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Description of the tadpoles of two endemic frogs: the Phu Luang cascade frog Odorrana aureola (Anura: Ranidae) and the Isan big-headed frog Limnonectes isanensis (Anura: Dicroglossidae) from northeastern Thailand

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Description of the tadpoles of two endemic frogs: the Phu Luang cascade frog Odorrana aureola (Anura: Ranidae) and the Isan bigheaded frog Limnonectes isanensis (Anura: Dicroglossidae) from northeastern Thailand Abstract We describe the external morphology of the tadpoles of two frogs endemic to Thailand: the Phu Luang cascade frog (Odorrana aureola) and the Isan bigheaded frog (Limnonectes isanensis) from the type localities in the Phu Luang Wildlife Sanctuary, Loei Province, northeastern Thailand. Morphological and genetic characters (16S rRNA) were used to identify specimen and match tadpoles to the adults. Detailed descriptions of external morphology and coloration in life are provided for both species. We provide a brief discussion of the ecology of these tadpoles and a comparison to previously published data from tadpoles of closely related taxa. Additionally, we provide evidence for the utility of larval morphology in resolving the taxonomic puzzles presented by cryptic species complexes.
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508
Accepted by J. Rowley: 16 Jun. 2015; published: 7 Jul. 2015
ZOOTAXA
ISSN 1175-5326 (print edition)
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Copyright © 2015 Magnolia Press
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http://dx.doi.org/10.11646/zootaxa.3981.4.3
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Description of the tadpoles of two endemic frogs: the Phu Luang cascade frog
Odorrana aureola (Anura: Ranidae) and the Isan big-headed frog Limnonectes
isanensis (Anura: Dicroglossidae) from northeastern Thailand
NATEE AMPAI
1
, ATTAPOL RUJIRAWAN
1
, JIRACHAI ARKAJAG
2
, DAVID S. MCLEOD
3
& ANCHALEE AOWPHOL
1, 4
1
Department of Zoology, Faculty of Science, Kasetsart University, Bangkok, 10900 Thailand
2
Phu Luang Wildlife Research Station, Wildlife Research Division, Wildlife Conservation Office, National Park, Wildlife and Plant
Conservation Department, Chatuchak, Bangkok, 10900 Thailand
3
Department of Biology, James Madison University, Harrisonburg, Virginia 22807, USA
4
Corresponding author. E-mail: fsciacl@ku.ac.th; Telephone: +6625625555 ext. 3283
Abstract
We describe the external morphology of the tadpoles of two frogs endemic to Thailand: the Phu Luang cascade frog
(Odorrana aureola) and the Isan big-headed frog (Limnonectes isanensis) from the type localities in the Phu Luang Wild-
life Sanctuary, Loei Province, northeastern Thailand. Morphological and genetic characters (16S rRNA) were used to
identify specimen and match tadpoles to the adults. Detailed descriptions of external morphology and coloration in life
are provided for both species. We provide a brief discussion of the ecology of these tadpoles and a comparison to
previously published data from tadpoles of closely related taxa. Additionally, we provide evidence for the utility of larval
morphology in resolving the taxonomic puzzles presented by cryptic species complexes.
Key words: amphibian, biodiversity, conservation, morphology, mitochondrial DNA, species complex
Introduction
Thailand is considered a global biodiversity hotspot and includes a tremendous diversity of amphibian species
(Myers et al. 2000). In Thailand, amphibians were reported more than 130 species and at least 32 species are
considered to be endemic (Frost 2015). Phu Luang Wildlife Sanctuary (PLWS) in Loei Province, northeastern
Thailand, is an economically and environmentally important forest conservation area covered by a mixture of dry
deciduous dipterocarp forest, mixed deciduous forest, hilly dry evergreen forest, montane evergreen forest,
coniferous forest, and tropical grassland (Phochayavanich 2007; Wanchai et al. 2012; Wanchai et al. 2013).
Elevations at PLWS ranges 400–1500 m above sea level (ASL). Several species endemic to Thailand, including
Limnonectes isanensis (Isan big-headed frog), Odorrana aureola (Phu Luang cascade frog), and Tylototriton
panhai (Salamandrid newt) have been reported from this site (Stuart et al. 2006a; McLeod et al. 2012; Nishikawa
et al. 2013). With respect to amphibian diversity, Phochayavanich et al. (2010) documented 22 species at PLWS
with greatest diversity occurring at lower elevations.
