Egyptian Journal of Forensic Sciences (2016) xxx, xxx–xxx
HO ST E D BY Contents lists available at ScienceDirect
Egyptian Journal of Forensic Sciences
journal homepage: http://www.journals.elsevier.com/egyptian-journal-of-forensic-sciences
CASE REPORT
Illegal trade of Indian Pangolin (Maniscrassicaudata): Genetic study from scales based
on mitochondrial genes
* Corresponding author at: Wildlife Forensic and Conservation Genetic Cell, Wildlife Institute of India, Post Box # 18, Chandrabani, D
248001, Uttarakhand, India. Fax: + 91 135 2640115.
E-mail addresses: [email protected] (V.P. Kumar), [email protected] (S.P. Goyal).
Peer review under responsibility of The International Association of Law and Forensic Sciences (IALFS).
http://dx.doi.org/10.1016/j.ejfs.2016.06.0082090-536X � 2016 The International Association of Law and Forensic Sciences (IALFS). Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Kumar VP et al. Illegal trade of Indian Pangolin (Manis crassicaudata): Genetic study from scales based on mitochondriEgypt J Forensic Sci (2016), http://dx.doi.org/10.1016/j.ejfs.2016.06.008
Ved Prakash Kumara,e, Ankita Rajpoot
b, Mukesh
c, Malay Shukla
d,
Dhyanendra Kumar e, Surendra Prakash Goyal a,*
aWildlife Institute of India, Post Box # 18, Chandrabani, Dehradun 248001, Uttarakhand, IndiabZoological Survey of India, NRC, Kaulagarh, Dehradun 248001, Uttarakhand, IndiacAmity Institute of Wildlife Sciences, Amity University, Sector 125, Noida 201 313, Uttar Pradesh, IndiadGujrat Forensic University, Gandhinagar 382007, Gujrat, IndiaeVeer Kunwar Singh University, Arrah 802301, Bihar, India
Received 2 November 2015; revised 26 March 2016; accepted 21 June 2016
KEYWORDS
DNA forensic science;
Manis crassicaudata;
Species identification;
Illegal trades and mitochon-
drial gene
Abstract Indian Pangolin (Manis crassicaudata), an endangered mammalian species under IUCN
red list. In India, small population of Chines Pangolin (Manis pentadactyla) are also present in the
eastern part and protected under the Wildlife (Protection) Act, 1972 as a schedule I species along
with Indian Pangolin. In national and international market the Indian Pangolin are highly in trade
principally for its meat and scales. In curbing illegal wildlife, trade of Indian Pangolin on national
and international level wildlife DNA forensics plays an important role in the identification of
species from the seized material. We received n= 15 wildlife seizures assumed to be the scales of
Pangolin from different state forest and the custom department for molecular analysis. The seizures
sequenced for two mitochondrial genes i.e. Cyt b and 16S rRNA, which are often used in wildlife
DNA forensic to species identification. The sequences thus generated from these seizures compared
to the NCBI database through blast search tool and our laboratory-generated references, yielding
100% similarity with Indian Pangolin. We identified twelve (n= 12) Cyt b and five (n= 5) 16S
rRNA species specific SNPs, fixed in Indian Pangolin. This study shows, the importance of authen-
ticated references, DNA sequence data availability, highlighting the application of DNA forensics
in identifying species from scales.� 2016 The International Association of Law and Forensic Sciences (IALFS). Production and hosting by
Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
ehradun
al genes,
2 V.P. Kumar et al.
1. Introduction
Pangolin is a mammalian species classified under the orderPholidota which includes only family Manidae. There are eight
species of Pangolin distributed predominantly in Asia andAfrica. These eight species are divided into three genus, Manis,Phataginus and Smutsia. Among these three genus, Manis has
four species and they present in the Asian subcontinent whilePhataginus and Smutsia have two-two species and are presentin African subcontinent.1 Among four species of AsianPangolin, two species are found in the Indian subcontinent
i.e. Indian Pangolin (Manis crassicaudata) and ChinesPangolin (Manis pentadactyla). The Indian Pangolin is
Figure 1 Distribution of Indian Pangolin (Manis crassicaudata) and
location.
