As much as any scientist before or since, Charles Darwin changed the way we view the world and ourselves. He showed us the continuity of life, from one part of the world to another and from one epoch to another. He also proved to be an excellent prophet.

It was 145 years ago that Darwin published his landmark work, The Origin of Species. In it he outlined his theory of natural selection as the means by which living things evolve, and he predicted that with this theory, "A grand and almost untrodden field of inquiry will be opened, on the causes and laws of variation, on correlation of growth, on the effects of use or disuse, on the direct action of external conditions, and so forth."

He was right. Not only was a grand field of inquiry opened, but it has continued to fascinate and confound Darwin's philosophical descendants to the present day.

Among them are eight members of the ECU biology faculty whose research is featured here: Dr. Jason Bond, Dr. Carol Goodwillie, Dr. Trip Lamb, Dr. Susan McRae, Dr. Jean-Luc Scemama, Dr. Edmund Stellwag, Dr. John Stiller and Dr. Kyle Summers. They study plants, reptiles, spiders and birds. Some work in fields Darwinwould recognize — taxonomy and morphology — using many of the same techniques, but with the added advantages of modern molecular science. Some look at what makes populations diverge. Others concentrate on survival techniques. Still others delve into the brand new field of evolutionary developmental biology, "evo devo" to the hipper biologists. It is not merely life on Earth that has evolved. Science has, too.

And because it has, Darwin's descendants worldwide are making advances at a mind-boggling pace: adding 2,000 new species a year to the botany world list alone, showing connections among living organisms never before imagined and tracing lineages nearer and nearer to the beginning of life on Earth. As so often happens, the more we know, the less certain we become.

"We had very nice, clear ideas about evolution 50 years ago," Stiller said. "It's much more complicated now. We are closer to understanding how nature works, but we have a much less defined story.

There are so many kinds of data coming in and so many ways of looking at them, that it will take a while. We're going into a period in which there will be lots of competing ideas, and it will take time to sift them all out. In our understanding of ancient evolution, I'd say we're back to many of the same questions we had before we started sequencing molecules, but at least now we have ways of addressing those questions.

THE POWER OF SAND

Ships' surgeons on foreign trips often sent specimens of flora and fauna back home to 19th century Britain. For a survey of South America, Capt. Robert FitzRoy proposed something different. He thought it would be enlightening to be accompanied by a full-time naturalist. Barely out of college and eager to see the tropics, Charles Darwin seized the opportunity. The journey of the H.M.S. Beagle stretched to nearly five years and allowed Darwin to explore the South American continent, the Galapagos Islands, New Zealand, Australia and parts of Africa. Though seasick for much of the time, Darwin never regretted his 40,0000-mile trip. "(I)t appears to me that nothing can be more improving to the young naturalist, than a journey in distant countries," he wrote in The Voyage of the Beagle.

Trip Lamb's journey has taken him to the dunes of the Namib and Kalahari deserts and the rocky coast of South Africa, all in search of lizards. Southern Africa supports as many endemic species of lizards as most whole continents can claim. They range from inch-long leaf-toed geckos to Angolosaurus, a herbivore measuring up to 18 inches in length.

Six years ago, Lamb and Aaron Bauer, a colleague from Villanova University, set out to discern whether the origin of these species could be traced to ancient geologic upheavals that separated ancestral populations. As they wrapped up their project recently, Lamb said they have learned a separate, equally important lesson: the power of sand.

At times together and at times on separate journeys, Lamb and Bauer collected close to 100 species of lizards for study. Some had not been observed in the field since the 1950s. Others were never before recorded in scientific literature. It's easy to understand why. Searching out desert dwellers, Lamb and Bauer once traveled off road across sand dunes for five days. During that time, they never saw another human being. Almost as hard to sight were some of the lizards. Angolosaurus in particular "is the most leery lizard I've ever encountered," Lamb said. "They can spot you a couple hundred yards away and dive a foot or more into the sand. You mark where you think they are and hope you can dig them up."

In a monograph being published by the California Academy of Sciences, Lamb and Bauer have rewritten the evolutionary history of southern Africa's geckos, showing close relationships in unexpected directions among these lizard families. Instead of sticky feet for clinging and climbing, the dune-dwelling geckos have modi- fied feet for digging. One genus, Palmatogecko, has developed extensive webbing between the toes to facilitate walking and burrowing in loose, wind-blown sands.