In recent years, a growing amount of attention has been given to species complexes and the underestimation of
amphibian diversity, particularly in Southeast Asia (Bain et al. 2003; Stuart et al. 2006a,b; McLeod 2008; McLeod
2010; Matsui et al. 2010a,b; Inger & Stuart 2010). In Thailand, the Odorrana livida complex (Stuart et al. 2006a,b)
and the Limnonectes kuhlii complex (McLeod 2008, 2010; Matsui et al. 2010b; McLeod et al. 2012) exemplify the
diversity that can be obscured by morphological similarities of postmetamorphic individuals. On the basis of
molecular data, Stuart et al. (2006b) identified seven species in the O. livida complex, two species of which occur
in Thailand. Similarly, more than 22 species (or candidate species) have been identified in the L. kuhlii complex
(Matsui et al. 2010b; McLeod 2010), of which four species occurs in Thailand.
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THE TADPOLES OF TWO ENDEMIC FROGS FROM THAILAND
The Phu Luang cascade frog, Odorrana aureola, was identified from within the O. livida complex on the basis
of morphology and molecular data and described from specimens collected at the PLWS and Phu Kradueng
National Park in Loei Province, Thailand (Stuart et al. 2006a). Recently, Chuaynkern et al. (2010) extended the
known range of this species and reported populations from Chiang Mai and Uttaradit Provinces, and northeastern
Phetchabun Province. Odorrana aureola is considered to be a Thai endemic and is listed as a data deficient (DD)
species by the IUCN Red List (2008). Beyond the distributional data, little is known about the natural history,
ecology, or reproductive biology of O. aureola.
Limnonectes isanensis, was identified with mitochondrial DNA data as “Lineage 11” by McLeod (2010) and
later described on the basis of morphology (McLeod et al. 2012). This species is closely related and
morphologically similar to L. megastomias and is currently known only from PLWS in Loei Province (McLeod
2008; McLeod et al. 2012). The IUCN Red List conservation status of L. isanensis is currently under review (J.
Rowley pers. comm). Like O. aureola, little is known about this species and informed conservation decisions are
difficult to make.
Despite recent progress towards elucidating diversity within amphibian cryptic species complexes, much of the
basic biology of these organisms remains unknown. Amphibians are characterized by their bimodal lifecycle, yet
much of the focus of taxonomic, phylogenetic, and ecological studies has been on the postmetamorphic forms
while disregarding the aquatic larval stages. Two endemic species, O. aureola and L. isanensis were originally
described solely based on adults, and larvae are unknown for both. The purpose of this study is to describe the
larvae of both O. aureola and L. isanensis in an effort to better understand these species and contribute to their
conservation.
Material and methods
Field collection of specimens. Adults and tadpoles of Odorrana aureola and Limnonectes isanensis were collected
from Phu Luang Wildlife Sanctuary, Phu Ruea District, Loei Province, northeastern Thailand (17°16'20.1"N
101°31'09.2"E; Fig. 1) between December 2012 and November 2013. Tadpoles of three species (Leptolalax sp.,
L. isanensis, and O. aureola) co-occurred in pools along the stream. For the purpose of this study, adults and larvae
of only two species (O. aureola and L. isanensis) were collected. Sampling was conducted over a six-day period
each month during December 2012 to November 2013. Environmental data (ambient air temperature, water
temperature, and relative humidity) were collected with a Kestrel 4000 Weather Meter, an Extech instruments
digital thermo-hygrometers, and a HM digital Waterproof pH meter. Tadpoles were collected with dip nets at night
from a variety of lentic and lotic habitats along the Kok Huai Toey Stream (described below). Adult frogs were
collected by hand at night. Live specimens of tadpoles were photographed with Nikon D80 and D300 cameras with
60 mm macro lenses and external flashes. Specimens were euthanized in the field with MS-222. Tadpole tail
muscle and liver tissue from postmetamorphic individuals were collected and preserved in 95% ethanol after
euthanasia. Specimens were fixed in 10% formalin and subsequently preserved in 70% ethanol (adult specimens)
or 10% formalin (tadpole specimens). All voucher specimens were deposited in the herpetological collection at the
Zoological Museum of Kasetsart University, Thailand (ZMKU).