Please cite this article in press as: Kumar VP et al. Illegal trade of Indian Pangolin (Egypt J Forensic Sci (2016), http://dx.doi.org/10.1016/j.ejfs.2016.06.008
distributed in South Asia from parts of eastern Pakistanthrough much of India, south of the Himalayas, SouthernNepal, Bangladesh and Sri Lanka,2,3 while the Chines
Pangolin present in the Himalayan foothills of Nepal, southernBhutan and northeastern India.3–5
In India, the wildlife is protected under the Wildlife
(Protection) Act 1972 (WPA), therefore due to rapidlydeclining Pangolin population in India, this species is kept inschedule I species under the WPA and also considered as
endangered species under the International Union for Conser-vation of Nature and Natural Resources (IUCN).6 Due tohigh demand for Indian Pangolin in international market itis listed in Appendix II of a Convention on International
Chinese Pangolin (Manis pentadactyla), red circle represent seizure
Manis crassicaudata): Genetic study from scales based on mitochondrial genes,
Table 1 Detail of used reference species Cyt b and 16S ribosomal RNA sequences retrieve from GenBank data base and detail of
wildlife seizures.
Species/Individual Common name Origin Accession No. Cyt b Accession
No. 16S rRNA
Conservation status
References detail
Manis crassicaudata/
n= 4
Indian Pangolin Asian/India KU9258021 to
KU9258024
KU925803 to
KU925806
Endangered
Manis pentadactyla/
n= 1
Chinese Pangolin Asian/India-
Chinese
JN411577.1 JN411577.1 Critically Endangered
Manis tetradactyla/
n= 1
Black-bellied Pangolin African KJ193388 AJ421454.1 Vulnerable
Manis tricuspis/n= 1 White-bellied Pangolin African KP306514.1| KP306514.1 Vulnerable
Manis javanica/n = 1 Malayan Pangolin Asian KP306515.1 X Critically Endangered
Manis gigantea/n= 1 Giant ground Pangolin African KJ193383.1 KJ193120.1 Vulnerable
Manis temminckii/
n = 1
Temminck’s ground
Pangolin
African X KP125951.1 Vulnerable
Specimen Suspected Origin Accession No. Cyt b Accession
No. 16S rRNA
Seizures detail
Seizure-1 Pangolin India/Haryana KU9258025 KU925807
Seizure-2 to 4 Indian Pangolin India/
Uttarakhand
KU9258026 to KU9258027 KU925808 to
KU925809
Seizure-5 Pangolin India/Uttar
Pradesh
KU9258028 KU9258010
Seizure-6 Pangolin India/Assam KU9258029 KU9258011
Seizure-7 and 8 Indian Pangolin India/Madhya
Pradesh
KU9258030 to KU9258031 KU9258012 to
KU9258013
Seizure-9 and10 Indian Pangolin India/
Chhattisgarh
KU9258032 to KU9258033 KU9258014 to
KU9258015
Seizure-11 Indian Pangolin India/Andhra
Pradesh
KU9258034 KU9258016
Seizure-12 and
Seizure-15
Indian Pangolin India/Karnataka KU9258035 to KU9258038 KU9258017 to
KU9258020
Table 2 (A) Most similarities in the Cyt b and 16S rRNA based on NCBI GenBank Blast search and (B) similarity based on ClustalW
(http://www.ebi.ac.uk/clustalw) compared with India Pangolin reference.
2A
Specimen Species with the highest similarity Query coverage (%) Similarity (%)
Cyt b 16S rRNA Cyt b 16S rRNA
Seizure-1–15 Manis pentadactyla 98 91 92 96
Seizure-1–15 Manis javanica 98 91 90 96
2B
Specimen Species with the highest similarity Len(nt) bp Similarity (%)
Cyt b 16S rRNA Cyt b 16S rRNA
Seizure 1–15 Manis crassicaudata 331 477 100 100
Seizure 1–15 Manis pentadactyla 331 477 88 96
Seizure 1–15 Manis javanica 331 477 88 X
Seizure 1–15 Manis tetradactyla 331 477 81 90
Seizure 1–15 Manis tricuspis 331 477 83 90
Seizure 1–15 Manis gigantea X 477 X 90
Seizure 1–15 Manis temminckii 331 477 82 90
Illegal trade of Indian Pangolin 3
Please cite this article in press as: Kumar VP et al. Illegal trade of Indian Pangolin (Manis crassicaudata): Genetic study from scales based on mitochondrial genes,Egypt J Forensic Sci (2016), http://dx.doi.org/10.1016/j.ejfs.2016.06.008
4 V.P. Kumar et al.
Trade in Endangered Species of Wild Fauna and Flora(CITES).7 Chines Pangolin in India, also is completely pro-tected being listed in Schedule I of the Wildlife (Protection)
Act 1972,8,9 critically endangered Pangolin species under theIUCN red data book and Appendix II specie in CITES.
