Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 April 2024 | 16(4): 25019–25028
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.8190.16.4.25019-25028
#8190 | Received 14
September 2022 | Final received 07 December 2023 | Finally accepted 25 March
2023
Mitochondrial CO1 gene haplotype
diversity of Sumatran Tiger Panthera tigris sumatrae (Pocock,
1929) (Mammalia: Carnivora: Felidae)
Ashrifurrahman 1, Saruedi
Simamora 2, Rusdiyan
Ritonga 3, Wilson Novarino
4, Djong Hon Tjong
5 , Rizaldi 6, Syaifullah
7 & Dewi
Imelda Roesma 8
1,4,5,6,7,8 Department of Biology, Faculty of
Mathematics and Natural Sciences, Andalas University,
Limau Manis, Padang 25175,
West Sumatra, Indonesia.
2 Dharmasraya Sumatran Tiger Rehabilitation
Center – ARSARI Djojohadikusumo Foundation (PR-HSD Yayasan ARSARI Djojohadikusumo), Dharmasraya, West Sumatra, Indonesia.
3 Conservation of Natural Resources
Office (KSDA) Padang 25173, West Sumatra, Indonesia.
1 ashrifurrahman@gmail.com, 2 saruedisimamora@gmail.com,
3 ritonga_yan@yahoo.co.id, 4 wilsonnovarino@sci.unand.ac.id,
5 djonghontjong@sci.unand.ac.id, 6
rizaldi@sci.unand.ac.id, 7 syaifullah@sci.unand.ac.id,
8 dewiroesma@sci.unand.ac.id
(corresponding author)
Abstract: Sumatran Tigers Panthera
tigris sumatrae inhabit
12 tiger conservation landscapes that stretch across Sumatra Island.
Conservation efforts for these species require robust, information-based
research, including a genetic approach. This study analyzed the haplotype
diversity of P. t. sumatrae based on the
mitochondrial CO1 (Cytochrome Oxidase Subunit 1) gene. Specifically, a
nucleotide guanine at position 121 was found, distinguishing P. t. sumatrae from other tiger subspecies. Among the 17
sequences of P. t. sumatrae, two haplotypes
were detected: 13 individuals were in haplotype 1 (Hap_1), and four individuals
were in haplotype 2 (Hap_2). Hap_1 individuals predominantly originated from
Riau and North Sumatra, while Hap_2 individuals were primarily from West
Sumatra. Haplotype diversity (Hd) (0.382±0.113) and
nucleotide diversity (pi) (0.00038±0.00011) confirmed the low genetic
diversity. Five seized samples exhibited Hap_2, suggesting they might have
originated from Riau and North Sumatra. However, this result cannot be
described as current due to the significant changes in P. t. sumatrae habitat. Further genetic studies, such as
whole-genome analysis, are needed to detect the origin and variation of P.
t. sumatrae across all landscapes.
Keywords: Forest lost, genetic diversity, Illegal
trade, mtDNA, PCR, population interactions, species
identification, Sumatran forest, wildlife genetic, wildlife forensic.
Bahasa: Harimau Sumatera Panthera
tigris sumatrae menempati 12 area lanskap Panthera tigris yang
berada di sepanjang Pulau Sumatera. Usaha konservasi spesies ini telah
banyak dilakukan dengan melakukan berbagai macam riset, termasuk dengan pendekatan genetika. Penelitian ini dilakukan untuk
menganalisis diversitas haplotipe dari P. t. sumatrae berdasarkan gen
Cytochrome Oxidase Sub Unit 1 (CO1) DNA mitokondria (mtDNA). Dua haplotip
ditemukan dari total 17 sekuen sampel P. t. sumatrae dengan komposisi 13 individu memiliki haplotip 1 (Hap_1) dan empat individu
memiliki haplotip 2
(Hap_2). Haplotip 1 (H_1) cenderung
terdapat pada individu-individu dari Provinsi Riau dan Provinsi Sumatera Utara. Haplotip
2 (H_2) cenderung terdapat pada individu-individu dari Provinsi Sumatera Barat.
Nilai diversitas haplotipe
(0.382) dan diversitas nukleotida (pi) (0.00038) menunjukkan
rendahnya variasi genetik dari semua
individu yang dianalisis.
