With a growing number of species going extinct or teetering on the brink, conservation biologists are concerned that even species that manage to increase their numbers will later succumb to sudden, devastating infections. Susceptibility to disease can be one consequence of genetic impoverishment — which may happen when a large, apparently recovered population has only a few forebears. But such low genetic diversity is thought to cause generally low species-fitness, resulting in lower reproduction and poor survival. This is why conservation genetics is becoming ever more important to scientists struggling to ensure the long-term persistence of endangered species.
A genetic bottleneck occurs when a species’ population numbers decline to such a level that they are insufficient to maintain genetic diversity. The UC Natural Reserve System provides research access for three different species that either have experienced or are currently experiencing such bottlenecks: (1) island foxes (Urocyon littoralis) at Santa Cruz Island Reserve off the coastline of Ventura County, (2) desert bighorn sheep (Ovis canadensis nelsoni) at the Sweeny Granite Mountains Desert Research Center in San Bernardino County,* and (3) northern elephant seals (Mirounga angustirostris) at the Año Nuevo Reserve in San Mateo County.
The science beneath the surface
A species’ genetic diversity is determined by the number of alleles — variants of specific genes within the overall genome — that are present across the population. Having multiple alleles available for each gene locus across a genome enhances a species’ ability to adapt to environmental changes. Even in individuals that are heterozygous at a dominant locus (where two different alleles are present at a single locus, but only one is expressed), the retention of the recessive allele is important because it provides flexibility. Populations that are genetically variable at a given locus are referred to as polymorphic; those that lack diversity are called monomorphic.
When scientists gauge the genetic diversity of a species, their standard practice has been to look at microsatellite site locales. These short, highly variable DNA sequences distributed throughout a genome have little impact on an animal’s fitness and therefore are under little or no selective evolutionary pressure. The combination of high variability with negligible selective pressure makes these sites ideal neutral markers for quantifying a species’ relative genetic variability.
More and more, scientists are also looking at the major histocompatibility complex (MHC), a set of genes that determines an animal’s disease resistance and influences kin recognition (and, therefore, reproduction). While microsatellite locales are neutral, because they have no impact on an animal’s fitness, MHC locales are highly adaptive, subject to strong selective pressure by variation in pathogens, and have a direct impact on fitness and survival.
There is no direct correlation between microsatellite variability and MHC variability. Some species considered monomorphic, based upon microsatellite studies, are nevertheless diverse at the level of their MHC. The island foxes on San Nicolas Island in the southern Channel Islands, for example, are considered the most monomorphic sexually reproducing animal population ever recorded, based upon microsatellite analyses. But when Andres Aguilar, then a graduate student at UCLA, looked at five loci in the MHC, he found remarkably high levels of variation. “Neutral markers are still important for showing variability,” Aguilar notes. “But in this post-genomics era, we’re going to have more markers that are possibly fitness related, and using those is going to be very important for conservation and maintaining genetic diversity.”
Captive breeders closely managed
The island fox (Urocyon littoralis), a scaled-down relative of the mainland gray fox, inhabits the six largest Channel Islands that lie off the southern California coast near Santa Barbara. Archaeological records and molecular genetic data indicate that foxes colonized the three northern Channel Islands (San Miguel, Santa Rosa, and Santa Cruz) about 16,000 years before present (B.P.), when a few gray foxes (Urocyon cinereoargenteus) washed ashore from the mainland. The animals adapted, as island species usually do, to the limited resources of their new habitat by becoming much smaller in size than their mainland ancestors. Many centuries later (between 4300 and 800 B.P.), Chumash traders, who were excellent seafarers, transported the now-diminutive foxes to the three southern Channel Islands (San Nicolas, Santa Catalina, and San Clemente). Over time, the fox populations on the different islands diverged sufficiently so that today each island has its own genetically distinct subspecies.
