Tiger (Panthera tigris corbetti) hair analysis from Uttarakhand, by S.K. Gupta, Scientist from Wildlife Institute of India, Wildlife Forensic Lab, Dehradun 2010.

A report on similarity test of two hair samples of Tiger (Panthera tigris)

Abstract:

Two tiger hair samples were sent by Frederique Lacraz, Society for Mahseer Conservancy, Ramnagar, Uttarakhand to Wildlife Institute of India. Those two tiger hair samples were collected in the same area (Tera village) where there has been two attacks on ladies by tigers. The reason for a DNA analysis was to identify, whether both the sample belonged to the same individual. Such issue can be dealt based on commonly used genotyping based genetic analysis. We describe here similarity test between two hair samples of Tigers (Panthera tigris). There are genetic markers used in similarity analysis are called Microsatellites. We used 5 fluorescent labelled microsatellite loci for analysis. After the comparison of all loci, the DNA typing indicates that in both hair samples alleles tested were not the same. We conclude the both the hair samples have different genotype i.e. Hair sample marked as F-1553 (174176, 130130, 182182, 124126, 166166) and Hair sample marked as R-1944 (174176, 132144, 182182, 124126, 162162) are not from the same individual.

Introduction:

DNA marker technologies have revolutionized molecular genetic techniques as they can contribute to the management of threatened species. However, despite a myriad of potential application, the usefulness of these techniques for making informed management decisions is generally underappreciated. Molecular genetic technique can be used to identify same individuals, parents, offspring and close relatives in captive and wild population (Liu and Cordes, 2004). Everyone, except identical twins, have a one-of-a-kind DNA genome. The DNA molecule is made of monomers called nucleotides, and the specific order in which nucleotides occur in a DNA molecule represents each individual’s unique genetic code. The analysis tests the DNA isolated to locate certain regions of chromosomes that are known to vary in length between individuals. These sites are tested; each site is called a “locus”, (“loci” – plural). Analysis of these sites in a large population reveals many different sized versions associated with each site.

Methodology

DNA fingerprinting determines whether two biological samples belong to same individual or not.

 Extraction of DNA

Both the hair samples marked as F-1553 and R-1944 were subjected to DNA extraction. Commercially available DNeasy Tissue Kit (QIAGEN, Germany) was used to extract DNA from these samples.

 PCR Amplification and electrophoresis

Samples were analysed using 5 polymorphic mcrosatellite loci developed for tiger. These primers (markers) are C6, F1, D6, FCA304 and Ple23.

Versions of a DNA sequence or a gene are called “alleles”. At one locus, there are two alleles, one come from father and other from mother. Genetic markers used in individual identification and parentage analysis are called Microsatellites, or Simple Sequence Repeats (SSRs), there are polymorphic loci present in nuclear and organelle DNA that consist of repeating units of 1-6 base pairs in length. They are typically neutral, co-dominant and are used as molecular markers which have wide-ranging applications in the field of genetics, including kinship and population studies.

Because each individual has two of each type of chromosome, one inherited from each parent and therefore has two alleles at each locus. These two alleles are sometimes identical called homozygous, but sometimes are not the same size called heterozygous. During similarity test on the basis of DNA fingerprinting, the analysis identifies the length of the two alleles found at each locus, by comparing the DNA profiles of the two different samples. In this report we document the similarity test undertaken between two hair samples of tiger sent by a NGO from Ramnagar, Uttarakhand and marked as F-1553 and R-1944.

Methodology

DNA fingerprinting determines whether two biological samples belong to same individual or not.

Extraction of DNA

Both the hair samples marked as F-1553 and R-1944 were subjected to DNA extraction. Commercially available DNeasy Tissue Kit (QIAGEN, Germany) was used to extract DNA from these samples.

PCR Amplification and electrophoresis

Samples were analysed using 5 polymorphic mcrosatellite loci developed for tiger. These primers (markers) are C6, F1, D6, FCA304 and Ple23.

