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ENDANGERED SPECIES RESEARCH
Endang Species Res
Vol. 30: 125–131, 2016
doi: 10.3354/esr00729 Published May 12
INTRODUCTION
Morphological and genetic studies of wild tigers
Panthera tigris have resulted in the designation of
6 extant subspecies: Amur P. t. altaica, Bengal P. t.
tigris, South China P. t. amoyensis, Sumatran P. t.
sumatrae, Indochinese P. t. corbetti and Malayan P. t.
jacksoni (Ma´zak 1981, Luo et al. 2004). Thailand is
one of 7 countries situated within the geographic
range of tigers and contains sufficient habitat to sup-
port 2 source sites and up to 250 tigers (DNP 2010,
Walston et al. 2010). The majority of tigers in Thai-
land are of the subspecies P. t. corbetti, and P. t. jack-
soni occurs in the south of the country separated by
the Isthmus of Kra (Luo et al. 2004). Most of the
remaining tiger habitat in Thailand is within the
Western Forest Complex (WEFCOM), a World Her-
itage Site which covers an area of approximately
© The authors 2016. Open Access under Creative Commons by
Attribution Licence. Use, distribution and reproduction are un -
restricted. Authors and original publication must be credited.
Publisher: Inter-Research · www.int-res.com
*Corresponding author: fvetwww@yahoo.com
NOTE
Mitogenome analysis reveals a complex
phylogeographic relationship within the
wild tiger population of Thailand
Waradee Buddhakosai1, Worata Klinsawat2, Olutolani Smith3,4,
Manakorn Sukmak5, Nongnid Kaolim6, Somphot Duangchantrasiri7,
Achara Simcharoen7, Boripat Siriaroonrat8, Worawidh Wajjwalku1,6,*
1Interdisciplinary Graduate Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkok 10900, Thailand
2Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, MN 55108, USA
3Division of Biology, Silwood Park, Imperial College London, Ascot, Berks SL5 7PY, UK
4Panthera, 8 West 40th St, 18th Floor, New York, NY 10018, USA
5Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Thailand
6Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsaen, Nakhon Pathom 73140,
Thailand
7Department of National Parks, Wildlife and Plant Conservation Department, Bangkok 10900, Thailand
8Zoological Park Organization, Dusit, Bangkok 10300,Thailand
ABSTRACT: We present the first study of the complete mitogenome of wild tigers Panthera tigris
in Thailand. Thailand has been recognised as one of the most important countries within the geo-
graphic range of P. tigris and is home to 2 subspecies: Indochinese P. t. corbetti in the north and
Malayan P. t. jacksoni in the south. We obtained samples from wild tigers in a large forest complex
in northern Thailand (Western Forest Complex, WEFCOM) and from locally confiscated individu-
als in southern Thailand close to the border with Malaysia. Our results support the occurrence of
both Indochinese and Malayan tigers in Thailand and reveal complex phylogeographic patterns
of the wild tiger population in Southeast Asia.
KEY WORDS: mtDNA · Panthera tigris corbetti · Panthera tigris jacksoni · Western Forest
Complex · WEFCOM · Large cats · Phylogenetics
O
PEN
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Endang Species Res 30: 125– 131, 2016
126
18 000 km2(Simcharoen et al. 2007). However,
despite government protection under the Wild Ani-
mal Reservation and Protection Act B.E.2535 (1992)
of Thailand, the tiger population here is still declin-
ing due to habitat loss and human persecution.
Due to the critical importance of regions such as
WEFCOM to tiger conservation in Thailand, National
Park staff have been monitoring the population
through regular camera trap studies, and they have
also begun to collect non-invasive samples (faeces
and hair) for genetic analysis. Recent genetic studies
of mitochondrial DNA variation in the WEFCOM
population and in southern Thailand (unpubl. data)
have revealed the presence of 2 new haplotypes that
have not been previously reported. One haplotype
(GenBank Accession No. KC879297) was discovered
in tiger samples from WEFCOM, and the other
(KC879296), which was detected in an individual
from southern Thailand, represents a new haplotype
of P. t. jacksoni.