Traditional taxonomy had pegged these geckos as distinctive genera, occupying distant branches of the gecko family tree. Lamb's DNA analysis tells a different story. All three genera of dune-dwelling geckos fall within a widespread lineage of "typical" geckos that climb on rocks and trees. Moreover, the dune dwellers are more closely related to species with whom they share little outward appearance than they are to each other.

The story is repeated for dune specialists in other lizard families. Modified feet, spadelike jaws for diving into the sand and scaled flaps to keep grit out of ears yield distinct forms long recognized as separate genera. In each case, the dune dwellers turn out to be closely related to non-dune species. Furthermore, the ancestors of these dune dwellers entered this harsh habitat at different times in history and evolved similar adaptations to the sand independently of one another.

The upcoming monograph, together with a series of other recent publications, depict newly redrawn family trees for five lineages of southern African lizards, each of which includes dune and non-dune species. In addition to expectations of divergence driven by geologic forces, Lamb and Bauer have documented remarkable examples of convergence. "One of the interesting things revealed in our study has been the powerful influence of the Namib and how it has similarly shaped the morphology of different lizard species," Lamb said. "There's just a limited number of ways to design a lizard, or anything else, for life in loose sand."

BACK TO BASICS

A century before Darwin, a Swede named Carl Linnaeus developed a system for naming, ranking and classifying living organisms. Although his Systema Naturae remains the basis of taxonomy today, the field for a time lost much of its luster. Gene splicing carried so much more panache than fitting beetles into a family tree. Harvard's E.O. Wilson believes the trend has come full circle. "Systematics has returned to the center of the action in biology," he wrote in The Future of Life. The molecular techniques that once led biologist away from taxonomy now bring them back, providing new tools to speed the discovery of organisms and to define relationships across species.

The trend has reversed none too soon for Jason Bond. "We're seeing the mass extinction of species around the world now, and it's caused by us, by human action," he said. "I'd like to think there's been a hard-core realization of what the crisis entails." Finding solutions, he said, focuses new attention on taxonomy. "Species and how we define species are at the core of any questions we might ask in biology and how we define ecosystems. É It would be impossible to develop conservation policy and management approaches if you have no idea what's there."

Bond has channeled his concern over such questions into the study the Mygalomorphae order of spiders, which includes tarantulas, funnelwebs and Bond's special interest, trapdoor spiders. He and a collaborator from San Diego State University are charged with documenting mygalomorph taxomony, diversity and kinship for the National Science Foundation's project Assembling the Tree of Life. They will sample up to 500 species of mygalomorphs worldwide, trying to clarify relationships to ensure that a family tree includes all related species and their most recent common ancestor. "Our classification system should reflect the evolutionary relationship," he said.

Mygalomorphs appear to date back 450 million years. More primitive in physiology and silk production than their web-spinning kin, they live relatively long lives — as much as 20 years for trapdoor females — but also fairly sedentary ones. Nearly all trapdoor spiders, for example, live in underground burrows that they cover with a hinged flap engineered out of silk and soil. They seldom change address.

Long lives and a lack of wanderlust make trapdoor spiders good subjects for evolutionary studies. "They have a strong tendency to become isolated so they're a well-designed model for looking at simple population subdivisions," Bond said. The same traits, however, put them at risk. One shopping center can wipe out the habitat for a whole species. "In Southern California, the diversity we're losing you could put a stopwatch on," he said.

Studying trapdoor spiders has its chalcally lenges. Whether he's in Australia or South America, South Africa or California, Bond spends long hours looking at the ground.

"They're incredibly cryptic," he said. He recalled hiking up and down rocky ravines to find one Australian species. After three hours in a driving rain, he gave up. "We got back to the car and there was a little bank not a meter from the car and they were all over the bank."

Even when found, trapdoor spiders don't give up easily. Sensing prey, they can spring the door for a lightning-speed capture, but in the face of danger, they seal the door so tight even a determined biologist can barely pry it open. Bond brings live samples back to the lab, to be photographed and measured. With a graduate student, he is trying to use mathematical models to help define spider shape. DNA sequencing will help reconstruct relationships, to show how characteristics have evolved, perhaps as far back as the breakup of continents. Such modern tools are dramatically changing the way biologists view species. "Morphological observations probably understate the extent of genetic diversity out there," he said. "We're seeing extreme molecular diversity." A few of Bond's specimens live in terrariums in the lab, but most are sacrificed. "The longer I do this, the harder that becomes," Bond said. "I limit the number I bring back for that reason. It takes me days to kill them, and I'm in a bad mood when I do, but I think it may be the only hope for the species."

