Geonomic Pursuit

For the African forest elephant and other difficult-to-track animals, DNA extracted from dung offers hope for survival.
By Melody Kroll

Genomic Pursuit: For the African forest elephant and other difficult-to-track animals, DNA extracted from dung offers hope for survival.
 

Southern ghana has seen its once-vast rainforests reduced to pockets of parkland, protected areas that, while representing just a fraction of that nation’s former forest cover, nevertheless remain among the most wild and scenic places on earth.

lori eggert;
forest elephants

New genetic census-taking tools are for the first time allowing scientists like Eggert to zero in on individual African forest elephants, recording their movement patterns and home ranges. The data will play a crucial role in developing strategies to save them.

Kakum National Park is one of these places, a terrestrial paradise populated by countless thousands of rare plant and animal species. One of the most magnificent of these, Loxodonta cyclotis, the African forest elephant, is also among its most elusive and endangered.

Forest elephants in Kakum spend their lives wandering restlessly beneath the canopy of the rainforest,foraging for fruit and edible plants as they wind their way through dense, mist-shrouded vegetation. Massive though they may be — adult males stand around 8 feet tall and weigh more than a ton — forest elephants have become adept at avoiding not only the farmers, loggers and ivory poachers who have reduced their numbers, but even those intrepid scientists who have devoted the better part of their careers to preserving them.

Count Lori Eggert among the latter class of elephant hunters.

Eggert, now an associate professor of biological sciences at MU, first traveled to Kakum a decade ago as a graduate student at the University of California, San Diego (UCSD). Her project involved joining the power of genetics with noninvasive sampling techniques to generate a more precise picture of Africa’s forest elephant populations, something previous researchers had struggled to accomplish.

“We didn’t really have a good way to count forest elephant populations,” she recalls. “You can’t see them. You can’t go out and look at a field of forest elephants. They’re hidden in the trees. So we use DNA methods. And I was in fact the first person to do that for elephants.”

Indeed, by the time she arrived in western Africa, Eggert was already one of only a handful of international experts to use noninvasive techniques to do genetic studies on whole animal populations.

MULTIMEDIA PRESENTATION: Genetic Science and Elusive Elephants

Section marker: flies
 

“The very first papers on this came out in 1995,” Eggert says, citing a Molecular Ecology study that used dung and hair samples collected from brown bears in Italy’s Adamello-Brenta Nature Park to estimate both the size of the remaining population and the genetic relationship of those bears to others in Europe. “When I read that paper, I jumped all over it and said, ‘I want to do this; I want to use noninvasive sampling and genetic methods to study populations of elusive species.’”

Thus committed, Eggert spent long hours in the lab perfecting methods for extracting genetic material from scat samples. The next step, naturally, was a trip to where the wild things were, or at least what passed for wild things in Southern California.

“I went down to the San Diego Zoo and collected some elephant dung, brought it back, and started testing methods for extracting DNA and testing different loci you could amplify out of it,” Eggert says. “I spent about a year developing all my methods.”

Persistence paid off, and it wasn’t long before Eggert found herself booking a plane ticket to Africa.

The goal was to use her newly developed methods to explore evolutionary relationships between forest and savanna elephant populations across west and central Africa. Her findings, published in the Proceedings of the Royal Society of London with her doctoral advisor David Woodruff, helped overturn the then-prevailing taxonomy suggesting all African elephants were of a like species. Instead, they suggested that forest and savannah elephants were more likely from separate and distinct genetic lineages.

Eggert’s study also brought added urgency to the movement to preserve the African forest elephant and its dwindling habitats, says Samuel Wasser, director of the Center for Conservation Biology at Washington State University.  It raised the ante, he says, by speaking to “the importance of taking extra effort to really try to conserve the remaining elephants in West Africa.”

what's left behind:
Collecting DNA from dung provides important advantages over more invasive forms of genetic material collection. Below, MU graduate student and Life Sciences Fellow Stephanie Schuttler collects samples in Gabon's Lopé National Park.

Eggert came to elephants via a circuitous route. Growing up in Dallas, she says, girls were told they could be one of  two things: a nurse or a school teacher.  She chose teaching, but after a year of college realized that it wasn’t for her. She dropped out, got married, and started a family. Eventually, she found herself in California.
Eager to resume her education, Eggert enrolled in the biology program at UCSD with the goal of later going to veterinary school. She soon discovered that vet med, like teaching, was not for her. Eggert refocused her attention on animal physiology and general biology, signing on for the master’s degree program in ecology at San Diego State University. There she worked with Oliver Ryder, an adjunct professor and conservation biologist whose lab is located at the San Diego Zoo. With Ryder’s guidance, Eggert found her lifelong focus: using genetic tools to unravel the mysteries of elusive animal populations. She finished her master’s degree and returned to UCSD as a doctoral student, turning her full attention to elephants. She completed her studies in 2001.