Historically, Indian Pangolin was distributed across
southwest China10,11 and in Myanmar12 but due to hunting,poaching13–15 and habitat destruction they are now extinct insome regions and now only present in South Asia (Fig. 1).
The Pangolin has been hunted for the last four decades forthe consumption of its meat as a protein source, and for itsscales for use in traditional medicines among other spiritualand ritualistic uses.16 It also continues to be hunted ritualisti-
cally, for example, during ‘Shikar Utsav’ in Eastern Indian
Table 3A Similarity of seizure with Asian and African Pangolin refe
site position with available Bos taurus (AY676873.1). Alignment o
represent in highlight.
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states, which poses a serious threat to the species5 while theflesh of the species is relished, by some tribal communities.17
Scales are the one of the most characteristic features of
Pangolin and of the reported eight species of Pangolin, therehas been very constricted knowledge of their morphologicalexamination at species level. However, these eight species are
morphologically distinct, but somehow the scales of Pangolindo not show clear distinction based on visual examination.18
Conventional taxonomist can identify the seized Pangolin spe-
cies based on morphological characteristics but this will nothelp further, once the Pangolin scales are removed from tradein the market. Consequently, a DNA based approach for spe-cies identification may prove to be a powerful and reliable tool
for wildlife law enforcement agencies.
rence sequences data. The number in table represents the variable
f Cyt b mtDNA gene and fixed SNPs sites in Indian Pangolin
(continued on next page)
Manis crassicaudata): Genetic study from scales based on mitochondrial genes,
Table 3A (continued)
Illegal trade of Indian Pangolin 5
In recent years, numbers of DNA based approaches havebeen developed for species identification, like DNA hybridiza-
tion, restriction enzyme digestion, random PCR amplification,species-specific PCR primer and DNA sequencing.19–24,17,25
The DNA sequencing is one of the most reliable and robust
techniques to identify species from the unknown sample inwildlife forensic. Mitochondrial gene is best suited for the iden-tification of species due to multiple characteristics in their cod-
Please cite this article in press as: Kumar VP et al. Illegal trade of Indian Pangolin (Egypt J Forensic Sci (2016), http://dx.doi.org/10.1016/j.ejfs.2016.06.008
ing region i.e. low level of mutation, high copy number in cell,maternally inheritance, etc. 12S ribosomal RNA (12S rRNA),
16S ribosomal RNA (16S rRNA), cytochrome b (Cyt b), andcytochrome oxidase I (COI) gene are the mostly used mito-chondrial gene for species identification in wildlife foren-
sic.26–29 Species identification depends on the reliablereference sequences availability.30 At present most of timeNCBI (http://www.ncbi.nlm.nih.gov/database) or BoLD
Manis crassicaudata): Genetic study from scales based on mitochondrial genes,
6 V.P. Kumar et al.
(http://www.barcodinglife.org) database that are publiclyavailable is used for species comparison. In the present study,for the first time in the world, we attempted to address the
molecular identification of Indian Pangolin from scales seizedunder the wildlife offence case and its application in wildlifeforensics and to frame a conservation action plan and strate-
gies for Pangolin conservation in India.
2. Materials and methods
Included fifteen independent consignments of scale samplesseized in India (Fig. 1) during year 2011–2014. The sampleswere seized under Wildlife (Protection) Act 1972, of India
Table 3B Similarity of seizure with Asian and African Pangolin refe
site position with available Bos taurus (AY676873.1). Alignment of 1
represent in highlight.
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and referred to Wildlife Forensic and Conservation GeneticCell (WFCG), Wildlife Institute of India (WII), Dehradunfor genetic study.
