By Holly Moeller | July 17th 2011 09:49 PM | 20 comments | Print | E-mail | Track Comments
Between Easter’s religious reminders and a molecular evolution class overdose of population genetics, I shouldn’t have been surprised to wake up yesterday from an unsettling dream about taking my midterm exam on Noah’s Ark. The ocean was rising, Noah was hustling animals aboard, and I was battling asthma (thanks, furry animal allergies). But what bothered me most about all this wasn’t that I’d forgotten the formula for heterozygosity. It was that there were only two animals of every kind.
Religious beliefs aside, today’s scientific consensus is that you need more than two individuals to save a species.
That’s because species survival isn’t just about baby-making potential. It also depends on genetic diversity: the number of traits (in humans, things like skin color, height or even intellectual aptitude) present in the breeding population. This diversity may be obvious, it may be subtle or it may be invisible, detectable only by extracting and sequencing samples of DNA.
The more genetic diversity a population has, the more resilient it tends to be. When the environment changes, the population faces new adaptive pressures. In a diverse population, it’s more likely that someone will have a set of traits well-suited for the new conditions. The idea also suits a well-developed NFL defense: the team practices a variety of plays so that it’s prepared for any offensive strategy.
As you might expect, the smaller your team (or your population), the slimmer your adaptive playbook. So unless they got really lucky, the pair of animals you picked for your modern-day ark probably wouldn’t be able to re-establish their species.
In genetics, we use the phrase “genetic bottleneck” to describe diversity loss in a shrinking population. If the breeding population gets too small, the negative effects of inbreeding may run rampant. Think of the Romanov family and hemophilia, for example, and you’ll begin to see some biological reasons for the social stigma surrounding “incest.”
If the population isn’t fraught with lethal genetic disorders, it may rebound to a substantial size
— but that doesn’t mean it’s safe. It takes millennia for genetic diversity to develop (through the

slow accumulation of changes to the DNA sequence), so even a large population may still bear the low-diversity signature of bottlenecks past.
That’s a major reason that cheetahs, for example, are hovering on the brink of extinction. Of course, cheetahs face the same human threats (habitat loss, poaching, etc.) that most African wildlife does. But while other species are recovering slowly under watchful conservation eyes, the cheetah isn’t sprinting back. The secret is written all over its DNA.
Where most mammals share about 80 percent of their genes with other members of their species, cheetahs share 99 percent — more than you or I have in common with even our closest relatives (save for identical twins). So the miraculous genetic reshuffling of sexual reproduction
— which evolved to produce varied offspring to meet a variable world — can’t help cheetahs claw back into synchrony with a changing environment.
The original cheetah bottleneck probably happened about 10,000 years ago, but other bottlenecks have been much more recent. The European bison, or wisent, population dwindled to 12 in the 1920s; California’s sea otters trace their ancestry to only 50 individuals alive in
1938. We may yet see the legacy of those bottlenecks in our conservation efforts.
But while most of us love animals, all of us depend on food. And the most troubling bottlenecks of all are those of our key crops, cut off from wild populations through domestication and further winnowed by breeding and (lately) genetic modification to just a few varieties.
In a stable environment (like the one we create with irrigation, fertilizers and pesticides), it makes sense to plant only the highest-yielding variety. But monocultures are risky: The Irish
Potato Famine killed one million people because single-variety crops were entirely vulnerable to disease.
Today, you can see the importance of crop diversity in eastern Ethiopia, where a hard year means a hard life (not just an expensive one). There, Romina Cavatassi and colleagues from the
UN’s Food and Agriculture Organization found that growers who’d switched to modern monocultures suffered more crop failures than their native-mixture-growing neighbors — even though, in a good year, modern seeds performed better.
Worldwide, though, agriculture is shifting from diverse crops to industrial monocultures. We’re losing the very varieties that spare Ethiopian fields as a result. At a time when climate change