Lima sampel yang berasal dari sitaan kasus
perdagangan ilegal memiliki haplotip 2 yang berarti dapat diasumsikan
cenderung berasal dari Riau and Sumatera Utara. Hasil ini
tentu belum dapat mendeskripsikan asal sampel P. t. sumatrae secara akurat dikarenakan keterbatasan sampel dan habitat P. t. sumatrae yang
luas. Selain itu perubahan fungsi
habitat yang berubah secara
signifikan mengharuskan perlunya dilakukan analisis DNA lengkap P. t. sumatrae dari individu-individu pada populasi di semua lanskap yang tersisa.
Editor: Mandar Paingankar, Government Science College Gadchiroli,
Maharashtra, India. Date of publication: 26 April
2024 (online & print)
Citation: Ashrifurrahman, S. Simamora, R. Ritonga, W. Novarino, D.H. Tjong, Rizaldi, Syaifullah & D.I. Roesma (2024). Mitochondrial
CO1 gene haplotype diversity of Sumatran Tiger Panthera
tigris sumatrae (Pocock,
1929) (Mammalia: Carnivora: Felidae). Journal of Threatened Taxa 16(4): 25019–25028. https://doi.org/10.11609/jott.8190.16.4.25019-25028
Copyright: © Ashrifurrahman et al. 2024. Creative Commons Attribution 4.0
International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: The Indonesia Directorate General of Learning and Student Affairs research grant (034/SP2H/LT/DRPM/2020).
Competing interests: The authors declare no competing interests.
Author details: Ashrifurrahman holds MSc from Andalas University. He is
experienced in Sumatran tiger genetic conservation, including for forensic
purposes with Barcode DNA. He was a volunteer in the Indonesia Covid testing Laboratory from 2020 with the quantitative
Real Time Polymerase Chain Reaction methods. Saruedi Simamora holds BSc from Udayana University and he is a veterinarian and had been
working in Dharmasraya Sumatran Tiger Rehabilitation
Centre. Rusdiyan Ritonga educational
background is environmental science. He has been working in Conservation of
Natural Resources Office (KSDA) of West Sumatra. Wilson Novarino holds a Doctor
from the IPB University. His conservation experience focuses on large mammal
conservation including Malayan Tapirs and Sumatran Tiger. He is experienced in
camera trapping for wildlife conservation. Now, Wilson Novarino
as one of Malayan Tapir assessment team in iucnredlis.
Djong Hon Tjong is
genetic and molecular lecturer in Biology and Medicine Department of Andalas University. He has made a significant contribution
in the field of amphibians and reptile on Sumatra island. Rizaldi educational background is
in biology and holds his Doctor from Kyoto University (Japan). He did his
specialization in primate, especially in the area of primate bahaviour. Syaifullah is a lecturer in the Biology Department of
Andalas University. He holds his Doctor from
University of Western Sydney (Australia). He is experienced in marine fish
genetic conservation. Dewi Imelda Roesma
is professor in the Department of Biology of Andalas
University. Her educational background is in biology with an focus on genetic
conservation. She hold a Doctor from Andalas
University and has over 30 year of genetic conservation study and experience, particulary sumatran’n freshwater
fish and sumatran tiger genetic diversity. Recently,
she has reported the barcoding DNA of freshwater with high throughput DNA
sequencing.
Author contributions: Ashrifurrahman contributed to sample
collection, methodology, formal data analysis and writing original draft. Dewi Imelda Roesma contributed to
designing ideas and methodology. Simamora Saruedi and Rusdiyan Ritonga contributed to sample collection. Djong Hon Tjong, Wilson Novarino, Syaifullah, Rizaldi contributed in designing ideas, improving data
analysis and manuscripts.
Acknowledgments: The authors would like to thank
to the Directorate General of Learning and Student Affairs who provided a
student research grant (034/SP2H/LT/DRPM/2020). Our thanks to Ir. Catrini Pratihari Kubontubuh, M.Arch. as the
executive director of Arsari Djojohadikusumo
Foundation's (YAD) and Dr. Erly Sukrismanto
as the head of Conservation of Natural Resources Office (KSDA) of West Sumatra
for helping and providing blood and preserved samples that used in this study.
Our appreciation also to Dyta Rabbani Aidil, S.Si for help in the
laboratory.
Introduction
The extinction of the Bali Tiger Panthera tigris balica and the Javan Tiger Panthera
tigris sondaica led to
the Panthera tigris
sumatrae being the only tiger subspecies
remaining in Indonesia (Seidensticker et al. 1999).