EVOLUTION IN ACTION

Just 100 million years ago, a monochromatic color scheme bathed the planet. Ferns, mosses and conifers supplied shades of cooling green. Then flowering plants appeared and, in what Loren Eiseley called "a soundless, violent explosion," blanketed the Earth with all the colors of the rainbow. Their seeds and fruits provided the energy to fuel the larger brains and higher metabolism of warm-blooded creatures. Birds took to the sky, and on the ground the age of reptiles gave way to the age of mammals. "Flowers changed the face of the planet," Eiseley wrote in The Immense Journey.

"Without them, the world we know — even man himself — would never have existed." As they aided the rise and diversification of other species, flowering plants achieved a remarkable diversity of their own. The diversity shows not merely in size, color and shape, but in the ways they reproduce. A few types, such as hollies, have separate male and female plants, but most species have both male and female parts in the same flower. Among these, some will fertilize themselves while others have evolved showy flowers, sweet nectars and easily dispersed pollen to help them exchange gametes with others of their kind.

Some clever flowers not only promote crossfertilization but recognize and reject their own pollen, which botanists refer to as self-incompatibility. In a tiny pink flower from northern California, Carol Goodwillie has discovered yet another reproductive strategy: transient self-incompatibility, a peculiar trait that may provide a glimpse of evolution in action. Goodwillie was a doctoral student at the University of Washington in 1995 when she and her adviser stumbled across Leptosiphon jepsonii, a previously undiscovered plant that has now been found in a several isolated pockets of the Napa Valley. Jepsonii hid its secret well. To all appearances, it could self-fertilize, just as its near relative Leptosiphon bicolor did. In one sense, self-pollination confers a tremendous advantage. When the plant no longer needs to attract pollinators, flower size can shrink over evolutionary time, conserving energy for other uses, including seed production. Just one little problem. "Some think that self-fertilization is a genetic dead-end," Goodwillie said. "You lose genetic variation and with it, the ability to adapt to change. So in family trees, you see selfing at tips of branches, usually not on the trunks. The idea is that it leads to extinction."

Goodwillie had moved to ECU and Goodwillie had moved to ECU and was in her eighth year of studying jepsonii when a series of unrelated experiments opened her eyes. On the first day of a bloom's four-day life span, it rejected its own pollen but accepted the pollen of others. Starting on the second day, it could self-fertilize.

"There was a clear-cut difference from day one to day two," Goodwillie said. "This had never been described before but I can easily imagine that someone could miss it." Jepsonii seems to have the best of both worlds. If its pollinator — the small, hovering beefly — is in abundance, genetic variation can be enhanced through cross-pollination. But if the beefly is scarce, as happens some years, the plant at least stands a chance of producing one more generation. Goodwillie, however, suspects this is a transition phase, not a stable adaptation.

By sampling different populations of jepsonii, she has found a full range of variation in the plant's ability to self-fertilize: some are fully self-fertilizing, some are self-incompatible and some display transient self-incompatibility. "So clearly, this is evolutionarily dynamic," she said. "We're looking at a slice of time. This is an opportunity to watch evolution in action."

Goodwillie's questions are almost as numerous as the jepsonii she has raised in her greenhouse. Early cross-breeding experiments indicate that a single recessive gene allows jepsonii's self-compatibility, but will this hold true for all of the populations? Are the different groups of jepsonii evolving in the same direction and at the same rate? Will she be able to locate the gene responsible for transient self-fertilization? Why, if jepsonii's transient self-incompatibility actually confers the best of both worlds, is its range so restricted?

Most of all, if this is a transition phase, is jepsonii on its way to fully crosspollinating or to fully selfing? If the former, the shortterm advantage that self-compatibility confers may spell doom for this small pink flower. "Evolution can't look forward," Goodwillie said. "It can't plan ahead. Evolution is always running to catch up. Any species or population is well-adapted to what's happened over the last hundred or thousand years, not necessary to now and not to the future. So as the environment changes, the ultimate fate could be extinction. Natural selection is just optimizing the fit for right now."

