Malays. Appl. Biol.

(2018) 47(1): 73–80

GENETIC VARIATION OF THE BORNEAN FANGED FROG,

Limnonectes kuhlii COMPLEX IN WESTERN BORNEO
(AMPHIBIA: ANURA: DICROGLOSSIDAE)

RAMLAH ZAINUDIN1,2* and NAJMI NAIM1

1Department of Zoology, Faculty of Resource Science and Technology,

Universiti Malaysia Sarawak, 94300 Kota Samarahan Sarawak
2Centre for Pre University Studies, Universiti Malaysia Sarawak,

94300 Kota Samarahan Sarawak

*
E-mail: zramlah@unimas.my

Accepted 1 February 2018, Published online 31 March 2018

ABSTRACT

The fanged frog Limnonetces kuhlii is known to be cryptic as seen in the dynamic of species delineation among the complexes.
Yet no historical demographic and genetic structure has been fully documented for this species. We investigate historical
events, diversification and dispersal of the Bornean Limnonectes kuhlii Complex via Mitochondrial DNA of partial 16S
rRNA. Haplotype graphical network, demographic history, neutrality test and population subdivision analysis were used to
assess population genetic of the species. Two haplogroups were detected distinct within population and panmictic from East
and West of Lupar gap (Batang Ai). The Lupar gap has become the geographical barrier that restricts gene flow from western
and eastern populations. Ragged multimodal mismatch distribution, long terminal branches and high mutational site of network
showing population in Sarawak and Sabah were rarely migrate and low gene flow. Surprisingly, most of L. kuhlii population
showed population constriction and presence of two or more mixed subpopulations. It can be concluded that high levels of
sequence divergence suggesting ancient DNA (lineage) and cryptic species hidden within the species. This study should be
extended in future with more samples and validate their true species status with morphological data.

Key words: Ancient DNA, cryptic species, genetic variation, mitochondrial (mtDNA), population genetic

INTRODUCTION al., 2016). According to Emerson et al. (2000),

Since introduction of Polymerase Chain Reaction
(PCR) in 1985 as a molecular technique for species
examination and identification, study and research
have increase exponentially on discovering and
uncovering cryptic species (Bickford et al., 2007).
McLeod (2010) stated that identification of these
cryptic species is critical to assess accurately
genuine biodiversity. This technique opens wide
opportunity for detecting and differentiating
morphologically similar species. Cryptic species are
known as two or more distantly genetically related
but possess conspicuously similarly morphological
characteristics that were recognized as a single
species (Pfenninger & Schwenk, 2007). Several
studies suggested that Limnonectes kuhlii repre-
sented a cryptic species complex (Emerson et al.,
2000; Evans et al., 2003; McLeod, 2010; Matsui et
broad ranging of L. kuhlii is as a matter of fact a
number of different species.
McLeod et al. (2011) suggested that the
diversity within the L. kuhlii complex was greater
than previously suspected. How much greater is the
diversity within L. kuhlii complex in Borneo? What
are the factors that caused high genetic divergence
within the population?. To address these and
related issues, a comprehensive assessment of the
diversity within this species complex is through the
population genetic context. Knowledge of genetic
structure of populations is very important for one
to understand the species evolution. Therefore, we
examine the historical events, diversification and
dispersal of L. kuhlii to apprehend on its ancient
polymorphism and genetic variation among L. kuhlii
populations from geographically isolated areas in
Borneo.

* To whom correspondence should be addressed.

