From tiger scat to DNA to — hopefully — survival

Tiger DNA expert Uma Ramakrishnan gets special permission to wander India’s protected forests on foot, following the same trails the big cats tread. While she enjoys coming across tigers and their cubs and watching them with binoculars, those sightings aren’t the treasure she’s after. What she loves most is to find tiger droppings — “almost like gold to me,” says the molecular ecologist at the National Centre for Biological Sciences in Bangalore.

Territorial tigers oblige by leaving scat regularly, as a warning to other tigers that this space is occupied. These nuggets contain genetic material that scientists like Ramakrishnan use to understand tiger populations: How many are there, and what kinds? Where did they come from, and how far do they travel?

It’s crucial information for conservation efforts. Tigers are endangered, with fewer than 4,000 wild ones roaming the lands of at least 10 nations, from Eastern Russia to the island of Sumatra. That’s down from an estimate of 100,000 in 1900. Human activity such as urban development, logging, farming and mining has fragmented and destroyed tigers’ forest habitats, and poaching is an ongoing problem in parts of Southeast Asia.

“If you’re going to have conservation management, you have to know what you’re dealing with,” says Stephen O’Brien, a geneticist at St. Petersburg State University in Russia and Nova Southeastern University in Fort Lauderdale, Florida. For example, genetic studies show that tigers are split into several subspecies, so conservationists may want to develop strategies to protect each group.

Over the past two decades, scientists have built up a picture of tiger evolution and ecology based on their DNA, as described this year in the Annual Review of Animal Biosciences. Early on, scientists could only look at a handful of spots in the tiger genome. Today, with the advent of inexpensive DNA sequencing and genomics that covers every bit of the instructions to make a tiger, experts are gaining a much broader picture of tiger biodiversity.

DNA analysis — which will help not just to save tigers, but also to preserve the range of genetic variety that they carry — “is one of the best tools we have,” says Byron Weckworth, conservation genetics director for Panthera, the global wild cat conservation organization in Missoula, Montana. That said, he adds, the application of genomic information to conservation is still in its infancy.

Here are some of the key questions that tiger genomes can answer.

How many tigers are there?

The nature of wild tigers — solitary, well-camouflaged and rare — makes it difficult to conduct a census. To that end, Ramakrishnan evaluated the potential of genetics to count tigers for a 2009 study in Biological Conservation. She and her colleagues collected 68 samples of tiger dung from Bandipur Tiger Reserve and National Park in the south of India, preserving them in alcohol. Back in the lab, they isolated the DNA inside to examine the genetic sequences — the strings of letters A, C, G and T — of certain parts of the tiger genome.

If a genome is like a book, most of the words will be identical from tiger to tiger. What geneticists look for are the places where the words can differ. Specifically, Ramakrishnan and colleagues analyzed places where the tiger genome repeats itself, and stutters — say, GATA-GATA-GATA-GATA-GATA. No two tigers have the same number of repeats at each spot, so scientists can use the differing counts to distinguish individuals.

The researchers got high-quality data from 38 of their scat samples, and together these represented 26 individuals. That is, some tigers had left Ramakrishnan multiple presents. By number-crunching the tigers appearing once or multiple times in these samples, she estimated that there were 66 tigers in the area.

Ramakrishnan updated the technique in an April 2019 study, this time using modern technology to read every letter in the genomes of 75 wild and captive tiger samples. Her team tested for even subtler genetic changes than the numbers of repeats, homing in on variations in the spellings of individual words — say, a place that had a CAA instead of a CAT. From this, the researchers devised a panel of 126 spelling variations that help to identify individuals, reporting their work in Methods in Ecology and Evolution.

The new test is fast, easy and inexpensive, Ramakrishnan says. She hopes that it will make tiger-typing (and tiger-counting) accessible to scientists in all nations that host the creatures. That’s important because, due to the Convention on International Trade in Endangered Species (CITES), researchers cannot send any tiger samples across borders.

What kinds of tigers are there?

Tigers are known as Panthera tigris to scientists, but there’s more to it than that. They can be divided into subspecies: groups that are isolated and might, over time, develop into new species, O’Brien says.

“It’s better to save tigers as a whole, but in doing that, we should be saving the subspecies wherever we can,” says John Goodrich, tiger program senior director and chief scientist for Panthera in Fort Collins, Colorado. Plus, Goodrich notes, regional governments are more likely to fund tiger conservation if they have a flagship subspecies of their own.

But how many subspecies are there to save? Estimates have ranged from just two to as many as nine (of which three are already extinct). Genomics can help.

Geneticist Shu-Jin Luo at Peking University in Beijing and colleagues sequenced the genomes of 32 samples of tiger blood and tissue. When they compared the millions of differing genetic spellings and built a tiger family tree, they found that living tigers fell into six distinct groups, described in a 2018 paper in Current Biology: Bengal tigers in India, Amur tigers in Eastern Russia, and South China, Sumatran, Indochinese and Malayan varieties.