SURVIVAL OF THE FITTEST FROGS

In Darwin's Ghost Steve Jones wrote: "Nature does not favor beauty, or strength, or ferocity; all it can do is to advance those best able to multiply themselves. Although its products include the most beautiful and most repulsive beings, there is no mystery to Darwin's machine; it is no more than genetics plus time." He makes it sound so simple and straightforward, yet mysteries abound. What is it that gives an animal, plant or bacterium the reproductive edge over those of its own kind? Clearly, the first struggle is to survive, against high odds, long enough to reproduce. After that, what does it take to attract a mate? Is it better to have a large number of offspring or to invest high levels of energy into a few, improving their odds of survival? Kyle Summers has focused his search for answers on poisonous frogs found in Central and South America. He began by studying patterns of parental investment, that is, how much time, energy and effort the frogs spend in producing and rearing their young, and how that varies by sex.

Among frogs, most of the parental care falls on the male, a pattern also common in fish. Summers has spent up to nine hours in the dense undergrowth of a rain forest observing a single frog as it carried tadpoles from the leaf litter where they hatched to pools of water where they could grow and mature. Parental care, Summers found, is not always benign. "A male frog will try to mate with as many females as possible, but the more offspring, the less attention he can give to each," he said. "In some cases, he will feed one clutch to another, for example, if he can't find enough separate pools of water in which to deposit them." Field experiments and observations helped Summers sort out what kind of pool parents prefer for their maturing offspring, how pool type affects offspring survival and whether tadpoles can recognize and avoid cannibalizing their kin. DNA sequencing enabled him to determine how these and other behaviors evolved. As he worked, new questions intrigued him. Prominent among them was the relationship between coloration and toxicity. For one project, he studied 21 species of frogs, comparing the level of toxicity with the intensity of color and plotting their family trees based on DNA analysis. The patterns he found supported theories that bright colors protect the frogs by advertising their unsuitability for the dinner menu to potential predators.

With Dr. Rainer Schulte, a collaborator in Peru, he then investigated a different color issue, mimicry. In the most widely recognized form, called Batesian mimicry, nontoxic species evolve to resemble a toxic one — benefiting from predators' recognition of the warning colors. One example is the Viceroy butterfly, a nontoxic species that looks nearly identical to the unpalatable Monarch.

Summers believes he has found a different type of mimicry in poisonous frogs. In three locations in north central Peru, Schulte had previously identified pairs of poisonous frogs that looked similar but were clearly different species based on traits such as egg color and mating calls. With DNA analysis, Summers and Schulte showed that three of the frogs — one from each set — belong to the same species, but in each locale they have taken on the coloration and pattern of a different, more distantly related poisonous frog. They suggest this is evidence of a phenomenon called Mullerian mimicry, in which toxic species provide mutual benefit by sharing the cost of training predators to keep their distance. "This (pattern) is only consistent with mimicry," Summers said. "Otherwise, it doesn't make sense."

AVIAN INSTINCTS

Animal instincts nearly stumped Darwin. How could natural selection explain why the European cuckoo lays its eggs in the nests of other birds? Failing to explain such instincts, he said, could overturn his whole theory. By the time he wrote The Origin of Species, Darwin had worked out his argument with the help of slave-making ants and bees that build geometrically perfect cells first time, every time. "No one will dispute that instincts are of the highest importance to each animal," he wrote. "Therefore I can see no difficulty, under changing conditions of life, in natural selection accumulating slight modifications of instinct to any extent, in any useful directionÉ. This theory is, also, strengthened by some few other facts in regard to instincts; as by that common case of closely allied, but certainly distinct, species, when inhabiting distant parts of the world and living under considerably different conditions of life, yet often retaining nearly the same instincts."

Susan McRae shared Darwin's interest in birds that lay their eggs in other birds' nests, a behavior called brood parasitism. Studying moorhens, she found consistent patterns of intraspecies brood parasitism but a range of responses to it. From England to Panama and Namibia, she observed females that would deposit eggs in other moorhens' nests.

The victimized English moorhens incubated the foreign egg along with their own, even when McRae painted the intruder's egg a bright red. Related species in Namibia and Panama, on the other hand, usually reject the foreign egg — the Namibian birds by burying the offender, the Panamanian hens by tossing it overboard. What accounted for the different instincts in closely related species?

"There was a fairly simple explanation, though it took a lot of work to figure it out," she said. "In England, parasitism isn't costly. Most of the parasitic eggs were laid after the host had finished laying her own clutch so most never hatched. The incubation period is not particularly costly — it's the feeding stage that takes so much energy — so if they don't hatch, it's not a problem.