74 GENETIC VARIATION OF THE BORNEAN FANGED FROG, Limnonectes kuhlii COMPLEX
MATERIALS AND METHODS
Sample description and collection locations
The study sites were located in known
distributions of L. kuhlii in Malaysian Borneo
(Supplementary material Appendix 1, Figure A1).
Batang Ai National is a large forest fragment and
hilly area consisti of primary and secondary forests.
Some parts of the forest were logged decades ago
according to the local people for swidden
agriculture. Santubong National Park, Kubah
National Park and Matang Wildlife Centre are
dominantly covered by primary forest due to gazette
as Totally Protected Area. However, perforated
fragmentation was observed nearby the sanctuary
of Matang Wildlife Centre. Saremas Oil Palm
Plantation, Miri occupied human settlements, High
Conservation Value Forest (HCV) fragmented area
and plantation area which indicates high human
disturbance.
Total of 19 individuals of the L. kuhlii (Supple-
mentary material Appendix 1, Table A1) were
collected via Visual Encounter Survey and all
ecological data were recorded on site. Muscle tissue
stored in 99% Ethanol. Additional sequences of
16S gene from McLeod (2010) were used for
complementary and assimilate material.
DNA extraction, Polymerase Chain Reaction
(PCR), and DNA sequencing
For molecular data, mitochondrial DNA was
extracted using CTAB protocol (Grewe et al., 1993).
Approximately 550bp of mtDNA region sequenced
is 16S RNA. Primers used to amplify the target DNA
fragments using the polymerase chain reaction
(PCR) were taken from Evans et al. (2003). Primers
of 16S RNA forward and reverse (16Sar-L 5’- CGC
CTC TTG CTT AAA AAC T-3', 16Sb-H 5’-ATG TTT
TTA AGC AAG AGG CG-3') were employed
according to Palumbi et al. (1991). PCR ampli-
fication reaction cycle parameters consist of
denaturation for 30 s at 95ºC, primer annealing
for 30 s at 50ºC and extension at 72ºC for 70 s
following McLeod (2010) for 35 cycles. Pre-
denaturation for 4 minutes at 95ºC and final
extension for 7 minutes at 72ºC were also used.
After successful PCR, the PCR tubes were sent to
1st Base Asia for DNA sequencing by using ABI 337
automatic sequencer.
Data analyses
CHROMAS version 1.45 software (McCarthy,
1996) was used for editing and eye checking of total
19 DNA sequences. A total length of approximately
550bp was successfully sequenced. ClustalX version
1.81 (Thompson et al., 1997) was used to perform
multiple alignment and aligning selected DNA
sequences. Molecular evolutionary and phylogenetic
analyses were performed using MEGA (Molecular
Evolutionary Genetics Analysis) version 6 (Tamura
et al., 2013). Bootstrap value performed were 1000
times randomization for each sequences. By using
Modeltest 3.7 (Posada & Crandall, 1998), Akaike
Information Criterion (AIC) was used to determine
the best-fit-model of sequence evolution in species
and consider loss of information. Maximum-
likelihood (ML) is based on the substitution model
and phylogenetic parameters General Time
Reversible model plus invariable sites (GTR+I)
identified as optimal by the Bayesian information
criterion (BIC) in Modeltest 3.7 (Posada & Crandall,
1998).
Minimum Spanning Network (MSN) of
haplotype was constructed to obtain a graphical
representation of 16S gene using Network 4.6.13
(Bandelt et al., 1999). Several tests were used to
examine weather haplotype frequency swerve from
evolutionary neutrality or support the neutrality
null hypothesis. The demographic history L. kuhlii
in the population of four regions was examined.
Mismatch distribution and frequency spectrum were
performed using DNA Sequence Polymorphism 5.1
(Rozas et al., 2009). Tajima’s Test of Neutrality, D
(Tajima, 1989), Fu and Li’s D* and F* (Fu & Li,
1993) and Fu’s Fs (Fu, 1997) were used. Neutrality
tests were conducted by using DnaSP 5.1 (Rozas
et al., 2009). Examination of gene flow such as
level of population subdivision (Fst), nucleotide
subdivision (Nst) and number of migrant per
generation (Nm) were also calculated using DnaSP
5.1 (Rozas et al., 2009).
RESULTS
A total of 471 base pairs of the 16S RNA gene of
31 sequences of L. kuhlii were completely aligned.
Total of 82 (17.4%) sites were variable, 386 (81.9%)
sites were constant, and of those, 74 (15.7%) were
parsimony informative and 11 (2.3%) were
singleton. Amount of parsimony informative was
relatively high, shows that this gene is sufficient
and acceptable to view phylogenetic relationship
and information about the species.
A total of 85 segregating sites were produced
from the 14 haplotypes distributed within and
among the four populations of L. kuhlii from the
total size of 31 individuals. Among these popula-
tions, 14 haplotypes were identified with no sharing
among populations. This indicates gene is highly
polymorphic within and among populations.
Maximum Likelihood tree topology shows
monophyly of Limnonectes kuhlii (100% of
bootstraps support), with respect to the outgroups,
Limnonectes laticeps and Limnonectes leporinus.
Two clades were observed, first clade namely
 GENETIC VARIATION OF THE BORNEAN FANGED FROG, Limnonectes kuhlii COMPLEX 75

