Chi Minh City, Linh Trung Ward, Thu Duc, Ho Chi Minh City, VIETNAM
*lttduong@hcmus.edu.vn
Abstract
Based on molecular identification, we described the
tadpole morphology of Dalat forest frog, Papurana
milleti. Twenty tadpole specimens of P. milleti (Gosner
stage 25–42) were collected from Di Linh plateau,
southern Vietnam. Detailed descriptions of external
morphological features, morphometric measurements,
color patterns in life and in preservation and
ecological notes are provided. Tadpoles of P. milleti
are distinguished from those of its congeners by having
yellowish brown body, LTRF 2(2)/3(1) and single
submarginal papillae from the lateral of row P-1 to
under row P-3.
Keywords: 16S rRNA, amphibian,
conservation, Hylarana, Southeast Asia
anuran
larval,
Introduction
With 41% of species threatened with extinction, the highest
rate among vertebrate classes, amphibians are facing a
conservation crisis21,23. Habitat change (destruction,
alteration and fragmentation) is considered the most
important factor contributing to amphibian decline,
particularly in tropical and subtropical regions of high
amphibian diversity and endemism21,22. Subsequently, the
urgent conservation action is to identify and to protect
important areas for amphibian species. Studying larval
stages of amphibian species is a useful approach to
determine their breeding habitat requirements.
The Dalat forest frog, Papurana milleti whose type locality
is at Dalat, Langbian Plateau, Southern Vietnam, is widely
distributed from Cambodia, Laos and Vietnam to Thailand,
Myanmar and China9,17. This species was recently renamed
from its previous name Indosylvirana milleti. Chan et al4
used genome-wide data to resolve the phylogeny of the
genus group Hylarana. Papurana milleti is a forestdependent species and is threatened by forest degradation
which is occurring throughout its distribution range. In this
study, we used molecular analysis to identify the tadpoles of
P. milleti and descrigrobed its tadpole morphology.
Material and Methods
Tadpoles of Papurana milleti were collected in Di Linh
plateau, Lam Dong province, Vietnam in July 2022. Eight
specimens were collected in a muddy pool (11.44412°N,
108.06244°E, Fig. 2H) while other 12 specimens were in a
slowly running stream section inside the mixed bamboo and
https://doi.org/10.25303/1808rjbt068073
broadleaf forest (11.42751°N, 108.06620°E). After being
photographed, tail muscle samples of tadpoles were taken
for DNA extraction and then tadpoles were preserved in 4%
formalin. All tadpole specimens were stored at the Zoology
Laboratory, Faculty of Biology and Biotechnology,
University of Science, Vietnam National University, Ho Chi
Minh City.
Genomic DNA of samples was isolated from tissue samples
using the kit with Omega BIO-TEK tissue kit following the
manufacturer’s recommendations. Approximately 550 base
pairs of the 16S rRNA were amplified using the primers AH16S_S and AH-16S_R12. The 40 µl PCR reaction mixes
included 20 µl of MyTaq PCR Mastermix (Bioline), 16 µl of
ultrapure water, 0.8 µl of BSA (Euromedex), 0.6 µl of each
primer (3µM) and 2 µl of DNA template. Amplifications
were performed in a PTC-100 BIORAD Thermal cycler. The
thermal regime consisted of an initial step of 2 min at 92 °C
followed by 35 cycles of 45 sec at 92 °C, 45 sec at 52 °C and
1 min at 72 °C followed in turn by 5 min at 72 °C and then
held at 4 °C. PCR products were visualized on 1–2% agarose
gels and the most intense products were selected for
sequencing by 1st BASE.
Electropherograms were visually checked using Chromas
2.6.6 and sequences aligned using MUSCLE implemented
in MEGA77,15. The new sequences were then checked on
BLAST (NCBI) to verify their approximate identity. DNA
sequences have been deposited in GenBank under accession
number OR095092 (DL38), OR095093 (DL40), OR095094
(DL51) and OR095101 (DL35). Sequence data obtained
from GenBank for 11 additional Papurana, Hylarana,
Sylvirana and two species Clinotarsus curtipes, Lankanectes
corrugatus was used as outgroup, since it is a closely related
to the genus Hylarana5,8,20. Phylogenetic inferences based
on Maximum Likelihood (ML) framework were made using
IQ-TREE through the IQ-TREE web server16,24.