In Africa, though, breeding is more synchronous because it's so dependent on rain so (without rejection techniques) the host birds could raise offspring that are not their own."

As McRae studied English moorhens, she noticed something peculiar. Although most family groups consisted of a single breeding pair and their young, occasional territories included a second breeding female. The second female was almost always the daughter of the first, and she mated with her father. "Incest is unusual in birds, and this might be a case of evolutionary lag," she said. "The population might be artificially dense so retention of offspring may be unusual and they haven't yet adapted incest avoidance."

The observations launched McRae on an inquiry into social evolution of birds: how the instinct for group living evolves, how the birds solve conflicts such as who gets to breed and how they avoid incest. African weavers, which live in groups and appear to have strong instincts against incest, offered a wealth of opportunity for study. They range from the white-browed sparrow weaver, whose adult offspring help raise the parents' new clutches, to the sociable weaver, whose apartment-style nests house as many a 100 birds, including several breeding pairs and their non-breeding, adult helpers.

With a colleague from Cornell University, McRae is studying the gray-capped social weaver of Kenya. Family groups in this species consist of the dominant pair and their sons, who take mates from outside the family. McRae is concentrating on the laboratory side of the project: teasing out the DNA fingerprints that allow her to determine paternity and, with that, to reconstruct the pedigree of the birds they're studying.

"We're interested in how they partition reproduction," she said. "Subordinates' nesting creates strife because of the limited resources. We've observed in the field some egg-tossing — birds throwing out each other's eggs — and we're just starting to look at who's doing it. It probably has a lot to do with conflicts over reproduction."

Eventually, she wants to do comparative studies of all the weavers, to understand the genetic, ecological and social factors that influence the degree to which they cooperate when it comes to building nests, breeding, raising young and establishing and defending territories. "What drives me at moment are the environmental influences," she said. "Group living is not something birds do under ideal circumstances. It's often the result of habitat saturation. When food or other resources are limited, it becomes a necessity. Sociable weavers, which have the apartment-style nests, live in the harshest area of southern Africa. Very few of other types live there. That's a clue. If you live in those areas, you have to adopt this strategy to survive."

EVOLUTION'S MASTER GENES

Little in the natural world escaped Darwin's interest, but morphology — with its questions about the form and structure of organisms — held special appeal. "This is the most interesting department of natural history, and may be said to be its very soul," he wrote in The Origin of Species. "What can be more curious than that the hand of a man, formed for grasping, that of a mole for digging, the leg of a horse, the paddle of a porpoise, and the wing of a bat, should all be constructed on the same pattern, and should include the same bones, in the same relative positions?"

What Darwin found curious has become all the more intriguing. Scientists now know the bones shaping those hands, legs, paddles and wings resemble each other because all these creatures descend from a common ancestor that lived hundreds of millions of years ago. They've also learned that a relatively few genes are involved in directing the placement of these and other body parts, and those genes bear remarkable similarities across species. Now, scientists are digging deep to understand what these similarities mean. Among them are collaborators Ed Stellwag and Jean-Luc Scemama.

Stellwag explained the larger question this way: "How do you think it is that humans evolved such that our arms are extensions from our shoulders? Why aren't they located somewhere else? Why do we have five fingers and not seven? Most evolutionary biologists will say, selection. We've been selected to have those. And I agree. What we're trying to get at are the relationships between developmental mechanisms and their role in the evolution of morphology."

Their focus is on what Scemama called the master genes in evolution. Dubbed Hox genes, their role in specifying morphology was first identified through research in fruit flies, where alterations in an individual gene resulted in dramatic and, after much study, predictable mutations in the structure of the fly. Simple mutations in a copy of one of these Hox genes, for example, would result in a fly with two midsections, each with its own set of wings, creating a fly with four wings instead of two. Other interesting characteristics also distinguish Hox genes. They are relatively few in number, occur in clearly defined clusters and appear in the order in which they are expressed during embryonic development. That is, the genes that direct development of the head will be at one end of the cluster, those dealing with midsection will be in the middle, and those specifying the posterior section will be at the other end, a highly unusual organization among genes. "These genes are unique in the way they are organized and have been organized throughout evolution," Scemama said.