The Convention on International Trade in Endangered Species of Wild Flora and
Fauna (CITES) categorizes P. t. sumatrae in
Appendix I, which means it is prohibited from being traded (Soehartono
et al. 2007). P. t. sumatrae naturally
inhabits the Sumatran forest ecosystem, exhibiting high adaptability supported
by prey availability and access to water sources (Nowell & Jackson 1996; Seidensticker et al. 1999). Additionally, factors such as
vegetation density and human activity play crucial roles in determining the
existence of P. t. sumatrae (Sanderson et al.
2006; Linkie et al. 2008; Suyadi
et al. 2012).
There are 76 tiger conservation
landscapes (TCLs) around the world, 12 of them being home to P. t. sumatrae is located on the island of Sumatra, covering
approximately 88,000 km2. These TCLs encompass various areas,
including Bukit Barisan Selatan, Tesso
Nilo, Kerinci Seblat, Bukit Balai
Rejang-Selatan, Rimbo Panti-Batang
Barat, Leuser, Berbak, Sibolga Kuala Kerumutan, Bukit Rimbang Baling, Rimbo Panti-Batang Timur, and Bukit Tigapuluh (Sanderson et al. 2006). Within these 12
landscapes of P. t. sumatrae, there are 18
conservation areas as well as other forested regions, including protected
forests and production forests (Soehartono et al.
2007). Human activities have been a significant cause of forest loss, posing a
severe threat to the sustainability of P. t. sumatrae
(Seidensticker et al. 1999; Linkie
et al. 2003; Wibisono & Pusparini
2010). Between 2000 and 2010, Sumatra saw a 3% loss in its forests (Wilcove et al. 2013), and deforested areas exhibited a 20%
lower occupancy rate for P. t. sumatrae
compared to areas that remained forested (Wibisono et
al. 2011). Empirical evidence has demonstrated that habitat fragmentation,
habitat loss, and isolation among populations can lead to changes in genetic
composition among living species (Keyghobadi 2007).
Consequently, genetic studies of P. t. sumatrae
have become increasingly important and are a key focus of research in the 21st
century.
Genetic diversity within a
population plays a critical role in determining a species’ ability to survive
and avoid extinction. Low or diminished genetic diversity can significantly
reduce a population’s capacity to adapt to environmental changes and succeed in
reproduction (Frankham et al. 2010). Several
parameters are used to assess genetic diversity, including genetic distance,
haplotype diversity, and nucleotide diversity. A haplotype refers to a group of
genes in organisms inherited from the same parent. It is defined as the
inheritance of a cluster of single nucleotide polymorphisms (SNPs), which are
variations In a single base within DNA sequences among individuals,
particularly within the CO1 gene (Frankham et al.
2010; Liang 2013). The CO1 gene is a protein-coding gene located in
mitochondrial DNA (mtDNA) and does not undergo
recombination because it is maternally inherited (Ladoukakis
& Zouros 2017). Consequently, individuals or
closely related species will exhibit a high degree of genetic similarity (Folmer et al. 1994).
Recent genetic studies within the
felid family have utilized microsatellite loci, as reported by Williamson et
al. (2002). They identified an ideal set of 16 microsatellite loci for
population genetic analysis. Another study successfully unveiled the
phylogenetic and evolutionary relationships among the six tiger subspecies
worldwide. This investigation employed three genetic markers, including
mitochondrial DNA spanning approximately 6.000 bp,
the class II gene DRB, and microsatellites. While these markers showed low
variation between subspecies, they exhibited significant distinctions in
partitioning among subspecies (Luo et al. 2004). The P. t. sumatrae, mitochondrial DNA study was developed with
discovered 7891 bp or 46.4% (Kitpipit,
Linacre, and Tobe 2009). Previously, Faizah (2008) conducted a study on the mitochondrial DNA
genetic markers (Cytochrome b and D-loop) of P. t. sumatrae.
The study involved designing primers based on the mitochondrial DNA of Felis catus.
Additionally, Kitpipit et al. (2012) reported the
identification of five single nucleotide polymorphisms (SNPs) specific to Panthera tigris,
three specific SNPs in P. t. sumatrae, and
three specific SNPs in P. t. tigris, based on
an approximately 15.000 bp mitochondrial DNA
sequence.