Identification. Adult specimens of O. aureola (ZMKU AM 01137) and L. isanensis (ZMKU AM 01139) from
the type locality (Phu Luang Wildlife Sanctuary for both species) were identified based on original descriptions of
the species. New molecular sequence data from adults and larvae were matched to previously published data
available on GenBank.
Tadpole morphology. Mensural data were collected for 15 morphological features of each tadpole, modified
from Grosjean et al. (2003) and McDiarmid and Altig (1999). Morphometric characters are abbreviated as follows:
body height (BH), body length: tip of head to the end of body (BL), body width (BW), internarial distance:
measured between centers of narial apertures (IND), interorbital distance: measured between centers of eyes
(IOD), distance from IOD to IND (IOS), maximum tail height: upper to lower edges at widest point (MTH), width
of oral disc (ODW), distance of tip of snout to upper corner of spiracle (SS), tail length (TAL), tail muscle width
(TMW), total length: head to tip tail (TL), height of upper tail fin (HUF), height of lower tail fin (HLF), tail muscle
height (TMH) measured at the plane of the end of body. Measurements of BH, BL, BW, TAL, and TL were made
with digital vernier calipers (Mitutoyo
®
, Japan) to the nearest 0.1 mm. All other measurements were taken with a
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stereomicroscope (Nikon SMZ445) with an ocular micrometer. Terminology of labial tooth row formula (LTRF)
follows those of McDiarmid and Altig (1999). Terminology of intestinal coiling follows that of Anganoy-Criollo
(2013). Description of external morphology and drawings were based on tadpoles at stages 30–38 (Gosner 1960).
For L. isanensis, the morphological measurements were compared with previously published data of L.
megastomias (See in Table 3 of McLeod 2008) using Mann-Whitney U-test in XLSTAT-Pro 2015 (Addinsoft
2015).
Molecular analyses. DNA sequences of tadpoles and adults were compared for homology. Total genomic
DNA was extracted with a Dneasy kit (QIAGEN, Inc.). A fragment of 553 bp (O. aureola) and 551 bp (L.
isanensis) mitochondrial DNA gene, 16s rRNA was amplified via PCR (94 °C, 45 s; 52 °C, 30 s; 72 °C 1 min) for
35 cycles with 16Sar and 16Sbr primers (Palumbi 1996). All PCR products were purified by Qiagen PCR
purification kit (QIAGEN, Inc.). Sequencing was performed on an ABI 3730 automatic sequencer. Sequences were
edited and aligned by Geneious v.5.4 (Biomatter, Ltd.). DNA sequences were deposited in GenBank (Accession
numbers: KT002161–KT002164).
FIGURE 1. Map of study site (star), showing the locality of Phu Luang Wildlife Sanctuary, Phu Ruea District, Loei Province,
northeastern Thailand.
Results
Odorrana aureola Stuart, Chuaynkern, Chan-ard, and Inger, 2006
Specimens examined. A single tadpole of O. aureola (ZMKU AM 01138) was collected from Kok Huai Toey
Stream, Phu Luang Wildlife Sanctuary, Loei Province (17°19'20.9"N 101°31'21.6"E, 1246 m ASL) by Attapol
Rujirawan and Natee Ampai on 19 September 2013 (2052 hr). A single adult specimen (ZMKU AM 01137; Fig.
2A) was collected at the same location (Fig. 2B) by Anchalee Aowphol, Attapol Rujirawan, Natee Ampai and
Somphouthone Phimmachak on 28 July 2013 (2100 hr).
Identification. Sequence data from the tadpole (ZMKU AM 01138) and adult (ZMKU AM 01137) were
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100% identical over a fragment of 553 bp. Morphology of the adult specimen of O. aureola matched the original
description (Stuart et al. 2006a).
Measurement of tadpole. Morphometric measurements (in mm) at Gosner (1960) developmental stage 30
(Table 1): BH = 3.5; BL = 8.8; BW = 4.7; IND = 1.8; IOD = 2.8; IOS = 1.5; MTH = 4.3; ODW = 2.5; SS = 6.6;
TAL = 20.9; TL = 29.7; TMH = 2.7; TMW = 2.4; HUF = 1.5; HLF = 1.2.
