Download Wildlife Genetics: Concepts, Tools, Applications

Wildlife Genetics: Concepts, Tools, Applications
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II.
Introduction
A. News of cloned animals like sheep and genetically modified (GM) crops like
corn and trees quickly attract the attention of the media, often casting
genetic research in a negative light. However, the current use of genetics in
wildlife conservation and management is facilitating insights into the
biology and ecology of wildlife species not seen since the development of
radiotelemetry in the 1960s.
B. Conserving the maximum amount of genetic diversity within a species is
important because within this diversity are contained the adaptations a
species possesses to survive local environmental conditions. Conserving
these adaptations increases the chances that the species will be able to
survive over the long term.
C. Many genetic studies of wildlife populations attempt to characterize the
amount of genetic diversity of a species on a molecular level and determine
the level of heterozygosity, which is a measure of the genetic variation
within a population. Other studies seek to determine familial relatedness
(i.e., who fathered whom and how closely related are the members of a
population?) and mating success (i.e., are a lot of males fathering the
offspring or is reproduction dominated by only a few males?).
1. Armed with data from genetics and other disciplines, such as
reproductive biology, management plans containing genetics
information can be developed and implemented.
2. In T&E species management, managers may decide to increase the
genetic diversity of a population by “forced” migration (i.e., moving
grizzly bears from northern British Columbia into Yellowstone National
Park) or set up captive breeding programs, such has been done for
black-footed ferrets and giant pandas.
Gene flow
A. Due to widespread habitat fragmentation, wildlife managers are interested
in the numbers of individuals moving between populations and whether
these individuals are breeding successfully.
1. Successful breeding by dispersing or migrating individuals would
indicate gene flow, which is the movement of genes between
populations.
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2. If little gene flow occurs between 2 populations, they may become
distinct enough that factors impacting 1 population will not affect the
other. It may also mean that different management strategies are
required for each distinct population.
B. Two examples
1. Eastern rat snake (Elaphe obsoleta)
a. Wildlife scientists have found that threatened Eastern rat snake
populations in Ontario, Canada, experience high gene flow between
populations that are located <4 miles (<6 km) from each other.
b. There is little gene flow, however, between populations separated by
>9 miles (>15 km).
c. These findings help clarify the management approach for this
threatened species. For example, there would be greater population
subdivision, increased risk of inbreeding, and loss of genetic diversity,
if barriers were created between populations that currently
exchange genes.
2. Grizzly bear (Ursus arctos horribilis)
a. Research illustrates how genetic data can determine patterns of
reproductive success in 2 grizzly bear populations: an Arctic
population and a more southerly population.
b. In the Arctic bear population, 49% of males ≥9 years old sired
offspring.
1) The most offspring sired by a single male was 11% of the cubs
produced for that year.
2) Because Arctic grizzlies are distributed sparsely and uniformly
across their range, a lot of males are able to successfully
reproduce.
c. To contrast, a southern population of grizzly bears congregate at
feeding sources during the breeding season (May through early July).
1) These interactions result in more male-to-male competition for
mates and aggressive male mating behavior in an attempt to
monopolize breeding females.
2) In such a situation, dominant males have much greater
reproductive success and contribute more to the local gene pool
(i.e., dominant males sire most of the cubs).
3) Therefore, the percentage of male Arctic grizzlies that successfully
reproduce appears much higher than in the southern population.
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III.
4) As a result of these findings, wildlife scientists have recommended
that Arctic grizzlies need a management plan that is unique to
their mating strategy.
5) In species that are hunted, this type of information could be
applied to hunting regulations (i.e., the effects of hunting big
males will affect the southern population much differently than
the Arctic population).
3. These examples illustrate the importance of genetic research in wildlife
conservation and management. The availability of genetic data has
opened doors for wildlife biologists to learn more about species that are
difficult, if not impossible, to determine through traditional field
methods.
What is Genetic Variation?
A. The phenotypic expression of almost all individual traits, whether it is
anatomical, physiological, and behavioral, is a function of their genotype
coupled with the environment to which the individual is exposed.
B. The genes that make up the genotype are sections of DNA located on
chromosomes in the nucleus of all cells.