The utilization of genetic
markers for P. t. sumatrae has been extended
to various applications, including the reconstruction of P. t. sumatrae pedigrees by targeting the D-loop region,
species identification, and phylogenetic analyses through the CO1 gene (Setianingsih 2013; Ashrifurrahman
et al. 2022). Additionally, Smith et al. (2018) analyzed the impact of habitat
loss and fragmentation on the genetic variation of P. t. sumatrae
using microsatellite markers. Their findings indicated that Sumatran forest
deforestation did not have a significant effect on the genetic variation of P.
t. sumatrae, mainly due to the maintenance of
heterozygosity. However, it is crucial to address the deforestation rate
promptly to mitigate future risks. In this study, we investigated haplotype
diversity and predicted the origin of tiger body part samples traded from three
provinces on Sumatra Island. We utilized blood and hair samples from P. t. sumatrae with known origins. This study provides
valuable information about the geographical origin of the CO1 haplotype, previously
reported by Luo et al. (2004) and Xue et al. (2015),
which lacked data on the sample origin.
Materials
and Methods
Five seized samples (PTS 1, PTS
3, PTS 5, PTS 6, and PTS 8) of P. t. sumatrae
from illegal trading were collected from the West Sumatra Natural Resources
Conservation Agency (BKSDA). These samples consisted of various body parts,
including hairs and bones, from P. t. sumatrae
that had been confiscated from illegal traffickers arrested by authorities on
Sumatra Island. In addition, we obtained nine blood samples (PTS 9, PTS 10, PTS
11, PTS 12, PTS 14, PTS 15, PTS 16, PTS 19, and PTS 20) and one hair sample
(PTS 4) from the Dharmasraya Sumatran Tiger
Rehabilitation Center (PR-HSD), as shown in Figure 1. The blood samples were
collected from P. t. sumatrae individuals that
had been evacuated from conflicts with humans in recent years in three
provinces (West Sumatra, East Sumatra/Riau, North Sumatra), except for PTS 11,
whose origin was unknown. Then, the tigers will be rehabilitated to be released
back into their habitat. All collected samples were placed into 1.5 ml
microtubes, appropriately labeled, photographed, and stored at room
temperature. For validation and comparison with previous studies, we utilized
sequence data (mtDNA) of P. t. sumatrae
as assessed by (Kitpipit et al. 2012).
Laboratory methods start with DNA
isolation for each sample using GeneAll Exgene Genomic DNA micro. Each type of sample used
different protocols according to the kit guide. Then, pairs of primers used to
amplify CO1 gene segments were performed using forward primers PTSF 5
‘AGTTACTGCCCATGCCTTTG 3’ and reverse primers PTSR 5 ‘CAGGCCTAGGAAATGCTGAG 3’ (Ashrifurrahman et al. 2022). The primers would amplify 1129
bp of the Sumatran tiger CO1 gene sequences. Finally,
PCR reactions were performed using 25μl reaction volume containing 11 μl supermix of bioline, 9 μl nuclease freewater, 1 μl forward primer, 1
μl reverse primer, 3 μl DNA
isolate. PCR machine was set to start from predenaturation
at 96oC for 1 minute to ensure complete denaturation, then
denaturation was carried out at 96oC for 30 seconds, annealing at 50oC
for 30 seconds, and elongation at 72oC for 1.5 minutes. The last
cycle at 72oC for 3 minutes, this PCR process runs for 40 cycles.
The amplification product was
sent to Firstbase Company in Malaysia to be purified
and sequencing reaction. The sequencing process used Applied Biosystems highest
capacity-based genetic analyzer platforms and used the BigDye®
Terminator v3.1 cycle sequencing kit chemistry. The forward and reverse DNA
sequences were then combined using the DNA STAR (Burland
2000). The P. t. sumatrae sequences were then
aligned using the Clustal X version 1.8. Polymorphism
sequence analysis was carried out using DNA sequence polymorphism 5.10. To
analyze the changes in the nucleotide base (haplotype), calculating the
haplotype diversity and nucleotide diversity (Rozas
2009). MEGA (Molecular Evolutionary Genetics Analysis) version 7 was used for
nucleotide base differences analysis (Kumar et al. 2016). The AMOVA (Analysis
of Molecular Variance) and FST (Population-based gene flow measures) analysis
was calculated with Arlequin 3.5.2.2 (Excoffier et
al. 2010).
Furthermore, the various haplotypes
identified in the genetic variation analysis were visually represented on a map
using QGIS 3.6. The known coordinates of P. t. sumatrae
were inputted into the QGIS software. Each P. t. sumatrae
specimen was labeled based on the specific haplotype type that had been
determined in the analysis of haplotype diversity. This mapping approach
provided a clear visual representation of the distribution of haplotypes among P.
t. sumatrae populations.