In the early 1990s, the fox populations on all three of the northern Channel Islands suddenly crashed. Scientists soon identified the problem: dramatically increased predation by a non-native golden eagle population that had moved over from the mainland (for more information, see Transect 20:2, [Summer 2002], page 1). The island territory became available to the golden eagles after the native bald eagles, which would have driven the golden eagles away, were wiped out by DDT contamination. The bald eagles, which fed heavily upon fish, were vulnerable to discarded DDT that infiltrated the marine food chain; the golden eagles preferred to feed on tender, easily caught island foxes.
Fox losses were dramatic. The Santa Cruz Island population plummeted from 2,000 animals in 1993 to less than 100 only seven years later — a survival rate of less than 5 percent. On San Miguel Island in a similar period, numbers of foxes fell from 450 to just 15 — approximately 3 percent survival. And on Santa Rosa Island, they fell from 1,500 in 1994 to 14 animals six years later — less than 1 percent survival. UCLA Professor Robert Wayne, a specialist in canid genetics, explains how the situation developed and what the effect was: “The foxes were very naïve. They lived on the islands for thousands of years and were not used to having aerial predators swoop down and eat them. And their numbers weren’t really large enough to recover from this kind of threat, in a genetic sense. It was clear that they would be extinct before they mounted some behavioral evolutionary response to this new threat.”
On the advice of a scientific panel that included Wayne, the National Park Service (NPS) acted to save the remaining populations. In 1999, NPS captured all of the remaining foxes on both Santa Rosa and San Miguel Islands and brought them into pens where the animals would be protected from golden eagle predation. Two years later, working with The Nature Conservancy (TNC), NPS initiated a similar program on the much-larger Santa Cruz Island, bringing 12 animals into captivity and allowing between 60 and 100 foxes to remain in the wild. The strategy on Santa Cruz Island, where the NRS reserve is located, was to give the fox populations a chance to recover, while other teams of wildlife biologists worked to move the predatory golden eagles from the islands and to restore the native ecosystem (by reintroducing bald eagles and by removing feral pigs).
Genetics was the primary consideration for the captive-breeding program. NPS biologist Tim Coonan directs the Island Fox Recovery Program: “It’s the primary factor in all three of our captive-breeding programs. It determines what animals we put together, and it will also determine who gets released into the Miguel Islands, where each founding population consisted of fewer than 20 individuals. The usual method is to avoid kinship pairings. As Bob Wayne explains: “You want to pick individuals to breed who are maximally unrelated. You want to decrease the amount of inbreeding in the population to preserve the maximum amount of genetic variation and manage the whole genealogy genetically. But it’s a very small captive colony they’re dealing with, so their genetic management choices aren’t that great. They’ll have to tolerate some level of inbreeding.”
Maintaining diversity was especially challenging in the instance of the island fox, because, as is the case with most island species, each individual carries only a small fraction of the total genetic variability of the parental population. According to a study by Melissa Gray (one of Wayne’s graduate students), while mainland gray foxes have 75 percent heterozygosity at specific microsatellite locales, island fox populations on the nearby northern Channel Islands have only between 16 and 30 percent heterozygosity — roughly, between 20 and 40 percent of the genetic wiggle-room possessed by their larger cousins.
The second genetic challenge stems from the fact that small populations lose alleles simply because their numbers are low and subject to major fluctuations. When a population dips, the loss of even a few key individuals can mean the total loss of important alleles.
Gray’s master’s thesis on the relatedness of the island fox populations provided important clues for the breeding program. “People often assume that all founding individuals in a captive-breeding population are unrelated because they’re from a wild population,” she explains. “But that’s actually not true, so you have to make sure you’re taking that into account when you’re doing your statistical analyses. In this case, we found that the founders of a subset were highly related, so we needed to make sure we were pairing them correctly.”
Using data assembled by Gray and other researchers, the park service established a detailed breeding strategy for each subspecies. The American Zoological Association, which keeps studbooks on endangered species held in zoos, also maintains a studbook on the island foxes. Each year they use a complex set of kinship formulas and analyses to identify the most appropriate matings.
