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22 Accepted by M. Vences: 24 May 2012; published: 24 Jul. 2012
ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Copyright © 2012 · Magnolia Press
Zootaxa 3398: 22–39 (2012)
www.mapress.com/zootaxa/Article
Detection of cryptic taxa in Leptobrachium nigrops (Amphibia, Anura,
Megophryidae), with description of two new species
AMIR HAMIDY1,2, MASAFUMI MATSUI1,3, KANTO NISHIKAWA1 & DAICUS M. BELABUT4,5
1Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan
2Museum Zoologicum Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, Gd. Widyasatwaloka, Jl. Raya
Jakarta Bogor Km 46, Cibinong West Java, Indonesia
4Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
5Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
3Corresponding author: E-mail: fumi@zoo.zool.kyoto-u.ac.jp
Abstract
We evaluated taxonomic relationships among allopatric populations of Leptobrachium nigrops Berry & Hendrickson from
Malay Peninsula, Singapore, Indonesia (Belitung), and Borneo (Sarawak). Phylogenetic relationships estimated from the
sequence data of mitochondrial 12S RNA, tRNAval, and 16S rRNA genes, and nuclear NCX1 and SLC8A genes revealed
presence of three distinct clades within L. nigrops: (1) true L. nigrops clade from Singapore and Malay Peninsula, (2) clade
from Belitung, Indonesia and coastal area of Sarawak, Borneo, and (3) clade from Kanowit, Sarawak, an inland area of
Borneo. Each of these three genetic clades is considered to represent distinct species because they are genetically highly
divergent and morphologically distinguishable. We therefore describe the populations from Belitung and coastal area of
Sarawak as L. ingeri sp. nov and the population from the inland area of Sarawak as L. kanowitense sp. nov. Ancestral L.
kanowitense seems to have invaded Borneo Island much earlier than ancestral L. ingeri, whose dispersion occurred during
the Pleistocene glacial periods.
Key words: Leptobrachium nigrops, new species, phylogenetic relationships, Malay Peninsula, Belitung, Borneo
Introduction
The megophryid genus Leptobrachium Tschudi consists of 30 species, occurring from Southern China and India to
the islands of the Sunda Shelf and the Philippines (Frost 2011; Sondhi & Ohler 2011; Stuart et al. 2011, 2012).
However, many more cryptic species await description even from Borneo alone (Hamidy et al. 2011). Among the
named species, eight occur in Sundaland including L. nigrops Berry & Hendrickson, which is the smallest species
of the genus, with body size of 35–37 mm for males and 37–47 mm for females (Inger 1966). This species is
widely distributed on the Malay Peninsula, Singapore, Sumatra, and Borneo (Iskandar & Colijn 2000), and on
some islands in the Karimata Strait such as Belitung (Matsui et al. 2010b) and Natuna Islands (Brown et al. 2009).
Like other congeneric species from Southeast Asia, L. nigrops was once treated as L. hasseltii Tschudi (Taylor
1962), although Taylor (1962: 314) doubted species identification of a specimen from Singapore by adding “?”
ahead of species name, L. hasseltii. It was Berry & Hendrickson (1963) who described the populations of L. has-
seltii from Singapore and Peninsular Malaysia as a distinct species, L. nigrops, with Singapore as the type locality.
They distinguished L. nigrops from L. hasseltii (type locality: Java), mainly based on the smaller adult body size
and different shape of larval oral disk. Later, Inger (1966) noted difference in the shape of finger tips between pop-
ulations of L. nigrops from Borneo and Malay Peninsula, but he retained the two populations as conspecific.
Recent molecular studies by Brown et al. (2009) and Matsui et al. (2010b) uncovered high levels of genetic
divergences among allopatric populations of L. nigrops. Matsui et al. (2010b) recognized three clades (populations
from Malay Peninsula, Belitung, and inland area of Borneo), but, like Brown et al. (2009), could not study the pop-
ulation from the type locality, Singapore. This strongly hindered the evaluation of the taxonomic status of those
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CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
clades that were phylogenetically distinct from each other. In this study, we examined samples of L. nigrops from
localities representing its whole range of distribution, including the topotypic sample from Singapore. By conduct-
ing molecular, as well as morphological analyses, we determined taxonomic relationships of the three clades of L.
nigrops originally reported by Matsui et al. (2010b), and describe two new species.
FIGURE 1. Map of Malaysia and western part of Indonesia, showing sample localities of the L. nigrops group that are used in
the molecular analyses (triangle) and morphological analyses (reversed triangle). Blue: L. nigrops. Yellow: L. ingeri sp. nov.
Red: L. kanowitense sp. nov.). Details of localities are shown in Table 1.
Material and methods
Molecular phylogenetic analyses. We newly obtained partial sequences of mitochondrial 12S rRNA, tRNAval, and
16S rRNA genes from 23 specimens of 10 Leptobrachium species, including L. nigrops from its representative
ranges of distribution, and two outgroup species, Leptolalax heteropus (Boulenger) and Megophrys nasuta
(Schlegel) (Table 1), and first resolved the evolutionary relationships of L. nigrops among Leptobrachium by
combining our new data with published data (Matsui et al. 2010b; Hamidy et al. 2011). We then compared more
detailed relationships within L. nigrops using both mitochondrial genes and nuclear NCX1 and SLC8A3 genes. For
nuDNA genes, we sequenced all the 14 specimens of L. nigrops studied for mtDNA, and each specimen of L.
hasseltii, L. smithi Matsui, Nabhitabhata & Panha, and L. chapaense (Bourret) as the outgroup species based on
results of the first analysis. Methods for DNA extraction, amplification and sequencing of the mtDNA and nuDNA
fragments are the same as those already reported (Matsui et al. 2010b; Shimada et al. 2011a; Hamidy et al. 2011).
Each alignment matrices of mtDNA and nuDNA was separately subjected to estimate phylogenetic relationships
using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). Pairwise comparisons
of uncorrected sequence divergences (p-distance) were also estimated from about 1.4 kbp of mitochondrial 16S
rRNA.
