Laxman Khanal was lost.
Chasing after a troop of elusive Assam macaque cliff monkeys along the dense forest of Api Nampa Conservation Area at the far western end of Nepal, he lost track of his local assistant. A mere glimpse of the monkey troop and he couldn’t hold back. He had to chase them. And why wouldn’t he? Three days of bumpy bus rides and another three spent on arduous jungle hikes had finally led to this moment.
After a desperate chase-off, he finally could rest and stare at this troop. The terrain was terrifying. One misstep and his body may tumble straight down to the cascading stream at the base of the mountain. Steep cliffs are the favourite spots of these monkeys — the best place to evade large carnivores like leopards. Somehow managing to stay still, he observes the monkeys; and as they depart, Khanal scoops up their fresh stool samples. It’s an absolute gold mine for his research.
“It was literally a do or die situation for me,” he said. “Either collect samples or your research is for nothing!”
The cost? Just minor bruises. “I could for sure take it. Come on, I am an ecologist,” he grinned. But for Khanal, a primatologist and molecular analyst at Tribhuvan University, scrambling for monkeys in unforgivable terrain was just a footnote of his two-year relentless quest. In early 2021, his work was published in the journal Zoological Research with an audacious claim: ‘Molecular research suggests Nepal population of Assam macaques (Macaca assamensis) to be a distinct species’. This could send shockwaves across the world of taxonomy.
The Nepal Population of Assam Macaques (NPAM) is a highly protected monkey species, not in the least because they look so similar to Rhesus Macaques, a common, urbanized, and occasionally aggressive monkey found across the Indian subcontinent. Assam Macaques are more elusive and shy, bigger, and heavier, with longer tails and genitals than the Rhesus.
It was based on these physical attributes that in the late 1990s, Mukesh K. Chalise, one of Nepal’s pioneer monkey researchers, speculated that Nepalese flocks of Assam Macaques may actually be a different species.
Research on Assam macaques has a big history. It was Brian Houghton Hodgson in 1840 who first collected a specimen of these monkeys from the Jiri area of central Nepal. When Chalise visited Chicago’s Field Museum in 1997, he noticed a specimen and its collection site. “It was defined as a monkey that looked like a rhesus macaque,” he said. Chalise, who had already been into primate research by that time, had also observed a similar kind of species in the field expedition at Gorkha in western Nepal in 1992. The museum specimen was the spark he needed. After returning from Chicago, he went through the reported literature of its sightings and planned out a research expedition. On a one-month expedition to the Sankhuwasabha in eastern Nepal, he happened to encounter a dead monkey. Measuring its shape and size and comparing it with the reported data of Assam macaques from India and Thailand, he was intrigued. “The difference in their bodily shape and size had convinced me that Nepal's population of Assam macaque may be a subspecies or entirely a different species”, he said.
Genetic analysis was certainly not an option back then. Khanal, who was then a student of Chalise, decided to follow the footsteps of his supervisor, and went for primate research, focusing on the macaques. His Ph.D. thesis involved the impact of geographical barriers like major river systems in creating potential genetic demarcation between the eastern or western population of Assam macaques within Nepal. The genetic difference between the two wasn’t significant. And he looked at their evolutionary history. “Their arrival in Nepal was recent, about 50,000 years ago,” said Khanal. Since NPAM was trapped within geographic boundaries for a significant time without mixing with any other subspecies, it could be a new species. Khanal’s Ph.D. research had brought up another quest.
For a reliable conclusion, Khanal needed as many samples as possible. Collecting stool samples from the entire distribution range of Nepal cost him almost two years. It took him from Api Nampa on the western Indian border to the Mulghat region of Dhankuta, less than a half-day drive from Bangladesh. Throughout this journey, he encountered 40 troops of monkeys and recorded 277 fresh fecal samples. Surprisingly plenty, given how few populations of macaques remain within the nation.
At each monkey chase, Khanal carried a bag with him: a camera, a binocular, a GPS tracker, a notebook, a few pencils and ball-pens, a tiny box of a lysis buffer, few tubes, and sterilized cotton swabs. The chemical preservatives protected the DNA samples without the need for freezing, a luxury not found in the remote Himalayan foothills.
Khanal soaked the mucous cells from the fresh stool sample through a cotton swab and released them in the lysis solution. He performed a tedious laboratory process of retrieving DNA from stool samples - amplifying, sequencing, and analyzing the mitochondrial and y-chromosomal loci. By studying the accumulated rate of mutation within mitochondria, the evolutionary distance, or how genetically similar individuals of two species are, can be calculated. Khanal found that the Nepal population of Assamese macaques were not closer to another eastern assamese subspecies (Macaca assamensis assamensis) but in fact closer to Arunachal macaques (Macaca munzala) and white-cheeked macaques (Macaca leucogenys). “It was something out of the box and I was pumped up!”
“The results are quite consistent with what I expected,” said Christian Roos, a primatologist at the German Primate Center who is not involved in the current study. Roos have studied museum specimens of Assam Macaques ranging from Vietnam, Myanmar, and Thailand in Southeast Asia to Nepal and Bhutan. These monkeys in Nepal, from Roos’ point of view, clearly have a recognizable distinction and a significant gap in their distribution. “We had recognized the difference, but could never carry out a proper study. I am glad that my colleagues finally came up with this,” he said.
“Molecular tools allow us to elaborate a much defined and specific look at the genome of the animal that directly impacts the taxonomic classification,” says primatologist Randall Kyes, a professor at the University of Washington and a co-author of the current study. “What this paper suggests is, based on the work that exists, there seems to be an evolutionarily distinct population in Nepal that in turn may qualify for re-evaluation and reassessment of the species.”
The genetic analysis also revealed that the Nepal-based population seemed to have lower genetic diversity than the monkeys from other parts, raising concern for the long-term viability of the species. “That’s why we suggested at the end that we may want to reevaluate their conservation status,” Khanal mentioned.
Taxonomists use a certain threshold for distinguishing a species. If a species has two percent or more of a genetic difference with another species then it qualifies to be distinguished as a separate species. But in addition to genomic data, a proper morphological and ecological assessment is indeed a necessity. For Khanal and his team, the way ahead is to do a complete genome sequence and integrate its genetic, behavioural, morphological, and ecological data to remove any doubt.
Khanal urges governmental bodies and conservation organizations to carry out comprehensive studies to draft proper conservation action plans for this newly-minted species. “It's a species that evolved in this region. It's our pride and we can’t let another species slip into oblivion right in front of our eyes”, he said.