The optimal partitioning models for the ML inference were
selected by ModelFinder in IQ-TREE using Bayesian
Information Criterion with the minimum score6,13. Partition
analysis suggested best fit models for ML inference
TIM2e+I+G4 (BIC = 5596.230, -lnL = 2598.786). Ultrafast
bootstrap (BP) analysis for 1000 iterations was carried out to
determine statistical support for the nodes in ML3. The trees
obtained from ML were visualized using Figtree v.1.4.3
(http://tree.bio.ed.ac.uk/software/figtree).
The staging of the tadpoles followed Gosner11.
Morphological measurements were taken to the nearest 0.1
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Research Journal of Biotechnology
mm with dial calipers and followed Altig and McDiarmid1
and Pezzuti et al19. Morphometric characters are abbreviated
as follows: Total length (TL): from the tip of snout to the tip
of tail; Body length (BL): from the tip of snout to the end of
body where the caudal muscle medial line touches body
wall; Tail length (TaL): distance between the level of the
caudal muscle medial line touches body wall and the tip of
tail; Maximum tail height (MTH): maximum distance
between the external ventral fin and dorsal fin; Interorbital
distance (IOD): distance between center of pupils;
Internostril distance (IND): distance between the medial
margin of nostrils; Tail muscle height (TMH): maximum
distance between ventral and dorsal edges of the tail muscle;
tail muscle width (TMW): maximum distance between
lateral edges of tail muscle; body width (BW): maximum
distance between lateral edges of the body; body width at
nostril position (BWN): maximum distance between lateral
edges of the body at nostrils level; body width at eye position
(BWE): maximum distance between lateral edges of the
body at eyes level; body height (BH): maximum distance
between dorsal and ventral edges of the body; eye–snout
Vol. 18 (8) August (2023)
Res. J. Biotech.
distance (ESD): distance from the center of eye to the tip of
snout; eye-nostril distance (END): distance from the center
of eye to the medial margin of nostril; nostril-snout distance
(NSD): distance from the medial margin of nostril to the tip
of snout; eye diameter (ED): maximum distance between eye
edges; nostril diameter (ND): maximum distance between
nostril edges; snout-spiracular distance (SSD): distance from
the tip of snout to the spiracle distal edge; oral disc width
(ODW): maximum distance between oral disc edges; dorsal
fin height (DFH): maximum distance between external and
internal edges of dorsal fin; ventral fin height (VFH):
maximum distance between external and internal edges of
ventral fin. Tooth formulas (LTRF) were determined
according to Altig and McDiarmid1.
Results and Discussion
Identification: The 16S rRNA phylogenetic topology
shows high support in maximum likelihood bootstrap
proportions over 70% in most nodes for a number of
different species of Papurana (Fig. 1).
Figure 1: Maximum-likelihood (ML) tree based on 16S rRNA mitochondrial gene sequences
for the recognized species of Papurana, Hylarana, Sylvirana in Vietnam and outgroups, ML bootstrap values (>50%)
shown on tree nodes
https://doi.org/10.25303/1808rjbt068073
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Vol. 18 (8) August (2023)
Res. J. Biotech.
All newly collected tadpole specimens from Di Linh plateau
were identical to specimen of Papurana milleti GenBank
accession number KR264108 which was collected in Gia
Lai, southern Vietnam by Oliver et al18 with high percent
identity 97.94%. The phylogenetic tree based on 16S rRNA
showed a close relationship between P. milleti and P. attigua
which was similar to finding of Gawor et al10 and to tadpole
morphological data provided by present study.
0.67 ± 0.14) with tapering tip. Dorsal fin intermediate height
(DFH/TAL = 0.1 ± 0.01), originating on the posterior third
of body, with slightly convex external margin at the middle
of trunk then tapering tip. Ventral fin low height (VFH/TAL
= 0.06 ± 0.01), originating at the level of the vent tube, with
slightly convex external margin at the middle of trunk then
tapering tip. Dorsal fin is higher than ventral (DFH/VFH =
1.75 ± 0.09).
Tadpole description: The following description of
Papurana milleti tadpoles is based on 4 specimens at stage
36 (Fig. 2, Table 1). In dorsal view (Fig. 2A, D): body
elliptical and depressed (BH/BW = 0.75 ± 0.04); snout
nearly rounded (BWN/BWE = 0.64 ± 0.03); eyes small
(ED/BWE = 0.25 ± 0.02), located dorsally (IOD/BWE =
0.64 ± 0.14) and directed dorsollaterally; nostrils rounded
opening, small (ND/BL = 0.01 ± 0.001), located dorsally,
directed rostrolaterally, positioned near snout than eyes
(NSD/ESD = 0.43 ± 0.04), with rim indistinct. The lateral
line system is visible as a series of light dots on the lateral
and dorsal surfaces of the body but invisible on the tail.