Convinced that no one would pay her to study elephant dung for a living, Eggert recalls feeling lucky to “fall over” a postdoctoral position at the Smithsonian Institution. The job involved using genetic techniques to study animals that are neither big nor dangerous: forest birds in Hawaii. But it wasn’t long before elephants stole the show.
“When I gave a talk, all [my colleagues] wanted to talk about was elephants. I had gone in talking about my bird work, and they wanted to talk about elephants,” she says.

Regardless, Eggert spent two years studying Hawaiian forest birds and avian malaria. It was exciting employment, she says, but admits that “her heart was in her elephant work.” As soon as she could, Eggert was off with elephants again — this time in Asia.

Like their African cousins, Asian elephants reside in dense jungle habitats, so obtaining accurate census data was nearly impossible. Impossible, that is, until Eggert arrived on the scene. “The methods I developed applied,” she says. “It was no problem using them to study populations of Asian elephants.”

Section marker: flies
 

Central to those methods are dung analyses, and Eggert is anything but shy about extolling its many virtues. “There’re just a million things you can do with it,” she says, adding that not all of these things require sophisticated lab work.

Consider what one can learn, for instance, from the size of an elephant’s excrement, pieces of which are referred to as “boli.” Bigger elephants produce larger boli. And because bigger elephants are older elephants, data on bolus size can yield insights into the age structure of elephant populations.

Dung is also a critical component in genetic census taking, particularly for forest animals that can’t be counted using visual identification during aerial surveillance flights. Conducting “dung counts,” a technique developed by another scientist formally at UCSD, is also an important technique for estimating populations of hard-to-see animals.

The method, as Eggert explains it, involves starting from a randomly selected point in the habitat then mapping out straight-line tracks, or transects, through the area. These transects become the focal points for what population biologists call an “index count” — the use of one thing, in this case elephant excrement, to estimate the population size of the creature they are interested in; that is, Loxodonta cyclotis.

Field staff count the number of dung piles on either side of these transects. Scientists then “correct” the count for certain variables and use a simple mathematical model to come up with a population number. Based on the number of dung piles found, an estimate of how many times an elephant defecates in a day, and how long the dung piles persist, researchers can tell roughly how many individuals comprise a population.

When done properly, dung counts have proven an accurate and effective way to count census-shy animals. But Eggert knew the method told her only so much. A dung count, for example, yields no information on sex ratios, age ranges, or genetic diversity — all critical in determining whether elephant populations are sustainable. Dung counts also say nothing about the animal’s diet, its stress levels, parasite loads, or whether there are hormones indicating pregnancy.

Most tellingly, as it passes through the intestines, the fecal material scrapes the sides of the intestines and picks up epithelial cells. It is from these cells that DNA can be extracted and, more importantly, the identity of the individual that left it behind can be determined. With data on individuals and repeated sampling, Eggert saw that she could get a more precise estimate of elephant numbers.

At Kakum National Park, Eggert scoped out a dauntingly ambitious plan calculated to take advantage of her genotype-building skills.

She first used latitude and longitude minute lines to divide the park into 15 equal-sized plots. She and a crew of five helpers then used hand-held GPS units to navigate their way along elephant trails in each plot. The team collected samples from fresh dung piles as they went, each time pausing to measure and record bolus circumferences.

Collecting DNA samples from dung, Eggert says, provided an important advantage over more invasive forms of genetic-material collection: Not a single animal would be trapped, drugged, poked, prodded or otherwise annoyed during the course of her investigation.

No doubt about it, Eggert says, noninvasive study has been a game changer for those interested in animal behaviors. “If you want to study the behavior of an animal and you are interacting with that animal,” she says, “you really need to think about whether your behavior is affecting the animal’s behavior.”

Few things, of course, affect an animal’s behavior more dramatically than shooting it, even if only with a tranquilizer dart. Forest elephants are particularly vulnerable.
When tranquilized, they may collapse in a recumbent position; that is, chest downward. If not shifted to their side quickly — something seldom possible in densely wooded forests — the elephant may suffocate before regaining consciousness.

Apart from the animal-welfare benefits, Samuel Wasser adds, it’s the frequency of getting access to samples that makes noninvasive sampling such a powerful technique.

“When you’re trying to cover a large area and get genetic samples from animals, the ability to get access to these genetic samples or other physiological products in the samples is key,” Wasser says. “Being able to dart an elephant to get a tissue sample is not an easy affair. It’s dangerous, and the elephants have a really good sense of smell and a good sense of hearing, and if you miss, you’re probably not going to get a chance at getting that elephant again.”

There are dangers, too, for researchers. Elephants, perhaps not surprisingly, resent being pricked with darts.

“It takes up to 2 kilometers for the elephant to fall,” says Eggert, who has witnessed this process firsthand. “The team is running to keep up with the elephant; it’s hard to keep everyone together, and you don’t know where the rest of the elephants in the herd are during the process.”

What you do know, she adds, is that “they are not happy animals.” 