Haplogroup 1 (HG 1) and second clade namely
Haplogroup 2 (HG 2). A similar result was obtained
from Minimum-spanning network analysis (Figure
1). MSN topology pattern was consistent with
topology of phylogenetic trees which showed
separation between populations of HG 1 (Santubong,
Kubah, Mendolong, and Kalimantan) and HG 2
(Miri, Bintulu, Crocker Range and Batang Ai).
These 14 haplotypes are categorized into five clades
correspond to biogeographical unit of Borneo
(MacKinnon et al., 1996) namely Western Sarawak
(Kubah and Santubong), Central Sarawak (Batang
Ai), Eastern Sarawak (Bintulu and Miri), Sabah
(Crocker Range and Mendolong) and lastly
Kalimantan population (Kutai).
Among populations, nucleotide diversity (π)
values of 5.3%–5.4%) and net nucleotide diver-
gences (Da) of 5.1%–3.1% were higher in popula-
tions between western Sarawak-central Sarawak and
western Sarawak-Sabah, indicating high genetic
differentiation among these three populations.
Lower π values of 3.8%–4.7% and net nucleotide
divergences (Da) of 1.7%–1.8% were seen among
eastern Sarawak-central Sarawak, and eastern
Sarawak-Sabah, indicating lower genetic differen-
tiation among these populations. Eastern and
western Sarawak populations, however, have higher
genetic differentiation among any other populations
with π value of 5.8% and highest Da value of 5.6%
(Table 1).
Neutrality test for overall populations suggested
shrinkage within all the L. kuhlii populations except
for central Sarawak population (Table 2). Tajima’s
D, Fu and Li’s F* and Fu and Li’s D* was negative
in central Sarawak indicating there were recently
derived haplotype and suggesting the presence of

Fig. 1. Relationships among the 14 haplotypes of Limnonectes kuhlii between the eastern population, central population,
western population and Sabah population. The networking was generated by using Network 4.6.13 (Bandelt et al., 1999).

Table 1. Measures of nucleotide (π) and net nucleotide divergence (Da) among population of Limnonectes kuhlii
analyzed by region

Distance Nucleotide Estimate of population Number of migrants

Region (km) subdivision (Nst) subdivision (Fst) per generation (Nm)

Central – Western 344.58 0.6151 0.6083 0.69

Central – Eastern 183.31 0.3758 0.3745 1.01
Central – Sabah 685.37 0.2647 0.2686 1.86
Western – Eastern 456.14 0.6543 0.6462 0.53
Western – Sabah 344.27 0.4341 0.4365 1.02
Eastern – Sabah 797.35 0.3210 0.3177 1.50
Overall – 0.4757 0.4721 0.55
76 GENETIC VARIATION OF THE BORNEAN FANGED FROG, Limnonectes kuhlii COMPLEX

Table 2. Summary statistics of 16S rRNA mtDNA sequence variation in four populations of Limnonectes kuhlii from Sarawak
and Sabah

Population N H S % sdiv h† π† K D Fs D* F* R

Central 4 3 29 3.3 0.833 0.0314 14.833 -0.643 3.299 -0.643 -0.674 0.416
(p > 0.10) (p > 0.10) (p > 0.10)