Morphological analyses. For morphological examination, we employed 31 specimens of L. nigrops from
Malay Peninsula, Sumatra, Belitung, and Borneo. These specimens are stored in Sarawak Research Collections
(SRC), Graduate School of Human and Environmental Studies, Kyoto University (KUHE), and Museum Zoologi-
HAMIDY ET AL.
24 · Zootaxa 3398 © 2012 Magnolia Press
cum Bogoriense (MZB). Measurements were made to the nearest 0.1 mm with a dial caliper under a binocular dis-
secting microscope following Matsui (1984) for 21 morphometric characters: 1) snout-vent length (SVL); 2) head
length (HL); 3) snout length (SL); 4) snout-nostril length (S-NL); 5) nostril-eye distance (N-EL); 6) eye length (EL,
including upper eyelid); 7) tympanum-eye length (T-EL); 8) tympanum diameter (TD); 9) head width (HW); 10)
internarial distance (IND); 11) intercanthal distance (ICD); 12) lower arm and hand length (LAL) from elbow to tip
of third finger; 13) third finger length (3FL); 14) first finger length (1FL); 15) outer palmar tubercle length
(OPTL); 16) inner palmar tubercle length (IPTL); 17) hand length (HAL); 18) tibia length (TL); 19) foot length
(FL); 20) hindlimb length (HLL); and 21) inner metatarsal tubercle length (IMTL). In addition, 22) interorbital dis-
tance (IOD); 23) upper eyelid width (UEW); 24) forelimb length (FLL); and 25) first toe length (1TOEL) were
measured for holotypes. In the univariate analyses among samples, SVLs were compared by the Tukey-Kramer test
(Sokal & Rohlf 1995). Next, we calculated percentage ratio (R) of each morphometric character to SVL, and made
comparisons using Dunn's multiple comparisons (Sokal & Rohlf 1995).
Of six qualitative characters, the development of toe webbing was recorded by the phalanges free of web on
inner (preaxial) and/or outer (postaxial) sides of the first to fifth toes (Savage 1975). For the other five qualitative
characters, we coded the following attributes to compare the frequency: 1) tibio-tarsal articulation (1 = reaching
between anterior and posterior borders of tympanum, 2 = beyond anterior border of tympanum); 2) finger tip (1 =
moderately pointed, 2 = markedly pointed); 3) dorsal marking (1 = disposed in longitudinal rows, 2 = asymmetric
blotches, 3 = absent); 4) tympanic dark mask (1 = present all over, 2 = present on upper half to two-thirds, 3 =
absent); and 5) ventral marking (1 = blotched, 2 = spotted, 3 = reticulated, 4 = absent). We used non-parametric
Dunn's multiple comparisons test and U-test to compare inter-group variation.
Acoustic analyses. For the acoustic data, we recorded frog calls in the field using a cassette tape recorder
(Sony TC-D5) with an external microphone (Sony ECM-23) or digital recorders (Zoom H2 or Olympus LS-11). At
the time of recording, we measured the temperature with a quick-recording thermistor thermometer (Takara A
600). Calls recorded were analyzed with the SoundEdit Pro (MacroMind-Paracom, Inc) software package on a
Macintosh computer, as described elsewhere (Matsui & Dehling 2012).
Results
Molecular phylogenetic analyses
In the first analyses, concatenating our data of mtDNA with GenBank data yielded 2,474 nucleotide sites (12S
rRNA: 933 sites, tRNAval : 71 sites, and 16S rRNA: 1,470 sites), of which 1,236 were variable and 906 were
phylogenetically informative. All analyses resulted in identical topologies as those already reported (Matsui et al.
2010b; Hamidy et al. 2011). The genus Leptobrachium formed a clade with respect to Leptolalax and Megophrys,
and divided into Sundaland and Thailand Clade and China and Indochina Clade. Sundaland and Thailand Clade
comprised three clades, and populations of L. nigrops formed a clade (MPBS=100%, MLBS = 99%, BPP = 1.00),
with a sister clade of L. hendricksoni Taylor and L. hasseltii.
In the second analyses, we obtained 2,426 nucleotides of mtDNA, comprising 918 sites of 12S rRNA, 69 sites
of tRNAval, and 1,439 sites of 16S rRNA. Of these, 957 were variable and 532 were phylogenetically informative.
The MP search recovered three most parsimonious trees of 1,571 steps (CI = 0.783; RI = 0.819). In ML and Bayes-
ian analyses, the best model for each partition of 12S and 16S rRNA was the GTR (Tavaré 1986) +G. For tRNAval
gene, the optimum model for ML analysis was J2_Homogenous (Jobb 2008), while HKY (Hasegawa et al. 1985)
+G was selected as the best model for Bayesian analysis. The ML and Bayesian analyses produced topologies with
ln L = –10160.524 and –10070.000, respectively.
For nuDNA genes, we obtained 1,004 bp for NCX1 and 983 bp for SLC8A3. In NCX1, nine haplotypes were
recognized. Haplotypes 1 (n = 3), 2, 3, and 4 (each n = 1) formed a clade (corresponding to Peninsular Clade shown
below), while haplotypes 8 (n=2), 5, 6, and 7 (each n = 1) (corresponding to Belitung and coastal Sarawak Clade
shown below) formed another clade. These two clades and haplotype 9 (n = 3, corresponding to Inland Sarawak
Clade shown below) were monophyletic, but showed unresolved relationships among them. In SLC8A3, seven
haplotypes were observed, and haplotype 4 included three individuals of the Inland Sarawak Clade. Haplotype 7 (n
= 4), 5 and 6 (each n = 1) tended to form a group (corresponding to the Peninsular Clade), but haplotypes 1 (n = 3),
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CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
2, and 3 (each n = 1) (corresponding to Belitung and coastal Sarawak Clade) did not form a group. In order to get
clearer phylogenetic information, we then combined the two genes. Of 1,987 bp of concatenated fragments, 83
sites were variable and 28 were phylogenetically informative. MP analysis yielded one most parsimonious tree of
97 steps (CI = 0.907; RI = 0.885). In the nuclear NCX1, the best model was TN 93 (Tamura & Nei 1993) +G for
ML analysis, and HKY (Hasegawa et al. 1985) +G for Bayesian analysis, while in SLC8A3 gene, the best model
was J2 (Jobb 2008) +G for ML analysis and GTR (Tavaré 1986) +G for Bayesian analysis. The ML and Bayesian
analyses produced topologies with ln L = –3369.377 and –3403.667, respectively.