and fuel shortages loom on the horizon, it’s never been more important to save that diversity.
Who knows which variety holds the key to drought tolerance? Or will resist the sweep of the next major pest?
While squirreling DNA away in freezers and seed banks has won favor amid desperate conservation efforts, in reality the best way to preserve genetic diversity is to preserve it in situ.
Today, we award huge grants to jet-setting scientists, who stock seed banks with 70-percent redundant collections. Instead, we should reward the farmer for planting his family’s heirloom varieties instead of Monsanto’s globalized products. And we should expand “biodiversity” to mean “genetic diversity,” and protect our wildlife (and our crops) from future genetic bottlenecks. Because a genetic bottleneck, like a real one, is nearly impossible to break out of.
This piece was originally published in the Stanford Daily on April 28, 2011.
Cheetahs have between 4 and 8 cubs each year. There is a 30% infant mortality before 1 month old. Only
50% of cubs make it to adulthood.
SARS in humans is similar to the coronavirus in cats. Domestic cats have a 1-2% mortality rate, but cheetahs have over 60% mortality.

99% of captive cheetahs get heliobacter gastritis (the same bacteria that cause ulcers in humans). It causes kidney, liver, viral problems (immunodeficiencies), decreased reproduction. Only 11% of cheetah in the wild get this. Since the normal home range for a cheetah is 800 square miles and the density in Namibia is now 4 cheetahs to every 1000 square miles, cheetahs are experiencing more problems.
The Genome Research Bank at the Smithsonian has the sperm of over 200 cheetahs. 80% of the samples are abnormal.
Why? The genetic bottleneck. Cheetah originated in North America. During the late Pleistocene era
(12,000-12,000 years ago) about 40 species went extinct in North America. This created a decrease in adaptation and a susceptibility to disease. Habitat loss is a major contributor to the current genetic bottleneck occurring. In Namibia, the cheetah population decreased from 100,000 to 10,000 since 1900.
That is a 90% population loss in 100 years. A loss of 90% of all cheetahs!
1. What is meant by a “genetic bottleneck”?
2. How is the bottleneck likely to affect the cheetah’s resistance to disease?
3. How are cheetahs important to the overall health of the ecosystem? (In other words, “why should we care?”)

Sources:
Tears of the Cheetah: And Other Tales from the Genetic Frontier By Stephen J. O'Brien
Thomas Dunne Books 2003 (ISBN 978­0312272869 )
Genetic Basis for Species Vulnerability in the Cheetah Author(s): S. J. O'Brien, M. E.
Roelke, L. Marker, A. Newman, C. A. Winkler, D. Meltzer, L. Colly, J. F. Evermann, M. Bush and
D. E. Wildt
Science Vol. 227, No. 4693 (Mar. 22, 1985), pp. 1428­1434
Stephen J. O’Brien
Cheetahs, like many African species, face extinction. Estimates of the total population in Africa range from 10,000 to 15,000 animals, but human encroachment on their habitat threatens these remaining animals. Attempts to breed cheetahs in captivity at zoos had been a problem with less than 17% of cheetah pairs having success conceiving and when successful the mortality of cheetah offspring was 30­40%. In the 1970s, the De Wildt Cheetah Breeding Center was established in Pretoria, South Africa to try to improve on these results. However, despite a more natural habitat at the breeding center, similar problems were encountered. The group at the cheetah breeding center sought the help of the head veterinarian at the National Zoo in
Washington, DC who was an expert in breeding in captivity. When sperm samples from the cheetahs at the breeding center revealed a high percentage of malformed sperm ­ a finding associated with male infertility ­ a possible genetic cause was sought. Dr. O’Brien’s Laboratory of Genetic Diversity at the NIH’s National Cancer Institute was contacted. Dr. O’Brien and his team of researchers had been investigating genetic diversity in general and in domestic cats, in particular, as part of their research into an infectious cause of leukemia and lymphoma in cats
(Feline Leukemia Virus). Dr. O’Brien describes how they approached the problem in 1980.
“Back in the 1960s, scientists developed a procedure called gel electrophoresis to