Here we describe a new study as an extension of
this work, to characterize the complete mitogenome
of tigers from WEFCOM and southern Thailand, and
to demonstrate the phylogenetic relationship be -
tween these haplotypes and tigers in other popula-
tions. The results of this and subsequent genetic
studies will act as the basis for a national genetic
database, which could be used for both research and
forensic applications in Thailand.
MATERIALS AND METHODS
Primer design
Most of the primers used in this study were newly
designed from the alignment of cymt (GenBank
DQ151150) and F2 numt sequences (GenBank
DQ151151) (Kim et al. 2006). Primers originally de-
veloped for Ursidae (Delisle & Strobeck 2002) were
used to amplify the region between the 2 ends of F2
numt (ATP6 − ND5). Full details of the primers used
are given in Table S1 in the Supplement, at www. int-
res. com/ articles/ suppl/n030 p125_ supp. pdf.
Samples and DNA extraction
Hair samples (n = 3) were collected from WEFCOM
during field surveys by National Park staff in 2009.
These 3 samples shared the same haplotype based on
aligned sequences from ND6, Cyt b and the control
region; we therefore chose only 1 sample for further
analysis (ID = WEFCOM). Three muscle samples
were also obtained from 3 tiger carcasses confiscated
by police from poachers in Narathiwat province,
southern Thailand, in 2009. One sample was obtained
from each individual, and 1 sample (ID = Ti2) was
identified as a new haplotype using the aligned se-
quences from ND6, Cyt b and the control region. This
sample was therefore chosen for subsequent analysis.
A map of Thailand showing the sampling sites is
shown in Fig. S1 in the Supplement at www. int-res.
com/ articles/ suppl/ n030 p125_ supp. pdf.
DNA extractions were performed following a mod-
ified method of Carter & Milton (1993). Briefly, the
hair or muscle was cut into small pieces, digested in
lysis buffer, and incubated overnight at 56°C. The
supernatant was then collected and mixed with silica
diatom. After shaking and centrifugation, the result-
ant pellet was washed with washing buffer. The
pellet was then dried and DNA eluted with elution
buffer.
PCR conditions
Each DNA template was amplified with a single-
direction PCR prior to specific fragment amplifica-
tion. PCRs were performed in a 20 µl reaction volume
containing 10 µl of 2× Phusion buffer (Finnzymes),
200 µM dNTP, 0.25 µl of each 1 µM reverse primer,
0.2 µl of Phusion High-fidelity DNA polymerase
(Finnzymes), 2 µl of DNA template (diluted to 5 ng
µl−1), and the relevant amount of purified water. PCR
cycling conditions were as follows: a pre-denatura-
tion step at 98°C for 3 min, then 35 cycles of 98°C for
30 s, annealing at 58°C for 30 s, extension at 72°C for
2 min, then 72°C for 5 min. The higher denaturation
temperature of 98°C is recommended for this high-
fidelity Taq enzyme.
The second round of PCR was performed in 50 µl
volumes containing 25 µl of 2× buffer, 200 µM dNTP,
0.1 µl of each of 10 µM forward and reverse primer,
0.5 µl of Phusion High-fidelity DNA polymerase
(Finnzymes), 1 µl of the PCR product from the first
round of single-direction PCR and purified water.
PCR was performed for 40 cycles under similar con-
ditions to those described above, apart from reducing
the extension time to 1 min. PCR products were puri-
fied using the silica diatom method as described
above (Carter & Milton 1993) and sequenced in
either forward or reverse directions. This 2-step PCR
protocol was repeated twice for each mitochondrial
region except the control region, which was repeated
3 times.