In both mtDNA and nuDNA trees, L. nigrops formed a clade (MPBS = 100%, MLBS = 99%, BPP = 1.00 in
mtDNA; 92%, 96%, 1.00, in nuDNA). In mtDNA tree (Figure 2 left), L. nigrops was divided into three clades, (I)
Inland Sarawak Clade (Kanowit: 100%, 100%, 1.00), (II) Peninsular Clade (Malay Peninsula and Singapore:
100%, 96%, 1.00), and (III) Belitung and coastal Sarawak Clade (100%, 99%, 1.00). Peninsular Clade and, Beli-
tung and coastal Sarawak Clade formed a clade (97%, 93%, 1.00), which was a sister group to Inland Sarawak
Clade. Similarly, in nuDNA tree (Figure 2 right), three mtDNA clades were confirmed (Inland Sarawak Clade:
99%, 99%, 1.00; Peninsular Clade: 93%, 98%, 1.00; Belitung and coastal Sarawak Clade: 77%, 92%, 1.00), but
their relationships were unresolved.
FIGURE 2. Bayesian trees of a 2426 bp fragments of mtDNA (left) and a 1987 bp fragments of nuDNA (right) for sample of
the L. nigrops group (for the sample numbers, see Table 1). Numbers above branches represent bootstrap support for MP and
ML, and Bayesian posterior probabilities (MPBS/MLBS/BPP). Asterisks indicate node with full bootstrap supports for MP and
ML inference (100%) and Bayesian posterior probabilities (BPP = 1.00).
Genetic divergences among the three clades of L. nigrops were much higher than those among other
congeneric species (Table 2). Uncorrected p-distances in 16S rRNA within a population was small (0.2–0.3% in
Inland Sarawak Clade; 0.0–3.1% in Peninsular Clade; and 0.0–0.2% in Belitung and coastal Sarawak Clade), but
the distances between allopatric populations were not small (2.2–3.1% in Singapore vs. Malay Peninsula
populaions of Peninsular Clade; 5.6–5.8% in Belitung vs. Sarawak coastal populations in Belitung and coastal
Sarawak Clade. Among the three clades, genetic distances were much larger (9.8–11.4% in Peninsular Clade vs.
Belitung and coastal Sarawak Clade; 12.1–12.4% in Peninsular Clade vs. Inland Sarawak Clade; 12.5–12.9% in
Belitung and coastal Sarawak Clade vs. Inland Sarawak Clade).
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CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
TABLE 2. Uncorrected p-distance (%) in 16S rRNA among species of Leptobrachium from Sundaland. Values shown in
parenthesis indicate intraspecific variation.
Morphological analyses
When the above results of the mitochondrial and nuclear DNA analyses are taken into consideration, specimens of
L. nigrops can be grouped (henceforth called the L. nigrops group), but split into three genetic clades that are
geographically well delimited. Therefore it would be pertinent to conduct morphological analyses based on this
grouping.
Univariate analyses. In all three clades, females tended to be larger than males in SVL, and variation ranges
did not overlap in Inland Sarawak Clade and Belitung and coastal Sarawak Clade (Table 3). However, probably due
to small sample size, significant difference was not detected in SVL between sexes in any of the three clades in
statistical comparisons (Tukey-Kramer test, P > 0.05). We therefore combined sexes in comparing ratio values
among the three genetic clades. As a result, significant difference was found in several combinations (U test, P <
0.05). Between the three clades, specimens of the Peninsular Clade were significantly larger than those of the
Inland Sarawak Clade in RSL and RIND, while specimens of the Inland Sarawak Clade had significantly larger
RHL and REL than those of the Peninsular Clade. Specimens of the Belitung and coastal Sarawak Clade were
significantly larger than those of the Peninsular Clade in REL, and of the Inland Sarawak Clade in RS-NL, RIND,
RICD, and RIMTL.
Qualitative analyses. Of the qualitative characters studied, the three clades differed in the development of toe
webbing. Significant sexual difference was detected only in one character of Peninsular Clade (males less
developed than females in inner side of the fourth toe: U test, P < 0.05). We therefore combined sexes in comparing
the characters among the three genetic clades. As a result, Belitung and coastal Sarawak Clade proved to have more
developed webbing than Inland Sarawak Clade in all points except for inner side of the second toe. Belitung and
coastal Sarawak Clade also had more developed webbing than Peninsular Clade in the outer side of the first toe,
inner and outer sides of the third and fourth toes, and inner side of the fifth toe. Peninsular Clade had more
developed webbing than Inland Sarawak Clade in outer side of the second toe (U test, P < 0.05).
In the remaining qualitative characters, no significant sexual differences were detected in all three clades and
we combined sexes for comparison among the three clades. In the point reached by the tibio-tarsal articulation
when hindlimb is bent forwards along body, the three clades did not differ significantly (U test, P > 0.05). In the
shape of fingertip, Belitung and coastal Sarawak Clade had more sharply pointed tips of fingers than the other two
clades (U test, P < 0.05). Both in dorsal and tympanum markings, the Inland Sarawak Clade lacked markings
significantly more frequently than the other two clades (U test, P < 0.05). In contrast, the three clades did not
significantly differ in the pattern of ventral marking (U test, P > 0.05).