Western 12 5 32 3.5 0.757 0.0336 15.848 2.236* 6.955 1.603* 2.015* 0.194
(p < 0.05) (p < 0.02) (p < 0.02)

Eastern 6 3 22 2.8 0.733 0.0276 13.000 2.190* 5.628 1.407 1.717 0.816
(p < 0.05) (p > 0.10) (p < 0.05)

Sabah 8 2 41 5.0 0.535 0.0466 21.964 2.082* 14.251 1.636* 1.941* 0.789
(p < 0.05) (p < 0.02) (p < 0.02)

Whole
population 31 14 85 6.6 0.927 0.0622 29.325 1.431 7.114 0.345 0.839 0.023
(p > 0.10) (p >0.10) (p > 0.10)

N = number of sequence; H = number of haplotypes, S = number of segregating sites, % sdiv = percentage of pairwise sequence divergence;
h = haplotype diversity; π = nucleotide diversity, K = average number of nucleotide differences; D = Tajima’s statistics (Tajima, 1989), Fs =
Fu’s statistics (Fu,1997), D* and F* = Fu and Li’s statistics (Fu and Li 1993); r = raggedness statistics (Harpending, 1994).
* Significance was calculated using coalescent simulation in DnaSP version 4.0 (Rozas et al., 2003).
† Sites with gap were completely excluded.

rare haplotypes or ancient test suggest the
occurrence of rare haplotypes, but insignificant to
show either genetic hitchhiking or polymorphism.
Neutrality population expansion (Fu, 1997).
Contradictorily, Tajima’s D and Fu and Li’s F* were
positive for western Sarawak, eastern Sarawak and
Sabah suggesting the populations are far from
neutral. Significant value of Tajima test suggests
a recent population contraction or selection that
maintains genetic variation (Fu & Li, 1993).
Furthermore, the hypothesis was significantly
supported by Fu and Li’s D* for western Sarawak
and Sabah populations.
By assuming constant population size, the
mismatch distribution of pairwise nucleotide
differences among 16S sequences for whole
population (Figure 2) of L. kuhlii exhibited a
ragged multimodal distribution supported by non-
significant value of Tajima’s D, rejecting the
expansion model. This indicates that the whole
population of L. kuhlii may experience population
change rather than population expansion and
experience a long-term demographic stability
(Zainuddin, 2009). There was smooth bimodal
distribution characteristic of pairwise differences
observed for central Sarawak and Sabah populations
which consistent with population expansion model
due to follow the expected models frequencies.
Kubah and Batang Ai populations showed
higher migrants per generation among other
localities, ranged from 0.27–0.49 and 0.14–0.52
respectively (Table 3), suggesting this population
is panmictic to each other. Overall, the analyses
from gene flow estimator revealed low levels of
migrants per generation showing high population
fragmentation. Only western Sarawak showed high
migrants per generations within region (Kubah –
Santubong). While eastern Sarawak (Miri – Bintulu)
and Sabah (Crocker Range – Mendolong) showed
low migrants per generation within region with
Nm=0.03 and Nm=0.00 respectively. However,
Santubong have high nucleotide subdivision and
population subdivision (Nst and Fst > 0.81) among
other localities except Kubah, showed that the
population was highly polymorphic.
DISCUSSION
Current study revealed local geographic structuring
of L. kuhlii with higher haplotype diversity and non-
sharing haplotype hence explained the complexity
of the species as well as highly polymorphism
among populations. This cryptic species problem of
L. kuhlii have been arise and issued in previous
studies (Emerson et al., 2000; Evans et al., 2003;
McLeod, 2010, Matsui et al., 2016). There are at
least four species subsumed under L. kuhlii which
are two in Borneo (Emerson et al., 2000). More than
22 distinct lineages and in those lineages, there are
eight endemic lineages distributed in Borneo
(McLeod, 2010). However, Matsui et al. (2016)
showed more devastating result, approximately 17
lineages distinguished in Borneo.
High levels of sequence divergence between
populations indicate the existence of cryptic species
among the population (Zainudin et al., 2010).
Previous study (McLeod, 2010) also revealed
 GENETIC VARIATION OF THE BORNEAN FANGED FROG, Limnonectes kuhlii COMPLEX 77

Fig. 2. Population expansion signatures in 16S sequences data among population of Limnonectes
kuhlii. Mismatch distribution of observed frequencies of pairwise differences among 16S sequences
and expected frequencies under the sudden expansion model and spatial expansion model.