In ventral view (Fig. 2C, F), gut tube is slightly visible as a
circular coil with a switchback point situated in the center of
the body. Rectus abdominis muscle is visible that blurs the
sight of the gut tube: Vent tube opening dextral of ventral
tail fin, fused to ventral fin and positioned at ventral margin,
ventral wall longer than dorsal.
In lateral view (Fig. 1B, E): body depressed (BH/TL = 0.16
± 0.01) with snout rounded. Spiracle sinistral, lateral,
directed posterodorsally, short, opening at posterior third of
body (SSD/BL = 0.74 ± 0.04), inner wall fused to body, with
distal portion free from the body. Tail intermediate height
(MTH/TAL = 0.26 ± 0.03); musculature robust (TMH/BH =
Oral disc is moderately wide (ODW/BW = 0.33 ± 0.04),
positioned anteroventrally, emargination present in lateral
sides of the oral disc, framed by a row of conical marginal
papillae except for a wide gap on the upper lip. Marginal
papillae row of lower lip was elongated. A row of
submarginal papillae presents from the lateral of row P-1 to
under row P-3 at the lower lip. A row with 3–4 bud-liked
submarginal papillae situated at lateral to jaw sheaths. Labial
tooth row formula (LTRF): 2(2)/3(1) with A-1>A-2; P-1>P2>P-3. Jaw sheaths are black, narrow, with fine serrated;
upper jaw sheath arch-shaped; lower jaw sheath V-shaped
(Fig. 2G).
Figure 2: External morphology of Papurana milleti tadpole (stage 36) in life (three figures above, left) and in
preservation (three figures above, right): (A), (D): dorsal view; (B), (E): lateral view; (C), (F): ventral view;
(G): jaw sheath; and (H): habitat of P. milleti tadpoles in Di Linh plateau, Lam Dong province, Vietnam
https://doi.org/10.25303/1808rjbt068073
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Color pattern: In life, the body is yellowish brown and
covered with gold-colored pigments. On the ventral side, the
chest and belly are transparent with bright pigments.
Musculature is yellow and tail fins are transparent. The tail
is covered with dark brown blotches.
In preservative, the body is semitransparent and is marbled
with dark brown blotches on the dorsal and lateral sides of
the body: Oral disc, throat and chest with brown blotches.
Ventral semitransparent without any brown spots. The tail
musculature is yellow with the upper and lower fin
transparent. The tail is covered with many irregular dark
brown pigments.
Variation within the series: Measurements of tadpole
series (Gosner stage 25–42, n = 20) are shown in table 1.
Proportions vary as follows: BH/BW 0.75 ± 0.06; BH/TL
0.17 ± 0.02; BWN/BWE 0.62 ± 0.04; ED/BWE 0.24 ± 0.04;
IOD/BWE 0.62 ± 0.16; ND/BL 0.01 ± 0.01; NSD/ESD 0.43
± 0.05; SSD/BL 0.74 ± 0.08; ODW/BW 0.34 ± 0.04;
MTH/TAL 0.27 ± 0.05; TMH/BH 0.63 ± 0.11; DFH/TAL
0.09 ± 0.02; VFH/TAL 0.07 ± 0.02; DFH/VFH 1.48 ± 0.28.
In ventral side, the gut tube is invisible because the rectus
abdominis muscle becomes more robust in stages 40 and 42.
In stage 42, keratinized labial teeth are absent; black jaw
sheaths absent except upper jaw sheath; marginal papillae
present with gap on both upper and lower lip; lateral line
system indistinct; light brown on dorsal of the body; a dark
brown line present from nostril to the end of the body in the
lateral side of the body; belly white; throat with many dark
small spots.
Ecological notes: Tadpoles of P. milleti were collected at
two muddy pools in permanent streams, next to the Highway
28 connecting Lam Dong and Binh Thuan province. One
pool is surrounded by bananas, grass and shrubs with many
aquatic plants and algae (Fig. 2H). The other pool is
surrounded by bamboo and broadleaf trees. Both pools are
relatively deep, more than 50 cm. Tadpoles were found in
the muddy bottom. Within the pools, the tadpoles of P.
milleti were associated with tadpoles of Rhacophorus
annamensis.
Comparisons with other Papurana tadpoles: Among the
described tadpoles of Papurana species (Table 2), the
tadpoles of P. milleti are most similar in morphology to the
tadpoles of P. attigua with LTRF 2(2)/31.