12345678910
1 L. nigrops (0–3.1)
2 L. ingeri 9.8–11.4 (0.0–5.8)
3 L. kanowitense 12.1–12.4 12.5–12.9 (0.2–0.3)
4 L. hasseltii 14.1–14.6 14.1–14.3 13.8–13.9
5 L. hendricksoni 14.1–14.6 14.2–14.3 14.5 9.7
6 L. lumadorum 16.1–16.4 15.8–16.4 16.1–16.3 11.8 12.4
7 L. gunungense 15.9–16.4 15.3–15.9 15.8–16.0 11.5 12.7 7.8
8 L. abbotti 15.8–16.3 15.2–15.8 15.5–15.7 11.5 12.7 8.0 3.2
9 L. waysepuntiense 16.0–16.6 16.2–16.8 15.9–16.1 12.4 13.8 9.3 5.5 6.1
10 L. montanum 15.9–16.6 15.9–16.7 15.6–15.8 12.3 13.4 8.8 5.5 5.8 2.7
11 L. smithi 17.2–17.6 17.5–17.6 17.3–17.4 13.7 14.1 13.3 13.6 13.4 13.7 13.0
HAMIDY ET AL.
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Systematics
The samples of the L. nigrops group examined here are genetically clearly split into three clades that substantially
differ from each other by large genetic distances (uncorrected p-distance in 16S rRNA of 9.9–12.9%), far
exceeding those usually observed among different species of frogs (Vences et al. 2005; Fouquet et al. 2007).
Moreover, congruent with genetic separation, specimens of these three clades were also separated in qualitative
morphological characters, although not clearly in morphometric ones. From the currently recognized lineage-based
species concept (Wiley 1978; Frost & Hillis 1990; De Queiroz 1998), each of the three allopatric clades should be
recognized as distinct species. Of these, the Peninsular Clade represents true L. nigrops, because it includes the
topotypic population from Singapore. Since the two remaining clades have no available names, we describe them
as follows:
Leptobrachium ingeri sp. nov.
Synonymy: L. nigrops: Inger 1966, p. 37 (part).
Holotype. KUHE 53848, an adult female collected from Matang Wildlife Centre (01°36’34’’N, 110°09’36’’E, 60
m a.s.l.), Kuching Division, Sarawak, Borneo, Malaysia, collected by K. Eto and K. Nishikawa at 20:00 h on 30
August 2010.
Paratypes. SRC 0001 (former KUHE 53833) and KUHE 53834, two adult males, and KUHE 53832, an adult
female, all collected from Santubong (01°44’37’’N, 110°19’11’’E, 60 m a.s.l.), Kuching Division, Sarawak, Bor-
neo, Malaysia on 29 August 2010, collectors same as for holotype; MZB Amp 11790, an adult female collected
from marshy area of Tanjung Padang, Belitung, Indonesia, collected by D. Gower, A. Hamidy, and K. Sanders on
December 2005.
Referred specimens. KUHE 12087 and KUHE 12099, two adult males, collected from Niah National Park
(03°49’N, 113°45’E, ca. 50 m a.s.l.), Miri Division, Sarawak, Borneo, Malaysia, collected by M. Matsui on 26
December 1990.
Etymology. The specific name is dedicated to Dr. Robert F. Inger of Field Museum, Chicago, who was the first
to notice morphological differences between populations of L. nigrops from Borneo and Malay Peninsula.
Diagnosis. A small-sized Leptobrachium of the L. nigrops group, with SVL of 39.3–47.1 mm in females and
28.6–36.8 mm in males; finger tip sharply pointed; toe web relatively well developed; inner metatarsal tubercle
large; dorsum brown with distinct dark brown markings; tympanum usually at least upper one-third covered by
dark brown marking; body laterally with black spots scattered from axilla to groin; belly and limbs heavily
blotched ventrally; limbs, including fingers and toes, distinctly barred dorsally; large dark blotches at groin con-
tinuing to ventral and posterior sides of thigh; femoral gland in a large white blotch.
Description of holotype (measurements in mm). Small sized (SVL 44.6); habitus moderately stocky, body
tapering to groin, head broad and depressed, slightly longer (HL 19.6: 43.9% SVL) than wide (HW 18.0: 40.4%
SVL); snout obtusely pointed, rounded triangle from above, truncate in profile, projecting beyond lower jaw; eye
large, obviously projecting from sides of head, slightly smaller (EL 6.8: 15.3% SVL) than snout (SL 7.3: 16.4%
SVL); canthus rostralis sharp, loreal region oblique, moderately concave; nostril lateral, below canthus, closer to
snout (S-NL 3.9: 8.7% SVL) than to eye (N-EL 4.1: 9.2% SVL); internarial distance (IND 3.4: 7.6% SVL) much
narrower than interorbital distance, (IOD 5.7: 12.8% SVL), latter slightly narrower than upper eyelid (UEW 6.0:
13.5% SVL); pineal spot absent; tympanum visible, diameter (TD 3.2: 7.2% SVL) almost half that of eye and sep-
arated from eye by about two-third of its diameter (T-EL 2.2: 4.9% SVL); no vomerine teeth; tongue heart-shaped,
without papillae, notched posteriorly.
Forelimb slender and long (FLL 31.3: 63.7% SVL), about three-fifth of hindlimb; fingers moderately slender,
unwebbed; first finger (1FL 5.9: 10.7% SVL) slightly longer than fourth and second, third much longer (3FL 8.9:
16.2% SVL); finger tip sharply pointed, slightly hooked, not swollen; inner palmar tubercle large (IPTL 2.5: 4.5%
SVL), not extending onto first metacarpal, and outer palmar tubercle smaller (OPTL 2.0: 3.6% SVL); subarticular
tubercles indistinct, replaced by low callous tissue.
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CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
FIGURE 3. Three species of the Leptobrachium nigrops group in life. (A) A female of L. nigrops from Malay Peninsula
(KUHE 42243); (B) A female paratype of L. ingeri sp. nov. from Santubong (KUHE 53832); (C) A female holotype of L.
kanowitense sp. nov. from Kanowit (KUHE 42575).
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FIGURE 4. Ventral views in males (top row) and females (bottom row) of (A) L. nigrops: KUHE 15658 (male) and KUHE
42243 (female); (B) L. ingeri sp. nov.: a paratype (KUHE 53834: male) and the holotype (KUHE 53848: female); (C) L.
kanowitense sp. nov.: a paratype (KUHE42574: male) and the holotype (KUHE 42575: female). Scale indicates 5 mm.