Table 3. Measures of nucleotide subdivision (Nst), population subdivision (Fst), and gene flow (number of migrants, Nm)
among 7 localities of Limnonectes kuhlii

Nucleotide Estimate of Net nucleotide Number of

Distance
Locality subdivision population divergence migrants per
(km)
(N st ) subdivision (Fst) (Da) generation (Nm)

Batang Ai – Santubong 199.207 0.8151 0.8108 0.0690 0.14

Batang Ai – Miri 258.242 0.5835 0.5879 0.0224 0.52
Batang Ai – Crocker Range 711.57 0.5385 0.5436 0.0187 0.47
Batang Ai – Mendolong 584.956 0.8185 0.8146 0.0691 0.19
Batang Ai – Bintulu 209.134 0.6839 0.6824 0.0383 0.35
Batang Ai – Kubah 119.564 0.6061 0.5995 0.0486 0.49
Santubong – Miri 346.338 0.9961 0.9958 0.0842 0.01
Santubong – Crocker Range 814.434 0.9957 0.9958 0.0778 0.00
Santubong – Mendolong 685.874 0.9948 0.9946 0.0651 0.01
Santubong – Bintulu 220.401 0.9743 0.9728 0.0884 0.02
Santubong – Kubah 22.544 0.6397 0.6381 0.0302 0.43
Miri – Crocker Range 464.478 1.0000 1.0000 0.0148 0.00
Miri – Mendolong 340.766 1.0000 1.0000 0.0934 0.00
Miri – Bintulu 49.104 0.9536 0.9524 0.0424 0.03
Miri – Kubah 367.988 0.7958 0.7930 0.0642 0.31
Crocker Range – Mendolong 178.718 1.0000 1.0000 0.0870 0.00
Crocker Range – Bintulu 514.217 0.9512 0.9500 0.0403 0.03
Crocker Range – Kubah 836.295 0.7681 0.7665 0.0550 0.27
Mendolong – Bintulu 385.708 0.9804 0.9790 0.0990 0.01
Mendolong – Kubah 707.691 0.7399 0.7396 0.0476 0.42
Bintulu – Kubah 244.574 0.7945 0.7895 0.0708 0.30

