Studying the dhole population in Wayanad using genetic markers

A new study reveals India’s first ever population estimates of the endangered dhole (Asiatic wild dog) in Wayanad Wildlife Sanctuary, Kerala.
India supports 23 percent of world’s carnivore species in around 2.3 percent of the global land area. But some of these species are facing the threat of extinction, even as their population remain unknown. Dholes are among such large threatened species.
In a new study scientists have collaborated to develop a scientifically robust method using genetic information and advanced population models to estimate the population of dholes.

In India, dholes, or Asiatic wild dogs, are mostly found in the Western Ghats, Central India and Northeast India. There are smaller populations in the Eastern Ghats and the Western Himalayas of Uttarakhand. However, there limited research and no organised method yet to study and estimate the actual population size of dholes, say experts. A recent study by a group of scientists claims to be the first to generate the estimates of dhole (Cuon alpinus) population and density in Wayanad, Kerala, India.

The study, titled, The truth about scats and dogs: Next-generation sequencing and spatial capture-recapture models offer opportunities for conservation monitoring of an endangered social canid, was published in the international journal, Biological Conservation. Scientists from Wildlife Conservation Society-India, National Centre for Biological Sciences, Kerala Veterinary and Animal Sciences University, University of Florida (USA), and Stanford University (USA), together developed a scientifically robust method — genetic information and advanced population models to estimate the population of dholes.

It all began in 2019 when the group of scientists started conducting multiple field surveys across 350 sq km of Wayanad Wildlife Sanctuary in Kerala, India. They collected dhole scats (fecal droppings), extracted DNA from the scats and used Single Nucleotide Polymorphisms (SNPs) to identify unique dhole individuals. They combined this with statistical methods called Spatial Capture-Recapture models, and with this, they were able to estimate and map dhole numbers and density across the sanctuary.

SNPs are a variation in genetic sequence or single mutations as a given physical location in the genome. “You can think of this data as one zero data (This the same as thinking of it as “yes/no” or “present/absent”). So an individual can be 1/1, 1/0 or 0/0. The important thing is that the genome is replete with SNPs. There are many more SNPs to choose from compared to other types of polymorphic markers. This is a reliable way to identify individual dholes from their fecal DNA,” explained Uma Ramakrishnan of National Centre for Biological Sciences, one of the authors of the study who has been working on endangered species and their genetics for over 15 years.

Elaborating on SNPs and Spatial Capture-Recapture models and their role in identifying dholes, co-author Arjun Srivathsa of Wildlife Conservation Society–India and University of Florida, USA, said, “Once we have assigned individual identities to the scats, we use statistical models that consider where these individuals have been detected and how many times they have been detected and predict how many individuals may have been missed during our surveys. The end result is that we get an estimate of the total population size, and we can also map their density– locations that have high and low densities of dholes.”

In addition to Srivathsa and Ramakrishnan, the authors include co-lead Ryan G. Rodrigues (Wildlife Conservation Society–India and National Centre for Biological Sciences), Kok Ben Toh (University of Florida, USA), Arun Zachariah (Kerala Veterinary and Animal Sciences University), Ryan W. Taylor (Stanford University, USA) and Madan K. Oli (University of Florida, USA).

Studying scat and hair for genetic information

Earlier, scientists were working with markers called “microsatellites” that were not just time and effort consuming but also, the results were subjective. It was then decided that they needed to make a switch to SNPs as this particular method of genotyping involves next-generation sequencing. “We started by developing these methods for tigers. The principle is that these methods are affordable, faster, easier and reliable. Also, they can be developed relatively easily for other species. We demonstrated the former in a 2019 study, and the latter in this dhole paper – we were able to quickly develop these methods. Overall, this approach requires a large set of polymorphic SNPs to choose from, and next-generation sequencing platforms,” explained Ramakrishnan.

Ramakrishnan talked about the robust scientific method to conduct this study, and said that in most cases, they work with non-invasive samples, like fecal (poop) samples, or shed hair. As fascinating as it sounds, the ability to work with such material is quite difficult. “DNA from such sources is degraded (broken up) and are in very low amounts. Doing this kind of lab work then requires a lot of patience and experience,” she said.

Dholes in Wayanad national park. Photo by Harsh Narasimhamurthy.

Meghana Natesh, postdoctoral researcher at Indian Institute of Science Education and Research Tirupati, who was not associated with this particular study on dholes, talked about the genetic methods being used in the study. The appeal of this method lies in the fact that a large number of target regions in the study species can be amplified (number of copies of gene sequence increased) simultaneously. The subsequent analysis of the genetic data is done bioinformatically, reducing subjectivity and bias in the interpretation. “The authors have also gone to great lengths to reduce noise from non-target regions and to ensure that their protocol is specific to dholes. The study also serves as a template for developing such genetic tools for other wild species, particularly when non-invasive sampling through feces or hair etc. may be the most accessible genetic material for such species,” she said.