PCR amplifications were performed in ABI thermal cycler GeneAmp® PCR System 2700, Applied Biosystems, Singapore) in a final volume of 10 μl, containing 25-50 ng of genomic DNA, 1X PCR buffer (applied biosystem), 2.0 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer and 1 units of AmpliTaq Gold DNA polymerase. Amplification conditions were 94°C for 10 min followed by 35 cycles at 94°C for 45s, 55 °C for 45s and 72°C for 1 min, with a final extension of 72°C for 20 min. Distilled water was taken as negative control to ensure there was no contamination in reaction mixtures. PCR products were genotyped using ABI Prism 3130 genetic analyzer.

Results

All the 5 fluorescent microsatellite were amplified successfully. Test in Table1 and Fig. 1 indicate multilocus 5 loci genotype obtained from both the hair samples. After the comparison of all loci, the genotype of both the samples is different at 2 loci. Therefore it is concluded that the hair sample marked as F-1553 having multilocus genotype (174176, 130130, 182182, 124126, 166166) is not the exactly same as the hair sample marked as R-1944 (174176, 132144, 182182, 124126, 162162).

Conclusion:

Five different loci were used as genetic markers in similarity test concluded to determine whether two tiger hair sample sent by Society for Mahseer Conservancy, Ramnagar, Uttarakhand are derived from the same individuals. The multilocus genotype data obtained from both the hair samples indicate that these hair are having the different allele at two loci out of five loci, therefore; both the hair samples are the derivative of two different individual. Genetic id or multilocus genotyping of both the hairs are as follows.

Hair I (F-1553): 174176, 130130, 182182, 124126, 166166

Hair II (R-1944): 174176, 132144, 182182, 124126, 162162

After landing and a bumpy but not too long drive to Tiger Camp at Corbett National Park, I was met by the most amazingly smiley faces, welcoming me at the reception with warmth and friendliness. As previously mentioned the wildlife I knew was going to be exotic and amazing but on my first day I never expected to be called into the ‘fun ranger’ Mikma’s hut to be greeted by the biggest lizard I had ever seen, a monitor lizard clinging on to the side of the hut with huge claws. Its beady eyes and body not moving an inch, it didn’t even look like it was breathing. Definitely a good start to this trip!

One of Vanghat's many pools

The next day we jumped into the back of a Gypsy we set off for the short drive to Vanghat, in the Ramganga Valley. On route we saw a crested serpent eagle soar above us and land on a nearby branch with a snake in its claws, as well as a lesser fish eagle rising in the airs hot vents. 

That evening at Vanghat we walked to a beautiful deep slow flowing pool hidden in the valley where we could see the huge golden mahseer swimming below us. On the walk we came across all types of fantastic birds, greater yellownape woodpeckers, crested kingfishers, slaty-headed and plum headed parakeets, pallas and lesser fish eagles and the stunning Asian paradise flycatcher with its long trailing tail feathers and most impressive a tawny fish owl,  which peered eagerly down at us.

Other mammal sightings included a growling wild boar as we disturbed its rummage in the bushes, a porcupine nuzzling in the undergrowth for food and Sambar and Barking Deer. It’s not just the sights of these animals that filled me with awe but the sounds were enlivening. From every angle birds chirped and mammals called.

Elephants in Dhikala

Well this is the trip I had been waiting for and I had heard many stories about people seeing the famous tigers of Corbett national park and little did I know that the next 3 days were going to be some of the best of my whole life. Dhikala was the area in the national park we were going into. The elephants were in abundance, for their size they moved so graciously and there herds were so protective of the tiny calves. Mothers and aunties would hide them as they crossed the track In front of us. One thing which I was astounded by with these huge creatures was the way they moved – silently, effortlessly you wouldn’t know they were there unless they made it really obvious! A big mugger swam at the area called high bank. No tiger today and I wasn’t holding my breath – ‘Do these elusive, fine-looking creatures really let you see them?’ I just couldn’t imagine it.

Heather in safari

Pied kingfishers dove in and out of the river collecting fish, a green and chestnut-headed bee-eaters in the grasslands, crested serpent eagles and the  huge pallas’s fish eagles scouring the river for prey. More exciting moments were to come when a large female elephant protecting its herd charged at us, was one of the scariest moments of the trip but highly exhilarating.

