The barking deer enigma

Most forests in India are home to the Indian muntjac, also called barking deer. Found across southern India, including the Western Ghats, in the dry forests of central India, the evergreen temperate forests of the Himalayas and the dense rainforests of northeast India, Indian muntjacs are a quiet and solitary species.

Relegated to a list of prey animals for carnivores like tigers, leopards and dholes in India, they are rarely studied for their own sake and so very little is known about this species. Indeed, scientists are not even sure if all Indian muntjacs are the same species. A number of scientific papers in the recent years have thrown light on the muntjac, but still more needs to be known about this shy animal, which makes a short barking sound like a dog when alarmed.

Muntjacs, or Muntiacus in Latin, are a group of leaf-eating deer species found in south and southeast Asia. Until the 1980s, only a handful of species were known from this ancient lineage. But towards the end of the 20^th century scientists and naturalists, aided by information from local communities, came across several species of muntjacs in China, Vietnam, Myanmar and eastern Arunachal Pradesh in India.

At present, the International Union for Conservation of Nature (IUCN), recognises 13 species of muntjacs. Most of these species are elusive and often restricted to small pockets of forests, except for the red muntjac group, which includes the Indian muntjac. For a long time, red muntjacs from northeast Pakistan, India, Bangladesh, Sri Lanka, southern China, Vietnam, Laos, Thailand, Malaysia and islands like Borneo and Sumatra, were all considered to be the Indian muntjac – Muntiacus muntjac.

In 1990, two taxonomists, Colin Groves and Peter Grubb suggested that red muntjacs were a group with several different species. In 2011, the taxonomists wrote that India itself likely had three different species. In northeast India, Nepal and parts of Myanmar, they suggested that it was a species they named Muntiacus vaginalis. This, according to them was distinct from red muntjacs in north-west and central India – Muntiacus aureus – and the red muntjacs in Western Ghats and Sri Lanka were a third distinct species – Muntiacus malabaricus. The original Muntiacus muntjac Groves and Grubb wrote was restricted to mainland Malaysia and the Sunda islands.

What was the basis for the split?

What makes a species unique and different from another is a complex question. But scientists largely agree that if a set of animals or plants can breed with each other and produce fertile offspring, they can be considered the same species. The same ancestor can give rise to several new species that are related to each other to varying degrees. This process is called speciation.

A drastic change in the environment or the discovery of a new food source can lead to some individuals of a single species changing their diet or behaviour. Physical barriers like a newly formed mountain or river can also separate two populations of the same species so that they are not able to mate and mix genes.

Over time (thousands or millions of years), the organisms that have adapted to the new situations, in appearance, behaviour or physical characteristics, from the original species. They become so different in their genetic makeup that they cannot have fertile offspring with each other. Biologists call this reproductive isolation.

To find out if two populations of an organism have become reproductively isolated, taxonomists usually compare their distribution to see if there is any barrier, their physical characteristics such as size, colour, dentition or skeletal system and their behaviour. In some species of muntjacs, these differences are easily noticeable. For instance, the size differences between the tiny Putao muntjac Muntiacus puhoatensis and the large giant muntjac Muntiacus vuquangensis.

Red muntjacs are more confusing. “The taxonomic uncertainty around (red) muntjacs comes greatly from the fact that they are one of the most widely distributed muntjac groups and have very similar ecological and morphological characteristics – the criteria used so far to describe species/sub-species within red muntjacs,” states Renata Martins, an evolutionary biologist from the Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany.

As Martins, who has studied the lineages of the red muntjacs as part of her PhD research, points out, in appearance and behaviour, red muntjacs across Asia are roughly similar. Their coats range from light pale brown to reddish in colour. Males have small tusks and very short antlers during the breeding season. Females have small bony knobs instead of antlers. Unlike some of the other related leaf deer species, red muntjacs are also not restricted to just rainforests. For example, in India, red muntjacs are found in the wet forests of the northeast and Western Ghats as well as the dry scrub forests of central India.

Varying number of chromosomes

It was not these external features that distinguished Muntiacus muntjac from Muntiacus vaginalis. It was something within their body – the number of chromosomes they had.

The muntjac group has the most diversity in their chromosome number among mammals. While the Chinese or Reeves muntjac has 46 chromosomes, the black muntjac and the Gongshan muntjac have eight chromosomes in females and nine in males and Fea’s muntjac has 13 chromosomes in females and 14 in males. In 1970s, it was discovered that the Indian muntjacs from Garo Hills, from the northern part of southeast Asia and southern China had just six chromosomes in females, seven in males, the lowest for any mammal.

Scientists estimate that their direct ancestor had 70 chromosomes and muntjacs are capable for rapidly fusing multiple chromosomes to make a few large chromosomes. Because the DNA within the chromosomes is not altered much – it is just the container – their appearance and behaviour are similar, but they are reproductively isolated. A Chinese muntjac cannot produce fertile offspring with an Indian muntjac.