Soon after the foxes were brought into captivity, however, a third problem emerged: not all of the foxes were breeding, and this further reduced the founding population. Coonan explains: “In captivity, you have some good breeders and some that are not so good. And the ones that are not so good are actually more important, because you don’t have their founder genes represented yet.” The question became why? Why were the nonbreeders not breeding? Was there a problem with the care the foxes were receiving? Was there a problem with how they were being confined? Or something else? Getting an answer quickly was crucial because nobody knew when the males would go into senescence and stop breeding entirely.
Fortunately, as adjustments were made, the previously uncooperative captive foxes did begin to breed. Then, as their numbers grew, a new question soon presented itself: which individuals should be released? Just as captive breeding is carefully thought out to minimize inbreeding, so too, releases must be carefully considered to prevent the loss of rare alleles. “Each year they’ll have a number of candidates for release,” Wayne says. “But the advisory committee has recommended that they only release animals whose genes are well represented by other animals in the captive population, so if they’re lost, at least no valuable unique genetic component is lost along with them.”
Meanwhile, a complementary relocation program had reduced the number of golden eagles on the islands, but some birds were still present and could still be deadly. Releases of island foxes were successful in 2003-04. Of the 17 foxes released that season on San Miguel and Santa Rosa Islands, eagles killed only one, on Santa Rosa. In 2004-05, however, things didn’t go as well. Although, once again, no foxes were taken on San Miguel, eagles killed 5 of 13 foxes released on Santa Rosa. Losing nearly 40 percent of foxes painstakingly bred in captivity and released into the wild prompted the park service to initiate a re-capture program. However, as biologists began to pull the released foxes back in, they discovered that some had already set up dens and were reproducing all on their own. The recall was cancelled.
Today the captive-breeding program for island foxes is in its seventh year and making slow but steady progress. Tim Coonan provides a brief report: “As of March 2006, there are 65 foxes for the San Miguel subspecies, including 39 in the wild. For the Santa Rosa subspecies, there are 68, including 34 in the wild. And on Santa Cruz, there are at least 150 in the wild — and that’s a conservative estimate; there may be more than 200.”
Even more significant, the foxes are not only surviving in the wild, but are also breeding in the wild on all three islands. Coonan, who has been working on this problem since 1999, is cautiously optimistic: “Depending upon how well they continue to breed, both in captivity and in the wild, the program could go on for six to eight more years on Santa Rosa and San Miguel. But on Santa Cruz, we might be able to get out of the captive-breeding business much more quickly.”
The island fox isn’t out of the woods yet. Even as their numbers increase, biologists will continue to monitor them. Wayne, Gray, and Coonan are currently collaborating on a test project that will allow them to estimate the populations by using scat samples. If their approach proves successful, the new process will allow them to estimate population sizes readily, without the time and expense involved in having to capture foxes and outfit each one with a radio collar.
For her part, Gray is currently expanding her study to include all subspecies and the entire genome. “I’m looking at the genetic basis for the fox’s dwarfism,” she says. “But, beyond that, I’m really interested in how this bottleneck has affected the foxes on a genomicwide level. Most studies look at only a handful of markers, between 10 and 20. My goal is to use the latest technology to go much larger, on the order of a couple of hundred markers, and get at what happens to a population overall. I’m definitely in this for the long haul.”
Taking the high ground
Desert bighorn sheep (Ovis canadensis nelsoni) inhabit a number of small, isolated mountain ranges throughout the Sonoran, Mojave, and Great Basin deserts of the U.S. Southwest. They live in small populations of usually less than 100 individuals. In California, biologists estimate the desert bighorn sheep population at about 4,300, spread across a territory that extends from the White Mountains east of the Sierras to the Mexican border. Their numbers are relatively stable, but during the last 60 years, 30 of 80 populations within the state have gone extinct. To maintain the species, scientists have moved sheep into seven ranges where extinctions have occurred. Three natural recolonizations have also been observed in recent years.
UC Berkeley postdoctoral researcher Clint Epps has spent much of the last six years tracking desert bighorn sheep throughout the California deserts. Epps often uses the Natural Reserve System’s Sweeney Granite Mountains Desert Research Center as his base of operations. “I started my genetics work, right across the road (Interstate 40) from the Granites in the Marble Mountains,” he says. “I had originally anticipated that I would sample maybe six populations, but as I discovered I could follow them with genetic tests, chasing the next ridge became a bit of an addiction. I ended up doing about 30!”