After the morning safari we opted for an afternoon on the watchtower. The watch tower was a large structure overlooking a river bed and many of the animals came here to drink especially in the 40’C mid day sun. Sitting there for 5 hours in the searing heat may not be everyone’s idea of fun but it was worth it, sighting a tiger one was going back into the bushes from the river. As well as wild boar having a dip to cool down we saw hog deer on the grasslands and jackals running playfully.

Once our time was up we were picked back up and headed back to camp for an elephant safari which was unfortunately cancelled due to some bad weather coming in but little did I know this was a good thing. Instead of the elephant ride we headed off again by gypsy for an evening safari. Weather was worsening so we weren’t hoping for many sightings as most of the animals would surely be sheltering from the storm? Divan our driver who had been brilliant the whole time suddenly spotted a tiger across the river heading back towards the long grass of the chaur. We stopped and looked through the binoculars and sure enough there it was leisurely strolling into the tall grass. It disappeared from view and bizarrely appearing a few seconds later in what looked like the same place stood a cheetal deer. Was it just this deer playing tricks on all our eyes or was it really a tiger!? After another 20 seconds of looking through the binoculars it was then that I spotted the tiger, watching the cheetal. It was going to kill. I said to those around me whatever you do keep an eye on that deer, its going to be prey!! We watched for what seemed like no time at all as the tiger slowly crept up behind the unsuspecting cheetal. Then the cheetals head darted up – it had heard the Tiger. With eye on the cheetal the tiger leapt through the grasses and with one swoop of its powerful paws it brought the cheetal down. With hardly a struggle the tiger had its jaws round its throat and in seconds the cheetal did not move. The tiger had it and I saw it!!! As if that wasn’t enough a 2nd Tiger emerged from the opposite side and walked towards the tiger and cheetal it had just killed. I must be dreaming I thought, this is surely too good to be true, a real life tiger kill, no one see’s this! With a gust of wind and dust flying in our faces we watched from a distance as the tigers took its prey and slinked off into the grasses! Now that was a lifetime experience!!

It was amazing to see and something that will stay in my mind forever. It was pretty gruesome but a fact of life, one which I understand has to happen to keep these extraordinary animals alive.

Elephant bath!

Next morning we had to leave the camp but not before we had our last safari. We headed off in a different direction to the routes we had taken previously and went to the reservoir where we saw basking Gharial & mugger crocodiles and woolly necked storks sitting on the banks looking for fish. We also saw black franklins on the grasslands, red headed vultures circling above us, ashy prinias and 3 brown fish owls sitting in one tree, perfectly camouflaged. Later we saw the beautiful Indian roller having a good old roll on the track in front of us and a jungle owlet swooping through the trees finding lizards for breakfast. I really don’t think I could have asked for more.

My time in Corbett National Park was now over. It’s not just the Tigers I came to see, that was just an added bonus, but the vast array of birds, mammals and reptiles. Every time I walked or was driven around the area, I was guaranteed to see something new. It fulfilled my lifetime ambition as well as giving me a new love for birding and exploration. I loved India and I will be back.

Women collecting wood

In India, a high density of people is dependant on forest products in order to cook, to heat the houses and to feed the cattle. This has been a tradition for decades if not centuries among Indian villagers. Wood is indeed a privileged energy source since it is free of cost and is, for now on, still available. But this wood collection is step by step destroying the ecosystems of the forests. The forest products collected which are bark, dead and green wood and grass all participate in the balance to keep forests in good health. The barks are protecting the trees from any aggression; the green wood is vital for trees and especially for young trees to grow; the dead wood, by decomposing, plays a major role in the richness of the soil, creating micro ecosystems and the grass gives wild herbivores a source of food. A degradation of any of those elements is putting the others in danger.