2.1. DNA extraction
The samples undertaken for the study i.e. scales were subjected
for DNA extraction, which were initially cleaned with ethanoland 10X Phosphate buffered saline (PBS) two times, outer sur-face (0.5 mm) of these samples was removed using a surgical
blade to remove possible surface contaminants and againwashed with 10X PBS. A small section of scales part was cutout, transferred to a 2 ll eppendorf tube using published
rence sequences data. The number in table represents the variable
6S rRNA mtDNA gene and fixed SNPs sites in Indian Pangolin
(continued on next page)
Manis crassicaudata): Genetic study from scales based on mitochondrial genes,
Table 3B (continued)
Seizure-1Seizure-2Seizure-3Seizure-4Seizure-5Seizure-6Seizure-7Seizure-8Seizure-9Seizure-10Seizure-11Seizure-12Seizure-13Seizure-14Seizure-15Manis crassicaudata-1Manis crassicaudata-2Manis crassicaudata-3Manis crassicaudata-4
Manis pentadactylaManis javanica
Asian pangolin
Manis temminckiiManis tetradactyla
Manis tricuspis
African Pangolin
100
99
99
100
0.02
Figure 2 Cytochrome b based phylogenetic similarity between the seizures and other pangolins, shaded box represent Indian pangolin
similarity with seizures.
Illegal trade of Indian Pangolin 7
Please cite this article in press as: Kumar VP et al. Illegal trade of Indian Pangolin (Manis crassicaudata): Genetic study from scales based on mitochondrial genes,Egypt J Forensic Sci (2016), http://dx.doi.org/10.1016/j.ejfs.2016.06.008
Seizure-1Seizure-2Seizure-3Seizure-4Seizure-5Seizure-6Seizure-7Seizure-8Sizure-9Seizure-10Seizure-11Seizure-12Seizure-13Seizure-14Seizure-15Manis crassicaudata 1Manis crassicaudata 2Manis crassicaudata 3Manis crassicaudata 4
Manis pentadactyla
Asian pangolin
Manis tetradactylaManis tricuspis
Manis giganteaManis temminckii)
African pangolin
98
76
100
100
0.01
(A)
Figure 3 16S ribosomal RNA based phylogenetic similarity between the seizures and pangolin, shaded box represent Indian pangolin
similarity with seizure.
8 V.P. Kumar et al.
ancient bone extraction protocol for further process.31 TheDNA concentration was quantified by absorbance measure-
ment at 260 nm. Absorbance measurements at 260 nm weredone using NanoDrop ND-1000 (Thermo Fisher ScientificInc., Waltham, USA) with 1 ll of samples.
2.2. PCR amplification
Partial fragments of the Cyt b gene (ca.350 bp) and 16S rRNAgene (ca. 511 bp) were amplified.32,33 All PCR reactions were
carried out on a Thermal Cycler 2720 (Applied Biosystem,USA) were set up in 15 ll of the reaction volume containing7.5 ll of 2� Qiagen Multiplex PCR Master Mix, 0.30 ll of10 lM of each primer pair, 1 ll of Q solution (supplied withkit), 2 ll of DNA elutant (approx. 20 ng) and remainingRNase-free water to make the final reaction volume of 15 ll.Primers, PCR conditions, and sequencing protocol were asdescribed in Kumar et al. (2014).34 Positive and negative con-trols were performed throughout all DNA extraction and PCR
amplifications.
2.3. DNA sequencing and analysis
The PCR products were purified using ExoSAP and finally
sequenced on an ABI 3130 genetic analyzer using a Big DyeTerminator v 3.1 Kit (Applied Biosystem, USA). Quality ofsequences was determined using Sequence Analysis v5.2 soft-
ware (Applied Biosystem). Apart from that CLUSTAL W
Please cite this article in press as: Kumar VP et al. Illegal trade of Indian Pangolin (Egypt J Forensic Sci (2016), http://dx.doi.org/10.1016/j.ejfs.2016.06.008
algorithm implemented in BioEdit version 7.0.5.335 was usedin the Multiple Sequence Alignments (MSA).The sequences
obtained from the fifteen different specimens were comparedwith other Pangolin sequences available on public domain atGenBank (http://blast.ncbi.nlm.nih.gov/) and wildlife forensic
reference databases (Table 1). For the species diagnosis, weconsidered the percentage similarity between query and refer-ence sequence pairs. To conform MSA result, we compareour data with phylogenetic analysis conducted using MEGA
v 636 for Cyt b and 16S rRNA using the neighbor-joiningmethod.
