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: tiwaryved@gmail.com (V.P. Kumar), goyalsp@wii.gov.in (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-

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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

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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.

References

1. Timothy Gaudin. The Phylogeny of living and extinct pangolins

(Mammalia, Pholidota) and associated taxa: a morphology based

analysis (PDF). J Mamm Evol 2008;16(4):71. http://dx.doi.org/

10.1007/s10914-009-9119-9, Retrieved 14 May 2015.

2. Schlitter DA. Order pholidota. In: Wilson DE, Reeder DM,

editors. Mammal species of the world: a taxonomic and geographic

reference. p. 530–1.

3. Srinivasulu C, Srinivasulu B. South Asian mammals. Their

diversity, distribution, and status. New York: Springer; 2012.

4. Tikader BK. Threatened animals of India. Calcutta, India: Zoolog-

ical Survey of India; 1999. p. 179–95.

5. Zoological Society of India. Pangolins (Mammalia: Pholidota) of

India. ENVIS Newsl 2002;9(1 and 2).

6. IUCN. The IUCN Red List of Threatened Species (2014). Version

2014.2. Available at: <www.iucnredlist.org>; [accessed 24 July

2014].

7. CITES. Manis crassicaudata, Manis pentadactyla, Manis javanica.

Transfer from Appendix II to Appendix I (India, Nepal, Sri

Lanka, United States). Prop. 11.13 (2000). Available at: <http://

www.cites.org/eng/cop/11/prop/13.pdf>.

8. The Wild Life (Protection) Amendment Act, 2006 (No. 39 of 2006,

[03/09/2006]).

9. The Wild Life (Protection) Amendment Act, 2002 (No. 16 of 2003,

[17/01/2003]).

10. Heath M. Manis crassicaudata. Mammalian Species 1995;513:1–4.

11. Smith A, Xie Y. The mammals of China. Princeton, New

Jersey: Princeton University Press; 2008.

12. Allen GM. The mammals of China and Mongolia Part I. Natural

history of central China. New York, USA: American Museum of

Natural History; 1938. p. 1–620.

13. Mishra M, Hanfee N. Pangolin distribution and trade in East and

Northeast India. TRAFFIC Dispatches 2000;14:4–5.

14. Challender DWS. Asian pangolins: increasing affluence driving

hunting pressure. TRAFFIC Bull 2011;23:92–3.

15. Mahmood T, Hussain R, Irshad N, Akrim F, Nadeem MS. Illegal

mass killing of Indian pangolin (Manis crassicaudata) on Potohar

Region, Pakistan. Pak J Zool 2012;44(5):1457–61.

16. Anon. Review of Significant Trade in Animal Species included in

CITES Appendix II. Detailed Review of 24 Priority Species.

Indian, Malayan and Chinese pangolin. CITES Animals Com-

mittee. CITES (1992).

17. Brown JR, Beckenbach K, Beckenbach AT, Smith MJ. Length

variation, heteroplasmy and sequence divergence in the mitochon-

drial DNA of four species of sturgeon (Acipenser). Genetics

1996;142:525–35.

18. Hsieh HM, Lee JC, Wu JH, Chen CA, Chen YJ, Wang GB, Chin

LC, Wang LC, Linacre A, Tsai LC. Establishing the pangolin

Manis crassicaudata): Genetic study from scales based on mitochondrial genes,

10 V.P. Kumar et al.

mitochondrial D-loop sequences from the confiscated scales.

Forensic Sci Int Genet 2010.

19. Hayashi J, Tagashira Y, Yoshida MC. Absence of extensive

recombination between inter and intraspecies mitochondrial DNA

in mammalian cells. Exp Cell Res 1985;160:387–95.

20. Irwin DM, Kocher TD, Wilson AC. Evolution of the cytochrome

b gene of mammals. J Mol Evol 1991;32:123–44.

21. Esposti MD et al. Mitochondrial cytochrome b: evolution and

structure of the protein. Biochim Biophys Acta 1993;1143:243–71.

22. Chow S, Clarke ME, Walsh PJ. PCR-RFLP analysis on thirteen

western Atlantic snappers (subfamily Lutjaninae): a simple

method for species and stock identification. Fish Bull

1993;91:619–27.

23. DeSalle R, Williams AK, George M. Isolation and characteriza-

tion of animal mitochondrial DNA. Methods Enzymol

1993;224:176–204.

24. Cano RJ, Poinar HN, Pieniazek NJ, Acra A, Poinar GO.

Amplification and sequencing of DNA from a 120–135-million-

year-old weevil. Nature 1993;363:536–8.

25. Burgener M, Hubner P. Mitochondrial DNA enrichment for

species identification and evolutionary analysis. Z Lebensm Unters

Forsch 1998;207:261–3.

26. Kocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S.

Dynamics of mitochondrial DNA evolution in animals: amplifi-

cation and sequencing with conserved primers. Proceeding of

National Academy of Sciences, United States of America

1989;86:6196–200.

27. Guha S, Kashyap VK. Molecular identification of lizard by

RAPD and FINS of 231 mitochondrial 16s rRNA gene. Leg Med

2006;8:5–10.

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

28. Hsieh HM, Chiang HL, Tsai LC, Huang NE, Linacre A, Lee JC.

Cytochrome b 264 gene for species identification of the conserva-

tion animals. Forensic Sci Int 2001;122(265):7–18.

29. Budowle B, Allard MW, Wilson MR, Chakraborty. Forensics and

mitochondrial DNA: applications, debates, and foundations. Ann

Rev Genomics Hum Genet 2003;4:119–41.

30. Ogden R, Dawnay N, McEwing R. Wildlife forensics-bridging the

gap between conservation genetics and law enforcement. Endang

Species Res. 2009;9:179–95.

31. Rohland N, Hofreiter M. Ancient DNA extraction from bones

and teeth. Nat Protoc 2007;2:1756–62.

32. Meyer R, Hofelein C, Luthy J, Candrian U. Polymerase chain

reaction-restriction fragment length polymorphism analysis: a

simple method for species identification in food. J Assoc Anal

Chem Int 1995;78:1542–51.

33. Mitchell SE, Cockburn AF, Seawright JA. The mitochondrial

genome of Anopheles quadrimaculatus species A: complete

nucleotide and organization. Genome 1993;36:1058–73.

34. Kumar VP, Kumar D, Goyal SP. Wildlife DNA forensic in

curbing illegal wildlife trade: species identification from seizures.

Int J Forensic Sci Pathol 2014;2(5):38–42.

35. Hall TA. BioEdit: a user-friendly biological sequence alignment

editor and analysis program for windows 95/98/NT. Nucleic Acids

Symp Ser 1999;41:95–8.

36. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6:

Molecular Evaluation Genetics Analysis version 6.0.Mol Biol Evol

2013;30:2725–9.

Manis crassicaudata): Genetic study from scales based on mitochondrial genes,