Reliably identifying or distinguishing individual animals in a population is often the first step in monitoring wild populations. Natesh said that this paper provides an alternative strategy to identify individuals in a species where camera-trapping is not a viable strategy, given that individuals do not have distinguishing stripes or marks. “It also goes on to use this individual information to estimate the population density of dholes from Wayanad. Population monitoring of wildlife is necessary to understand long-term population dynamics, to assess threats, and even to understand whether any conservation measures that may have been taken are effective.” Methods such as this, therefore, allow for population monitoring – both, through demographic estimates, as shown here, or even through genetic monitoring over the long-term, she added.

The favourable landscape of Western Ghats that contributed to the dhole population

From field experience, the scientists knew that the Western Ghats landscape likely supports a good population of dholes. Before starting to test out any new method, the team needed to make sure that they got enough data to see if their approach was feasible, reliable and easy to implement or not. Another important reason for conducting the study in Kerala’s Wayanad is that the Kerala Forest Department was very enthusiastic and encouraging about the prospect of obtaining the population estimates of an endangered species. “Wayanad is part of a larger complex of protected areas– Nagarahole, Bandipur and Mudumalai. Together, this complex is well-protected, has mixed- to dry-deciduous forests which in turn have high diversity and densities of prey animals. All these factors are likely to make it very conducive for supporting high density of dholes,” said Srivathsa.

Dholes are carnivores. Typically, scientists use camera traps to estimate carnivore populations. “But this method works when we can distinguish between individuals based on their physical appearance in the camera trap photographs,” said Srivathsa. Tigers have stripes whose patterns are unique to each individual. Leopards have spots and rosettes. “Dholes do not have any patterns on their fur. So we cannot use camera traps to count their numbers. It is because of this issue that there were no population estimates of dholes in the past. We are hopeful to replicate this study in other parts of Kerala and eventually across India in the future,” he added.

The study found that Wayanad has 12–14 dholes per 100 sq km, with around 50 individuals estimated within the sanctuary’s administrative boundary and also supports high densities of the dhole. “Recent nationwide tiger surveys showed that the sanctuary also has a relatively large tiger population, with 11–13 animals per 100 sq km. The fact that two large carnivores can co-exist in such high densities is indicative of an abundant prey base and high-quality habitat. It is also a testament to how well the sanctuary is managed by the Forest Department,” said Srivathsa.

The average lifespan of a dhole in the wild is around 10 years. However, nothing can be said about the age of animals based on a study. “Since this is the first such study to estimate their populations, we cannot claim that it is only in this area that there are high densities. We need information from many locations to know more about population sizes and potential explanations,” said Srivathsa.

The first objective of this study was to implement the methods in one location to test whether it works or not. “Now that we have completed that, we will implement it in other locations. When we have information from multiple locations and across multiple years, we can get some interesting insights on how populations fluctuate, are individuals moving across protected areas, and if numbers are stable, or going up or down,” informed Srivathsa.

Dholes or Asiatic wild dogs are understudied in India. Photo by Diinesh Kumble.

The authors recommend that the methods developed and demonstrated in the study should be used as a standard protocol for estimating dhole numbers and for conservation monitoring of their populations in other protected areas in India and across the species’ distribution range.

Population size (or density) is the most basic metric we need to gauge how well a species may be faring in its natural habitat. Srivathsa says that their study offers the first step in this direction for dholes, and the species will benefit if the methods they propose are adapted and implemented across all important dhole landscapes in India and in other dhole-range countries. “Once we have this kind of information, we can make science-based recommendations for management. Dholes are endangered carnivores. In some sense, they signify the health of forest systems in south and south east Asia. Ensuring that dhole populations are protected would indirectly help conserve forest ecosystems in these regions.

Science and challenges of researching a new species

While researching a new species, and science itself, is a challenge, Ramakrishnan is just excited that the team could turn bits of brown smelly poop into SNPs and then an estimate. “This never ceases to amaze me. Having said that, ordering and keeping a constant flow of reagents is always challenging because it takes time.”

The laboratory processes are also relatively new for the scientists. Srivathsa said that the team dealt with a fair bit of trial and error in the beginning. Besides the challenges in laboratory techniques, the other issues were raising funds to support this kind of study and statistical models that could be applied for dholes. “It is incredibly difficult to raise fund support for species like the dhole, especially when the study involves exploring a new aspect that has not been done before. Second, the statistical models are being developed and refined over the past few years. The one we have used is ideally suited for dholes at this point, but we anticipate that there will be newer and better models in the future that will help refine our estimates even further,” said Srivathsa.