Among vertebrates, more interesting developments have occurred. The closest living relative to the common ancestor of all vertebrates is a small wormlike sea creature commonly called a lancelet. The lancelet has one cluster of 14 Hox genes. As vertebrates diverged from their common invertebrate ancestor during evolution, they duplicated the original Hox cluster. Lineages comprising mammals have four, for example, and the zebrafish has seven — but so far, no other species studied has retained all 14 genes in each cluster, and species vary in which genes they've retained in their different clusters.

Two hypotheses could explain this phenomenon, Scemama said. One is similar to redundant duplication: two genes sharing the work that one originally did so that together, they equal the one original gene. The second theory he called neofunctionalization. "It could be one kept the function of the ancestor gene and the other duplicate took a new function," he said. "It is complicated to understand because the common ancestor that existed prior to the gene duplication no longer exists."

Also curious is why the major lineages of vertebrates have the number of clusters they do.

Early on, scientists speculated that the number of clusters would be related to morphological complexity. Since fish seem to fall between the wormlike lancelet and a mammal in terms of complexity — a fish has fins, which the lancelet lacks, but not a mammal's seemingly more complex limbs — it was expected to have an intermediate number of clusters. Instead, it has more.

"Everybody's still wondering what that means," Stellwag said. "The only hypothesis anyone has come up with is that maybe Hox genes have a role not only in the determination of morphological complexity but also in the capacity for evolutionary diversification. That is, the more of these genes a lineage has, the more readily it can diversify its morphology, not in a developmental sense, but in an evolutionary sense. The only support for this theory is that the lineage represented by the ray-finned fishes is thought to have a greater number of species than does the lineage to which mammals belong."

Unraveling the mystery of vertebrate Hox genes means tackling a broad range of questions, from when and how they are expressed in the development of an embryo to whether the corresponding genes in different species — whether fish, mouse or man — have the same role in development. "Obviously, fish morphology doesn't look like human morphology, but that doesn't mean it couldn't be specified in a similar way," Stellwag said. "Or maybe, if the gene expression is different, that's why the morphology is different. In fish, maybe the region of cells that is specified by a particular Hox gene is greater than or less than the region that is specified in humans, and that would influence the structure."

As in most areas of science, the big questions must be tackled in small segments. For their segment, Stellwag and Scemama decided to study striped bass development and compare it with the best known of fishes, the zebrafish. They narrowed it even further to the development of the jaw.

They soon discovered a difference between the two fishes, which diverged from their common ancestor 150 millions years ago. Where the zebrafish had two copies of a specific gene involved in making the jaw, the striped bass had three.

A photograph taped to Scemama's lab door provides insight into the next step of the process. It shows a vibrant green glow in the otherwise transparent outline of a fish embryo. They had successfully "tagged" a gene they were studying with a fluorescent copy. As the embryo's cells multiplied and migrated to form different parts of the fish, they could follow the action directed by this gene.

"The presumption is that Hox genes specify the destination of cells, like a postal code," Stellwag said.

Then they ran into difficulty. To fully understand the gene's function, they need to be able to alter its expression in the developing embryo — blocking it in some cases and in others stimulating the expression in a region where it does not normally occur — and study the resulting malformations in the adult fish. Striped bass, however, proved particularly difficult subjects for genetic manipulation, and because they spawn only once a year, progress would be slow in coming.

So the researchers recently switched their focus to tilapia, which breeds year round and is more easily manipulated. Although tilapia is a close relative of striped bass, they will have to step back and duplicate their initial studies in identifying and tagging genes.

Eventually, they hope to transfer genes from one species to another, to see whether and how they affect the jaw structure of the fish. "That's not a trivial step," Stellwag said. "Just because we put a gene in a zebrafish doesn't mean it will be expressed because all the other mechanisms necessary for expressing the gene may have been lost evolutionarily. But when it works, you can get valuable and sometimes dramatic results."

He pointed to experiments with fruit flies and mice. Mutant flies were created that were deficient in a particular Hox gene. When the corresponding Hox gene from a mouse was inserted into the fruit fly, it recovered its normal function. The reverse also held true — corresponding genes from a fly could function in the mouse.

"That was shocking when it was done," Stellwag said. "This really goes back to show these genes are descended from a common ancestor, and they have retained over enormous lengths of evolutionary time certain deep, ancestral functions."

