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Winter 1995

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Following Evolution's Footprints Through Nature
by David Hart

The whole thing started innocently enough. A handful of research projects on unrelated species in the Southeast -- sparrows, horseshoe crabs, pocket gophers, oysters and bluegills, for example -- independently turned up the same pattern.

What all these animals share is a distinct genetic break between Atlantic and Gulf Coast regions. Most populations weren't different enough to be considered separate species, but analysis of the animals' cells revealed that the Atlantic and Gulf Coast "families" rarely intermixed and probably hadn't done so for hundreds of thousands or even millions of years.

So far, UGA genetics research professor John Avise and graduate students in his lab have detected this pattern in about two dozen Southeastern species that live in ocean, freshwater and land environments. Not all the studies started out looking for this pattern; nonetheless, there it was.

They had found an answer to a question no one had asked before. In fact, the data they turned up didn't really fit into the language of any existing scientific discipline. So Avise and his students made one up -- or at least coined a term to describe the field and the subset of evolutionary questions that seemed to belong to it.

"It wasn't the case that everybody sat around thinking, 'We need to have a field called phylogeography. Let's go out and develop some molecular tools to do it,'" Avise said. "It wasn't that way at all.

"Molecular tools came along and provided data of a sort that had never been available before," said Avise, who has been named to the American Academy of Arts and Sciences and the National Academy of Sciences for his work with molecular markers. "This got people thinking along new lines and led to the development of this term and this whole field it encompasses, which is a historical approach to population biology."

To put it another way, phylogeography is the study of how geography plays a part in phylogeny, an organism's evolutionary history. Avise and his students track these geographical changes by the footprints they leave in the animals' mitochondrial DNA (mtDNA) -- the DNA in cells' power plants.

In the case of the Southeastern marine species, for example, Avise suggests a scene like this: Beginning a couple of million years ago during the Pleistocene Epoch, advancing and retreating glaciers changed climates and sea levels, which combined to isolate the Gulf of Mexico and the Atlantic Ocean populations of animals.

That's one possible reading of the story recorded in just a few molecules. In this new field, not only does the past unfold in mtDNA studies, but with just a small twist of logic, Avise and his former graduate students use the same sort of analyses of mtDNA and other molecules to find ways to protect endangered species today.

Molecular Conservation

The phylogeographic patterns detected by Avise and his students provide glimpses into the heavily debated question of speciation: When are two organisms different enough to be separate species?

It's a hot potato of a technical topic, but it has down-to- earth implications for conservation. Whether a population of animals is a bona fide separate species or merely a regional variant affects how that animal is treated by conservation measures.

Genetic differences on top of visible physical differences between organisms may support the naming of a new species. "The more lines of independent evidence you have, the better chance you have for declaring a new species," said Trip Lamb, one of Avise's former graduate students who detected the Gulf-Atlantic separation in diamondback terrapins. "It's unbelievably contentious. It shouldn't be, but it is."

Slight differences in mtDNA also can help unravel the mystery of how geography affects a species' survival. In fact, geography may play a much more important role than previously thought. Current approaches to conservation, such as the U.S. Endangered Species Act, often focus on one species at a time; however, considering Florida panthers, green turtles or seaside sparrows in isolation is probably not the best way to proceed, Avise said.

"Genetics can contribute by revealing evolutionary patterns of  [organisms], whether they're endangered or not, geographically across an area," said Avise, whose latest project is a book, Conservation Genetics: Case Histories from Nature, about genetic perspectives on conservation. "It's a developing viewpoint that I think is going to be increasingly widely adopted."

Many former graduate students from Avise's lab also have taken molecular analysis into conservation fields. Kim Scribner looked at similar species of Southeastern mosquito fish which normally don't mate to form hybrids. He and Avise wondered whether the species would intermingle if geographic barriers were eliminated. They experimented in the lab, in natural environments and even in Biosphere 2. In all three cases, the fish hybridized in unexpected patterns -- patterns which were reflected in their genetic makeup.

"Without the molecular markers, we would not have been able to do the study at all," Scribner said. "The use of molecular markers was absolutely critical."