Smokeless chulah

But the villagers can not be blamed so easily. The government is given almost no alternative to this wood collection. Liquefied Petrol Gas can be purchased at low price (≈200INR) but for poor villagers, this alternative is still too much expensive. The only way to get energy supply and moreover for free is by collecting wood. The best approach to reduce the wood consumption and therefore the wood collection is to provide sustainable alternatives to the villagers for cooking like smokeless chulah and solar ovens. The introduction of those alternatives may be hard at the beginning but they are necessary to change the wood consumption pattern and reduce the impact of the villagers on the forests. Another way to reduce the wood consumption is to give the opportunity to the villagers to raise their monthly income and make them able to afford energy supplies else than wood. The Lantana furniture making project serves this aim.

Woman making wood bundle

The wood collection is, one must never forget, a hard job. Villagers (mostly women) go early morning and walk sometime several kilometres inside the jungle in order to find wood supply. They spend around 2 hours collecting and cutting branches with a dharati. After that, they make wood bundles and tie them with green barks. They walk then back home with loads of dozens of kilograms on their heads. Sometime, they go twice a day in the jungle to get more wood if the weather is good, if more wood is needed or if children do not go to school. When asked, the women often say that they do not like going inside the jungle. First because they are afraid of tigers but also because it enable them to do work at home.

Wood bundles are to be sold

The collection of wood serves two purposes. One is for the use in the houses, the other one is to have it sold. For the second one, the problem is even bigger since the collection of wood can be seen as an usual job, and the forests as money suppliers. The more you take from the forests, the more money you will get. This excessive collection can only be stopped or at least reduced if an economic alternative is proposed to those villagers.

Woman attacked by a tiger in June 2009

Those ladies are incredibly brave, especially considering the fact that the collection of wood happens in areas where potentially lethal animals (tiger, leopard, elephant, bears, snakes, etc) can be met. Since December 2008, four attacks on villagers by tigers have been encountered, one leading to death. Those attacks are mostly due to the fact that villagers, by going inside the jungle, are stepping on big cats territories. That intrusion is sometime severely punished. When they are moving in groups (more than 4 persons), the villagers are safe from tiger attacks. But when they separate on the collection point, they are much more vulnerable and can not always look around for their safety since they have to search for wood.

Tiger’s pug or pawprint indicates the age and health of the animal

We glimpse a lone sambar deer, more timid than the chital; the canine bark of the barking deer reaches us from a bend ahead. Porcupines and wild boar have dug up the ground in many places. Tiger scat, examined by expert eyes, reveals fur and crushed bones from its last kill: that is as close as I’ll come to a face-to-face encounter with the king of the forests. Tiger hunting has long since ceased, but the ‘king of Indian sport fish’ still draws anglers from around the world. Esteemed by pukka sahib sportsmen since the nineteenth century, the yellowfin or golden mahseer remains abundant here; catches are released.

At a once-abandoned hamlet on the river flat, ecologist Sumantha Ghosh, in partnership with local communities has established Vanghat River Lodge, a wilderness and fishing lodge just outside the reserve.

Vanghat trains and employs young villagers as housekeeping staff and gillies. Poaching and dynamite fishing have virtually ceased, and catch sizes are rising noticeably. The hamlets and their garden beds are shielded by solar-powered electric fencing, for protection from predatory leopards, browsing elephants – and the occasional tiger.

Vanghat’s stone and mud-brick cottages have been fitted with comfortable beds and ensuite bathrooms: far more welcoming than India’s scruffy government resthouses. Drinks are offered around the campfire before dinner materialises.

Barry Abbott, a retired Briton who has fished all over the world, declares himself well pleased with his first day’s tally: a 15lb mahseer, with a 25-pounder slipping off the hook. My own catch is considerably less impressive, but the intangible rewards include a glimpse of two very large otters slithering across the river-worn pebbles.

“Tiger is giving us dodge”, declares wildlife guide Hem Bahuguna, calling a halt near some tell-tale pug marks (pawprints) and scrapings. As the engine cools, then stills, we hear only the birds, the soft breeze and the distant chattering of monkeys. From time to time, another jeep materialises, stopping to exchange a few words. Otherwise, here in India, most crowded of nations, there is perfect peace.