Hindlimb slender and relatively short (HLL 65.6: 119.2% SVL); heels not meeting when legs held at right
angles to body; tibia distinctly longer (TL 20.0: 36.3% SVL) than foot (FL 17.0: 30.9% SVL); tibiotarsal
articulation of adpressed limb reaching to middle of tympanum; third toe longer than fifth; toe tips similar to those
of fingers; toe webs rather well developed, webbing formula I 1–11/2 II 1/4–23/4 III 11/4–32/3 IV 32/3–11/2 V; inner
metatarsal tubercle oval, low but large (IMTL 2.2: 4.9% SVL) more than half of first toe (1TOEL 3.2:7.2%); outer
metatarsal tubercle absent; subarticular tubercles obscure, elongate, replaced by low callous tissue.
Skin above nearly smooth, scattered with minute granules on top of head, especially around interorbital region,
granules denser on upper eyelid; ventrum weakly granular, particularly on belly; low supratympanic ridge from
posterior corner of eye to axilla; low dermal ridges in longitudinal rows on upper arm; flat pectoral gland at medial
border of axilla behind arm insertion; femoral gland small, concentrated in one cluster on right side, but diffused on
left side.
Colour. In life, dorsally brown with dark brown blotches in interorbital region, medial one followed by
continuous longitudinal band and lateral ones by more or less interrupted bands; dorsal brown laterally with
distinct dark spots fading to lighter towards belly; loreal region, anterior to eye, covered by dark brown; upper one-
third of tympanum and supratympanic ridge covered by dark band; groin marked with irregular dark blotches;
ventrum cream heavily blotched all-over; limbs with distinct black bars dorsally and irregular dark blotches
ventrally; posterior thigh banded by black bars, banded pattern continuing to posterior flank; iris totally black,
surrounded by light blue sclera visible in maximally opened eye. In preservative, aspects of the colour pattern
remain, but dorsal brown colour has been darkened.
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CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
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FIGURE 5. Ventral sides of thigh (top row), hand (middle row), and foot (bottom row) of (A) L. nigrops (KUHE 42243), (B)
the holotype of L. ingeri sp. nov. (KUHE 53848), and (C) the holotype of L. kanowitense sp. nov. (KUHE 42575).
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FIGURE 6. Spectrograms (above) and wave form (below) of advertisement call of (A) L. nigrops (KUHE 15430) from Kuala
Lumpur, Malay Peninsula and (B) L. ingeri sp. nov. (KUHE 53834) from Santubong, Sarawak.
Variation. Morphological variations are shown in Table 3. The male paratype (KUHE 53834) is smaller in
body size, and has an internal vocal sac and a pair of vocal sac openings. The female paratype (KUHE 53832) is
larger in body size with much darker throat than the holotype. It has distinct white dots around vent and posterior
thigh. The female paratype from Belitung (MZB Amp 11790) is smaller than holotype. Although the color slightly
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faded, blotches on groin and posterior thigh can be still recognized. Specimens from Belitung have larger femoral
gland and more rugose dorsal skin than specimens from Borneo. Like the holotype, tibio-tarsal articulation in one
female reaches only to middle of tympanum, but it reaches to points beyond anterior border of tympanum in the
remaining one female and all males.
Calls. The calls recorded at Santubong on 29 August 2010 (air temperature = 26.2°C; Fig. 6B) had the duration
from 305–763 (mean+SD = 485.9+243.8, n = 3/1 male) ms, and emitted intermittently. The call consisted of 5–9
(6.3+2.3, n = 3) short notes, each with the duration of 16–33 (20.7+5.1, n = 18) ms, and the note gap of 88–102
(93.7+3.8, n = 15) ms, with the note repetition rate of 11.8–16.4 (13.7+2.4, n = 3) per s. Each note is composed of
4–11 (6.1+2.0, n = 18) fine pulses. Frequency bands spread over 1.5 to 2.2 kHz range, with the dominant from
1,650 to 1,950 (1,792+75, n = 18) Hz. Each note showed weak frequency modulation, with the initial and middle
frequencies (1,650 to 1,950 [1,792+75, n = 18] Hz) falling towards the end of the note (1,600–1,900 [1,760+99, n =
18] Hz).
The calls recorded on 26 December 1990 at Niah (air temperature = 25.9°C) were basically similar in temporal
structure (call duration 539–697 [618.2+111.7, n = 2/1 male] ms; number of notes 8–10 [9.0, n = 2]; note duration
21–39 [25.8+5.1, n = 18] ms; note gap 72–105 [76.2+7.9, n = 16] ms; note repetition rate 14.3–18.4 [14.6, n = 2]
per s; number of pulse per note 5–10 [7.0+1.5, n = 18]). However, frequencies were lower than in Santubong calls,
with the dominant from 1,300 to 1,500 (1,392+62, n = 18) Hz. Lowering of the initial frequency (1,350 to 1,600
[1,450+132, n = 3] Hz) towards the end of the note (1,250–1,350 [1,300+41, n = 4] Hz) was similar to the Santu-
bong calls. Three harmonic frequencies were present at approximately 1,875–1,930, 2,250–2,300, and 3,900–4,000
Hz. Comparisons. Leptobrachium ingeri is placed in the L. nigrops group by having small body size. This
character distinguished it from all other species of Leptobrachium, except for the other two species of the group (L.
nigrops and L. kanowitense). Leptobrachium ingeri has relatively larger eye than L. nigrops (15.0–18.5% SVL vs
.12.0–17.3% SVL in L. nigrops), and relatively larger values than L. kanowitense in snout to nostril length
(7.9–10.4% SVL vs. 7.0–8.5% SVL in L. kanowitense), internarial distance (7.2–8.7% SVL vs. 6.2–7.0% SVL),
intercanthal distance (17.6–20.7% SVL vs. 16.9–18.5% SVL), and inner metatarsal tubercle length (4.8–6.3% SVL
vs. 4.3–4.4% SVL). In addition to morphometric differences, L. ingeri differs from L. kanowitense in more
developed toe webs: phalanges free of web L. ingeri samples were 1–13/4 on the outer side of first (vs. 13/4–2 on L.
kanowitense), 1/4–2/3 on the outer side of second (vs. 11/4–13/4), 22/3-3 on the inner side of third (vs. 3–31/4), 11/4–13/4
on the outer side of third (vs. 2), 32/3 on the inner side of fourth (vs. 33/4–4), 32/3–33/4 on the outer side of fourth (vs.