Epps’s population genetics tests were conducted on the animals’ fecal samples, which contain DNA. “I was really interested in the question of metapopulation dynamics, extinction, and colonization,” he explains. “Colonization and dispersal have been difficult to study, because they’re relatively rare. It doesn’t take many individuals moving back and forth to maintain these processes, and the odds of detecting such movements are not very high. With genetics testing, we can match relatives and track individuals. In two cases, I detected the same sheep in two different nearby mountain ranges at different times, just from their droppings.”
In studying the flow of genes across the desert, Epps discovered that genetic diversity varies greatly between populations (based on results at 14 microsatellite loci). The Sweeney Granite Mountains bighorns, for example, are quite diverse and thus serve as a source population for recolonizing neighboring ranges. “The Granites are high elevation, which makes them ideal territory for desert bighorns,” Epps points out. “And they’re well connected with the Providence Range, which, in turn, is connected to another small chain of populations. Taken together, this area is one of the most genetically diverse areas in the desert.”
In the Newberry Mountains just outside of Barstow, however, the population’s genetic diversity was found to be just half of that in the Granite Mountains population. Epps attributes this circumstance both to recent bottlenecks and to that population’s geographical isolation. The Newberry Mountains animals live on an extremely remote range at the western edge of the territory, surrounded by long stretches of flat desert. Epps has found that desert bighorns will cross about 5 to 10 kilometers of flat ground, but seldom go much further. Such isolation can be devastating to small populations.
Epps has identified two major human-created obstacles that interrupt the movement of animals between populations: freeways and canals. Freeways are often fenced to prevent cattle from wandering into oncoming traffic, but they also prevent bighorn sheep from finding one another as well. A freeway has cut off the Newberry Mountains sheep population from its only known source of new animals. Even in the Granite Mountains, the heart of bighorn sheep territory, freeways have had an impact, as Epps discovered early in his study.
“I found populations just to the south of the Granites that should have been within easy dispersal range,” Epps recalls, “but an interstate built through the area in the 1960s cut off any interaction. And when I started working up my genetic samples in the lab, I noted that there was a surprising amount of genetic distance between these populations across the interstate and that the populations to the south were less genetically diverse. That’s when I really started getting interested in the impact of the interstates. One reason I sampled so widely was to increase my sample size so I could make these comparisons.”
Global warming is another major factor affecting the movements of bighorn sheep and their ability to intermingle dispersed populations. Over the last century, the mean annual temperature in southwestern U.S. deserts has risen by 1.8 degrees Farenheit, while annual precipitation in California’s deserts has dropped 20 percent. Epps believes these conditions are already affecting the region’s bighorn sheep populations. “Losing 30 populations over the last 60+ years is an unsustainable rate of population extinction,” he points out. “And those extinctions weren’t random — they especially hit populations in lower, dryer ranges that lacked dependable springs. All of those are parameters related to climate.” If global warming continues or even (as is often predicted) accelerates, the desert bighorn sheep population will become increasingly fragmented.
Epps is working on a number of possible solutions for the immediate problem of bighorn mobility. “One thing I’d like is to see what we can do about restoring the connectivity between the Granites and the Marbles to the south. The ideal thing for desert bighorn sheep would be a big overpass, but the odds of that happening are pretty low. There are some large culverts in the pass where the interstate cuts across the north end of the Marbles, but they’re fenced. I’m hoping CalTrans can open those back up.”
“The Granites and the Providence Mountains and that region are really key,” he continues. “To ensure the long-term persistence of desert bighorn sheep, you have to maintain those core areas and the connectivity with the more outlying areas. You don’t want to give up on those outlying areas. If disease or mountain lions hit the higher, wetter populations, those peripheral populations might be the saving grace.”
Epps also hopes land managers will use his research and that of his colleagues to guide their handling of the different populations of desert bighorn sheep. By understanding the flow of animals and genes between populations, land managers will be able to react quickly when a local bighorn population goes extinct. If the area is still connected, they can wait for it to be recolonized naturally. If the area is not still connected, they can bring in bighorns to get the population going again.