Now, as project leader in the molecular biology lab for the National Biological Survey in Anchorage, Alaska, Scribner uses molecular tools to study waterfowl, fish and marine mammals. "Basically, almost all the work I'm doing now is involved with conservation of natural resources," he said.

Lamb, in addition to finding the genetic break in diamondback terrapins, also studied hybrids. By using different tools to examine proteins, in addition to mtDNA, he was able to distinguish hybrids of two species of tree frogs. Normally, the two species wouldn't mate in the wild, but because of altered environments, they had. Molecular markers allowed Lamb to identify the hybrids, even though they didn't necessarily look like hybrids and the mating behavior was never witnessed firsthand.

Now an assistant professor at East Carolina University, Lamb has set his sights on the threatened gopher tortoise. He detected two phylogeographic divisions in that species -- one break at the Apalachicola River and a smaller break in south Florida. Knowing these boundaries can help conservation workers decide where to relocate tortoises that have lost their habitats to human development, he said.

While these studies have a regional impact, Avise and Brian Bowen, another former student, have shown that the secrets in mtDNA can echo around the world.

Rescuing Sea Turtles

Because sea turtles start life as tiny hatchlings on isolated beaches and spend 20 or 30 years in the ocean before reaching reproductive age, the logistics of sea turtle research have always been tricky. Early studies of tagged females found that they returned to the same nesting beaches each year. This led researchers to suggest that sea turtles "natally home" -- that is, return to the beach where they hatched -- to lay their eggs.

The problem was proving it. Molecules came to the rescue.

Avise and Bowen, now director of the conservation genetics core at the University of Florida's Biotechnology for Ecological, Evolutionary and Conservation Sciences lab, realized that mtDNA held the answer to this question. Mitochondrial DNA has two properties that make it ideal for studying sea turtle behavior, as well as phylogeographic patterns. First, it evolves rapidly, relatively speaking. Second, it is passed along strict maternal lines. In other words, mtDNA records an organism's maternal family tree; it is analogous to a female surname. So straightforward logic allowed Avise and Bowen to address the question.

"If it's true that female turtles natally home, almost every nesting population ought to be genetically distinguishable from every other one with respect to any genetic traits transmitted through females, such as mitochondrial DNA," Avise said. "On the other hand, if female family names are all mixed across nesting colonies, that would be evidence against natal homing."

The logic wasn't complicated, but even with molecular methods, the logistics were a big hurdle.

"Way back in the Dark Ages of genetics -- the mid-'80s -- you needed fresh tissue for the lab  [procedures]," Bowen said. That was a problem when they were collecting samples on remote Costa Rican beaches, 20 miles from the nearest road, and had to get those samples to the lab in Georgia. "All it needed was the do-or-die determination that only a graduate student could give it," he said.

They solved the problem by collecting lethargic hatchlings who wouldn't have escaped the nests on their own -- the first of the 99.9 percent of hatchlings fated never to reach maturity. (Today, getting to remote beaches is still tough, but the development of the polymerase chain reaction allows Bowen to use a single drop of blood as his sample, and collecting live samples is not an issue.)

The molecular evidence, when all was said and done, pointed to natal homing. It turned out that every beach had its own set of family names -- mtDNA markers -- which typically appeared at no other beach. Since earning his doctorate, Bowen has continued his molecular research on sea turtles, with results that could shake up international conservation efforts.

Each year between 20,000 and 50,000 loggerhead turtles are snared in fishing nets in the Mediterranean where they feed; 20 percent to 50 percent of them die. The mtDNA markers gathered by Bowen and Luc Laurent of France at the feeding sites matched many of the markers previously collected by Bowen, Avise and others. The markers showed that 57 percent of the turtles at Mediterranean feeding sites had originated from nesting beaches in the Western Atlantic.

Besides adding further support to the natal homing theory, these results give the United States a legal hook for protecting these endangered animals. According to a 1982 United Nations convention, because the United States provides the necessary nesting habitat, it has some jurisdiction over protecting migratory turtles at any stage of the life cycle.