CONNECTING THE TWIGS

Back when Darwin explored aboard the Beagle, living things were easily classified. They were animal or plant, with little confusion between the two. Since that time, whole new categories of life have been discovered, more numerous and distinct from one another than Darwin's generation ever dreamed of. Other discoveries have muddied waters, showing similarities where once there were clear distinctions. "The new taxonomy has transformed the tree of life into an exotic plant," Steve Jones wrote in Darwin's Ghost. "Men and chimps are indeed more related than are men and bananas, but humans, insects, and plants are, the DNA shows, all mere twigs on the same branch."

John Stiller has begun to take a hard look at this branch, which includes life's most complex organisms, in hopes of finding one of the smoking guns of evolution. With support from an NSF Faculty Early Career Development Award, he is looking a billion years back in time for that moment, metaphorically speaking, that organisms developed a new way to regulate genes. With this ability, their cells can differentiate, assuming different shapes and functions, even changing throughout their lifetimes. Organisms from the other major branches retain relatively uniform cell shape and function.

"Even red algae that are large, multicellular organisms don't differentiate tissue so that one type of cell is an eye or a foot or a leaf or a root," Stiller said. "They have weird ways of making cells twist together and form outwardly complex forms, but they don't differentiate cell type."

The reason, he suspects, is that they evolved before his smoking gun was fired. The gun in question has to do with something called the C-terminal domain, a protein structure found on an enzyme that transcribes genes into their biochemical messages. This domain serves as a platform for binding many other proteins involved in the process. Those proteins in turn are responsible for controlling complex maneuvers, such as putting an eye in its proper place or telling a seed to sprout a root on one side and a shoot on the other.

"The fact that the dominant organisms that we know are the same ones that have this C-terminal domain, I don't think is coincidental," he said. "One of the reasons we have evolved such complexity, and such a diversity of complexity, is because we have a series of key underlying mechanisms that arose during our evolutionary history. I think this is one of them."

Stiller wants to learn when and how the C-terminal domain, or CTD, originated and what that has meant for the evolution of more complicated forms of gene expression. As part of his search, he has been crafting evolutionary trees that identify which organisms have the Cterminal domain and which don't. As he looks at those trees, one thing jumps out. Organisms that evolve the CTD-based gene expression keep it, without exception.

"It's surprisingly easy to lose functions in evolution," he said. "A fish moves into a cave and loses its eyes. Take something as fundamental as an arm, lose it and evolve a wing instead if that's more adaptable. Very complicated structures are lost repeatedly in evolution. But nothing we have looked at so far has lost this domain. So something profound happened to lock the CTD into the basic core functions of gene expression. Once you have taken this step, whatever it is, you can't go back, you can't lose the CTD. Once it was there, the CTD proved tremendously useful. You can lay all kinds of proteins on it and make thousands of different complicated organisms from very similar genetic material. So here (in the step that locked CTD in place) is the foundation for the development of a lot of complex organisms, as well as tremendous evolutionary diversity, and I want to know what that is."

Organisms with the C-terminal domain vary considerably, so to find the domain's original, core function, Stiller is trying to identify the biochemical properties they have in common and that are missing from organisms lacking the domain. Dr. Zhenhau Gua, a postdoctoral researcher in his lab, has identified one likely set of proteins. These proteins are critical to the CTD's ability to initiate action and appear to have evolved about the same time as the domain itself. "Have we demonstrated the C-terminal domain was locked in at that point because of this co-evolution? No, but I think we are on to something," Stiller said. "Will we fully understand this 'smoking gun' during my lifetime? Probably not, but it is a good start." In time, Stiller said, understanding the evolution of the C-terminal domain will further define relationships among the major groups of organisms. "One example is this issue of red algae," he said. "I have been involved in a philosophical and scientific debate over last five or six years, based on different interpretations of different molecular analyses, as to whether red algae and green plants are related to one another. É It's been hard to pin down." But similarities and differences that don't appear when looking at the external structure may become apparent at the molecular level. "If we can say to other researchers, look, we have a C-terminal domain along with the following critical functions that are absolutely conserved in plants and animals, and red algae just don't have them, doesn't that mean that plants are more closely related to animals than they are to red algae?" he said. "Then we will have defined evolutionary relationships based on something that's both very complicated and clearly shared, and it will be difficult to argue it's just an aberration in the data." EDGE

---

East Carolina University News Bureau 1001 East Fifth Street Greenville, North Carolina 27858 252.328.6481 | 252.328.6300 fax

Contact Us