Section marker: flies
 

Back in the lab, Eggert was able to successfully extract genotypes from 124 of the 205 samples her team collected in the Kakum study. Of the 124 samples that she genotyped, 86 were from individual elephants, while 38 represented “recaptured” dung from the same animal.

Eggert works with “microsatellites” — tandem repeats of DNA — that allow her to build a genotype for each animal. “Microsatellites form a sort of DNA fingerprint,” Eggert says, adding that these DNA fingerprints “are so polymorphic” — meaning they contain distinct heritable traits — “that with as few as four or five of them, you can normally differentiate all the individuals in a population.”

The 86 individuals Eggert was able to identify yielded an unprecedented amount of information about the Kakum herd. Eggert was able to confirm a population number obtained by an earlier study: about 225 elephants. She determined that the ratio of males to females was close to one-to-one, just where it should be. She found, using the bolus measurements, that the age ratio was also healthy: about 80 percent adult to 20 percent juvenile. This meant that the park herd was probably self-sustaining, at least over the short term.

Less reassuring was Eggert’s examination of the elephants’ genetic diversity. Here she found that the fragmented nature of Ghanian parkland was taking its toll on the forest elephants’ ability to retain what are called “rare alleles” — less-common forms of single DNA strands that allow genetic traits to be passed from parent to child, or in this case, from elephant to calf.

Over three generations, Eggert found, rare alleles had declined by 20 percent. “This may signal that we are losing genetic diversity and that this population is suffering from the effects of isolation,” she reported.

Still, she says, these same tools that helped identify genetic problems may well help overcome them. Genetic markers employing microsatellites can, for example, help wildlife officials monitor animals in a genetic version of the catch–release–re-catch method.

“You can find out how isolated they are, how diverse each individual is, and how diverse that population is with respect to others. You can also characterize each individual and figure out its movement patterns and estimate its home range,” Eggert says.

The technology is advancing rapidly too. “My whole Master’s degree was done on 30 birds. I could now genotype 30 birds in less than a week. It took me two years to do that before.”

Such technological advances are making it easier for researchers like Eggert to think in big-picture terms of sequencing and comparing entire genomes with an eye toward identifying genes that lead to adaptation to different environments. On the flip side, the ease in genotyping means questions can be asked at a much finer scale. Instead of queries about whole populations, for instance, scientists can now track individual animals across their entire range.

Section marker: flies
 

These days eggert is focused on an aspect of this big-picture analysis: namely, how the social structure of elephants and other animal populations can be used to explain and predict “genetic structure” over ecologically relevant timescales.

Genetic structure, Eggert says, is broadly defined as the geographic distribution of genealogical lineages; the idea, in other words, that genetic relatedness occurs over both time and space.

“As populations are separated in space or in time, vicariance and dispersal are normally invoked as mechanisms for change and for producing genetic structure,” she says. Vicariance describes the process by which a large population splits into more than one population, generally because of some geological feature such as a large body of water or range of mountains. Dispersal is the process by which a portion of a population splits off to colonize a new area, as when a few individuals disperse from a mainland and colonize an island.

Over short periods of time, or over short distances, other factors come into play. With elephants, social structure is perhaps chief among these factors. Elephants live in matrilineal societies. Females tend to stick around their birthplaces, whereas males roam. This social behavior, says Eggert, is reflected in their genetic makeup. “If you look at their mitochondrial DNA, which is inherited solely from the mother, what you are going to see is much more differentiation than you would if you looked at their nuclear DNA,” she says.

A current study in this vein is focused on forest elephants that visit patches of savanna, or forest clearings. “It has been suggested that these may be places where forest elephants meet and interact to form higher order social groups,” Eggert says. “But currently there is no genetic data to support this hypothesis.”

It also may be that forest elephants behave differently in the savannahs than they do in the forests. Eggert and her lab team are seeking to sort all this out by collecting dung across parts of the landscape in areas well used by elephants. “We’re looking to see if we have any evidence for a cryptic social structure.”

Although Eggert’s specialty is elephants, she and her students use the same approach to ask similar questions about the genetic structure of a number of other animal populations, including many in our own backyard: most notably black bears, raccoons, otters, and hellbender salamanders.

By studying the influences on genetic structures of these different animals, Eggert’s research is shedding light on fundamental ecological principles. “What you learn about hellbenders can give you insight into bears, and what we learn from otters can give us insight into hellbenders.”

Wasser says these larger insights are what truly make Eggert stand out. “She’s a thinker,” he says. “She asks big questions on a continent-wide scale, and there are not a lot of people doing that.”

For Eggert, it’s these big questions that make her research enjoyable. “The really cool thing about my research is I get to ask really neat questions about really neat species,” Eggert says, adding that she prides herself on applying the cutting-edge tools of molecular biology to thorny questions about non-model species. “I take what has been developed in model species and apply them to wild populations to ask really neat questions in ecology.” *

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