Buddhakosai et al.: Phylogeographic relationship of wild tigers in Thailand 127
Data analysis
Amplified mtDNA fragments were assembled
into complete mtDNA sequences using BioEdit
(Hall 1999). Haplotypes identified in this study
were then analysed jointly with haplotypes of
4078 bp of concatenated sequence from extant
tiger subspecies (Luo et al. 2004); see Fig. 1. Three
felid species related to tigers, viz. snow leopard
Panthera uncia (GenBank NC_010638), leopard P.
pardus (GenBank EF551002) and clouded leopard
Neofelis nebulosa (GenBank DQ257669), were
used as outgroups in the analysis (Table S2 in the
Supplement). Phylogenetic relationships between
the 2 tiger samples in this study and published P.
tigris haplotypes were assessed using neighbour-
joining (NJ), maximum parsimony (MP), maximum
likelihood (ML) and Bayesian inference. The
Bayesian information criterion implemented in
jModelTest v2.1.4 (Posada 2008) was used to select
the best-fit substitution model for ML and Bayesian
analysis. The Hasegawa-Kishino-Yano model +
gamma distribution (base = [0.3250 0.2911 0.1338],
nst = 2, tratio = 20.6989, rates = gamma, shape =
0.1520, ncat = 4, pinvar = 0) was selected as the
best-fit model. Using these model parameters,
Bayesian inference was executed in MrBayes
v3.2.5 (Ronquist & Huelsenbeck 2003, Ronquist et
al. 2012) for 2 000 000 generations with trees sam-
pled every 100 generations and the first 25% gen-
erations discarded as burn-in. NJ, MP and ML
analyses were performed in PAUP* v4a142 (Swof-
ford 2002) with 2000 repetitions for MP and NJ,
and 100 repetitions for ML. Heuristic search with
random additions of taxa and tree-bisection-recon-
nection branch swapping was employed. Trees
were then viewed and manually annotated with
FigTree v1.4.2. The median-joining phylogenetic
network was computed using Network 4.6.1.2
(Bandelt et al. 1999) to analyse the relationship
between tiger haplotypes from this study and
those from Luo et al. (2004).
Longer sequences of 10 140 bp (excluding the
control region and the fragment between positions
7399 and 12 707) were also evaluated following
alignment of complete mtDNA sequences from
Zhang et al. (2011), Kitpipit et al. (2012) and Sun et
al. (2015). The Tamura-Nei model + gamma distri-
bution (base = [0.3243, 0.2612, 0.1509] nst = 6, rates
= gamma, shape = 0.1280, ncat = 4, pinvar = 0) was
the optimal model. A similar method to that
described above was used to construct the tree (see
Fig. 2).
RESULTS
The size of the complete mitogenome in the
WEFCOM and Ti2 individuals (GenBank KJ508412
and KJ508413) was 16 954 and 17 008 bp, respec-
tively (Table S3 in the Supplement). The difference
in mitogenome size between this and other studies
was mainly due to variation in the tandem repeats
TACACACG and TATACACG in the repetitive
sequence box 3 of the control region (Fig. S2 in the
Supplement). Thirty variable sites were identified in
the whole mitogenome excluding the control region
(21 C/T and 9 A/G nucleotide substitutions), and
there were 22 new variable sites (11 in WEFCOM
and 11 in Ti2) when comparing our results to other
studies (Luo et al. 2004, Driscoll et al. 2009, Zhang et
al. 2011, Kitpipit et al. 2012, Mondol et al. 2013, Sun
et al. 2015, Xue et al. 2015) (see Table S4 in the Sup-
plement). The WEFCOM haplotype carried 1 Pan-
thera tigris altaica-specific single nucleotide poly-
morphism (SNP; 14711A) as described by Luo et al.
(2004), whereas Ti2 contained no subspecies-specific
SNPs.
In each sample, we found 2 distinct sequences with
numt contamination in the region of COX2–ATP6.
No heteroplasmy was found in either sequence. The
first sequence (amplicon 9.1) generated from primers
described for Ursidae (Delisle & Strobeck 2002) con-
tained an F2 numt sequence from the COX1 to COX2
gene as found in the report by Zhang et al. (2011) and
could not be assembled with the flanking fragments.
