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The plot could come straight from a casebook from TV's CSI. Two men are the prime suspects in the murder of their mother. DNA from a single hair left at the scene of the crime ties them to the matricide, but which brother did it? The men happen to be identical twins, so their DNA is identical too, and neither brother is talking. Then the diligent CSI team uncovers a crucial difference between them: Brother A is religiously vegan, while Brother B abhors greens and eats only meat. Running the precious hair through a handy mass spectrometer, a device that measures stable isotopes, reveals the killer is a carnivore. Case closed.

By analyzing an animal's stable nitrogen isotopes, scientists can determine if it is a carnivore, like this lion, or an herbivore, like the zebra it preys upon.

Sound far-fetched? Although this case is pure invention, it's not implausible. In the past ten years, scientists have learned a lot about how measuring stable isotopes in tissues such as hair, feathers, and blood provides insights into an animal's diet and its place in the food chain. And scientists are applying this knowledge to solve a wide array of problems, from determining whether Neanderthals were scavenging or hunting carnivores, to tracking crop-raiding African elephants, and, yes, telling whether a person is a vegan or not.

Scientists at the Smithsonian Migratory Bird Center (SMBC) at the National Zoo are using stable-isotope analysis to follow the routes of migratory birds between northern breeding grounds and southern wintering areas. As a result, they are making remarkable advances in understanding how birds' ecology and behavior during one part of the year influences those during other times—and providing crucial information for migratory bird conservation.

Stable isotopes demonstrate the truth of the old adage, "you are what you eat."

Stable isotopes are forms of an element that work identically in chemical reactions but differ in mass because they have different numbers of neutrons in the atoms that make up the element. For instance, there are two stable isotopes of carbon, referred to as ¹³C and ¹²C; ¹³C has one more neutron than ¹²C. Nitrogen also has two stable isotopes, called ¹⁵N and ¹⁴N. For reasons explained later, these are the stable isotopes, along with those of hydrogen and oxygen, most commonly measured by ecologists. Because the stable isotopes of a particular element differ in mass, it is possible to separate and measure their relative amounts in a sample. This is what a mass spectrometer does.

Turning from chemistry to biology, plants use one of three different methods, or pathways, to convert carbon dioxide to carbohydrates during photosynthesis. Depending on which of these the plants use, the relative amounts of stable carbon isotopes in their tissues differ. Plants that use what is called the C₄ pathway are primarily grasses and have a carbon-isotope signature distinct from plants that use the C₃ pathway, which are mostly trees and shrubs. The third, or CAM, pathway is used primarily by desert plants such as cacti. Further, but for other reasons, marine plants have their own carbon-isotope signature distinct from terrestrial plants.

Pears are C3 plants.

Now here's where this starts to get interesting. Carbon-isotope signatures are passed up the food chain, so that, for example, a grazing animal that eats grasses will have the terrestrial C₄ plant carbon-isotope signature in its tissues, while a browser that eats shrub and tree parts will have the C₃ signature. The carbon-isotope signature of an animal that eats a mixed plant diet will be intermediate. Further, carnivores—both meat and insect eaters—have carbon-isotope signatures that match those of their prey.

Nitrogen-isotope signatures are revealing too, but in a different way. The heavier isotope, ¹⁵N, increases as you go up a food chain. Carnivores have a higher ratio of ¹⁵N than herbivores, and herbivores have a higher ratio than the plants they eat. (Omnivores that eat both meat and plants, surprisingly, have the lowest ¹⁵N ratios, rather than being intermediate between carnivores and herbivores.) So, to go back to our CSI case, the higher ¹⁵N in the evidentiary hair nailed the murderous meat-eating twin.

In a scientific study published in 1999 in the American Journal of Physical Anthropology, T.C. O'Connell and R.E.M. Hedges of Oxford University looked at the nitrogen-isotope signatures in hair samples from people who were vegans, ovo-lacto-vegetarians, and typical omnivores (people are rarely strict carnivores like the hypothetical Brother B). They found a significant difference between the vegans and the other two groups, but not between omnivores and ovo-lacto-vegetarians, because the latter do not eat meat but do eat animal products—milk and eggs—which have the same isotope signatures as beef and chicken.

Scientists have also identified other predictable relationships between stable-isotope ratios and environmental variables. For instance, carbon-isotope ratios vary with latitude and between species living in open versus forested habitats. Terrestrial animals that live in dry habitats have different carbon- and nitrogen-isotope ratios than those living in moist habitats. Stable isotopes of hydrogen and oxygen vary in the rain that falls over different parts of the globe, and thus in the local water available to animals; the isotope ratios found in animal tissue are closely related to those of the local water. Hydrogen and oxygen ratios also vary with latitude, and as you move inland from coastal areas.