"What starts out as an esoteric natural history study provides the foundation for the conservation of animals on the high seas," Bowen said. His current research is using these same molecular techniques to ask similar questions, again with a strong conservation slant.

For example, Cuba harvests hawksbill turtles from its reefs for their shells. To avoid conflict with the Convention on the International Trade in Endangered Species (CITES), Cuba assumes a model in which only turtles nesting on Cuban beaches feed in Cuban reefs.

"We think that's hokum," Bowen said. A survey of seven of the largest Caribbean nesting sites, in collaboration with Dr. Anna Bass at Louisiana State University, identified genetic markers for Caribbean turtles, all of which were found in Puerto Rican feeding sites (since Cuba refused access to its reefs).

"Population data firmly refute their harvest model," he said. "They're impacting species from around the Caribbean." These results were brought up at the recent CITES meeting last November to force Cuba to halt its turtle harvests. At the meeting, Cuba backed off from turtle harvesting, and instead announced an initiative for sea turtle ranching, Bowen said.

Catching a turtle on the reefs will always be cheaper than raising one, he added, but "with genetics techniques, it's possible to tell when non-ranch turtles enter the marketplace."

Such techniques weren't available even five years ago, but it's no surprise that Bowen, Avise and others have turned these methods towards conservation goals.

"It's always been an underlying theme in my lab," Avise said, "to retain a cognizance of how whatever we find might relate to conservation biology, at what I see as a real critical time in human history, when we're losing biodiversity at an almost unprecedented pace."

Evolution Revolution

Which sea turtles feed where, whether they return to the beaches where they hatched, and other questions of conservation biology and phylogeography are just some of many applications in molecular evolution that Avise describes in his recent book Molecular Markers, Natural History and Evolution. The book chronicles how studies of DNA and proteins are revolutionizing the way scientists view the world.

In the same way they helped create phylogeography, analyses of mtDNA and other molecules have formed the basis for the field of molecular evolution. Do these novel approaches mean researchers should just toss out all the research since Darwin and start over?

"That's what I think we're struggling with as a discipline right now," Avise said. "We, meaning molecular evolutionary biologists, should not lose sight of our broader roots, which go back to natural history studies, ecology, biogeography and a host of other disciplines that lay the foundation for where we are today."

Instead, molecular methods provide new tactics for attacking old questions. For example, systematics and population genetics have long staked separate claims to the study of evolution above and below the species level, respectively. But the division had always been artificial -- imposed by scientists, not by the nature of organisms, Avise said. By approaching questions from a different route, molecular techniques bypass the conventional wisdom on what can be asked.

"I think there's a real integration of these disciplines taking place now and I would argue that that's largely been due to the leading influence of molecular approaches," Avise said. "Mitochondrial studies have been particularly influential in that regard because the nature of the data literally forces you to think phylogenetically even at the within-species level."

That just wasn't done before. Molecular methods offer ways to cross the species boundary seamlessly. In the same way that Avise and his lab use mtDNA to study an organism's recent evolutionary history, related molecular methods can uncover details about the organism's place on the tree of life.

"The different tools and the genetic systems they're applied to -- the interaction of those two things -- determine your window of resolution," Avise said. "Whenever you're dealing with a particular problem, you have to adjust the focus of your lens."

Since the 1960s, a slew of methods for focusing on the genetic innards of plants and animals, including mtDNA analyses, has been added to the scientific toolbox. It's hardly surprising that a scientific revolution occurred once scientists began getting a closer look at the messages encoded in DNA molecules.

"Indeed, almost all the really insightful molecular applications involve not just molecular examinations, but integrating that information in some innovative way with pre- existing information from other sources," Avise said. "And that's where the real breakthroughs occur.

"The techniques themselves really precipitated this revolution, but it has become more than that," he said. "It's become a revolution in the way people look at evolution, because we have new windows on the world -- new ways of viewing nature through molecules."

For more information please check out http://www.genetics.uga.edu/faculty/bio-Avise.html or e-mail avise@bscr.uga.edu


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