Results
In total, 17 samples of P. t. sumatrae were sequenced for a 999 bp
segment of the mtDNA CO1 gene. The analysis revealed
the presence of two distinct mtDNA haplotypes:
haplotype 1 (Hap_1), consisting of 13 individuals, and haplotype 2 (Hap_2),
which was found in four individuals. Hap_1 included PTS 4, PTS 9, PTS 10, PTS
11, PTS 12, PTS 19, PTS 1, PTS 3, PTS 5, PTS 6, and PTS 8, while Hap_2 was
identified in PTS 14, PTS 15, PTS 16, and PTS 20. The accuracy of these
haplotypes was verified through a thorough examination of the electropherograms
obtained during the sequencing process. The analysis was conducted using the
MEGA 7.0 program, and DNA-to-protein translation was applied for amino acid
translation. It’s worth noting that the sequence variability observed in other
research studies corroborates the mutations found at these specific sites.
Additionally, to ensure the accuracy, several amino acid sites were carefully
examined and corrected, with the best frame selected from multiple frames
generated by the DNA-to-Protein Translation program. Confirmation was also
obtained from NCBI data with accession number AEJ88608.1. Lastly, as part of
the DNA-to-amino acid translation process, the initial two nucleotide bases
(TT) and the final nucleotide base (A) were removed for consistency and
accuracy.
The amino acids were obtained
from the translation of the 996 nucleotide bases of P. tigris
sequences (332 amino acids) with eight various sites (Table 2). Notably, all
variations observed in the amino acids were synonymous mutations and
transitional mutations. The substitutions detected in the nucleotide base
sequences analyzed served to differentiate between tiger subspecies.
Specifically, based on subspecies-specific nucleotides, P. t. amoyesis is characterized by guanine at position 17,
adenine at 121 and 302, and thymine at 422. On the other hand, P. t. altaica and P. t. corbetti
share the same specific nucleotides: adenine at 121, cytosine at 825, and
thymine at 920. P. t. corbetti does not have a
specific site for this study. Of particular significance is the discovery of a
specific nucleotide, guanine at position 121, which serves as a distinctive
marker distinguishing P. t. sumatrae from
other tiger subspecies (Table 3).
The genetic variation in the P.
t. sumatrae population was characterized by two distinct
haplotypes: Haplotype 1 (H_1) and Haplotype 2 (H_2), each distinguished by a
single nucleotide site. Haplotype 1 (H_1) is characterized by the guanine
nucleotide base at position 842, while Haplotype 2 (H_2) features an adenine
base at the same position (Table 3). This result aligns with findings by Luo et
al. (2004), who reported one nucleotide base variation in eight P. t. sumatrae samples based on a 409 bp
segment of the CO1 gene sequence. Five of these individuals had guanine at
position 7382 bp, while the remaining three had
adenine bases. Furthermore, Xue et al. (2015)
examined five P. t. sumatrae museum samples
using the same primers as Luo et al. (2004), with four samples having guanine
bases and one sample featuring adenine bases in the same order as previously
reported. Despite the reporting of haplotypes, the geographical origin of these
haplotype samples within P. t. sumatrae has
not been previously documented. A map illustrating the distribution of these
haplotypes is presented in Figure 2.
Haplotype diversity throughout
the population of P. t. sumatrae in this study
was low at 0.382 ± 0,113 (Table 1). Haplotype diversity values range from 0 to
1, with values exceeding 0.5 indicating high haplotype diversity, while values
below 0.5 suggest low diversity (Curry et al. 2015). The nucleotide diversity
(pi) value of the 17 sequences was 0.00038±0,00011 From several previous reports, other tiger
subspecies also have low mtDNA nucleotide diversity,
including P. t. tigris 0.000355±0.000256, P.
t. jacsoni 0.00118±0.000670, respectively.
Discussion
The low levels of haplotype
(0.382±0,113) and nucleotide diversity (0.00038±0,00011) were found in P.t. sumatrae from
this research. Recent reports on Felidae mtDNA
diversity show comparable values. For example, Panthera
pardus saxicolor
exhibited comparably low diversity levels in haplotypes (0.247) and nucleotides
(0.00078) (Farhadinia et al. 2020), Puma concolor mtDNA diversity
(0,006) (Caragiulo et al. 2013), In the case of Panthera
tigris in the Sundarbans, haplotype diversity was
0.50, and nucleotide diversity was 0.00266 (Aziz et al. 2022). Previous
research by Luo et al. (2004), involving the analysis of several gene sequences
in mtDNA (4078 bp),
consistently reported low nucleotide diversity values (0.00717±0.00444). The
levels of genetic variation, whether high or low, as determined by the CO1 gene
play a significant role in determining the genetic relatedness between
populations and taxa. Lower genetic variation indicates a closer relationship
among individuals or populations of living organisms, especially in the case of
tigers. This condition has implications for the geographic isolation of tigers,
suggesting that they were separated approximately 100,000 years ago (Luo et al.