separate blood proteins based on their electrical charge. Blood enzymes... can be subjected to electrophoresis and stained with colored dyes that identify particular enzymes. For most species, common variants in the genetic regions responsible for encoding these enzymes cause mobility shifts that can be monitored easily on an electrophoresis gel. Different electrophoretic patterns indicate subtle differences in the
DNA sequence of the gene that coded the individual enzyme. The variants of a single gene found in a population are called alleles, so the enzyme’s genetic differences were called allozymes, short for allelic enzymes ...
“By the time the cheetah blood samples arrived at our laboratory, close to one thousand population surveys of allozyme genetic variation had been reported on all sorts of species, from flies to plants to bacteria to birds to cats. Between 20% and 50% of sampled allozyme genes had discernible genetic differences. This means that an individual is heterozygous (i.e. has two different alleles at the same enzyme gene inherited from different parents) at between 5% and 20% of his allozyme genes.
“In our ongoing effort to develop genetic tools to study cats, my students and I had already developed testing procedures for some sixty cat allozyme genes. As soon as we could, we thawed the frozen cheetah blood tubes, prepared cell extracts rich in cheetah enzymes, and began a population genetic survey of the cheetahs from De Wildt. We expected to find a modest level of genetic variability and to report that the cheetahs had normal levels of genetic diversity. But that is not what we found.”
In fact, they found that every cheetah sample had an identical allozyme pattern. This was totally unexpected as this degree of allozyme similarity is usually only found in deliberately inbred strains of mice. Because this was so unexpected Dr. O’Brien’s lab sought to confirm this lack of variability in other parts of the cheetah genome.
“We turned our attention to a gene group that we knew a lot about called the major histocompatibility complex, or MHC. The MHC is a cluster of some 225 genes that occur together along a short chromosomal segment in the DNA of humans, mice, cats and other mammals. About a dozen MHC genes encode proteins that coat the surface of cells where they present small peptides (short stretches of amino acids) from invading viruses as a prelude to immune­mediated demolition. Most of the MHC genes are extremely variable; some have over two hundred different alleles in outbred populations of mammalian species...
“That the MHC was extremely variable between individuals was well known to organ transplant surgeons because the MHC gene products, proteins expressed on nearly all cell surfaces, are themselves antigens ­ that is, proteins that can elicit a powerful immune reaction. Kidneys or livers transplanted between unrelated people rapidly lead to graft rejection, an attack by the immune system on foreign MHC antigens of the implanted organs. A tissue match between unrelated people is very rare, less than one in ten thousand...”

Dr. O’Brien’s lab developed the ability to do skin grafts on their domestic cat colony as a way of assessing MHC variability. When a skin graft was performed between unrelated domestic cats, the grafts were rejected 12­14 days after surgery. This indicated that the cats’ immune system recognized the MHC molecules in the graft as foreign and rejected the graft. They undertook to use this system to evaluate the genetic diversity of cheetahs and contacted the Pretoria Zoo where they had a group of cheetahs in captivity who were unrelated. Six cheetahs were chosen for the experiment. A skin graft between unrelated cheetahs was performed (called an allograft ­ think of allozyme) and at the same time skin was grafted next to the allograft from the cheetah itself (an autograft). At 14 days the zoo reported to Dr. O’Brien’s lab that BOTH the autograft and allograft were doing fine. And this finding persisted so that by 6 weeks hair was beginning to grow from all grafts. This finding would only be expected in genetically identical animals like cloned mice. http://www.wildlifesafari.net/cheetah.html
Dr. O’Brien needed confirmation of these remarkable results and sought out a site in the US that was breeding cheetahs. Wildlife Safari in Winston, Oregon was founded in 1972 and had successfully produced a litter of cheetah cubs the year following. They had a dedicated staff with an interest in the biology of the cheetah and when Dr. O’Brien informed them of the findings from the Pretoria Zoo skin graft experiment, they were interested in helping to test the hypothesis that cheetahs had lost their MHC diversity ­ a finding, if true, that had important implications for the species’ survival.
Because there had been no rejection of either the autografts (not expected) or the allografts
(expected) in the Pretoria Zoo experiment, another control was needed to be sure the cheetah’s immune system was able to reject a skin graft. Thus, skin from a domestic cat from Dr.
O’Brien’s lab named Heidi was also transplanted to two of the cheetahs in the Wildlife Safari experiment. This is called a xenograft as it is from a different species. Results from two cheetahs (one with a xenograft) are shown in the image. The Table shows the results from the 8 cheetahs from Wildlife Safari, 2 from Johannesburg Zoo and 4 from De Wildt (Total = 14).
Image above and the Table below are from Science 1985;227:1428­1434.