The information scientists can gather from measuring stable isotopes in animals has enabled them to resolve some once intractable problems.

Hunter or Scavenger?
Stable-isotope analysis has been a godsend to anthropologists trying to understand the subsistence strategies of early humans and their relatives, including Neanderthals. Were they active hunters who got their meat from herbivores such as deer and wild cattle they killed themselves, or were they mostly skulking omnivorous scavengers who opportunistically stole the kills of other carnivores but relied on plant food most of the time? Stone and bone tools and the remains of herbivores in archeological sites clearly indicate that they hunted at least sometimes, but this is not evidence that they hunted all of the time. Moreover, the association of tools with herbivore remains could as easily be a sign of scavenging as a sign of hunting. And because plant foods decay so rapidly, they rarely appear in archeological deposits.

Isotope ratios in bones reveal the diets of extinct hominids.

To get at the question of what Neanderthals ate regularly, scientists measured stable isotopes in a protein called collagen that was extracted from Neanderthal bones. Isotope ratios in collagen reflect an individual's diet over several years, while an array of butchered bones are merely evidence of a single meal. Studies of stable isotopes in Neanderthal remains from different parts of Europe demonstrate that Neanderthals were pretty strict carnivores, with isotope signatures comparable to those of the carnivores they coexisted with. This means that, like wolves and lions, Neanderthals were active hunters of meat on the hoof.

Zeroing in on Crop Raiders
The body replaces collagen slowly, so collagen's isotope ratios reflect an individual's diet over the course of several years. Hair and fur grow and are replaced rapidly, so analyzing isotope ratios along a strand of hair reveals short-term dietary changes. One woman in the 1999 Oxford study described above switched from an omnivorous to a vegan diet, and changing nitrogen-isotope ratios began to register in her hair within just a few months. She moved from the United States to England at about the same time, which was registered in a similar change in carbon isotopes—U.S. diets tend to be higher in C4 grass plants than northern European diets because Americans consume more corn (a grass) and more meat, eggs, and dairy from corn-fed animals.

A team of scientists, led by Thure E. Cerling of the University of Utah in Salt Lake City, reported using a similar analysis of isotope ratios in hair to study elephants in a 2006 paper in the Proceedings of the National Academy of Sciences. The researchers collected hairs from African savanna elephants (Loxodonta africana) for stable-isotope analysis to see how a population of elephants resident in Kenya's Samburu National Reserve shifted its diet and habitat seasonally. But they were more interested in how the behavior of a migratory male, an old bull that visited the park several times a year, compared with that of the resident elephants.

An elephant tail hair grows at a constant rate of about half a millimeter to just over one millimeter a day. Because each tail hair grows to a length of 500 or more millimeters (almost 20 inches), it records information about an elephant's diet and habitat changes over the course of a year or more.

African elephants' tail hairs tell their dietary history.

Savanna elephants are primarily browsers that eat C₃ plants—trees and shrubs—most of the year. During the rainy season, however, they consume the succulent new grasses—C₄ plants—that sprout with the rain. Over the course of 18 months, isotope ratios measured in the hair of the resident elephants and the migratory bull pinpointed three periods of increased grass consumption during rainy seasons, as expected. But unlike the hair of the residents, the migratory bull's hair recorded a fourth period of grass-eating during the dry season. This was when the old bull was moving out of the forest and into the adjacent croplands to feast on corn.

The ability to identify crop-raiding elephants and predict when they might cause trouble for farmers may help to alleviate the growing conflict between people and elephants, which threatens human lives and livelihoods as well as the survival of elephants.

Tracking Migratory Birds
Work by Russell Greenberg, head of the SMBC, and Peter Marra, also of the SMBC, provides a simple, elegant example of the value of using stable isotopes to answer a question that has long plagued those who study migratory birds: Where do you start to look when the wintering grounds of a migratory bird are unknown? Other work at the SMBC uses stable-isotope analysis to get at an equally challenging problem: If you cannot track individual birds between their summer and winter habitats, how can you determine how events in one season affect those in another?

For migratory mammals and large birds, it is possible to use radio or satellite telemetry to follow short- and long-distance movements, but the required transmitters are too heavy to be attached to small birds like sparrows and warblers. Biologists have also banded an immense number of birds in hopes of sighting the same birds again on their summer or winter grounds. But for small birds with potentially vast geographic ranges at either end of the migratory route, this is largely wishful, "looking for a needle in a haystack" thinking.