Biologists theorise that this sort of rapid change in chromosome numbers was the cause for so many different species of muntjacs even though in ecology and behaviour they are largely similar. This theory has especially gained credence with red muntjacs.

All red muntjacs are not the same

For a long time, it was simply assumed that all red muntjacs across Asia had the same karyotype (the number and appearance of chromosomes in the nucleus) as that of the Indian muntjac. But Groves and Grubb analysed a single female red muntjac from Malaysia and found that it had eight chromosomes. A different karyotype meant there were two populations and they were reproductively isolated.

They asserted that this difference in karyotype along with some differences in body size, antler size and colour, was enough to show that the Indian muntjac was at least two different species. In 2014, the International Union for Conservation of Nature (IUCN) tentatively accepted Groves and Grubb’s classification of two red muntjac species and renamed them as northern red muntjac (which includes populations in south Asia, Myanmar, Vietnam, China and Laos) southern red muntjac (southern parts of southeast Asia including Malaysia and the Sunda region).

But the scientists, who assess species’ risk for the IUCN, urged more studies pointing out that the karyotypes of many red muntjac individuals across the species range would be needed to be really certain. “There appears to have been no significant further investigation of this taxonomic split since 2008, and thus the 2014 reassessment largely for the sake of status quo maintains this taxonomic treatment,” the IUCN website noted.

As for Groves and Grubb’s contention that Indian muntjacs in central India (Muntiacus aureus) and Western Ghats and Sri Lanka are (Muntiacus malabaricus) were also distinct species, the evidence seems even shakier. They point out the difference in the coat colour of muntjac populations from different parts of the country.

Muntiacus vaginalis, the Indian muntjac from the eastern parts of the country, is described as dark red, with brown to grey limbs antlers ranging from nine to twelve centimetres in size. The muntjacs from Western Ghats, M. malabaricus, according to Groves and Grubb are much paler, almost light brown and antlers are less than 9.5 centimetres, while M. aureus from central India is apparently the smallest species and palest, almost yellow in colour with antlers that are less than 10 centimetres.

Groves and Grubb give no information about how many individual muntjacs they examined across the country and how consistently they found these differences, which is problematic because these colour differences don’t appear to be uniform. The IUCN points out that they don’t consider any alternative explanations for the differences in physical characters across the country.

The international body does concede that the Indian muntjac might indeed be multiple species. However, the documentation of such species, especially for the sake of taxonomic stability, requires the burden of proof to be placed on those authors attempting taxonomic splits,” notes the IUCN website. Although Groves and Grubb have furnished no new proof, Martins’ doctoral work found there might be something to the whole thing.

Analysing the mitochondrial DNA to trace the maternal line

Biologist Martins and her colleagues extracted mitochondrial DNA from red muntjac parts (such as skulls and antlers) collected from across Asia and preserved in natural history museums in Europe, as well as fresh DNA from poached animals in Vietnam. Most of the samples came from southeast Asia (including China, Vietnam, Thailand, Malaysia and the Sunda islands).

In south Asia, nine samples were used from north India (only Himachal Pradesh), east India and south India and one from Sri Lanka and one from Nepal. They also pooled in DNA information archived from the Western Ghats. There are two types of DNA in an organism. Nuclear DNA that is passed down by both parents to an offspring and is present in the nucleus (the control system of a cell) and mitochondrial DNA that is exclusively passed down from one parent, usually the mother to the child and is stored in the mitochondria (the energy producing part of the cell).

Most of the genetic information about a species is present in the nuclear DNA. Mitochondrial DNA only has a very small part of that information. But because mitochondrial DNA can only be passed on from mother to child, it allows us to trace the entire maternal line of an individual.

The study revealed that all the red muntjac samples they examined fell into three distinct maternal lineages. The oldest lineage was from the Sri Lanka-Western Ghats population. The red muntjacs from north India and northern SE Asia fell into one maternal lineage which the authors called the Mainland lineage and the red muntjacs from Malaysia and the Sunda islands formed another lineage.

The latter two lineages were similar to the IUCN’s classification of northern and southern red muntjacs, but the Sri Lanka-Western Ghats lineage seem completely distinct. The results suggest Groves and Grubb’s assertion of a separate Sri Lanka -Western Ghats species must at least be investigated.

“Indeed, this was a very important finding of our study,” stated Martins. “Unfortunately, we were not able to obtain more samples from these regions in order to be able to assess the genetic variability among the populations present there,” she added. “However, we did find strong evidence that these are very distinct populations from the remaining red muntjacs.”

In India, there are several species that are only found in the rainforests of the Western Ghats in Southern India and the rainforests of northeast India but nowhere in between. One theory is that when India first merged with Asia, the entire Indian landmass was probably a wet evergreen forest. But an intense cooling period, may have dried out Central India, leaving some species trapped in small patches of wet forests in the Western Ghats and Northeast India. This is why these two regions have several species or at least related species in common such the Asian Fairy Bluebird or the flying lizards.

