The ABC’s of Elephant DNA

My previous blog brought up how difficult forest elephants are to see, and therefore study. Much of the research on forest elephants has actually been on their dung to obtain information about the elephant.

Forest elephants defecate roughly 17-20 times a day, making it an accessible source of information. Traditionally, dung has been used to study diet. Forest elephants consume hundreds of species of plants, either as fruits, bark, or leaves, and sorting through dung piles gives scientists’ detailed information on what they are eating. More recently, scientists have used dung to obtain DNA. But how do scientists get DNA from dung?

DNA is found in nearly every cell in an individual’s body. The best sources of DNA come from tissue and fluids. Scientists who study amphibians will often cut off a portion of an individual’s toes (“toe clipping”) to get a DNA sample. For many species (forest elephants included), sampling body parts simply will not work. Tissue can be accessed though from dead animals, which is important in forensics cases to combat poaching and illegal wildlife trade. Collecting blood, although less invasive than tissue collection, still requires capture, which is stressful, and for many species, anesthesia, which is costly, and a risk to the individual. Within the past few decades, methods have been developed that allow researchers to collect DNA samples without ever even coming into contact with the animal. This is called non-invasive genetic sampling and uses sources such as hair, feathers, shed skin, egg shells, and feces to obtain DNA. It is more difficult to obtain DNA from non-invasive samples than tissue or blood, but it is worth it because it has no impact on the animal you are studying.

For elephants, the best way to get DNA is from their dung (feces). As mentioned before, they defecate often, making it easier to collect a large number of samples. We obtain DNA from dung samples, but the DNA is not in the dung per say. Rather, the DNA is located in the cells that have been sloughed off onto the dung. When the dung passes through the intestines inside the elephant’s body, it scrapes along the walls. When the elephant defecates, some of these cells will be stuck to the dung bolus and fall off with it. That is why it is best to have fresh dung (we use look for dung that is 24 hours old or less). When scientists find fresh dung, they put a small portion of the dung inside a collection tube. It’s easy to tell fresh dung apart from older ones; it has a stronger smell, sheen around it, and is usually intact as a bolus (unless an animal stepped on it or went through it; insects can break it apart and red river hogs will go through it). For importation into the U.S., the sample needs to be boiled within the tube in a bath of hot water to make sure that any pathogens are killed. To preserve the DNA for long-term storage, a liquid buffer is added, turning it into “dung slurry.” These samples are stored in a cooler, dark area of the field station for the duration of the trip.

Once the samples make their way through customs and into the lab, they need to be turned from dung into just the DNA. This involves an extraction process that takes several hours. Briefly, the samples go through a series of steps that involve breaking open the cells (the DNA is inside the cell) and removing the parts of the cells and the sample that are not needed. The sample contains large non-DNA parts including grasses or seeds from fruits the elephant has eaten, and also insects that may have been on or inside the sample when it was collected. The extraction process removes all of this. People often think it’s gross to work with dung samples, but after extractions, you are only working with the DNA, which is basically colorless, odorless liquid that resembles water inside a tiny tube.

In my research, I was able to use dung to identify patterns of sociality in forest elephants. When I found more than one dung pile together and of the same freshness, the elephants were likely part of the same group. The DNA from the dung allowed me to uniquely identify each individual. Therefore, I could keep track of who was hanging out together without ever even seeing them. I found that forest elephants were mostly in groups of individuals of the same matriline (their mother’s ancestry), which is also seen in African savanna elephants. Also they have larger associations than what is observed just from their group sizes – a hidden social network.

The diagram above is a network of forest elephant associations collected from dung samples. Individuals (symbols) are connected to one another by lines if their dung samples were ever collected together. Darker lines mean they were collected together more often. The symbols are a circle if the elephant is a female, and a square if a male. Each color represents what matriline (mother’s ancestry) the elephant belongs to.

DNA is a powerful tool and allows you to answer questions about animals without ever even seeing them. Some of the ways that scientists use non-invasive DNA include species identifications (finding new species or detecting if a species is present in a certain area), population estimates, the connectivity of populations across the landscape (are animals moving between populations?), and inbreeding. These findings are not only important contributions to science, but often critical in the management and conservation of threatened species.