Results of Allograft and Xenograft Skin Transplants in Cheetahs:
Rapid rejection (R in the table) is due to incompatibility at the major MHC loci. Slow rejection (S) is seen with minor allelic differences at minor cell surface genes. Acceptance (A) was judged by the ability of allografts to produce hair and to be similar in appearance to autografts at the time indicated.
1. Differentiate between allografts and xenografts.
Use the information in the above data table:
2. What is the percentage of rapid rejection (R) of allografts in the cheetahs? ______________
3. What percentage of slow rejections (S) occurred in allografts at Wildlife Safari? _____________
4. What is the importance of the presence of the 2 rapid rejections (R) of the xenografts in Tamu and Kali?

5. Comparing the results in the cheetahs with the result you would expect from cloned animals, which hypothesis below best summarizes the results of the cheetah experiment? a. The cheetahs are likely to be clones. b. The cheetahs are very similar genetically but not identical. c. The cheetahs are able to mount an immune response like cloned mice but prefer not to most of the time. d. The cheetahs are identical genetically.
A CASE HISTORY (quoted from Science 1985;227:1428­1434.)
“The cheetah breeding program at Wildlife Safari is among the most successful in the world. Since 1973,
73 off­spring have been born from 18 litters, with an infant mortality rate of 24.7 percent.
“In May 1982 a clinically healthy 8­year­old female cheetah developed severe icterus (jaundice), depression, elevated temperature, and diarrhea 5 weeks after being transferred to Wildlife Safari. Despite aggressive therapy, including diuretics, antibiotics, vitamins, corticosteroids, and forced feeding, the animal deteriorated and died within 1 week. Pathological and serological analysis at necropsy revealed that the animal had the effusive form of feline infectious peritonitis, a generally fatal disease of domestic cats that has only occasionally been reported in exotic felids. Feline infectious peritonitis (FIP) is caused by a coronavirus, an immunogenic virus with an RNA genome. The feline coronaviruses are not well understood, but at least three forms of associated disease are recognized: (i) the effusive or wet form of
FIP, which is characterized by fibrinous peritonitis or pleuritis and which is always fatal; (ii) the noneffusive or dry form of FIP, which does not have the fibrinous peritoneal deposition and which is also fatal; (iii) a feline enteritis, which is usually mild (often subclinical) in manifestation. The three conditions are each accompanied by increasing titers of antibody against coronavirus. In domestic cats the fatal form is rare (approximately 1 percent of cats), and it seldom affects more than 10 percent of cats in a group under the worst conditions...
“A retrospective survey of sera from 42 cheetahs at the Oregon colony in 1982 and 1983 revealed that each animal was negative for circulating antibodies against coronavirus before June 1982. By January
1983 every cheetah had developed antibodies (titer, 1:100 to >1:1600) against coronavirus. During 1983 clinical signs (intermittent and chronic diarrhea, weight loss, and depression) and mortality were apparent in over 90 percent of the cheetahs. Even with aggressive clinical therapy, 18 cheetahs died...
This is, to our knowledge, the most extreme response to an FIP virus infection reported to date in any feline species. For whatever reason, the Oregon cheetahs reacted to the virus in a homogeneously sensitive manner.”
Dr. O’Brien, the paper’s author, goes on to report that transmission of the virus from the infected cheetahs to domestic cats (in an experiment) did not result in fatalities. And, in addition, African lions (also at the
Wildlife Park), who developed antibodies suggesting they had also been exposed to the virus, did not suffer any ill effects. This suggests that the virus itself was not more deadly than the usual strain of FIP.
Thus, it suggested that the high fatality rate in cheetahs was related to an increased susceptibility of the
Wildlife Safari cheetahs to FIP.

1. What is a necropsy?
2. What is another type of virus that infects humans that is a coronavirus?
3. What body parts are affected by pleuritis and peritonitis?
4. The antibody test in the cheetahs and lions mentioned in the Case History above evaluates a single antibody to establish that the animals are infected by FIP. This test is not able to tell us about the presence or absence of other antibodies to FIP. Despite the presence of this one antibody, the cheetahs died at a dramatic rate whereas lions did not suffer even a mild infection. Therefore multiple different antibodies to the FIP virus are needed to be made to combat the disease. Knowing that the cheetahs at Wildlife Safari had a high fatality rate from the FIP outbreak, what would you predict about the diversity of antibodies produced by these 2 groups?
5. How does the information above relate to the data from the skin graft experiment?