Table 1
Morphometric measurements (average ± SD, in mm) of Papurana milleti tadpole series from Di Linh plateau, Lam
Dong province, southern Vietnam.
Gosner
stage
Number
of
samples
(n)
TL
BL
TaL
MTH
IND
IOD
TMH
TMW
BW
BWN
BWE
BH
ESD
END
NSD
ED
ND
SSD
ODW
DFH
VFH
25
28
29
30
31
34
35
36
40
42
n=3
n=2
n=1
n=2
n=1
n=2
n=2
n=4
n=2
n=1
16.23 ±
3.61
28.46 ±
2.66
10.38 ±
0.13
18.08 ±
2.53
5.30 ± 0.21
2.00 ± 0.06
4.02 ± 0.02
2.95 ± 0.4
2.33 ± 0.21
6.92 ± 0.08
3.89 ± 0.17
5.82 ± 0.1
5.35 ± 0.48
3.74 ± 0.11
1.97 ± 0.02
1.71 ± 0.08
1.20 ± 0.03
0.09 ± 0.02
8.08 ± 0.08
2.42 ± 0.10
1.79 ± 0.15
1.31 ± 0.25
19.01
27.67 ±
0.17
38.08
38.03 ± 0.05
35.68 ± 2.68
42.20 ± 2.90
37.99
12.67 ± 0.37
11.96 ± 1.42
13.73 ± 0.15
11.46
25.35 ± 0.32
6.94 ± 0.14
2.41 ± 0.14
3.54 ± 1.5
4.43 ± 0.57
3.61 ± 0.38
7.94 ± 1.03
4.00 ± 0.18
6.26 ± 0.17
6.32 ± 0.71
4.16 ± 0.3
2.52 ± 0.21
1.86 ± 0.18
1.87 ± 0.32
0.14 ± 0.003
8.83 ± 1.17
2.67 ± 0.46
2.46 ± 0.11
1.56 ± 0.10
23.72 ± 1.41
6.10 ± 0.65
2.10 ± 0.56
4.08 ± 1.00
3.90 ± 0.75
3.18 ± 0.57
7.73 ± 0.51
4.08 ± 0.71
6.40 ± 0.89
5.81 ± 0.34
4.15 ± 0.48
2.29 ± 0.52
1.80 ± 0.34
1.62 ± 0.11
0.14 ± 0.02
8.89 ± 1.07
2.58 ± 0.39
2.42 ± 0.28
1.34 ± 0.16
28.47 ± 3.05
7.36 ± 0.53
2.57 ± 0.01
5.38 ± 0.33
3.88 ± 0.08
4.20 ± 0.27
9.26 ± 0.11
4.75 ± 0.27
7.55 ± 0.26
6.89 ± 0.10
5.07 ± 0.08
2.92 ± 0.03
2.26 ± 0.13
1.87 ± 0.12
0.16 ± 0.01
10.87 ± 0.24
2.72 ± 0.15
2.51 ± 0.30
1.46 ± 0.06
26.53
4.90
2.01
5.31
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
2.76
6.21 ± 0.62
10.01 ± 3.9
3.56 ± 0.27
1.52 ± 0.14
2.66 ± 0.16
1.79 ± 0.22
1.46 ± 0.01
4.31 ± 0.52
2.28 ± 0.16
3.67 ± 0.32
2.93 ± 0.19
2.45 ± 0.20
1.36 ± 0.04
1.18 ± 0.16
0.91 ± 0.04
0.06 ± 0.01
5.01 ± 0.98
1.64 ± 0.14
1.04 ± 0.48
0.75 ± 0.2
9.00
10.01
4.31
1.19
1.54
2.27
1.34
4.92
2.36
4.05
4.09
2.53
1.34
0.99
0.80
0.09
4.72
1.51
1.10
1.45
9.80 ± 0.27
17.87 ±
0.10
4.72 ± 0.54
1.72 ± 0.01
1.81 ± 0.5
2.66 ± 0.06
2.24 ± 0.35
5.39 ± 0.37
2.95 ± 0.23
4.81 ± 0.03
3.99 ± 0.12
3.20 ± 0.17
1.50 ± 0.27
1.21 ± 0.14
0.86 ± 0.03
0.10 ± 0.02
6.67 ± 0.20
1.86 ± 0.07
1.70 ± 0.27
1.27 ± 0.23
https://doi.org/10.25303/1808rjbt068073
12.62
25.46
6.76
2.40
4.76
3.52
3.61
8.15
4.72
7.14
6.29
4.33
2.52
1.92
1.55
0.12
9.79
3.27
2.16
1.50
34.04 ±
4.31
10.85 ±
1.96
23.19 ±
2.35
5.72 ± 0.37
1.86 ± 0.57
3.24 ± 1.89
4.01 ± 0.48
3.16 ± 0.29
6.67 ± 1.17
3.41 ± 1.08
5.77 ± 1.03
5.31 ± 0.77
3.49 ± 0.82
1.99 ± 0.37
1.51 ± 0.64
1.23 ± 0.34
0.12 ± 0.02
8.40 ± 1.34
2.15 ± 0.66
1.99 ± 0.43
1.48 ± 0.19
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Table 2
Diagnostic features and color patterns of Papurana milleti tadpoles in comparison
with known tadpoles of other Papurana species.