The second sequence (amplicon 9.2), amplified by
the newly designed primers in this study, contained
F2 numt contamination in the COX2 to ATP8 genes,
similar to the results of Kitpipit et al. (2012) (see
Table S1 and Figs. S3 & S4 in the Supplement), and
perfectly assembled to the adjacent fragments. The
last domain of sequences covering part of the ATP6
gene also differed (Figs. S3 & S4). We therefore
excluded the mitochondrial fragment between posi-
tions 7399 and 12 707 (based on the WEFCOM
mitogenome) from further analysis as it may have
contained additional numt sequences.
Results from the MP, NJ and ML trees and the
Bayesian inference analyses on 4078 bp of con -
catenated mtDNA indicate that the WEFCOM
haplotype is nearly equidistant to the fixed haplotype
of P. t. altaica (8 different nucleotides) and a haplo -
group consisting of all reported P. t. corbetti mtDNA
haplotypes (9 different nucleotides; Fig. 1A). The
high Bayesian posterior probability and bootstrap
support of this monophyletic cluster suggests a
recent common ancestor of tigers now distributed in
Endang Species Res 30: 125– 131, 2016
128
Fig. 1. Phylogeny of mtDNA haplotypes of extant tiger Panthera tigris subspecies based on 4078 bp of concatenated sequences
following Luo et al. (2004). (A) Results from maximum parsimony (MP), neighbour-joining (NJ), maximum likelihood (ML) and
Bayesian analyses were similar, so only the MP tree is shown here. The numbers above each branch represent the statistical
support in percentages for the MP, NJ, ML, and Bayesian analyses, respectively. Dashes represent statistical support values
<50. The numbers below each branch represent the number of changes. (B) The median-joining network demonstrates the
evolutionary relationship between our 2 samples and previous reports (Luo et al. 2004). Each circle represents a haplotype, and
the marks on each branch indicate mutation points. Circle size is not proportional to the haplotype frequency. Both the MP phy-
logenetic tree and the median- joining network support an additional Indochinese haplotype (WEFCOM), which evolved from a
common ancestor between P. t. altaica and P. t. corbetti. The new Malayan haplotype (Ti2) descended from the same historic
ancestor as P. t. jacksoni
Buddhakosai et al.: Phylogeographic relationship of wild tigers in Thailand
Indochina and Far Eastern regions of Asia, and sub-
stantial population genetic substructure within
Indochinese tigers. The southern haplotype, Ti2,
clustered with JAX1 and JAX2 haplotypes and
formed a monophyletic group within the Malayan
tiger subspecies P. t. jacksoni. These results were
supported by the median-joining network analysis
and suggested a complex phylogeographic pattern
within P. t. corbetti in that the WEFCOM haplotype is
different from other P. t. corbetti haplotypes, and that
the Ti2 haplotype belongs to P. t. jacksoni (Fig. 1B).
The analysis of the longer mtDNA sequence
showed that the WEFCOM haplotype appears to be
more closely related to P. t. altaica than to other P. t.
corbetti haplotypes with high statistical support in all
algorithms. However, the number of changes from
the WEFCOM haplotype to the haplogroup of P. t.
altaica (15 nucleotides) is larger than that to the
haplo group of P. t. corbetti (13 nucleotides; Fig. 2).
This result further indicates a close and complex
relationship between the 2 subspecies. All analyses
using the MP, ML and Bayesian approaches con -
sistently supported the finding that the lineage of
P. t. jacksoni diverged earlier than the splitting of P. t.
corbetti and P. t. altaica.
DISCUSSION
Tiger populations with a sufficient number of
breeding individuals to support the wider landscape
are currently thought to occur in only 8 countries,
including Thailand (Goodrich et al. 2015). Therefore,
it is vitally important to understand the dynamics of
the wild tiger population in Thailand. In the present
study, we provide the first report of the complete
mitogenome of the Malayan tiger and of novel haplo-
types in the wild Indochinese tiger population in
Thailand. We also demonstrate the phylogenetic
relationship of these 2 subspecies to other tiger pop-
ulations in Asia.