TABLE 1. Morphometric measurements (mm) of Odorrana aureola tadpole (ZMKU AM 01138; n = 1) and
morphometric variations of Limnonectes isanensis tadpole series (ZMKU AM 01140; n = 3, ZMKU AM 01141; n = 1,
and ZMKU AM 01142; n =1) from Phu Luang Wildlife Sanctuary, Loei Province, northeastern Thailand.
Description of tadpole. Body small and streamlined (Figs. 3, 4). From above, body oval, wider anteriorly than
posteriorly, snout acutely rounded; in profile body dorsoventrally depressed, snout rounded, sloping gently from
the plane of the eyes, oral disc anteroventral. Nares small with elevated rim, anterodorsally positioned, closer to
snout than eye. Eyes positioned at the anterior one-third of the body, dorsolateral, not visible from ventral view.
Interorbital distance almost twice internarial distance. Single, sinistral, tubular spiracle opening just below body
axis posterior to midpoint of body (Fig. 4C); SS = 6.6 mm. Vent tube dextral, opens midway between muscle and
edge of ventral fin. Tail musculature well developed, round shaped, end rounded. Fin beginning at root of tail,
margin of dorsal fin higher than ventral fin and slightly convex, ventral fin parallel to caudal fin, fin tapering to
pointed tip, TMH = 2.7 mm.
Color pattern. Color in life (Stage 30, Fig. 4): body brown to russet with dense dark brown pigmentation on
the dorsum from snout to distal third of tail, clusters of melanophores become less dense towards the venter giving
the sides of body and tail a spotted appearance. Dorsum, sides of body and tail flecked with small golden spots
arranged almost linearly. Distal third of tail musculature dark brown, tip transparent. Fins flecked with dark brown
pigment near musculature, fading to transparent at margins and distal tip of tail. Oral disc pale white. Venter
translucent revealing internal organs and centro-sinistral intestines. Coloration in preservative similar to that in life
with dark brown pigmentation remaining the same, but body pale grayish, and spots pale golden.
Oral disc. Oral disc (Fig. 3B) anteroventral, emarginated with uniserial array of blunt marginal papillae and 5–
6 submarginal papillae at intersection of anterior and posterior labia. Upper and lower jaw sheathes medium and
serrated with black margins, LTRF: 5(2–5)/4(1).
Species Odorrana aureola Limnonectes isanensis
ZMKU number ZMKU
AM 01138
ZMKU
AM 01140
ZMKU
AM 01141
ZMKU
AM 01140
ZMKU
AM 01142
Gosner stage 30 30 32 35 38
Number of tadpole (n) n=1 n=1 n=1 n=2 n=1
Body height (BH) 3.5 3.8 5.6 5.9±0.1 5.9
Body length (BL) 8.8 9.6 12.5 12.8±0.8 13.4
Body width (BW) 4.7 6.0 7.4 7.8±0.1 7.9
Internarial distance (IND) 1.8 1.5 2.4 2.5±0.1 2.8
Interorbital distance (IOD) 2.8 3.0 3.8 3.4±0.7 4.0
Distance from IOD to IND (IOS) 1.5 1.7 2.5 2.5±0.1 2.6
Maximum tail height (MTH) 4.3 5.2 6.2 6.3±0.1 6.8
Width of oral disc (ODW) 2.5 2.0 2.2 2.5±0.1 2.4
Distance of snout to spiracle (SS) 6.6 6.2 7.2 7.6±0.1 7.7
Tail length (TAL) 20.9 17.6 19.2 19.5±0.1 26.9
Total length (TL) 29.7 27.1 31.7 32.3±0.2 40.3
Tail muscle height (TMH) 2.7 2.2 2.9 3.0±0.1 3.1
Tail muscle width (TMW) 2.4 1.9 2.3 2.8±0.1 3.2
Height of upper tail fin (HUF) 1.5 1.9 1.9 2.1±0.1 2.2
Height of lower tail fin (HLF) 1.2 1.1 1.3 1.4±0.1 1.5
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FIGURE 2. (A) Adult male of the Phu Luang cascade frog, Odorrana aureola and (B) The habitat of O. aureola at Phu Luang
Wildlife Sanctuary, Loei Province.