“It’s crucial for each of these small populations to have new individuals come in from other populations,” Epps explains. “Otherwise, you will eventually lose that population. That’s a standard feature of metapopulation models. The population may hang on for a long time, but if you don’t make it possible for new animals to move in, you’ll eventually lose these areas. You’ll always have populations winking out here and there, but the system as a whole should remain pretty stable as areas are recolonized. Disrupt that, however, and you run the risk of breaking down the entire system. The harsh environment inhabited by desert bighorn sheep already has them walking on a knife’s edge. It doesn’t take much to push them off. The bottom line is that more than one-third of the population that was once known is now gone, and we could lose them all.”
A monomorphic profusion
Burney Le Boeuf has been conducting research at the Año Nuevo Island Reserve, off the Santa Cruz coast, for almost four decades. It was 1968, just a few years after the northern elephant seals began having pups on the island, that he and a few other scientists started their investigations. Over the next nearly 40 years, Le Boeuf, his colleagues, and his students would lead the way in demystifying the lives and lifestyles of these giant animals who spend months in the open ocean. “They really live in two worlds,” Le Boeuf explains. “For much of the year, they’re in the open ocean, diving to great depths, dealing with high pressure, darkness, and cold. At other times, they come ashore to reproduce on land.”
Long-term studies conducted by Le Boeuf and his colleagues have revealed much about the elephant seals — their history, feeding patterns, mating strategies, vocalizations, annual travels, factors that determine the health of pups, and ways they deal with oceanic changes. But for all of these scientists’ successful accomplishments, one common area of biological research has always been closed to them: they’ve never been able to determine kinship or conduct parenting studies.
Le Boeuf seems wistful as he recalls this long quest: “It goes back to a seminal 1974 study by one of my former graduate students, Michael Bonnell, who worked with Robert Selander on what was, at the time, a state-of-the-art paternity study. I was interested in their work because northern elephant seals are one of the most polygynous of all the mammals … just a few males do the majority of the breeding. And I wanted to document whether the discrepancy of paternity in the colony was similar to the monopoly that we saw in mating behavior. Were the few animals you see mating also siring all the pups? ”
Unfortunately, Selander and Bonnell’s study revealed it was impossible to determine a pup’s parentage, because, at the genetic level, all pups look pretty much identical. This makes a lot of sense when the species’ history over the last couple of centuries is considered. European seal hunters arrived in the Pacific Ocean to find about 300,000 northern elephant seals living in colonies from Mexico into Canada. The seals were highly valued for their oil, and, throughout the 1800s, sealers decimated them. By the 1880s, almost all of the elephant seals were gone. Scientists estimate their effective population during that nadir at less than 40 individuals, perhaps only 20 to 30, all of which were located on Guadalupe Island off the coast of Mexico.
Yet, from this extreme bottleneck, the elephant seals have made a dramatic comeback. Their numbers are currently estimated to exceed 175,000. Año Nuevo* provides a perfect example. In 1968, scientists counted 140 breeding females on the island; now each year, between 2,500 and 2,600 breeding females come ashore — and even more can be found on the nearby mainland beach. Moreover, elephant seal rookeries dot the coast from Mexico to central California, most recently the Farallon Islands and Point Reyes. Le Boeuf notes that the only thing that has delayed this northward spread is the lack of suitable islands along the coastlines of northern California, Oregon, and Washington. He’s confident, though, that the process will continue. “I wouldn’t be surprised to see them breeding on Vancouver Island in the near future,” he says. “Maybe even further north.”
However, while the numbers of elephant seals have increased dramatically, scientists fear the vulnerabilities of their genetic bottleneck remain. “Clearly, they lost some variability when the axe fell in the 1800s,” says Le Boeuf, “but what’s hard to know is whether there were similar events that reduced variability before that bottleneck.”