Corbett is India’s first, perhaps finest, Tiger Reserve and is buffered by surrounding tracts of country. You can spend days chasing tigers or cast a line to tempt the golden mahseer. The Ramganga Reservoir provides year-round water for the animals and spawning grounds for the golden mahseer, which migrates upstream.

Tall stands of teak, with its strikingly wide leaves, and the equally imposing sal clothe the hillsides, and wide gravel river beds traverse expanses of waving grass. Gharial, the snouted crocodile, coexist alongside the mugger crocodile and the otter. Sambar, chital or spotted deer and the solitary muntjac or barking deer are all readily spotted, especially when browsing in the grasslands. Less visible, the wild boar, sloth bear and tiger all record their passing with spoor – paw prints and droppings.

The eco-friendly Vanghat River Lodge in the Corbett Tiger Reserve

Panthers, although endangered, continue to be sighted in the hill country – but, Bahuguna admits gloomily, Indian authorities have recently confiscated quantities of contraband skins.

Macaques or rhesus monkeys, the males’ buttocks comically inflamed during the present mating season, together with the larger langurs, enliven otherwise still forests.

At least, 500 of India’s 1,300 known bird species are recorded at Corbett: a hoopoe browses boldly; a lone rose-winged parakeet stands out against bare boughs.

Bahaguna has set up a pre-dawn rendezvous at Amdanda Gate, outside the town of Ramnagar. A pallid pink orb begins to burn through the mists which rise above forest and grassland as the jeep reaches Bijrani camp, where day-visitor facilities operate.

By noon, we have jolted across innumerable gravel river beds, wound up into the dappled shade of sal forest and back down again, and climbed a watchtower on the edge of a broad river valley. We have examined the bark torn and chewed by elephants, noting the bushes trampled by these huge and demanding creatures. Tiger pug marks and droppings beside the track indicate the age and health of the animal.

Tigers often prove elusive, but park director Rajiv Bhartari will explain why this is no cause for concern. At Corbett, an estimated 143 tigers range across 1,218 square kilometres of rugged terrain. This population density is considerably lower – and therefore healthier – than at some of the better-known reserves in western India.

Scientists are conducting a tiger census, and the numbers are coming in well above expectations.

Villagers in the community established by celebrated tiger hunter Jim Corbett

Jim Corbett, author of the best-selling Man-Eaters of Kumaon, became a larger-than-life figure in the Himalayan hill country before World War II. The British hunter tracked down and killed 50 man-eating tigers and more than 250 leopards which had terrorised local villagers, but believed that a taste for human flesh was developed only by ageing or wounded tigers. His concern for the tiger’s survival led to the reservation of what would become today’s Corbett National Park: the starting point in 1973 for the groundbreaking Project Tiger.

The hill people of Kumaon remember Jim Corbett not only by the reservations which bear his name, but for his dedication to the welfare of his tenant farmers for whom he created a model village at Kaladhungi. Choti Haldwani, Corbett’s bungalow where the life-long bachelor lived with his sister Maggie, has been preserved as a museum, whilst a walking trail meanders through the mustard seed and sugar canefields of his former estate.

Discipline and dedication sum up Subrati the forester. Subrati joined forest department in 1988 and ever since didn’t look back. He was honest and hard working person. He had good knowledge of wildlife as well as meticulous office work. Be it anti-poaching of fire-fighting he was often the groupleader. On the fateful evening of 09 September 2006 Subrati passed away in the line of duty and the tiger lost one of it’s most dedicated guards.

Also known as The Tiger Man of Corbett Jolly has witnessed when India’s finest national park Hailey National Park was renamed as the Ramganga National Park and then in 1956 as the Corbett National Park and finally he saw Project Tiger being launched from the  Corbett Tiger Reserve in 1973.

Jolly has never known fear and never will. While serving with the forest department he has caught several poachers and has caught hunting army soldiers, a member of legislative assembly. In the Mohan forest he caught the son of a serving judge and cleared his misconception that he was above the law, he even booked a Wildlife Warden.