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FIGURE 3. Odorrana aureola tadpole (ZMKU AM 01138; Gosner stage 30); (A) lateral view, (B) oral disc. Scale bar = 1 mm.
FIGURE 4. Odorrana aureola tadpole (ZMKU AM 01138; Gosner stage 30) in life; (A) Dorsal view, (B) ventral view, and (C)
lateral view. Scale bar = 1 mm.
Ecology. The tadpole was discovered at the bottom of a small, shallow pool in a permanent stream with an
accumulation of dead leaves and rocks <40 cm in diameter (Fig. 2B). The tadpole was buried within the gravel
substrate on the bottom of the pool. Sampling area in the stream was 2.4 m
2
with a maximum depth of 16 cm water.
Tadpole was collected at 2052 hr on 19 September 2013. Ambient air temperature = 21.4°C, relative humidity =
85.9%, surface water temperature = 19.8°C, water temperature at 16 cm = 18.9°C, and stream current was slow
(0.2 m/s). Stream pH taken at 1036 hr on 20 September 2013 was 8.6 (weak base), ambient air temperature =
22.8°C, water temperature was not collected. No egg masses were found during the surveys. When disturbed, the
tadpole moved quickly and hid within gravel, rock crevices, and dead leaves. Adult males of O. aureola were often
found 20–30 cm above the water perched on large rocks. When disturbed, adult frogs escaped to other rocky
perches or took cover at the bottom of the stream.
Comparisons. The external morphology of some tadpoles in the genus Odorrana has been described
previously. Inthara et al. (2005) reported LTRF 5(2–5)/4(1) for Odorrana livida from Loei Province but did not
address other oral features. Songchan (2007) described coloration and body morphology of a Gosner stage 35
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tadpole of O. livida from Chiang Dao, Chiang Mai Province, northern Thailand and reported a LTRF consistent
with Inthara et al. (2005). Unfortunately, these studies are confounded by the taxonomic confusion over the identity
of O. livida complex members and the fact that some species occur in sympatry in Thailand. The lack of genetic
data in Inthara et al. (2005) and Songchan (2007) precludes the use of molecular sequence homology to validate the
identification. We found the labial tooth row formula of O. aureola to be the same as that of O. livida in Songchan
(2007) and Inthara et al. (2005), but we found marginal papillae arranged in a single row not two rows as described
by Inthara et al. (2005) and Songchan (2007). A distinctive larval feature of O. aureola appears to the presence of
small golden spots arranged almost linearly along the entire brown to russet colored body. This is differs from
previous descriptions of O. livida tadpoles, which are described as having scattered small golden or yellow flecks
on a gray or dark colored body with some dark spots on the tail muscle. We suggest that the tadpoles described by
Inthara et al. (2005) and Songchan (2007) may be those of O. chloronota, but additional studies are required to
confirm this.
Limnonectes isanensis McLeod, Kelly, and Barley, 2012
Specimens examined. Five tadpoles of L. isanensis (ZMKU AM 01140 [n=3], ZMKU AM 01141 [n=1] and
ZMKU AM 01142 [n=1]) were collected from Ron Ton Son Stream, Phu Luang Wildlife Sanctuary, Loei Province
(17°17'53.2"N 101°31'22.1"E, 1,361 m ASL). Specimens were collected by Anchalee Aowphol, Attapol
Rujirawan, Natee Ampai, Korkhwan Termprayoon, and Somphouthone Phimmachak on 28 July 2012 (2100 hr)
and 11 December 2012 (2045 hr). A single tadpole specimen (ZMKU AM 01142; stage 38) was photographed in
life and illustrated (Figs. 6–7). A single adult specimen (ZMKU AM 01139; Fig. 5A) was collected at Nam San
Noi Stream, Phu Luang Wildlife Sanctuary, Loei Province (17°20'01.0" N 101°30'35.0"E, 915m ASL) by Anchalee
Aowphol, Attapol Rujirawan, Natee Ampai and Somphouthone Phimmachak on 26 September 2012 (2030 hr).
Identification. Tadpole (ZMKU AM 01140) has an uncorrected pairwise divergence of 0.18 % from an adult
specimen (ZMKU AM 01139). Morphology of the adult specimen of L. isanensis matched the original description
(McLeod et al. 2012).
