Wildlife forensics in dealing with wildlife crimes in

Wildlife forensics in dealing with wildlife crimes in India using DNA techniques Dr. Vipin Sharma, Dr. Vinita Sharma, C. P. Sharma and Dr. S. P. Goyal Wildlife Forensic & Conservation Genetics Cell Wildlife Institute of India, Dehra Dun

Wildlife Forensics is a science which employs modern molecular genetic techniques to help not only in the identification of commercial products and body parts of protected species , but also in linking the victim to the suspect and the crime scene.

Conservation threats to natural resources Habitat fragmentation, Loss of genetic diversity Inbreeding due to smaller sized of Protected Areas Poaching

Why poaching: Traded wildlife parts and their price in an international market Sr. Illegally smuggled wildlife No. articles Estimated value 1. Tibetan antelope (Shawl) $1200 -$20, 000 per shahtoosh shawl 2. Rhino horn 4945 -$50, 000 per kilogram of rhino horn 3. Bear gall bladders $250 -$8, 500 per gall bladder 4. Elephant Ivory $121 -$900 per kilogram of ivory 5. Tiger bones and skins $1, 300 -$20, 000 per tiger skin and $3, 300 -$7, 000 per set of tiger bone 6. 7. Snow leopard skin Reptiles and Insects (often live) 8. Exotic Birds (often live) 9. Great Apes (often live) $1, 300 -$20, 000 per skin $30, 000 per oenpelli python; $30, 000 per komodo dragon; $5, 000 -$30, 000 per plowshare tortoise; $15, 000 per Chinese alligator; $20, 000 per monitor lizard; $20, 000 per shingleback skink; $8, 500 per pair of birdwing butterflies $10, 000 per black palm cockatoo egg ($25, 000 -$80, 000 per mature breeding pair); $5, 000 -$12, 000 per hyacinth macaw; $60, 000 -$90, 000 per lear macaw; $20, 000 per Mongolian falcon $50, 000 per Orangutan * Wyler L, Sheikh P (2008) International illegal trade in wildlife: threats and U. S. policy. CRS Report for Congress, March 3, 2008, 49 pp.

Do we know implications of poaching on conservation ? Observed sex in seized items in wildlife offences Affecting Random Mating and role of as an evolutionary unit Caused changes in social structure, movement, reproductive dynamics, ecological interactions Affecting overall evolutionary and ecological process

A new beginning in India WII-U. S. FWS Collaborative project (1995 -2001) “Establishment of Forensic Capacity at the Wildlife Institute of India” Objectives: (i) Prepare a perspective plan for the development of wildlife forensic technology in India, (ii) Create the required infrastructure, (iii) Become proficient in species Identification of vertebrate using external morphological characteristics, and other techniques (iv)Begin accumulating and storing tissue samples for eventual use in biochemical forensic analysis and develop techniques

Wildlife offence sent WII

Genetic tracking of wildlife offences Parentage Species Diet / prey Sex Population Genetic Structure and variation Advances in use Non-invasive genetic sampling (NGS)- A better insight Individual identity Detection of hybrids Presence, distribution and population estimation Taxonomic relationship Movement between populations

Steps involved in identification DNA analysis I. Isolation of DNA II. PCR amplification of mitochondrial genes (Cytochrome b, 16 S r. RNA and MCB) III. Sequencing of PCR product IV. Comparison of sequence with reference data base.

I. Isolation of DNA Quality of the DNA depends on the sample type & quality Sample type Quality Good Not good Poor Meat Fresh Old Cooked/ with fungus Blood Fresh/ dried Blood on soil/ rock Blood on weapons Bone Fresh Old Burnt bones Faecal sample Fresh Old Weathered Hair With root Without root

Different Protocols for Isolation of Genomic DNA 1. Qiagen DNA extraction kits (Qiagen, Valencia, CA, USA) for Tissue Blood Stool Hair 2. GUCN and Silicon dioxide method for Hard tissues like Bone Tooth Claws

II. PCR amplification of mitochondrial genes Genes: Designed by aligning the following mitochondrial genes of different mammal. The one primer binding sites lies in the highly conserved region of mt. DNA. Cytochrome b, 381 bp (Kocher et al. , 1989) MCB, 472 bp (Verma & Singh, 2003) 16 S r. RNA gene, 550 bp (Mitchell et al. , 1993)

II. PCR amplification of mitochondrial genes PCR conditions 1. Cyt b, 381 bp 1 2 3 M 4 2. MCB, 472 bp 3. 12 S r. RNA, 350 bp 4. 16 S r. RNA, 550 bp

III. Sequencing of PCR product §Amplified PCR products are purified using QIAquick® PCR purification kit (Qiagen, Germany). §Cycle sequencing PCR is performed for purified PCR products with their respective primers and PCR products are cleaned up using alcohol precipitation method. §After cleanup these products are subjected to sequencing on 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Dendrogram generated based on NCBI data for 16 S r. RNA sequences

