Whose scat is this: tiger or leopard?

How do scientists estimate the number of tigers in the wild? Shikaris traditionally used paw prints or ‘pugmarks’, but more recently scientists use remote cameras to photograph individual tigers, which are identified from their unique stripe patterns. However, deploying cameras in tiger habitats is not always feasible due to logistical problems.

Another method involves obtaining DNA from tiger poop — scientifically known as ‘scat’ — and amplifying them using species-specific primers (short sequences of DNA unique to each species). If designed properly, the species-specific primers should act like unique keys that only ‘unlock’ the DNA of their target species. However, if the primer is designed poorly, false ‘hits’ are possible, that can lead to wrongly identifying other species in the wild.

In a recent study, a team led by Professor Y V Jhala, Scientist at the Wildlife Institute of India in Dehradun, has found evidence to suggest that often used species-specific primers are not be as accurate as previously assumed. While conducting the country-wide tiger population estimation, the team was mandated to estimate the minimum number of tigers in areas where camera trapping was not possible through scat DNA. “As part of this project, when Pranay Maroju and Sonu Yadav (joint first authors of the study) were standardising already published and commonly used primers on samples where the species identity was already known, we came across species-specific primers that cross amplified in other species also,” says Vishnupriya Kolipakam, conservation geneticist at the Wildlife Institute.

Tigers and leopards both belong to the genus Panthera and are therefore genetically similar. They inhabit the same geographic area and both produce similar looking scat that cannot often be distinguished in the field. The short sequences of DNA used to identify each species varies only by a small amount, a few base pairs.

This means that the primers for amplifying DNA of tigers and leopards need to be accurately designed — the tiger-keys need to fit the tiger-locks perfectly and not fit the leopard-locks. Furthermore, DNA obtained from scat is often degraded, increasing the chances of false hits with poorly designed primers.

DNA amplified

In their study, cleverly titled ‘Schrodinger’s scat: a critical review of the currently available tiger (Panthera tigris) and leopard (Pantherapardus) specific primers in India, and a novel leopard specific primer’, the team tested the specificity of the DNA primers often used for the identification of tiger and leopard DNA.

They found that aside from amplifying the DNA of the target species, the primers also amplified the DNA of other species to varying degrees. For instance, a commonly used tiger-specific primer amplified the DNA of tiger, leopard and lion. In other words, these primers were not behaving like unique keys to unlock their specific DNA sequence, as they should have, if properly designed. What are the implications of these findings?

The authors point out that erroneous primers have been used in many prestigious publications. Due to the possibility of leopard scats being misidentified as tiger, this could lead to inflated tiger population estimates.

For instance, the historical population of tigers south of the Narmada has been estimated at around 50,000 in one study using these primers. Given that these studies were conducted with the erroneous primers, this number may need to be revisited.

Species misidentification from scat DNA also has other harmful effects.

“Incorrect inference of species has an impact on inferring conservation consequences of gene-flow and connectivity.

It gives us a false sense of security because of a mistake at the basic species identification step,” says Professor Jhala, further highlighting the need for stringent quality checks on primers before publishing scientific studies. The study was published in the journal BMC Genetics.