Measurement of tadpole. Morphometric measurements (in mm) at Gosner (1960) developmental stage 38:
BH = 5.9; BL = 13.4; BW = 7.9; IND = 2.8; IOD = 4.0; IOS = 2.6; MTH = 6.8; ODW = 2.4; SS = 7.7; TAL = 26.9;
TL = 40.3; TMH = 3.1; TMW = 3.2; HUF = 2.2; HLF = 1.5.
Variation within the series. Mensural and ratio values for external morphology of the tadpole series (Gosner
stages 30–38; n = 5) is provided in Table 1. External morphology of the tadpoles remains generally consistent
throughout the developmental stages in this series. In all specimens the sinistral spiracle and intestine coils were
visible in lateral and ventral views. In early stages 30–35, the tail is weakly mottled whereas in later stages (e.g.,
Gosner 38), the tail is much more densely mottled.
Description of tadpole. Body moderate (Fig. 7A). From above, body oval, length twice width, wider
anteriorly than posteriorly, snout rounded; in profile body depressed, sloping steeply from level of eyes, oral disc
anteroventral. Nares are small, rim slightly elevated, anterodorsally positioned, closer to the snout than eye. Eyes
dorsolaterally positioned at the end of the anterior quarter of body (closer to nares than spiracle), widely spaced
relative to nares (IND/IOD = 0.70), not visible from ventral view. Single, sinistral, tubular spiracle, opening below
body axis near midpoint of body SS = 7.7 mm (Fig. 7A–B). Vent tube dextral, opens midway between muscle and
edge of ventral fin; both of fins slightly convex (Fig. 7B). Tail musculature well developed, tail tapering gradually
to pointed tip. Fin beginning at root of tail, margin of dorsal fin slightly higher than ventral fin and convex, ventral
fin parallel to dorsal fin, TMH = 3.1 mm.
Color Pattern. Color in life (Stage 38, Fig. 7): body pale brown to yellowish with slightly darker brown
pigment. Many small, thin, golden-yellow stripes on the sides of body and on dorsum of tail musculature. Dorsum
and tail with many dark brown to black spots. Oral disc pale white. Venter of body nearly transparent, lightly
flecked with gold; centro-sinistral intestine visible. Ventral surface of tail is semitransparent to white; caudal
musculature faintly pale brown in dorsal view, with the upper and lower fins transparent. In preservative, coloration
similar to specimen in life, pale yellowish stripes on ventrolateral body, dark brown pigmentation on tail remains
distinctly visible.
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FIGURE 5. (A) Adult male of the Isan big-headed frog, Limnonectes isanensis, (B) The habitat of L. isanensis at Phu Luang
Wildlife sanctuary.
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FIGURE 6. Limnonectes isanensis tadpole (ZMKU AM 01142; Gosner stage 38); (A) lateral view, (B) oral disc. Scale bar = 1
mm.
FIGURE 7. Limnonectes isanensis tadpole (ZMKU AM 01142; Gosner stage 38) in life; (A) Dorsal view, (B) ventral view, and
(C) lateral view. Scale bar = 1 mm.
Oral disc. Oral disc (Fig. 6B), anteroventral, 5–6 marginal papillae at corners of upper labium, papillae on
upper labium larger than lower lip, upper and lower jaw sheathes medium, slight medial convexity, margin
serrated, black. Separation between individual denticles narrow, LTRF: 2(1)/3(1); first row without gap, second
row sub-equal left and right. The first lower labial tooth row with narrow median gap. Third lower labial tooth row
nearly half the length of first and second rows.
Ecology. Tadpoles were collected from a shallow pool (12–15 cm deep) at the edge of a permanent stream (4 m
wide, 60 cm deep) at 0845 hr on 11 December 2012 (Fig. 5B). Ambient air temperature = 22.8°C, surface water
temperature = 17.9°C, water pH = 6.14, and stream current was slow (0.1 m/s). Tadpoles were abundant at the
study site during the study period. During the daytime, tadpoles were occasionally seen in the water column, but
were predictably found buried in the gravel substrate or hiding beneath clumps of leaves at the bottom of the pools
in the stream. When disturbed, tadpoles were observed to swim quickly to new hiding places. At nighttime,
tadpoles were active in the water column. Egg masses containing 35–40 ova with transparent external jelly were
found in clumps stuck to rocks in stream or to stream bank at the same location as the tadpoles during December
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(2012) and September (2013). Individual eggs encased in a jelly capsule measured 4–5 mm. Adult males of L.
isanensis were found perched either on the ground amidst leaf litter, or on rocks on the bank of the stream at this
site.