33/4–4), and 11/2–12/3 on the inner side of fifth (vs. 2) toes. Leptobrachium ingeri further differs from the other two
species in much more sharp finger tips, and in more distinct markings on dorsum and ventrum than the other two
species.
In calls, L. ingeri closely resembles L nigrops from Malay Peninsula: Calls of L. nigrops, recorded on 30
December 1992 (air temperature = 26.4°C) and on 21 January 1993 (air temperature = 25.9°C) at the campus of
University of Malaya, Selangor, West Malaysia were short trills (Fig. 11). The call varied in length from 618–851
(617.9+131.4, n = 3) ms and consisted of 8–12 (mean+SD = 9.7+2.1, n = 26/2 males) short notes, each with 4–26
(8.4+5.2, n = 29) pulses. The note duration varied from 14–85 (29.8+17.8, n = 29) ms, and the note gap from
72–111 (78.6+10.4, n = 26) ms. The note repetition rate varied from 12.9–14.3 (13.8+0.7, n = 3). Frequency band
spread over 1,700 to 1,950 Hz range, with the dominant from 1,700 to 1,950 (1,852+75, n = 29) Hz. Within a note,
the initial frequency (1,800–1,900 [1,840+39, n = 10] HZ) slightly lowered at the end (1,700–1,950 [1,777+82, n =
11] Hz). These characteristics overlap those of L. ingeri, and the two species cannot be differentiated acoustically.
Range. The coastal area of Sarawak in Malaysian Borneo (Santubong, Matang [Kuching Division], and Niah
[Miri Division]); Belitung Island, Indonesia (Tanjung Padang) (Figure 1).
Natural history. Larval, and other ecological data are lacking, but breeding season seems to include late
August and late December, as evidenced by calling males. Matang, Santubong, and Tanjung Padang, where the
type series of L. ingeri were collected, are lowland (<60 m a.s.l.). The holotype was collected under a dead tree in
an old trail near a small river in secondary forest. Other species found on this trail were: Limnonectes kuhlii
(Tschudi), Limnonectes leporinus Andersson, Limnonectes kenepaiensis (Inger), Fejervarya limnocharis (Graven-
horst), Ansonia leptopus (Günther), Meristogenys jerboa (Günther), Hylarana signata (Günther), Hylarana rani-
ceps (Peters), Polypedates leucomystax (Gravenhorst), Rhacophorus fasciatus Boulenger, Kalophrynus
heterochirus Boulenger, and Microhyla borneensis Parker.
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CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
Leptobrachium kanowitense sp. nov.
Holotype. KUHE 42575, an adult female collected from upper part of Kanowit River (02°07’59.7’’N,
112°08’48.1’’E, 60 m a.s.l.), Sibu Division, Sarawak, Borneo, Malaysia, collected by M. Matsui, K. Nishikawa,
Daicus M. Belabut, Norhayati Ahmad, and Chan Kin Onn on 21 November 2008.
Paratypes. SRC 0002 (former KUHE 42587), KUHE 42588–42590, four adult females, KUHE 42574, an
adult male, collectors and locality same as for holotype.
Etymology. The specific name refers to the place where the type series was collected.
Diagnosis. A small-sized Leptobrachium of the L. nigrops group, with SVL 38.6–44.8 mm in females and
32.9 mm in males; finger tips weakly pointed; tympanum visible, uniformly dark brown; dorsum uniformly dark
brown, lacking conspicuous markings; laterally light brown with few dots near groin; ventrum whitish dotted with
black particularly on belly and on throat; chest and ventral side of limbs whitish with only few black dots; thin
black bars on dorsal side of limbs including digits; clear markings only at groin and posterior thigh; ventral thigh
whitish with some dots; femoral glands large in white blotch.
Description of holotype (measurements in mm). Small sized (SVL 44.8); habitus moderately stocky, body
tapering to groin, head wider than body, broad and depressed, longer (HL 19.8: 44.2% SVL) than wide (HW 17.4:
38.8% SVL); snout obtusely pointed, rounded triangle from above, truncate in profile, slightly projecting beyond
lower jaw; eye large and obviously projecting from sides of head, longer (EL 7.4: 16.5% SVL) than snout (SL 7.2:
16.1% SVL); canthus rostralis sharp, loreal region oblique, moderately concave; nostril lateral, below canthus,
distinctly closer to snout (S-NL 3.8: 8.5% SVL) than to eye (N-EL 4.3: 9.6% SVL); internarial distance (IND 3.1:
6.9% SVL) much narrower than interorbital distance, (IOD 5.2: 11.6% SVL), latter slightly narrower than upper
eyelid (UEW 5.8: 12.9% SVL); no pineal spot; tympanum visible, diameter (TD 3.2: 7.1% SVL) less than half that
of eye and separated from eye by more than half of its diameter (T-EL 1.7: 3.8% SVL); vomerine teeth absent;
tongue heart-shaped, without papillae, notched posteriorly.
Forelimb slender and long (FLL 31.7: 70.8% SVL), about three-fifths of hindlimb; fingers moderately slender,
unwebbed; first finger (1FL 4.8: 10.7% SVL) slightly longer than fourth and second, third much longer (3FL 6.6:
14.7% SVL); finger tips weakly pointed; inner palmar tubercle large (IPTL 1.8: 4.0% SVL), not extending onto
first metacarpal and larger than outer palmar tubercle (OPTL 1.5: 3.4% SVL); subarticular tubercles indistinct,
replaced by low callous tissue.