2004; Xue et al. 2015).
A total 17 individuals were
analyzed there were nine individuals have known wild origin. The nine
individuals are spread across three provinces on the island of Sumatra. There
are four individuals from West Sumatra Province (PTS 12, PTS 15, PTS 16, PTS
20), four individuals from Riau Province (PTS 14, PTS 10, PTS 4, PTS 9), and
one individual from North Sumatra Province (PTS 19). These individuals can
serve as representatives to determine the distribution of haplotypes forensic
samples that unknown origin (PTS 1, PTS 3, PTS 5, PTS 6, PTS 8) and GeneBank data sequences (P. t. sumaterae_JF357969.1
and P. t. sumaterae_JF357970). All P. t. sumatrae
forensic samples showed that conceivable from Riau and North Sumatra.
Haplotype 2 (Hap_2) was found in
all seized samples of poaching and illegal trade in P. t. sumatrae, suspected from Riau and North Sumatra
provinces. However, this assumption is not entirely accurate because Hap_2 also
exists in individuals from West Sumatra. This suggests the possibility that
confiscated tiger samples could originate from other populations on the island
of Sumatra.
For the current number of
samples, there was a propensity for all districts to have the same haplotype
variation, particularly Riau and West Sumatra. The haplotype distribution
indicates the sharing of haplotypes by individuals from West Sumatra and Riau
Provinces. Furthermore, the sharing of haplotype 1 (H_1) from Riau and North
Sumatra Provinces come to pass. The distribution of haplotypes shown in Figure
2 is not significant indicating the specific haplotypes from each province.
Haplotype distribution in this
study indicates that no specific grouping formed between these three provinces.
The AMOVA and FST (Population-based gene flow measures) analysis was calculated
with Arlequin 3.5.2.2 (Excoffier et al. 2010). FST
was not calculated between areas within the North Sumatra population due to
contributing only one sample that would have skewed the result. AMOVA analysis,
run with each of the main areas within Riau group and West Sumatera group,
resulted in an FST of 0.2. In line with Smith et al. (2015) reported on
microsatellite analysis of 37 samples of P. t. sumatrae
originating from the North, West, East and South of Sumatra. The data showed
inconsistent group separations between regions using three different software.
First, structure analysis shows two subgroups, Northern Riau and the island of
Sumatra in general. Second, Tess’s analysis shows two subgroups, namely the
Southern Way Kambas group and the Sumatra Island
group in general. Finally, Geneland’s analysis
indicates four subgroups, namely Northern Sumatra, Eastern Sumatra, mixed
East-West Sumatra, and Southern Sumatra. Gene flow values from west to east are
0.20. This value indicates the existence of a migration history of P. t. sumatrae is quite high from the west to the east. At
the same time the value of 0.06 gene flow from the main area of Sumatra to the
southern region indicates the low gene flow of P. t. sumatrae
to South Sumatra.
The mitochondrial CO1 haplotypes
presented here show historic connectivity between Riau and West Sumatra. The P.
t. sumatrae sharing haplotype in this study is
due to the maternal lineage between populations in each province. Any
population of P. t. sumatrae seems to be
bordered by a mountain range from South to North sumatra.
The mountain range might not be a barrier for each population to interact. The
adaptability and roaming abilities of the P. t. sumatrae
are among the factors that support the possibility of interaction between
populations in each province. Franklin et al. (1999) reported that the
territory of adult male P. t. sumatrae is 110
km2 and for adult females around 50–70 km2. Griffiths
(1994) also reported home ranges of adult male tigers of about 180 km2
at altitudes ranging 100–600 m, 274 km2 at altitudes of 600–1,700 m.
Mitochondrial DNA CO1 genes are inherited maternally. The sharing of the
haplotypes of each population from this study shows the distribution of
individual females carrying specific haplotypes from the original population.
The distance from the origin of the discovery of P. t. sumatrae
in West Sumatra Province to P. t. sumatrae in
Riau and North Sumatra Provinces is in the range of 200–400 km. Geographical
facts support the possibility of interactions or encounters between
populations. Priatna’s (2012) research reinforces
that female P. t. sumatrae can have a home
range of 376.8 km2.