substantial levels of sequence divergence of 16S as
the mean corrected sequence divergence was 10.9%.
Limnonectes kuhlii possesses high genetic diversity
within L. kuhlii as there is within all of Limnonectes
where the pairwise sequence divergences are 10.9%
and 12.7% respectively (McLeod, 2010). Besides,
high level of sequence divergence suggesting
likelihood of ancient lineage within species. The
genetic divergence between Mendolong – Miri and
Mendolong – Bintulu were as high as 9.9%. There
is variation in morphology between Sarawak and
Sabah populations in terms of their pigmentation,
78 GENETIC VARIATION OF THE BORNEAN FANGED FROG, Limnonectes kuhlii COMPLEX
skin surface and size (Inger, 1966). These traits
might cause high genetic variation among these
populations.
Based on phylogenetic analysis, Sabah
population were separated as Mendolong clade into
HG 1 while Crocker Range clade into HG 2. This
supported by bimodal mismatch distribution, where
there are two separated populations of Mendolong
and Crocker Range. No gene flow shows between
Mendolong and Crocker Range implies the
population is growing separately. According to
Mora et al. (2007), bimodal distributions are two
isolating populations caused by geographical
barrier. Besides, bimodal also indicates either, two
population undergone expansion or presence of
two or more mixed populations that have sub-
sequently expanded (Jalil et al., 2008). Placement
of haplotypes from Sabah into both HG 1 and HG 2
presumed of two of apparent species or cryptic
species might be present within these populations.
The hilly terrain may cause these populations to
diverge. Borneo geographical landscape produced
a greater barrier than the event of Pleistocene
(Zainudin et al., 2010). The outcome of this event
might be more profound exclusively to this species
complex as they prefer hilly and mountain area.
Related incident of Meristogenys genus which
inhabit hilly torrents possessed several significantly
different sympatric lineages (Shimada et al., 2008;
Matsui et al., 2010; Shimada et al., 2011) thus
warranted three new described species at Sabah by
means of larval morphology and latitude differences.
Slatkin (1987), geographic variation forcing to
generate genetic differentiation and this event
produce low genetic similarity. In the event that, no
migration between fragmented populations, the
probability of genetic differentiation within species
increases. The confine of isolation and mixing
between populations can attribute higher genetic
differentiation eventually speciation occurs.
Closely distance of central Sarawak and western
Sarawak populations showed event of genetic break
between western and eastern Sarawak. The levels of
migrants per generation were the smallest among
western and eastern supported with population
subdivision value. The populations were separated
by the presence of Lupar Line. The Lupar Line is a
major suture which has resulted from movement of
plate that largely resolved the Cretaceous to
Paleogene history of Borneo (Haile at al., 1994).
According to Hutchinson (1996), the Lupar Line
resulted by Lupar River 10-15 Mya during the
tectonic evolution. The populations of eastern and
western Sarawak were evolving separately since
then. Matsui et al. (2016) also emphasized
divergence of closely related lineages are estimated
to have arisen during Late Mieocene and Early
Pleocene. The barrier restricting the movement of
anurans due to Batang Lupar are surrounded by
swampy area and some frogs cannot adapt with high
acidic and salinity of sea water (Zainudin, 1998).
Moreover, according to Zainudin (2009) frogs are
sensitive to environment, especially to high acidity
of water. Their skin is very permeable to water for
breathing and need suitable acidity of water to breed
as their life cycle include aquatic stage which is
tadpole. Phylogeographic studies show that the
Lupar Line is a factor of changes in genetic structure
within the species. It has been proven Lupar gap has
become barrier for CO1 gene in Limnonectes kuhlii,
Hylarana erythraea and Limnonectes leporinus
(Zainudin, 1998; Deka, 2007; Zainudin et al., 2010)
as well as in fishes (Yuzine et al., 2007). Ryan and
Esa (2006) also revealed the existence of two
different Hampala bimaculata (fishes) lineages of
southern and central part of Sarawak assigned as
H. bimaculata Type A; with northern Sarawak and
the west coast of Sabah assigned as H. bimaculata
Type B.
CONCLUSION
High level of sequence divergence indicating high
rates of 16S evolution therefore suggesting there
might be ancient lineages and cryptic species
hidden within the species. More extensive studies
of L. kuhlii on morphological and molecular
disciplines are needed to validate species status.
Comprehensive data collection on microhabitat
characteristic for forthcoming research would be
better and helpful to examine and relate the factor
contributing to high genetic divergence among
populations. This finding is important for con-
servation value of L. kuhlii complex in the future
for the purpose of having a better conservation plan
and generating new data about this species.
ACKNOWLEDGEMENTS
This project was funded by Niche Research Grant
Scheme NRGS/1088/2013(02) and FRGS/06(14)/
704/2009(20). My deepest gratitude to Faculty of
Resource Science and Technology, Universiti
Malaysia Sarawak especially Department of Animal
Science and Resource Management for providing
me with all the sufficient equipment and facilities
during laboratory works, assisting me during
fieldworks and provides good research environment.
This writing is part of dissertation of Bachelor
Degree in 2015. Special thanks to Sarawak Forestry
Department for the research permit (NCCD.907.4.4
(Jld.12)-1106; No.236/20154) and Sarawak Forestry
Corporation for field accommodation.
 GENETIC VARIATION OF THE BORNEAN FANGED FROG, Limnonectes kuhlii COMPLEX
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