Comparison of the sample with data available at Wildlife Forensic Cell for different species

Dendrogram (MEGA 3. 1, S Kumar et al, 2004)

Project initiation : 2004 (Panthera tigris genome: Implications in wildlife forensics) Ascertain source of origin of wildlife seized materials of these key species

Genetic tracking of tiger offences 1. Mitochondrial DNA (mt. DNA) genes (550 bp four genes) 2. Microsatellite DNA genotyping Tiger scat – source of DNA for genotyping

(Observed haplotypes in tigers of Indian subcontinent based on mt. DNA genes ) 65° 75° 85° 95° 105° 35° Chitwan NP North Western India (TAL) Buxa TR TIG*2 a, b TIG*3 a 25° TIG*4 a Western India North East India TIG*5 a, b TIG*6 a, b TIG*7 b TIG*8 b TIG*10 b TIG*11 b TIG*12 b 15° TIG*13 b Central India 0 TIG*14 b Southern India N 200 65° 15° TIG*15 b a; Luo et al. , 2004 b; This study, Sharma et al. , 2008 400 km 75° 85 ° 95° 105°

Summary: it is possible to assign poaching case with 100 per cent certainty to the tiger population of Northern India, Peninsular India (including Central and Southern India), North East and Sunderbans. N=30 NE

Genetic tracking of tiger offences Microsatellite DNA genotyping Establish unified and standardized multi locus genotyping (n=20) data base across 12 tiger reserves.

Additional insight from genetic data: Gene diversity among tiger populations Zoos Pench Bandahvgarh Kanha Sunderban Ranthambhore Rajaji Dudhawa Corbett

Additional insight from genetic data: Gene flow among tiger populations of Terai Arc Landscape Corbett TR FST = 0. 071 Chilla Sanctuary FST = 0. 079 Dudhwa TR FST = 0. 111 High gene flow, FST = 0 – 0. 04 Moderate gene flow, FST = 0. 05 – 0. 14 Less or no gene flow, FST = >0. 15

Population assignment of tiger seizures having TIG 7 haplotype to tiger populations of Northern India using Structure 2. 3. 2 (Bayesian based using Marko Chain Monte Carlo) Dudhwa Corbett Assignment level Rajaji Wildlife offences = , Male= 3 ; Female= 2 Case sample having TIG 7 haplotype

Additional insight from genetic data: Gene flow (Pair wise population FST) between tiger populations of central India Panna TR 0. 10 2 02 Ban dhav 6 ga rh TR 0. 085 0. 059 3 0. 064 9 0. 06 Pench TR High gene flow, FST = 0 – 0. 04 Palamau TR 7 0. 057 0. 041 95 0. 0 Kanha TR Moderate gene flow, FST = 0. 05 – 0. 14 Less or no gene flow, FST = >0. 15

Assignment level Structure 2. 3. 2 used to assign seizures to central India tiger population (Bayesian based using Marko Chain Monte Carlo) Seizures (n=21) RTR Panna Palamau Bandhavgarh Pench Kanha

Few products of elephant tail hair found in trade 1. 2. 3. 4. 5. Bracelets Tooth pricks Necklaces, Rings Flywhisks Type of elephant tail hair A. Thick B. Medium C. Thin

Sex Identification based on zfx & zfy sex primer AE 1 M ♂ AE 2 ♀ AE 3 ♂ AE 4 ♀ AE 5 ♂ AE 6 ♀ AE 7 ♂ AE 8 -Ve ♂ 420 bp (Zfy-M) 230 bp 200 bp (Zfx-F) 100 bp 5 were from males where as 3 were from females

Challenges:

Challenges: Quail species Black partridges

A Need of Research and Development in Wildlife Forensics : Relatively a new science and need a very strong data base and standardized protocols. Different kind of Items are in wildlife trade and requires different approaches for identifying species

Wildlife Forensics in India: Way Forward Need to establish other areas of Wildlife forensics Forensic toxicology, ballistics, ascertain cause and time of death, Forensic entomology and use of Stable isotope facility, Marine forensics Immediate attention is needed Examine scope and gaps in “Wildlife Crime scene investigation” and proper collection of biological samples” in relation to species’ biology and ecology and use of state-of-the-art technology

Wildlife Forensics in India: Establish genetic ID of tigers in India Way Forward Tiger Skins Are Like Fingerprint But Requires “Genetic ID” for dealing items like bones, nails, whisker canine , blood stains etc which constitute ca. 60% of tiger seizures A Beginning : Proposal submitted to Uttarakhand Forest Department for “Establishing genetic ID of tigers and its significance in conservation and wildlife forensics: A pilot study from the state of Uttarakhand” and to NTCA

Sample preservation for DNA analysis 95% Ethanol Silica gel Saline EDTA veils for blood Note: Never use Formalin for sample preservation Whatman FTA Bloodstain Cards

Thanks.