Swamp sparrows are fairly common birds that range across the northern portion of North America, from Alaska to Labrador and south through the Appalachians. They nest in freshwater wetlands and migrate to spend the winter throughout the southern United States. In 1951, however, scientists discovered an unusual subspecies, known as the coastal plain swamp sparrow (Melospiza georgiana nigrescens), that nests only in brackish tidal marshes in Maryland, Delaware, and New Jersey. But where birds of this subspecies wintered was a mystery; no one had ever seen one except on the nesting grounds. Yet this information is critical to protecting these birds, whose small nesting distribution and specific habitat requirements make them vulnerable to loss of habitat and other environmental changes. Greenberg found that the subspecies is already disappearing from parts of Maryland, and estimates its total population at fewer than about 28,000 pairs.

Originally, coastal plain swamp sparrows were reported to be year-long residents of the Delmarva marshes. Over the years, Greenberg and others visited the marshes of the Chesapeake and Delaware bays in the winter months in search of the coastal plain subspecies. But they came up dry. They found lots of swamp sparrows, but none of the coastal variety. Clearly, they needed a better way to narrow down their search, so they turned to—you guessed it—stable-isotope analysis.

Isotope analysis helped Zoo scientists track a subspecies of swamp sparrow. (J & K Hollingsworth/USFWS)

Swamp sparrows molt, or lose and replace feathers, twice a year. They molt all of their feathers in late summer or early fall before leaving their breeding grounds for more southerly wintering grounds. In addition, they molt only their crown feathers in late winter or early spring before leaving their wintering areas. This means that the crown feathers collected when the birds first appear in their summer habitat reflect the stable-isotope ratios of the food they eat in the winter, and thus might provide a clue to the location of their winter habitat.

In the summer of 2001, Greenberg's field team collected crown feathers from coastal plain swamp sparrows in Delaware and sent them to the Alaska Stable Isotope Facility at the University of Alaska Fairbanks for analysis. (The SMBC does not have an in-house mass spectrometer, but the scientists there lust after one of these half-a-million-dollar machines!) The carbon and nitrogen signatures suggested a coastal habitat, and the hydrogen signature—remember, hydrogen ratios vary with latitude—pointed to somewhere between North Carolina and Georgia.

The following winter, Greenberg and Marra led a team of ornithologists to scour the coast of Virginia and North and South Carolina, determined to discover the birds' winter whereabouts. Based on the carbon-isotope signatures, Greenberg bet that the sparrows would occupy habitats similar to those on the breeding grounds—the fringe of brackish marsh between the pine forest and the open marsh dominated by salt-tolerant shrubs and grasses. So this is where Greenberg started his search, in Cedar Island National Wildlife Refuge near Beaufort, North Carolina. And he won: The first bird he saw as he waded into the marsh was a coastal plain swamp sparrow.

The team then found 16 more coastal plain swamp sparrows mixed in with other swamp sparrows in North Carolina and southern Virginia, but none in South Carolina. Returning to North Carolina the following December, the team caught and banded a dozen more. These preliminary results suggest that the subspecies makes a short, 150- to 200-mile migration to escape the harsh conditions of the mid-Atlantic marshes.

Redstart Reproductive Success
American redstarts (Setophaga ruticilla) are small insect-eating warblers that breed in moist second-growth deciduous forests over a vast area from southeastern Alaska to Newfoundland, and southward to Utah, Louisiana, and Georgia. They winter over a huge area too, from Mexico, Central America, and the Caribbean to northern South America, where they occupy both moist forests and dry scrub habitats. These birds spend six to seven months on their tropical wintering grounds, two to three months on their breeding grounds, and another two to three months migrating between them in spring and fall.

Peter Marra has been studying redstarts for many years, in the breeding season at his study sites in New Hampshire and Ontario, and during the winter in southwestern Jamaica—but he does not study the same redstarts year-round because where the birds that breed in New Hampshire and Ontario go for the winter is unknown. With redstarts, like many migratory birds, declining, Marra is interested in identifying the factors that influence their survival and reproductive success, and thus their population numbers.

Scientists know a fair amount about how various factors on summer breeding grounds, such as food abundance and predation rates, affect birds' reproductive success. They are also beginning to learn more about how the availability of habitat in winter areas may limit the overwintering survival of birds, and about how both food and habitat are related to birds' ability to survive migration. The missing link, however, has been understanding how events in one period of the birds' annual cycle affect them in the rest of the year because of the difficulty of tracking the same birds throughout the year.

Some large-scale effects that do not require studying the same birds in both winter and summer have been documented. For instance, in a study published in Science in 2000, the SMBC's Scott Sillett and his colleagues used long-term demographic and climate data to determine that the overwinter survival of black-throated blue warblers (Dendroica caerulescens) in Jamaica is low in El Niño years but high when the insects these warblers eat are more abundant in wetter La Niña years. And this effect carried over into the warblers' breeding season in New Hampshire, when fledglings were heavier, and thus more likely to survive, in La Niña years, which produced more of the caterpillars that form the bulk of their summer diet.