“It has major implications on how we do conservation,” says study coauthor Dale Miquelle, director of the Wildlife Conservation Society’s Russia program and coordinator for the society’s tiger projects. For example, zoos breed animals to keep subspecies bloodlines pure. One day, says Miquelle, conservationists might return some of those animals to the wild, infusing natural tiger populations with diverse genes that exist only in captives today.

In places where tigers roam no more, it might be feasible to introduce animals that genomics identify as most related to the extinct populations, suggests O’Brien, also a coauthor of the study. For example, Amur tigers are a close match for the extinct Caspian subspecies.

How have tigers changed over time?

Fossils indicate that tigers roamed China and Java 2 million years ago. Based on their tiger family tree, Luo’s group predicts that between 1 million and 110,000 years ago, tiger populations contracted into a small group, located in modern-day China and Southeast Asia. This was probably the result of the cool climate at the time, a feature of the Earth’s most recent ice age.

From this small group sprang all modern tigers, which spread across much of Asia. They developed genes best suited to their lifestyles in locales ranging from the tropics to the tundra.

For example, Sumatran tigers are smaller than others. They range from 165 to 308 pounds, compared to about 550 for Bengals, the biggest subspecies. Miniaturization is a common island adaptation for large animals, says Yue-Chen Liu, a geneticist who recently earned a PhD in Luo’s lab and plans to start work at Harvard University soon. Tigers may have encountered smaller prey when they reached the island of Sumatra, and through evolution, shrank to match.

Size is controlled by many genes, so Liu was surprised to find one in particular that might control Sumatran tigers’ size. The animals differ from other tigers in a gene for a protein — called ADH7 — that affects metabolism. Mice with mutations in this gene are small; the same might have happened in the tigers.

It’s just this kind of genetic diversity that conservationists ought to protect, says Liu.

Where does poached or illegally traded tiger material come from?

These endangered beasts are victims of their own majesty, as practitioners of traditional Chinese medicine believe their body parts are powerful restoratives, and others purchase the gorgeous pelts for decoration. One estimate valued the tiger-poaching industry at $19 billion per year. Unpermitted transport of tigers or their parts from one country to another is a crime, so identifying the source of the material can help prosecutors.

Luo’s group developed a test they call tigrisPlex. It uses 22 genetic repeat sequences to identify tiger subspecies and individuals. Reporting in the journal Integrative Zoology in 2015, they used the test to identify 12 confiscated skin or muscle specimens as being from six Amur tigers.

TigrisPlex can also help zoos maintain subspecies. In 2013, Luo got a call from a zoo in Osaka, Japan. The zoo was thinking of importing a tiger cub from China, but wanted to pick one that would be a good fit for the Amur tiger population they already had. Luo confirmed that the cub was an Amur, and that tiger is now living — happily, Luo is told — in Osaka.

How do tigers travel?

Since tigers maintain solitary territories, young animals leaving their mothers must find a new place to live. Individual tigers may travel more than 600 miles.

That’s difficult if tigers live mainly in protected parks, and human towns or roads get in the way of migration between their havens. To study tiger transit, Ramakrishnan’s group collected 289 tiger scat samples from 11 Indian parks, as well as areas beyond the parks. These samples represented 116 individuals.

Based on the DNA in the scat, the researchers found that tiger populations were less likely to mix when separated by roads, human settlements or nonwild areas such as pastures. Reporting in the journal Biological Conservation in 2018, they used computer models to predict what might happen to tiger populations under this kind of isolation, and it wasn’t good: Tigers would become less diverse, and the smallest populations were likely to disappear.

Conservationists are trying to stop that from happening. For example, the National Highways Authority of India recently expanded National Highway 7, which runs between Pench Tiger Reserve and Kanha Tiger Reserve in the middle of India, from two to four lanes. Environmentalists used Ramakrishnan’s data to argue that tigers need safe crossings.

The Highways Authority ended up constructing underpasses. And there’s evidence that tigers do use them: Early in the morning of February 23, 2019, a camera trap in one underpass outside the Pench Reserve caught a tiger strolling by.

What will become of tigers?

Genomicists hope that their data will be used to perform a sort of conservation triage, identifying the subspecies and populations most in need of help, or most likely to survive with assistance.

Today, the outlook is mixed for tigers, Goodrich says. They are doing well in southern Asia, and likely in the northeast, though he doesn’t have good population estimates for eastern Russia. In Southeast Asia, poaching remains a huge problem. And climate change is causing droughts and rising waters, further endangering tiger habitats, he adds.

Miquelle is optimistic about tigers’ long-term prospects. He hopes that as human populations stabilize and people move from the countryside to cities, there will be more room for wild things like tigers.

“One hundred years from now, there could be many more tigers in Asia than there are today,” he says. “There may be light at the end of the tunnel.”