*This post was originally featured on the African Wildlife Foundation

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Windows into the Forest

Elephants are the largest land animals, so it comes as a great surprise that for one species, we really don’t know that much. How can an animal so large be studied so little? Despite their size, African forest elephants are actually very difficult to see.

As their name suggests, they live in the forests of Central and West Africa, and can be easily hidden by vegetation (see photos for proof!). You can spend a whole day walking through the forest and never see them (although they probably see you). The canopy of forest trees also prevents viewing from overhead, making aerial surveys ineffective. Their dense habitat, large ranges, and overall difficult access for humans in the forest make it very difficult to study this species, and are the reasons why we know so little about their basic biology.

 

Despite these difficulties, there is one way to see this species and much of our knowledge about forest elephants comes from these types of observations. Within the forest, there are places called bais, a word from the Mbuti pygmy people, which describes a natural clearing in the forest with large deposits of minerals. Similar to a natural salt lick, the minerals attract all types of wildlife for consumption. The clearings are created and maintained by the heavy foot traffic from animals such as gorillas, sitatunga, and bongo in addition to elephants. Some bais, such as Dzanga Bai in the Central African Republic and Langoue Bai in Gabon are very large and attracted tens, and in Dzanga’s case, hundreds of elephants at a single time. Researchers have set up viewing platforms to observe elephants safely as they come into the bai and forage for minerals.

For my research in Lopé National Park, Gabon, I initially set up a platform at a saline, which is similar to a bai, but runs along a water bed. Although I saw some elephants, I quickly realized that the best place to see forest elephants was in the savanna clearings in the northeastern part of the park. Although Lopé is deep within the forest zone, there are patches of savannas, which are remnant from the Pleistocene Epoch and have been maintained by humans for thousands of years through prescribed burns. These savannas attract elephants as they forage for savanna fruits and grass, which is especially palatable after the burns.

At first, all of the elephants look similar, but once you start watching them, you will start to notice individual differences between them. The first elephant identification projects took place in Kenya, and researchers used the tears elephants get on their ears to identify individuals. Savanna elephants live in drier habitats with more thorny vegetation and therefore have prominent ear tears. Forest elephants tend to have far fewer ear tears than savanna elephants, and for many, no ear tears at all. Therefore, it takes a keen eye and plenty of photographs to be able to tell individuals apart. There are a series of steps that one must go through to deduce which individual is which:

  • First, it is essential to identify the sex of the elephant. The easiest and most obvious cue is behavioral. If the elephant has calves around it, it is very likely a female, especially if it is the only adult in the area. Females are hardly ever seen alone, while males are almost exclusively seen alone. If the individual is seen solitarily, it is probably a male, but look at the overall body and tusk size to make sure. Males are larger and have more girth with thicker tusks.
  • The most obvious difference and the best place to start identifications are with the tusks. Tusks vary from short to long, thick to thin, crooked to straight, and some individuals are even tuskless. Forest elephants tend to have straighter tusks, but variation still does exist and you can find individuals with more curvy tusks similar to savanna elephants.
  • Next is locating ear tears if they are present. Individuals may get ear tears from vegetation, but also predators (this mostly applies to calves, which can be predated on by leopards), and from fighting with each other. Ear tears can range from very small to large, and individuals can also have holes through their ear.
  • If individuals have no ear tears, you can start comparing the ears themselves. Just like humans, elephants have differences in their bodies. Some ears are very large, small, or circular, with prominent to non-existent ear lobes. You can also look at the vein patterns on their ears to help. This requires good photographs and light, and also depends on the elephant. Some individuals have prominent ear vein patterns that can be seen in almost any light and from a far distance.
  • ​Finally tailbrushes can be used to help separate similar-looking individuals. Individuals will have a varying amount of hair on their tailbrush from sparse to full. Some individual lack a tailbrush completely and there are even individuals in the population that have had their tail damaged or cut-off.

Each unique individual is entered into database program called Portfolio (by Extensis), which is actually used by art galleries to sort paintings, sculptures, and other artwork. The reason why it works so well for animals is because it provides a photo for each individual and allows you to input a code that can be used to sort individuals by their characteristics. For example, an elephant coded “FEHLETXRECGTL2F5GTB” would be female (FE), have a high tear in its left ear (HLET), no tear in its right ear (XREC, right ear “clean”), have a longer right tusk than left tusk (GTL, right tusk longer) with a left tusk in the range of 11-20 cm (2F) and a right tusk in the range of 41-50 cm (5G), and a thin tailbrush (TB). These codes allow you to search photos of individuals quickly for certain characteristics. Since 1999, 177 unique adult females and 53 males have been identified in the park.