Hindlimb slender and relatively short (HLL 56.4: 125.9% SVL); heels not meeting when legs held at right
angles to body; tibia distinctly longer (TL 17.6: 39.3% SVL) than foot (FL 14.3: 31.9% SVL); tibiotarsal articula-
tion of adpressed limb reaching to beyond anterior edge of tympanum; third toe longer than fifth; toe tips similar to
those of fingers and hooked; toe webs very poorly developed; webbing formula I 13/4–21/4 II 11/4–3 III 2–33/4 IV 33/
4–2 V; inner metatarsal tubercle small (IMTL 1.6: 3.6% SVL), low and oval; outer metatarsal tubercle absent; sub-
articular tubercles obscure, but elongated, replaced by low callous tissue.
Skin above nearly smooth, with granules scattered posteriorly, particularly around sacrum; ventrum slightly
granular, especially on belly; very low supratympanic ridge from posterior corner of eye to axilla; indistinct low
dermal ridges on upper surface of limbs; flat pectoral gland at medial border of axilla behind arm insertion; femoral
gland on posterior surface of thigh in white spot.
Colour. In life, brown dorsally with large dark brown blotches on interorbital and parietal regions; laterally
brown fading to whitish on ventral side, where black dots scattered, especially dense on belly; loreal region widely
black, with some space before eye; tympanum uniformly brown, bordered above by very thin black line of
supratympanic ridge; forelimb dorsally light brown with faint bars; chest and ventral side of forelimb whitish;
throat darker mixed with guanophores; hindlimb ventrally whitish, spotted with black, particularly near groin and
anterior thigh, with blue ground color; dorsolateral sides of fingers and toes light brown; iris black surrounded by
light blue sclera visible in maximally opened eye. In preservative, dorsal ground colour darkened, but aspects of
colour pattern remained.
Variation. Morphometric variations are shown in Table 3. The male paratype (KUHE 42574) is morphologi-
cally similar to holotype, but has smaller body, more rugose dorsal skin, and more distinct dark crossbars on dorsal
side of thigh. It has an internal vocal sac and a pair of vocal sac openings. The female paratypes (KUHE
42587–42590) have smaller body, more densely dotted black on ventrum particularly on belly, and on lateral side
between armpit and groin, and have more hooked tips of fingers and toes than the holotype. In three females,
HAMIDY ET AL.
36 · Zootaxa 3398 © 2012 Magnolia Press
including holotype, and one male, tibio-tarsal articulation reaches to points beyond anterior border of tympanum,
but in the remaining two females it reaches only to middle of tympanum.
Calls. At Kanowit, we heard the calls of L. kanowitense, but failed to record them. Calls were very similar to
those of L. nigrops and L. ingeri to the human ear.
Comparisons. Leptobrachium kanowitense tends to have relatively longer head (43.0–45.8% SVL) and larger
eye (15.4–16.5% SVL) than L. nigrops (41.2–45.3% SVL and 12.0–17.3% SVL, respectively). Morphometric and
qualitative differences between L. kanowitense and L. ingeri are as noted above. Leptobrachium kanowitense is
differentiated from L. ingeri by much less developed toe webs. In addition, the new species is differentiated from L.
ingeri by having narrowly pointed finger tips. Leptobrachium kanowitense is similar to L. nigrops in the
development of toe webbing, but on the outer side of the second toe, web is less developed in L. kanowitense (11/
4–13/4 phalanges free of web) than in L. nigrops (
1/2–12/3, mostly 1 phalange free of web). Leptobrachium
kanowitense differs from L. nigrops and L. ingeri, by having whitish ventrum dotted with black (vs. usually
reticulated in L. nigrops, and blotched in L. ingeri). Dorsally, L. kanowitense lacks conspicuous blotched pattern,
unlike L. nigrops and L. ingeri that have distinct blotches. In addition, L. kanowitense has uniformly dark brown
tympanum whereas whole or upper half of tympanum is black in L. nigrops and L. ingeri.
Range. The inland area of western Borneo, so far only known from Kanowit, Sibu Division, Sarawak (Figure 1).
Natural history. Type series was collected from a marshy area in a jackfruit plantation near a long house
(traditional Iban house). Larval, and other ecological data are unknown, but the male paratype was found
intermittently emitting calls, indicating late November is within the breeding season. Frog species observed
associated with the type series were: Limnonectes paramacrodon (Inger), Limnonectes ingeri (Kiew), Fejervarya
limnocharis (Gravenhorst), Occidozyga laevis (Günther), Ingerophrynus quadriporcatus (Boulenger), Hylarana
baramica (Boettger), Hylarana raniceps (Peters), Hylarana erythraea (Schlegel), Hylarana glandulosa
(Bouleger), Hylarana signata (Günther), Polypedates colletti (Boulenger), Polypedates macrotis (Boulenger),
Polypedates leucomystax (Gravenhorst), Polypedates otilophus (Boulenger), Rhacophorus appendiculatus
(Günther), Rhacophorus pardalis Günther, Nyctixalus pictus (Peters), Chaperina fusca Mocquard, and Microhyla
malang Matsui.
Discussion
Amphibian diversity in Borneo is very high, and the exact frog species diversity is still unknown, because new
species have been described steadily from this island every year (e.g. Kalophrynus [Dehling 2011; Matsui &
Nishikawa 2011]; Ingerana [Iskandar et al. 2011], Meristogenys [Shimada et al. 2011a, b)] in addition to 150
named species reported nearly a decade ago (Inger & Stuebing 2005). The increase in frog diversity is mainly due
to both extensive field surveys in these decades and adoption of molecular tools. The application of various
molecular analyses has detected presence of cryptic species within a taxon that are morphologically difficult to
differentiate. Such examples range from species with wide distribution within and around Borneo like the
Limnonectes kuhlii complex (Matsui et al. 2010a, c; McLeod et al. 2011), to a Bornean endemic group such as the
genus Meristogenys (Shimada et al. 2008, 2011a). In the genus Leptobrachium, recent molecular studies have
suggested the presence of many cryptic species in many regions including Borneo (e.g. L. chapaense: Zheng et al.