Gosner stage
BW/BL
TaL/BL
Number submarginal
row of papillae in
lower lip
Marginal papillae
size in lower lip
LTRF in stage 36
LTRF
Lateral line organs
Gut tube
Coloration in
preservative
Coloration in life
Reference
Papurana milleti
25–42
0.60–0.70
1.86–2.17
Papurana attigua
35–41
0.51–0.68
1.93–2.46
Papurana daemeli
32–40
-
Papurana waliesa
25–40
0.64
1.72
Papurana
supragrisea
25
0.62
1.71
1
1
0–2
1
2
Elongated
Elongated
Elongated
Uniform size
Uniform size
2(2)/3(1)
2(2)/3(1)
Visible
Slightly visible
(except stage 40, 42)
Body marbled with
dark brown blotches.
Ventral
semitransparent
without any brown
spots. Tail is covered
with many irregular
dark brown pigments.
2(2)/3(1)
2(2)/3(1)
Visible
2–3(2,3)/3(1)
2–3(2,3)/3(1)
Visible
4(2–4)/3
4(2–4)/3 or 3(2–3)/3
-
4(2–4)/3
-
Slightly visible
Visible
Invisible
Visible
Body dark brown.
Venter straw,
heavily stippled
with brown on
body, clear under
tail.
Body and tail
musculature pale
straw. Tail fins white.
Dark brown flecks
present on body, tail
musculature and tail
fins. Ventral pale
white.
Body yellowish
brown with goldcolored pigments.
Ventral is transparent
with bright spots
This study
Body with dark
brown and green
pigments.
Venter
semitransparent to
white. Tail with
irregular dark round
flecks.
Body greenish
brown with goldcolored patches.
Ventral transparent
to white.
Gawor et al10
However, the tadpoles of P. milleti can be distinguished
from tadpoles of P. attigua by body yellowish brown, belly
transparent with bright spots vs. body greenish brown, belly
is transparent to white without any light spots; single
submarginal papillae runs from lateral of row P-1 to under
row P-3 vs. only under row P-310. Tadpoles of P. milleti
differ from tadpoles of three other Papurana species by the
keratodont row formula: 2(2)/3(1) vs. 4(2–4)/3 or 3(2–3)/3
in Papurana waliesa; 4(2–4)/3 in P. supragrisea and 2–
3(2,3)/3(1) in P. daemeli.
In addition, tadpoles of P. milleti have a row elongated
marginal papillae on lower lip vs. uniform sized marginal
papillae on both lower and upper lips in P. waliesa and P.
supragrisea tadpoles; one row of submaginal papillae is
under last keratodont row in lower labial vs. absent or two
rows in P. daemeli2,14.
Conclusion
DNA barcoding can be used to identify tadpoles quickly and
accurately. The similarity in tadpole morphology of
Papurana milleti and P. attigua is consistent with their
closeness of genetic relationship. The tadpole description of
https://doi.org/10.25303/1808rjbt068073
-
Body black or dark
brown or oliveyellow with gold
iridophores. Ventral
translucent grey
with scattered fine
iridophore cluster.
Anstis2
-
Kraus and Allison14
Body and upper half
tail olive green, lower
half tail light brown.
Black spots all over
tail.
Kraus and Allison14
Papurana milleti provided in this study could be important
background data for subsequent studies on life history,
behavior, ecology, distribution and phylogeny of the species.
Acknowledgement
This research was funded by Vietnam National University,
Ho Chi Minh City (VNU-HCM) under grant number 5622022-18-06. The Di Linh Forestry Company, Lam Dong
province, Vietnam, kindly facilitated surveys and issued a
specimen
collection
permit
(permit
number
0025/GGTKHTN). Do Tran Phuong Anh assisted with DNA
sequencing. We are very grateful to all.
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