The results of our phylogenetic analyses are in
agreement with the subspecies classification previ-
ously described (Luo et al. 2004), but indicate phylo-
genetic differences between the WEFCOM haplo-
type and other Panthera tigris corbetti haplotypes
found across Asia. We found that the WEFCOM
haplotype contained the 14711A SNP of P. t. altaica,
a common SNP shared among P. t. altaica and P. t .
corbetti (15756C), and no subspecies-specific SNPs
described for P. t. corbetti (Luo et al. 2004). Our
results suggest that the WEFCOM haplotype is
129
Fig. 2. Maximum parsimony (MP) phylogenetic tree based on 10 140 bp of mitochondrial sequence (Zhang et al. 2011, Kitpipit
et al. 2012, Sun et al. 2014) excluding the control region (CR) and the numt-contaminated fragments. The numbers above each
branch represent the bootstrap values for the MP, neighbour-joining, maximum likelihood and Bayesian analyses, respec-
tively. Dashes represent bootstrap values <50. The number below each branch represents the number of changes. The
placement of the WEFCOM haplotype was consistent in all methods with high statistical support
Endang Species Res 30: 125– 131, 2016
closely related to P. t. altaica and that there may be
more complex genetic structure in Indochinese tigers
than previously thought. As expected, the Ti2 haplo-
type sampled in southern Thailand formed a mono -
phyletic group with JAX haplotypes and indicates
that P. t. jacksoni diverged from a common Indo -
chinese ancestor earlier than the divergence between
P. t. corbetti and P. t. altaica.
A recent genetic study of tigers in the Sunda region
suggests that the tiger subspecies now found in much
of southeast Asia and the Russian Far East (P. t. cor-
betti, P. t. jacksoni and P. t. altaica) most likely
evolved from a common ancestor in a second wave of
expansion and divergence from P. t. amoyensis in
China (Xue et al. 2015). Amur tigers (P. t. altaica) and
Indochinese tigers (P. t. corbetti) have also been
shown to be sister taxa separated by only a few mito-
chondrial steps. The close phylogenetic relationship
between 1 wild P. t. altaica (GenBank KF297576)
from China (Sun et al. 2015) and the WEFCOM indi-
vidual in this study provides further support for a
recent common ancestor for these 2 haplotypes, and
hints at further local Indochinese substructure. Thus,
tiger populations in northern Thailand may contain
some of the haplotypes present in the ancestral pop-
ulation when tigers expanded out of China and into
Russia and mainland Asia. Thailand is now the only
country in the region with a reasonable population of
P. t. corbetti tigers, as they have largely been poached
from other neighbouring countries such as China,
Laos and Cambodia. Thus, the existence of new hap-
lotypes in Thailand also highlights its critical geo-
graphic position between the populations of P. t .
altaica in the Russian Far East and P. t. jacksoni in
Malaysia.
The discovery of 2 distinct sequences containing
numt in this study is notable, as it suggests the pres-
ence of additional numt in multiple fragments, as has
been reported in species such as the domestic cat,
human and chimpanzee (Lopez et al. 1994, 1996,
Tourmen et al. 2002, Antunes et al. 2007, Hazkani-
Covo & Graur 2007). Therefore, future studies
involving the complete mitochondrial genome of
tigers must take care to check for confounding
sequences between the 2 ends of F2 numt, which
should be excluded from any downstream phylo -
genetic analysis.
CONCLUSIONS
Here, we have provided an account of the com-
plete mitogenome of Panthera tigris corbetti and P. t .
jacksoni, and have provided further information on
the phylogenetic relationship between these 2 tiger
subspecies in Thailand. Our results also provide evi-
dence for the close relationship between P. t. altaica
and P. t. corbetti, and support the importance of
WEFCOM to the Asian tiger population.
Acknowledgements. We acknowledge the Graduate School,
Kasetsart University, Thailand, for supporting this project.
We are also grateful to all the project collaborators in the
Zoological Park Organization under the Royal Patronage of
His Majesty the King, the Department of National Parks,
Wildlife and Plant Conservation and associated field re -
searchers who provided the samples. We also thank
Anyalak Wachirachaikarn and Phasit Charoenkwan for
their advice on the bioinformatics analysis.
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Cardiff, UK
Submitted: December 11, 2014; Accepted: March 17, 2016
Proofs received from author(s): April 25, 2016
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