After his retirement as a Deputy Ranger 1993 he continued serving the jungles with more vigour by joining the Corbett Foundation. As the head tracker his job is to find cattle that have been hunted down by Tiger or Leopard and offer compensation to villagers under the instant relief scheme. This programme is the single most reason for very few cases of carcass poisoning by local inhabitants around Corbett and healthy Tiger and Leopard numbers not just in the Corbett Tiger Reserve but in the adjoining and vast reserve forests.

Respected and loved by villagers and forest staff he is a key link and has assisted numerous researchers. Those who accompany him believe that he speaks to Tigers and Leopards, his understanding of Tiger psychology is legendry. While inspecting kills on foot he has been charged by Tigers and has saved the lives of those accompanying him by calming them down and holding ground.

This grand old man will be difficult to replace from the web of life. To know more about the organisation that Jolly the Tiger man works for please log on to www.corbettfoundation.org.

She ended up with very important and alarming results such as:

- Tiger pugmarks are seen almost every morning on the nulla, proof that at least one tiger inhabits the area and uses this nulla

- Tigers and Elephants regularly use the nulla and game tracks in the vicinity of Ringora village

- The speed limit of 40 kilometers is seldom adhered to and the highway takes a heavy toll on wildlife. Mongoose, porcupine, primates, cheetal or spotted deer and numerous reptiles are commonly run down. In the past 3 leopards have died and a tiger was severely injured by such vehicular accidents.

Laurie’s work was one of the first ever meticulous assessment of the wildlife corridor between Ramnagar and Dhikuli and brought our alarming facts regarding the ever increasing traffic on NH 121 and impacts of mindless tourism.

Since her childhood Laurie had a passion for wildlife and especially big carnivorous fauna like wolves and tigers. It is very important to her to act for their conservation and protection. She graduated in sciences with a specialty on Biology Ecology and now, she is following a two year course in Management and Protection of the Nature and the Environment (High National Diploma) at the University les Barres (France).

For her Corbett was very motivating because it has a extraordinarily rich wildlife and different emblematic species to preserve. She also interacted with foresters and naturalists to help her in her research, which was indeed very interesting. Moreover during this three months period, she got the opportunity in term of human relationship to learn, meet people and discover a new culture. It was a fascinating experience to her, to be close to tigers, to feel their presence, to follow their pugmarks… it really was a dream. Laurie is now committed to act for the protection of endangered species especially carnivorous and to follow a course on mammals’ behavior, also called ethology.

A legendary veteran of Corbett, he gave us valuable inputs on conservation of the Ramganga  River and on its monarc the Golden Mahseer and ethical ways of fishing. Brijender Singh, or just Brij has been the Honorary Warden of Corbett Tiger Reserve since 1982. He was recently awarded the Esso Award. The award recognizes the efforts of an individual or an organization for bravery, valor and lifetime dedicated to Tiger Conservation. Popularly known as Raja sahib he belongs to the royalty of Dada Siba in Himachal Pradesh and Kapurthala. Influenced by the well known hunter Colonel ANW Powell (Author of, The call of the Tiger) he was an avid hunter till the late sixties but he gave it up and instead became a staunch protector of wildlife and a keen nature photographer, this time much like the legendary Jim Corbett.

In the mid-1980s, he was also appointed as a member on the Indian Board for Wildlife, and the Steering Committee of Project Tiger and is its longest serving member. He was instrumental in many of the expansion plans of the Project Tiger Reserves, national parks and sanctuaries throughout the country, and trans-location of the great one horned rhinos from Assam to Dudhwa. During his tenure Corbett Tiger Reserve increased from a 520 sq km to its present 1,319 sq km with the addition of the Sonanadhi Sanctuary and adjoining buffer areas. This has ensured safe future for many migratory species particularly elephants. He has been instrumental in established Benog in Mussoorie, though sanctuary may still be hosting the Himalayan Quail a bird mysteriously lost to science.

He spends most of his time patrolling and organizing logistics in the Corbett Tiger Reserve. His wife Dawn has started a small school at Dhikala.