Studying forest elephants, as in any elephant species takes time. Elephants are long-lived animals (about 60 years) and even studying them for years can only be a snapshot into their lives. Because of the nature of bai and savanna clearing observations, you can’t choose who you study, and you may only see an individual elephant once, and therefore we are still limited on the data collected. Therefore, long-term studies, and complementary, but different approaches, such as genetics and satellite telemetry, are needed to more fully understand this elusive species.

*This blog was originally posted on the African Wildlife Foundation.

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Elephants of Central Africa: Giants in the Dark

Nighttime in a Central African forest reveals a suite of completely new, mostly acoustic, experiences from the day.

Sleep is often interrupted by the clamoring within chimpanzee groups and the screams of a tiny primate, the bush baby (which gets its name from sounding like a baby crying), all above the drone of the regular nocturnal insects and frogs. But nothing is more remarkable than the forest elephant because night allows one the rare opportunity to experience them closely. Feeling protected by the cover of darkness and the absence of humans, they forage right next to inhabited cabins.

I was studying African forest elephant social structure at the Station D’Etudes des Gorilles et Chimpanzes, a field station developed for primate research in the northeastern section of Lopé National Park, Gabon. African forest elephants are a separate species from the well-known savanna elephants of Eastern and Southern Africa. They are less accustomed to tourism and therefore much more elusive than their sister species. In Lopé, with the exception of two males named Billy and Senegal, elephants visit the field station exclusively at night. They are attracted to the mowed lawn surrounding the field station, which serves as a constant source of soft, palatable grasses, in addition to several preferred fruiting trees on site.

Outside my window was a Nauclea diderichii tree, a tree bearing one of the forest elephants’ favorite fruits. When night falls, the elephants slowly emerge from the forest surrounding the field station and crunch loudly on these brown, orange-sized fruits. On nights with full moons, the sky becomes lit, enabling you to see the elephants in detail. Standing behind my window, only a screen, a few planks of wood, and approximately three feet of space separated me from these multi-ton animals. Their gray skin, dry and ridged, would glow in the moonlight; the gentle sounds of huffing and swishing as they pulled grass with their trunks made them sound almost like giant horses grazing in a field.

One night, something deviated from this typically serene event. I was awakened by elephants, but could immediately sense they were behaving strangely. From my bedroom I listened as they moved brusquely about the station. I heard the splitting and tearing of branches as they were ripped off fruiting trees and the clatter of garbage cans being broken into. Although this behavior wasn’t normal at Lopé, it was not outside the realm of elephant behavior. After an hour, the elephants were still circling the main building of the station. It was then that I heard something distinctly unusual: the sound of breaking glass. The elephants were breaking the windows of the main building.

The ruckus of the agitated elephants continued around the site, and it had been nearly two hours since I was first awakened by their presence. It was at this time that I heard the unnerving sound of the elephants closing in on the cabin I was in, which consisted of four bedrooms and a bathroom. The twisting sounds of metal pipes being bent and the dull thumping of their bodies against the wooden walls indicated that they were breaking into the bathroom. Outside of my room an elephant tried to climb the stairs leading up the front porch. It pushed my door in with its trunk, but the door held, luckily, being locked from the inside.

The thumping was approaching my bedroom walls, and the reality of the flimsy planks of wood standing between the several highly agitated, multi-ton elephants and me suddenly closed in. I decided it was no longer safe to stay in bed. I leapt out of bed to the opposite side of the room. There was no time to grab a flashlight and the moon was dull, so I only saw darkness. I heard wood breaking and falling, but the noise was coming from inside my room. There were two other researchers in the rooms next to mine and I called out “They’re coming in my room!”