The interaction among P. t. sumatrae populations on the Sumatra island was
estimated to have occurred tens to hundreds years ago. The anthropogenic
influence was not great enough to fragment the P. t. sumatrae
habitat. Currently, it is very unlikely that interactions and breeding between
populations naturally because of the fragmentation and reduction of forest
areas on the island of Sumatra. From 1985–1997, it was recorded that 61% of the
total forest on the island of Sumatra had disappeared (Holmes 2002). Genetic
studies of the P. t. sumatrae population with
microsatellite markers show that the genetic structure of the P. t. sumatrae population is still good with preserved
heterozygosity values to minimize the risk of genetic drift. However, the high
rate of forest fragmentation and loss will accelerate the risk of decreasing
genetic variation (Kenney et al. 2014; Smith et al. 2018).
Despite the limitations of our
sample size, this study generated the first report of CO1 genetic datasets for P.
t. sumatrae population in several origin
locations. The CO1 mtDNA haplotypes exhibited here
show historic connectivity, and maintain genetic connectivity within both East
and West Sumatra. The Initial overview of P. t. sumatrae
gives a basic picture of how the genetic structure (Smith et al. 2018).
Furthermore, to detect the confiscated sample origin and accomplish the case of
illegal trade with genetic forensic tools, ideally reveal the complete mtDNA and nuclear markers of P. t. sumatrae
with most recent technology.
Conclusion
Haplotype diversity and sharing
haplotypes showed the possibility of interaction by each population in the
past. Evidenced by the haplotypes distribution in several regions (West, North,
East Sumatra). One variation in P. t. sumatrae
is the important data and supports the previous studies. The results of this
study can also determine the origin of unknown samples, although not too
significant. Other genetic studies on the entire population of P. t. sumatrae with geological time observation are needed.
Table 1. Haplotype variation,
haplotype diversity value (Hd) and nucleotide
diversity (Pi) in the Panthera tigris sumatrae sequences.
|
|
Haplotypes |
Samples |
Origin |
Haplotype diversity (Hd) |
Nucleotide diversity (Pi) |
|
1 |
Hap_1 |
PTS_1 |
Unknown |
0.382±0,113 |
0.00038±0,00011 |
|
2 |
PTS_3 |
Unknown |
|||
|
3 |
PTS_4 |
Riau |
|||
|
4 |
PTS_5 |
Unknown |
|||
|
5 |
PTS_6 |
Unknown |
|||
|
6 |
PTS_8 |
Unknown |
|||
|
7 |
PTS_9 |
Riau |
|||
|
8 |
PTS_10 |
Riau |
|||
|
9 |
PTS_11 |
Unknown |
|||
|
10 |
PTS_12 |
West Sumatra |
|||
|
11 |
PTS_19 |
North Sumatra |
|||
|
12 |
JF357969_1_P.t.sumatrae |
Unknown |
|||
|
13 |
JF357970_1_P.t.sumatrae |
Unknown |
|||
|
14 |
Hap_2 |
PTS_14 |
Riau |
||
|
15 |
PTS_15 |
West Sumatra |
|||
|
16 |
PTS_16 |
West Sumatra |
|||
|
17 |
PTS_20 |
West Sumatra |
Table 2. Variations in the amino
acid of Panthera tigris
cytochrome oxidase subunit 1 gene.