However, unlike the fairy bluebird, red muntjacs are today found in the dry forests in south and central India as well as forests in the north. So, what explains the distinct lineage in the Western Ghats – Sri Lanka group?

Martins speculates that during one of the many climatic shifts that happened in the region, some population of red muntjacs must have colonised the dry areas. Others must have remained in the Western Ghats-Sri Lanka region for long enough to become genetically different, perhaps changing in their karyotype.

“Muntjacs are widely studied for their extreme chromosomal variations. Species with very different karyotype are unable to produce viable offspring. Therefore, if indeed such barriers to gene-flow as different karyotype were created during the isolation of Western Ghats and northern Indian populations, then it could explain why they are still genetically isolated after the species return to Central India.” But she emphasises caution while theorising. “However, as far as we know karyotype studies on individuals from Western Ghats or Sri Lanka are still lacking.”

Does this mean Central India could have its own sub-species or species, a Muntiacus aureus as Groves and Grubb suggested? As the study did not use any samples from this region, Martins refuses to speculate adding, “I believe that part of the taxonomic uncertainty with this group stems from speculations based on very small samples (sometimes, single individuals) to the larger group.”

“Sampling other regions, such as mountainous regions or the dry zones of India, will be paramount to the clarification of the taxonomy of red muntjacs and to possibly finding distinct and unique populations that are hidden today within the large M. vaginalis and M. muntjac,” she states.

“You have to sample more,” agreed Uma Ramakrishnan, an evolutionary biologist from the National Centre for Biological Sciences (NCBS), Bangalore. Ramakrishnan, who is not part of any of the studies discussed here cautions against using only mitochondrial DNA which comes only from the mother and doesn’t capture the full picture. “Ideally, if you can get specimens, you should look at morphology as well as DNA and you want to get more than the mitochondrial genome.”

Martins is hopeful that biologists will be able to do this in the future. “With the advances in sequencing technology we can expect, and see already, easier access to nuclear DNA. The study of nDNA will have important implications in understanding species adaptations and help resolve intriguing phylogenetic relationships.”

Ramakrishnan also pointed out that to truly resolve the taxonomic confusion of the species, DNA samples would have to be collected from most of its range. But this is where the ubiquitous nature of red muntjacs may be a stumbling block. There are challenges for sampling in so many different places across the red muntjac’s range.

One option she pointed out is getting cell samples from red muntjacs in captivity. This seems like a viable options in India at least. There are 141 Central Government zoos in the country. At least 63 of these have red muntjac exhibits and they are located across the country, including states like Himachal Pradesh in the north, Ooty in the Western Ghats and Madhya Pradesh in central India and Arunachal Pradesh in the northeast.

But does any of this matter? What will having multiple species of barking deer achieve?

This might depend on how we see the business of identifying and classifying species. Taxonomy, on the one hand, satisfies the age-old human need to place the world (in this case the living world) into neat categories. In that sense identifying species and sub-species might seem like a rabbit hole of organisation. On the other hand, sprawling family trees can show us how far organisms have travelled in the evolutionary sense, accumulating extraordinary adaptations and mutations along the way. In this way, they can tell the story of the planet itself.

“I would love to see more research on the taxonomy of this/these species,” stated Martins. “Red muntjacs are truly a fascinating group, from an evolutionary point of view (with for example the karyotypic differences) but also a great model to study how past climatic changes affected the evolution of mammals in a biodiversity hotspot.”

Ramakrishnan, who is a conservation geneticist, suggests that there could be some practical value in resolving the taxonomy issues as well. “It is useful if you’re looking at evolutionarily significant units for conservation. For example, suppose muntjacs become extinct in some location, how should we manage? Should we re-introduce them? Where should we re-introduce them from? And that might also inform things like significant units of management,” she asked.

This might be especially urgent with the Western Ghats-Sri Lanka population according to Martins. “Taking into account the genetic distinctiveness of this population, their spatial restriction and the human pressure on their habitats, it is safe to say that further assessment and evaluation of their conservation status is granted.”

CITATION:

Martins, R. F., Fickel, J., Le, M., Van Nguyen, T., Nguyen, H. M., Timmins, R., … & Wilting, A. (2017). Phylogeography of red muntjacs reveals three distinct mitochondrial lineages. BMC Evolutionary Biology, 17(1), 34.

Timmins, R.J., Steinmetz, R., Samba Kumar, N., Anwarul Islam, Md. & Sagar Baral, H. (2016). Muntiacus vaginalis. The IUCN Red List of Threatened Species 2016: e.T136551A22165292. http://dx.doi.org/10.2305/IUCN.UK.2016-1.RLTS.T136551A22165292.en. Downloaded on 05 April 2019

Groves, C., & Grubb, P. (2011). Ungulate taxonomy. JHU Press.

Wang, W., & Lan, H. (2000). Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny. Molecular Biology and Evolution, 17(9), 1326-1333.

Groves, C. P., & Grubb, P. (1990). Muntiacidae. In Horns, Pronghorns, and Antlers (pp. 134-168). Springer, New York, NY.