Comparisons. McLeod et al. (2012) described the PLWS endemic, L. isanensis, based on adult morphology,
but provided no tadpole information. Our description of the tadpole of L. isanensis closely match the general
characteristics of genus Limnonectes tadpoles described by Altig & McDiarmid (1999). McLeod (2008) provided
morphometric data from a series of tadpoles and the description of the external morphology of a Gosner stage 40
tadpole of L. megastomias from Sa Kaeo Province, in eastern Thailand. Limnonectes megastomias is the sister
taxon of L. isanensis and adults of both species are morphologically very similar (McLeod et al. 2012). Despite this
overall adult similarity, the tadpoles of L. megastomias and L. isanensis differ notably in several features.
Measurement proportion of two species is shown in Table 2. In overall size, the tadpole of L. isanensis seems to be
smaller than L. megastomias, particularly during early stages of development (TL = 27.1–40.3 mm and 31.3–39.8
mm, respectively). Nevertheless, relative to total length, the body of L. isanensis appears to be larger than L.
megastomias. When comparing tadpoles of the same developmental stage, Limnonectes isanensis was found to
have a significantly greater body height (BH), the ratio of body height (BH) to body length (BL) and consequently
in the ratio of body width (BW) to body length (BL) than L. megastomias (P<0.05). Oral disc structure and pattern
of pigmentation on the body tail were also different in two species. Marginal papillae on the upper labium in L.
isanensis are arranged in single row with about 5–7 papillae per side, two rows of papillae on the lower labium
with about 20–22 papillae per side, a very narrow gap in the posterior marginal papillae, and the occurrence of two
submarginal papillae ventrally. Tadpoles of L. megastomias have a single row of upper labial papillae, about 11–13
thick papillae of upper labium per side and about 28–30 papillae per side on lower labium arranged in two rows
with narrow median interruption (but broader than in L. isanensis). The caudal musculature and fins are more
heavily pigmented in L. isanensis (dark brown to black on posterior half of tail) than in L. megastomiasis (faint
brown spots scattered over length of tail). Additionally, tadpoles of L. megastomias have a median dark-brown
horizontal bar through eye, which L. isanensis lacks.
TABLE 2. Comparison of morphological measurements and ratio between Limnonectes isanensis and Limnonectes
megastomias, and P-value from Mann-Whitney U-test (* significant difference lower than P < 0.05).
Characters Limnonectes isanensis Limnonectes megastomias P-value
Gosner stage 3032353830 323640
Body height (BH) 3.8 5.6 5.9 5.9 2.6 3.5 3.6 3.5
0.02
*
Body length (BL) 9.6 12.5 12.8 13.4 10.7 11.2 13.8 13.7 0.56
Body width (BW) 6.0 7.4 7.8 7.9 5.3 5.4 7.6 7.9 0.47
Internarial distance (IND) 1.5 2.4 2.5 2.8 1.9 1.9 2.3 2.2 0.25
Interorbital distance (IOD) 3.0 3.8 3.4 4 2.5 2.7 3.0 3.5 0.11
Maximum tail height (MTH) 5.2 6.2 6.3 6.8 6.7 5.5 7.3 7.0 0.25
Tail length (TAL) 17.6 19.2 19.5 26.9 20.6 22 23.2 26.1 0.25
Total length (TL) 27.1 31.7 32.3 40.3 31.3 33.3 37.0 39.8 0.56
Tail muscle height (TMH) 2.2 2.9 3.0 3.1 2.8 3.0 3.5 3.5 0.31
BH/BL 0.40 0.45 0.46 0.44 0.24 0.31 0.26 0.26
0.02
*
BL/TL 0.35 0.39 0.40 0.33 0.34 0.34 0.37 0.34 0.38
BW/BL 0.63 0.59 0.61 0.59 0.50 0.48 0.55 0.58
0.02
*
IOD/BW 0.50 0.51 0.44 0.51 0.47 0.50 0.39 0.44 0.14
IND/IOD 0.50 0.63 0.74 0.70 0.76 0.70 0.77 0.63 0.24
TAL/TL 0.65 0.61 0.60 0.67 0.66 0.66 0.63 0.66 0.38
TAL/BL 1.83 1.54 1.52 2.01 1.93 1.96 1.68 1.91 0.39
TH/BL 0.54 0.50 0.49 0.51 0.63 0.49 0.53 0.51 0.56
TH/TL 0.19 0.20 0.20 0.17 0.21 0.17 0.20 0.18 0.88
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Discussion
Amphibians are a diverse group of vertebrates characterized by a bimodal life cycle with aquatic larvae undergoing