|
|
Species |
G (Glycine) |
G (Glycine) |
L (Leucine) |
A (Alanine) |
L (Leucine) |
L (Leucine |
I (Isoleucine) |
|
GGA |
GGG |
CTG |
GCC |
TTA |
TTG |
ATC |
||
|
15/5 |
120/40 |
300/100 |
420/140 |
823/275 |
840/280 |
918/306 |
||
|
1 |
PTS 1 |
- |
- |
- |
- |
- |
- |
- |
|
2 |
PTS 3 |
- |
- |
- |
- |
- |
- |
- |
|
3 |
PTS 4 |
- |
- |
- |
- |
- |
- |
- |
|
4 |
PTS 5 |
- |
- |
- |
- |
- |
- |
- |
|
5 |
PTS 6 |
- |
- |
- |
- |
- |
- |
- |
|
6 |
PTS 8 |
- |
- |
- |
` |
- |
- |
- |
|
7 |
PTS 9 |
- |
- |
- |
- |
- |
- |
- |
|
8 |
PTS 10 |
- |
- |
- |
- |
- |
- |
- |
|
9 |
PTS 11 |
- |
- |
- |
- |
- |
- |
- |
|
10 |
PTS 12 |
- |
- |
- |
- |
- |
- |
- |
|
11 |
PTS 14 |
- |
- |
- |
- |
- |
TTA |
- |
|
12 |
PTS 15 |
- |
- |
- |
- |
- |
TTA |
- |
|
13 |
PTS 16 |
- |
- |
- |
- |
- |
TTA |
- |
|
14 |
PTS 19 |
- |
- |
- |
- |
- |
- |
- |
|
15 |
PTS 20 |
- |
- |
- |
- |
- |
TTA |
- |
|
16 |
P. t. amoyensis_HM589215 1 |
GGG |
GGA |
CTA |
GCT |
- |
- |
- |
|
17 |
P. t. sumatrae_JF357969 1 |
- |
- |
- |
- |
- |
- |
- |
|
18 |
P. t. sumatrae_JF357970 1 |
- |
- |
- |
- |
- |
- |
- |
|
19 |
P. t. corbetti_JF357972 1 |
- |
GGA |
- |
- |
CTA |
- |
ATT |
|
20 |
P. t. altaica_KF297576 1 |
- |
GGA |
- |
- |
CTA |
- |
ATT |
|
21 |
P. t. altaica_MH124080 1 |
- |
GGA |
- |
- |
CTA |
- |
- |
|
22 |
P. t. altaica_MH124110 |
- |
GGA |
- |
- |
CTA |
- |
ATT |
|
23 |
P. t. altaica_MN624080 1 |
- |
GGA |
CTA |
ATT |
Table 3. Specific nucleotide
bases in Panthera tigris
sequences.
|
No |
Sample |
|
Site |
||||||
|
Luo et al. 2004 |
6479 |
6583 |
6764 |
7130 |
7287 |
7304 |
7382 |
||
|
mtDNA (NC_010642.1) |
6543 |
6647 |
6828 |
7194 |
7351 |
7368 |
7446 |
||
|
CO1 (NC_010642.1) |
263 |
367 |
548 |
668 |
1071 |
1088 |
1166 |
||
|
CO1 in this study |
17 |
121 |
302 |
422 |
825 |
842 |
920 |
||
|
1 |
PTS 1 |
|
A |
G |
G |
C |
T |
G |
C |
|
2 |
PTS 3 |
|
. |
. |
. |
. |
. |
. |
. |
|
3 |
PTS 4 |
|
. |
. |
. |
. |
. |
. |
. |
|
4 |
PTS 5 |
|
. |
. |
. |
. |
. |
. |
. |
|
5 |
PTS 6 |
|
. |
. |
. |
. |
. |
. |
. |
|
6 |
PTS 8 |
|
. |
. |
. |
. |
. |
. |
. |
|
7 |
PTS 9 |
|
. |
. |
. |
. |
. |
. |
. |
|
8 |
PTS 10 |
|
. |
. |
. |
. |
. |
. |
. |
|
9 |
PTS 11 |
|
. |
. |
. |
. |
. |
. |
. |
|
10 |
PTS 12 |
|
. |
. |
. |
. |
. |
. |
. |
|
11 |
PTS 14 |
|
. |
. |
. |
. |
. |
A |
. |
|
12 |
PTS 15 |
|
. |
. |
. |
. |
. |
A |
. |
|
13 |
PTS 16 |
|
. |
. |
. |
. |
. |
A |
. |
|
14 |
PTS 19 |
|
. |
. |
. |
. |
. |
. |
. |
|
15 |
PTS 20 |
|
. |
. |
. |
. |
. |
A |
. |
|
16 |
JF357969 1 P t sumatrae |
|
. |
. |
. |
. |
. |
. |
. |
|
17 |
JF357970 1 P t sumatrae |
|
. |
. |
. |
. |
. |
. |
. |
|
18 |
JF357972 1 P t corbetti |
|
. |
A |
. |
. |
C |
. |
T |
|
19 |
MH124110 1 P t altaica |
|
. |
A |
. |
. |
C |
. |
T |
|
20 |
MN624080 1 P t altaica |
|
. |
A |
. |
. |
C |
. |
T |
|
21 |
KF297576 1 P t altaica |
|
. |
A |
. |
. |
C |
. |
T |
|
22 |
MH124080 1 P t altaica |
|
. |
A |
. |
. |
C |
. |
. |
|
23 |
HM589215 1 P t amoyensis |
|
G |
A |
A |
T |
. |
. |
. |
FOR FIGURES
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