As genetic techniques have improved over the last 20 years, a number of other scientists have attempted paternity studies. All have proven unsuccessful. “We pursued it in the hope that DNA fingerprinting would allow us to do that,” Le Boeuf explains. “That didn’t work, because we didn’t see sufficient differences among males to separate one potential father from another. Next we tried mitochondrial DNA, but found nothing there either.”
Figuring that the problem stemmed from simply needing more probes, another one of Le Boeuf’s graduate students, Michelle Weinstein, began a study on both the northern elephant seal and its close relative, the southern elephant seal. As Le Boeuf recalls: “She ended up doing her thesis totally on the southern elephant seals. She didn’t do the northern elephant seals, because she couldn’t find differences between the males. And that situation still exists today. We could never do that study.”
Out of frustration, Le Boeuf’s group resorted to nongenetic techniques for their paternity study. Le Boeuf confirms that now they “estimate paternity using probability and other techniques, and the answer is yes, in both northern and southern elephant seals, the male who is doing most of the mating is responsible for most of the paternity.”
The most surprising aspect of this particular genetic bottleneck is that lack of variability doesn’t seem to have reduced the elephant seal’s fitness. Some scientists suggest the species might even have become adapted to low genetic diversity. But Le Boeuf finds that idea hard to fathom: “It doesn’t make much sense to me that they would be adapted to low genetic variability. I think it’s much more reasonable that the variability has been reduced because of their history.”
Genetic theory says that the best hedge against an uncertain future is to produce genetically variable offspring because, as conditions change, some individuals will do better than others. But the northern elephant seal has not diversified its portfolio of genetic investments. So far, that strategy has worked. Will this be true in the future? Le Boeuf is philosophical: “When we were dumping DDT off the southern California coast, the elephant seals and other deep-diving marine mammals weren’t affected. We did see high levels of mercury and lead, but that didn’t seem to bother them. I guess it just depends on what kind of pollution, and who knows? — there might be a by-product of something we start using tomorrow that goes into the ocean and hits them hard. You don’t know.”
Individual species — individual solutions
Although past human disregard for the survival of individual species is responsible for the present endangered status of the island fox, desert bighorn sheep, and northern elephant seal, the question of how to support these species so they may persist in the world of the future presents a different scientific challenge in each case.
The island fox’s genetic bottleneck was brought on when humanmade disruptions in their Channel Island habitat led to increased predation by another species that might never have become a problem in a healthier, more balanced environment. Now the foxes survive through carefully monitored rescues and captive breeding designed to encourage genetic diversity. The current solution to this genetic bottleneck requires constant monitoring of predation and survivorship of each subspecies.
Some hands-on stewardship has also been applied successfully to the desert bighorn sheep. It was discovered, however, that the bighorns, despite living in small herds in isolated mountainous areas spread across the Mojave Desert, make an effort to maintain their own genetic diversity: individual animals cross miles of lowland desert to reproduce with new herds. Scientists who follow the flow of genes across the desert have learned that they may be able to best help this species by informing management decisions that, in turn, either remove obstacles (such as freeways and canals) from bighorn reproductive migration routes or enable the animals to circumnavigate the obstacles. Scientists can also help by influencing the decisions that corporations and nations make that affect global warming.
Finally, the northern elephant seals, hunted to near-extinction during the nineteenth century, have rebounded on their own into the twenty-first century. At this point, they seem well adapted. However, because the entire huge population is descended from a very few founders, scientists worry that lack of genetic diversity may eventually doom the species. All that can be done in the case of the elephant seals is to monitor and study them in order to understand better how they have managed to recover and even to thrive — and to hold the intention to protect the species, should further threats present themselves. — JB
For more information, contact:
The National Park Service
Island Fox Recovery Program at www.nps.gov/chis/naturalresources/IslandFox/Fox.htm
Clinton W. Epps, Environmental Science, Policy and Management
College of Natural Resources, University of California Berkeley, CA 94720
Email: buzzard@nature.berkeley.edu
Burney Le Boeuf, Associate Vice Chancellor for Research, and Research Professor of Biology
283D Clark Kerr Hall, University of California Santa Cruz, CA 95064
Email: leboeuf@ucsc.edu
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