3. Result and discussion
The obtained genomic DNA concentration in all seizures wasgood and ranged from 10.90 ng/lL to 12.84 ng/lL. In PCR,
used mitochondrial genes i.e., Cyt b and 16S rRNA genes weresuccessfully amplified in all seizures. The seizures yielded read-able sequences for Cyt b and 16S rRNA genes of length ca.
331 bp and ca. 477 bp, respectively. The sequences obtainedwere submitted as independent entries in BLAST analysis,Cyt b gene indicated that the all seizures were 92% similar
to the Chinese Pangolin (Manis pentadactyla) and 91% withMalayan Pangolin (Manis javanica) (Table 2A) where the16S rRNA mitochondrial gene sequences showed 96% similar-ity with Chinese and Malayan Pangolin. In public domain, sin-
gle short Cyt b sequence of Indian Pangolin was available.Therefore these fifteen sequences of both mitochondrial genes
Manis crassicaudata): Genetic study from scales based on mitochondrial genes,
Illegal trade of Indian Pangolin 9
show higher similarity with Chinese and Malayan Pangolinsbut, when we are comparing the gene sequences of all seizureswith the reference sequence, available at WFCG,WII refer-
ences repository, the seizures showed high similarity (100%)to Indian Pangolin (Manis crassicaudata) (Table 2B). In aCyt b gene, twelve (n = 12) species specific fixed SNPs were
detected in Indian Pangolin, while in 16S rRNA gene, five(n= 5) species specific fixed SNPs were detected(Table 3A & 3B).The neighbor-joining phylogenetic tree
showed that CLUSTAL W analysis is similar in Cyt b geneand 16S rRNA (Figs. 2 & 3). All generated sequences weresubmitted in GenBank and the assigned accession number isgiven in Table 1 for future use. To find out the nucleotide posi-
tion of variable sites we align our data with available completemitochondrial genome of Bos taurus (AY676873.1).
4. Conclusion
The present study is a small step forward to understand thedynamics of poaching of Indian Pangolins and the illegal trade
of Pangolin scales using the blend of molecular biology andforensics to effectively deal with such offence cases. Once thePangolins killed and their scales are removed from body deter-
mine the exact species is challenging, but the present studyindicates that the Pangolin can be differentiated and identifiedusing molecular tools and techniques. Samples from all fifteen
seizures were identified to be of Indian Pangolin based onmitochondrial genes viz. Cyt b and 16S rRNA. The obtainednovel sequences of Indian Pangolin of both mitochondrialgenes are very helpful in solving the wildlife forensic cases
related to Indian pangolin in future analysis. This result showsthe importance of reference samples to species identification inwildlife forensic and the utility of DNA-based analysis in the
implementation CITES. It is necessary to establish and pro-mote the use of such DNA profile database of mitochondrialDNA to put an insight into actually tracking the poaching
hubs and thus nailing down poachers for better enforcementand conservation. This study also indicates that, the exact pop-ulation of India Pangolin is unidentified; therefore, we recom-
mend the Indian population around Indian subcontinentshould be investigated in further genetic studies in combina-tion with a good sampling strategy to investigate species biol-ogy (including patterns of genetic diversity, relatedness and
population connectivity) and status for conservation program.
Source of funding
The funding for this research is supported by the Ministry ofEnvironment and Forest, Govt. of India through Grant-in-Aid to WII.
Conflict of interest
None to declare.
Informed consent
The used sample in present study was collected from alreadydead animal.
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Ethical approval
Necessary ethical approval was obtained from the instituteethics committee.
Acknowledgments
The authors are thankful to the Director, Dean and ResearchCoordinator, WII, Dehradun, for their strong support alongwith the Nodal Officer of Wildlife Forensic and Conservation
Genetics, Cell. We would like to thank all the researchers andstaff of the Wildlife Forensic and Conservation Genetics Cell,WII for providing scientific and technical assistance whileundertaking the work.
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