One of the researchers answered back and we decided I should move to his room. I listened carefully outside my door to make sure no elephants were there and quickly snuck into the room next to mine. The researcher didn’t have a flashlight so we lit a candle. As we were whispering what to do, the elephants indicated their displeasure with several rumbles (deep growling noises) and trumpets. We blew the candle out immediately to try to appease them. A few seconds afterwards one of the elephants let out a long, deep rumble. We remained quiet, frozen in place, and then after a minute, they finally left for the night.

The next morning we surveyed the damage. They broke several windows including the panels and screens of mine, tore the shower curtain and rod off, pulled on pipes, and pulled items out of rooms. In the history of the field station’s 25 years, elephants have never tried to break into any of the buildings. There didn’t seem to be any patterns. If they were interested in food, they would have spent more time in the kitchen or have tried to break into the pantry. Elephants can also be interested in scents, for example those in shampoos or soaps. Although they tore into the bathroom, they didn’t ingest or open toiletries, and seemed to be equally destructive around the rest of the field station. While I don’t know why this particular incident happened, I do know that the elephants in Lopé are aggressive. When walking in the Lopé forests, guides stop frequently, listening and smelling for signs of elephants, and anticipating charges around every bend in the trail.

Hypotheses about aggression in elephants in Lopé center around the park’s history in poaching. As Lopé elephants have been poached heavily in the past, and although they are still poached, threat has alleviated in the northeastern section of the park because of research and tourism (although this currently may longer be true). Elephants may have become more aggressive as they still associate humans with poaching, but are less fearful and now stand their ground, a behavior also observed in gorillas. Another hypothesis is that many of the Lopé elephants may have lost their mothers to poaching when they were younger, and were exposed to the trauma and/or may not have been properly raised. For example, in savanna elephants, when only younger males were relocated to a park in South Africa, they began killing rhinos. Adding older males to this population ended this because they were able to suppress the hormones causing these behaviors in the younger individuals (Slotow & van Dyk 2001).

Regardless of the reason for aggression, poaching is a severe threat not only to elephant numbers, but also elephant society. In savanna elephants, populations that have been poached have higher stress levels, lower numbers and survival of calves (Gobush et al. 2008), and are less able to effectively respond to the calls of predators such as lions (McComb et al. 2011). These groups usually lose the matriarch of the family, which is the eldest female and desirable for poachers because tusks grow larger with age. As elephants are long-lived species, and have good memories, losing this individual affects the whole group, as generations’ worth of knowledge about the ecosystem is lost.

Poaching has reached its highest levels in recent years because demand for ivory is increasing, especially as more people moving into the middle class in China and are able to display their wealth with ivory statues or jewelry. Although my night with the “crazy” elephants was the scariest moment of my life, I respect the elephants and advocate for their conservation. Previous to my research in Gabon, I worked in Kenya for a year. There, I was able to see some of the most tolerant elephants in the world in Amboseli National Park, Kenya, where they have been exposed to tourism on a daily basis and have been researched for over 30 years. These elephants are so easygoing that you can park your car right next to a group and they will remain there, casually eating, so close that you can almost reach out and touch them.

The elephants are merely responding to the situation they are in, and their situation is becoming progressively worse. Even the Amboseli elephants are no longer immune to the effects of poaching with several having been killed this year. The demand for ivory has become so high, that 62% of all forest elephants have been lost in only the short period of 2002-2011 (Maisels et al. 2013). The methods of poaching have also exacerbated. Now elephants are killed by well-regulated militia with AK-47s and poachers closely tied to terrorist organizations. With elephants living in such a heightened and violent state, their only hope for survival is to run, hide, and fear people if we are not there to protect them.

*Originally posted on the African Wildlife Foundation’s website

Gobush KS, Mutayoba B, Wasser SK (2008) Long-term impacts of poaching on relatedness, stress physiology, and reproductive output of adult female African elephants. Conservation Biology 22, 1590-1599.

Maisels F, Strindberg S, Blake S, et al. (2013) Devastating decline of forest elephants in Central Africa. PLoS ONE 8, e59469.

McComb K, Shannon G, Durant SM, et al. (2011) Leadership in elephants: the adaptive value of age. Proceedings of the Royal Society B 278, 3270-3276.

Slotow R, van Dyk G (2001) Role of delinquent young “orphan” male elephants in high mortality of white rhinoceros in Pilanesberg National Park, South Africa. Koedoe – African Protected Area Conservation and Science 44, 85-94.

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