Marra's burning question concerned events on a finer scale. The two habitat types American redstarts occupy in winter are significantly different in quality. Moist forests, such as mangroves in Jamaica, support more insects for a greater part of the season than dry scrub habitats. Marra discovered that redstarts compete for territories in lush, moist forests, where older, bigger dominant males win most of the battles. Less dominant males and most females end up eking out a meager existence in the dry zone. By the end of the season, birds living in the better habitat are in better body condition and leave earlier on spring migration than those stuck in the scrub. It stands to reason that this might affect redstarts' future success during the breeding season, but without the ability to match birds from winter to summer, Marra couldn't test this.

Then stable-isotope analysis came to his rescue. In two studies, Marra and his colleagues collected blood samples from redstarts just as they arrived in New Hampshire and Ontario, so carbon-isotope signatures in the samples would reveal the birds' winter habitats. (They tested blood rather than feathers because American redstarts molt just once a year, in the late summer.) Throughout their winter range, redstarts occupy one or the other of these habitats. So even though Marra couldn't look at the same individual birds in both winter and summer, he could tell in which habitat birds he followed in the summer had spent the previous winter. The results, published in Science in 1998, were profound.

Using carbon-isotope ratios from the birds arriving on the breeding grounds, Marra found that males that lived the good life in winter arrived on their breeding grounds as many as two weeks earlier than their less fortunate fellows. This is huge because in most migratory bird species, early birds enjoy greater reproductive success than late-comers, and this was borne out in Marra's redstart studies. Compared to late-comers, early-arriving male redstarts paired with females (which generally start arriving a week or so later than males) sooner, and more often paired with females coming from high-quality winter habitats, suggesting they enhance their success by mating with females in the best condition. Down the road, this head start translates into more offspring that fledge sooner in the season. This gives their youngsters more time than others to fatten up before undertaking their first, grueling migration in the fall. And, if these birds get to their wintering areas in better condition as a result, they may have a better shot at snagging high-quality winter territories. But this remains to be tested.

Why is this important? The coastal mangrove forests and tropical lowland forests that give some American redstarts such a winter boost are rapidly being lost to human uses. As the best wintering habitats disappear, more and more redstarts will be forced to struggle for a living in the scrub habitat left to them. This, in turn, will lead to population declines because birds wintering in scrub have lower overwinter survival rates and produce fewer young during the breeding season.

Victims of Success
As every human parent knows, raising kids, however rewarding, is also a costly, stressful affair. It is no different for bird parents. In a study published in Science in 2004, Marra and his student Ryan Norris demonstrated this for American redstart fathers, which share parental duties, including feeding young, with their mates.

American redstarts molt once a year, after the breeding season. Molting is expensive—imagine replacing your entire wardrobe annually—for birds in terms of energy instead of dollars. And it turns out that male redstarts that invest most heavily in producing young have less energy to spend on replacing their feathers.

Marra and Norris looked at stable hydrogen-isotope signatures in the feathers of known males at his Ontario study site to determine where these males molted the previous fall, which was impossible to find out any other way. Sixty percent of the males molted on the breeding grounds before embarking on migration. Forty percent, however, molted during migration, forcing them to stop for about a week along the way—and these were the guys that had raised the most young during the breeding season. Further, males that raised their young late in the season molted farther south than the early birds.

The upshot of all this is that male redstarts that molt during migration may arrive later in the tropics and find the best winter habitats—moist forests buzzing with bugs—already spoken for. This is bad news because, as we learned earlier, spending the winter in the low-rent scrub district doesn't bode well for a male's reproductive prospects the next summer.

And there's another twist. Male redstarts sport red-orange feathers that reveal their quality as mates and fathers to females. In many birds, females prefer to mate with the most brilliantly colored males—those that can afford to produce the most pigments that paint their feathers. Marra and Norris found that the farther south a male redstart molted, the paler his red-orange feathers, further diminishing his hopes of future breeding success.

Stable-isotope studies like these are shedding unprecedented light on previously obscure aspects of the lives of many animals and providing much-needed information to aid in their conservation. New forensic applications are also emerging. Scientists can trace the geographic origins of cocaine, for instance, to help them identify trafficking routes, and may similarly be able to pinpoint where microbial biological warfare agents were produced in the event of a terrorist attack. Stable isotopes sound as dull as wooden knives, but their analysis is among the sharpest cutting-edge tools in the modern biologist's kit.

—Susan Lumpkin is the editor in chief of ZooGoer and the director of communications at Friends of the National Zoo.

ZooGoer 35(3) 2006. Copyright 2006 Friends of the National Zoo. All rights reserved.