2008, Rao & Wilkinson 2008, Matsui et al. 2010b; Philippine L. hasseltii: Brown et al. 2009; L. montanum: Matsui
et al. 2010b, Hamidy et al. 2011; L. abbotti: Hamidy et al. 2011).
In the case of the L. nigrops group, presence of morphological variation was noticed nearly half a century ago
(Inger 1966), but presence of high genetic divergence among populations have never been recognized until recently
(Brown et al. 2009; Matsui et al. 2010b). Matsui et al. (2010b) clarified presence of high genetic divergence
comparable to species level in L. nigrops from Malay Peninsula, Belitung, and Borneo. The results of this study
using not only mitochondrial, but also nuclear genes, confirmed the previous finding and we recognized three
species within frogs traditionally identified as L. nigrops. In contrast to their large genetic divergence, the three
species are generally similar in morphology, although they can be differentiated through careful examinations.
Leptobrchium nigrops is now restricted to Malay Peninsula and Singapore, but the species possibly occurs on
eastern part of Sumatra, because two MZB specimens from Riau (eastern Sumatra) had finger tips similar to L.
nigrops. Unfortunately, they are not available for DNA analysis. Although in mtDNA data, the genetic distances
Zootaxa 3398 © 2012 Magnolia Press · 37
CRYPTIC TAXA IN MALAYSIAN MEGOPHRYID
between two allopatric populations of L. ingeri from Belitung and costal area of Borneo are not small (5.5–5.8%),
nuclear data show that the two populations should be recognized as a single species. One GenBank sequence, reg-
istered as L. nigrops from Borneo without exact locality data (Brown et al. 2009), was nested in our L. ingeri in the
comparison of short sequence data of mtDNA.
As far as we know, L. ingeri and L. kanowitense are allopatric on Borneo, and results from nuclear DNA
rejected hybridization or gene introgression between them. Unfortunately, we failed to record the calls of L.
kanowitense, but they were very similar to calls of the other two species to the human ear. Similarly, no tangible
difference was detected in acoustic characteristics between L. ingeri and L nigrops. Some allopatrically occurring,
putative sister species in other frog lineage possess very similar call characteristics (e.g. Rhacophorus owstoni
(Stejneger) and R. moltrechti Boulenger: Matsui, 1994). It is surmised that such acoustic similarities in closely
related and allopatrically distributed species may result from their isolated distributions from the past, where
selection did not act to differentiate their calls. Isolation would have permitted retention of ancestral acoustic traits
in the two lineages in separate regions.
Although not unambiguously supported in nuclear DNA tree, mtDNA tree indicated early divergence of ances-
tral L. kanowitense from the common ancestor of L. nigrops and L. ingeri. This means the separation of Peninsular
and Bornean L. nigrops group involves multiple events. Because current distribution of the L. nigrops group in
Borneo is restricted to lowlands of western regions of Sarawak, just opposite to Malay Peninsula, it is reasonable to
consider that multiple invasion of ancestral stocks occurred from the peninsula to Borneo. The first invasion was by
the ancestor of L. kanowitense that split earlier than the divergence of L. ingeri and L. nigrops. Inger (2005) argued
that the long-term continental connection between Borneo and Malay Peninsula (50–5 MYBP) was interrupted
twice, one in the mid Miocene (15 MYBP) and another in the early Pliocene (5 MYBP). The first event might have
isolated the ancestor of L. kanowitense from a common ancestor of L. nigrops and L. ingeri, as hypnotized by Mat-
sui et al. (2010b). The second event would have separated the ancestor of L. ingeri from the ancestor of L. nigrops.
Current distribution pattern of two Bornean species, with L. kanowitense inhabiting inland area and L. ingeri in
coastal area, conforms to the above scenario. If this is the case, the estimated migration route of L. ingeri is from
Borneo to Belitung, because the latter island seems to have appeared later than Borneo (Voris 2000). This event
most probably occurred during the Pleistocene glacial periods when all Sundaland islands were connected as one
landmass, as already proposed by Inger and Voris (2001). In order to test these hypotheses, examination of more
samples, especially from Sumatra and other islands in Karimata Strait such as Natuna islands, are necessary.
Acknowledgements
AH thanks Prof. P. Ng, K. Lim, and D. Gower for providing tissue, I. Sidik and S. Hartini for helping to borrow
MZB specimens, and the Monbukagakusho for scholarship funding. Permission to conduct fieldwork by MM in
Sarawak between 1989 and 1991, and in Peninsula between 1992 and 1993 were kindly provided by the Socio-
Economic Research Unit of Malaysia and the State Government of Sarawak. The Forest Department, Sarawak and
University Malaya provided all the facilities for conducting research. The late A. H. Abang, K. Araya, T. Hayashi,
T. Hikida, K. Eto, A. H. Kassim, J. J. Kendawong, D. Labang, H. Ota, L.-H. Seng, and N.-S. Wong are
acknowledged for help in field trips. For the fieldwork in 2008, research passes were kindly issued by Economic
Planning Unit, Prime Minister’s Department (40/200/19/2285) and The Forest Department, Sarawak
(NPW.907.4.2(III)-68). We are grateful to Datu Haji Len Talif Salleh, L. Chong, K. Het, A. Norhayati, and H.-S.
Yong for providing all help for conducting research. We thank an anonymous reviewer, S. Mahony, and M. Vences
for improving the manuscript. This study was partly supported by grants from TORAY grant (MTSF1019-2007A)
to DB. Field trips were made possible by grants under The Monbusho International Scientific Research Programs
(Field Research, Nos. 01041051, 02041051), grants from The Monbusho through the Japanese Society for the
Promotion of Sciences (JSPS: Field Research, No. 20405013), and Universiti Kebangsaan Malaysia (UKM-OUP-
PLW-14-59/2008) to MM.
HAMIDY ET AL.
38 · Zootaxa 3398 © 2012 Magnolia Press
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