a dramatic metamorphosis into terrestrial adults. The larvae of anurans (tadpoles) are often overlooked in scientific
research programs and are thus poorly understood. Previous studies have demonstrated that an understanding of
anuran larval morphology contributes to many aspects of research including phylogenetic systematics, habitat
assessments, rapid inventories of complex biological systems, interspecific competition studies, and conservation
efforts (Inger 1966; Bain et al. 2003; Stuart et al. 2006a,b; McLeod 2008; Haas & Das 2008). A few studies have
dealt with the larvae of frogs in Thailand, but much of the basic biology of these taxa remains unknown (Smith
1916; Heyer 1971; Inthara 2000; Noikotr 2001; Taksintum 2003; Inthara et al. 2005; Meewattana 2005; Stuart et
al. 2006a; Songchan 2007; McLeod 2008; Danaisawat et al. 2010; McLeod et al. 2012).
Whereas this study represents a first step towards understanding the natural history, ecology, and conservation
status of these two frogs endemic to Thailand, much more remains to be studied. At present we know very little
about the reproductive biology of these frogs and the ecological and morphological diversity among their larvae. In
light of the relatively recent attention given to cryptic species complexes, particularly among amphibians, there
seems to be a great deal to be gained by understanding interspecific differences in the larvae of these animals. The
example presented herein of two closely related species in the L. kuhlii complex demonstrates that both quantitative
and qualitative differences in larval morphology can be used to distinguish these taxa one from another. In this
study, O. aureola and O. livida differed in both coloration and the arrangement of labial papillae on the oral disc.
With respect to the limitations of our small sample size (n=1) it is likely that analysis of additional samples of O.
aureola would elucidate other features that distinguish these two species from each other. We also found the
tadpoles of L. isanensis to be similar in gross morphology to the closely related L. megastomias. Nevertheless, our
data suggest that L. isanensis is greater in some characters (BH, BH/BL, and BW/BL) with different coloration,
and a unique arrangement of the papillae on the oral disc.
Clearly, more work remains to be done to fully understand these and other amphibian species complexes. As
more information on tadpole morphology (e.g., buccal structure), ecology, patterns of development, and natural
history is available, we expect that it will be possible to use larvae to more rapidly assess species distributions,
identify cases of sympatry between members of a species complex, and to identify the unique niche requirements
that define amphibian species in both their larval and adult life phases.
Acknowledgments
This work was supported by grants from the Graduate School Kasetsart University and the Partnerships for
Enhanced Engagement in Research (PEER) Science program (PGA-2000003545), which is a partnership between
the U.S. Agency for International Development (USAID) and the National Science Foundation (NSF). We are
grateful to Bryan Stuart for his improvement an early draft of this manuscript and his expertise on the Odorrana
livida complex. Jodi Rowley and Ronald Altig provided helpful critical reviews. We acknowledge National Park,
Wildlife and Plant Conservation Department for permission to collect the data and research work; head of Phu
Luang Wildlife Sanctuary, Chainarong Doodurm for granting permission to conduct this research at Phu Luang
Wildlife Sanctuary, Loei Province. Amphibian and Reptile Ecology Laboratory members: Korkhwan
Termprayoon, Siriporn Yodthong, Somphouthone Phimmachak and Vattikorn Sophonrat. Staff of Phu Luang
Wildlife Research Station: Nopporn Juttano, Stapron Tongpan, Pratinnakorn Juttano, Wittawat Bunchit and
Wutthichai Bhudprom for assistance during fieldwork.
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