1. The location of a gene on a chromosome is called a locus (plural loci).
2. A gene can vary in the specific sequences of DNA nucleotides that
comprise it; different forms of a gene are called alleles. For example,
there are several alleles that determine coat color in felids.
C. In sexually reproducing (diploid) organisms, like almost all vertebrates, half
of the genome is received from 2 separate parents. That means that 1
allele at each locus is inherited from each parent. If the 2 alleles at a locus
are the same, the individual is homozygous for that gene; if they are
different, then the individual is heterozygous for that gene (Figure 1).
1. The concept of heterozygosity is commonly extended to a population
(i.e., the fraction of individuals in a population that are heterozygous for
a particular locus).
2. It can also refer to the fraction of loci within an individual that are
heterozygous.
D. While heterozygosity describes variation in how genes occur at each locus,
several other terms describe the number of alleles at each locus.
1. A gene is considered polymorphic if >1 allele is detected at a locus
across all individuals sampled; otherwise the gene is monomorphic.
2. Allelic diversity or richness describes the mean number of alleles/locus.
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Figure 1. Homologous chromosomes, alleles, and gene loci.
3. Homologous chromosomes are a pair of chromosomes having the same
overall genetic composition and sequence. In diploid organisms, each
chromosome inherited from 1 parent is matched by an identical (except
for mutational changes) chromosome—its homolog—from the other
parent.
E. Cheetahs, lions, and black-footed ferrets (to name a few) suffer from low
allelic diversity.
1. Because of habitat loss, cheetah populations have been declining for the
past 25 years. Existing populations consist of closely related individuals.
2. A population of closely related individuals exhibits low genetic variability
(their genes are very similar because they share a common ancestry).
3. This is especially critical—and dangerous—if environmental conditions
change and the population does not have the genetic variability to cope
with the change.
F. Thus far we have only mentioned nuclear genetic variability based on loci
with distinct, identifiable alleles contained within the nucleus of the cell.
However, another form of genetic variation occurs in mitochondrial DNA
(mtDNA).
1. Mitochondria are the powerhouses of cells: they generate ATP. The
genes in mitochondria are different than those in the nucleus: mtDNA is
circular, 16,000 base pairs in length, and codes for 37 genes that control
cell machinery functions only (with a few exceptions). mtDNA is
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IV.
maternally inherited; thus, mitochondrial genes are haploid: they have
only 1 form of the gene, not 2 as in nuclear genes). mtDNA accumulates
mutations 5-10 times faster than nuclear genes, which is an important
feature that it very useful for applied wildlife ecology (see pages 39 and
40 in Mills 2007).
2. There are many ways to measure genetic variability both within a
population, between populations, and between different but closely
related species. We will discuss some of these methods next.
Genetic Markers Used in Wildlife Science: Measuring Genetic Diversity
A. Protein electrophoresis
1. Beginning in the 1960's, protein electrophoresis was the technique
commonly used by geneticists to describe and quantify genetic variation
in wild and captive populations. This technique looks at allelic variation
in charged proteins as they migrate through a ¼ inch-thick slab of gelatin
composed of starch, cellulose, or polyacrylamide to which an electrical
current has been applied. This was the first tool that provided biologists
the opportunity to compare the levels of variation found in wildlife
populations. Protein electrophoresis is still used today because of its
ease of use and low cost.
2. To compare proteins from individual animals or animals from different
populations, it is necessary to separate the proteins from each other so
they can be examined separately.
a. To do this brain, liver, or heart tissue, which contains a mixture of
protein molecules, is extracted from an animal, ground up in a
blender, and the resulting sludge is then placed at one end of a
gelatin slab.
b. The gel slab is then placed in a strong electrical field. All of the
proteins are pulled toward the positive (+) pole of the electric field.
c. Because big proteins travel more slowly through the gel than small
proteins do, the proteins become separated according to their size.
d. Proteins can also be separated along a pH gradient.
3. The banding pattern of proteins that forms on the gel is distinctive for
each set of proteins studied.
a. In fact, comparison of the same protein in different species can show
different banding patterns for different species.
b. Using this sort of distinguishing information we can, for example,
identify the species to which an individual belongs.
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4. Figure 2 shows the results of an electrophoretic study of 2 "forms" of a
fern, Notholaena candida.
a. The dark spots in the picture are proteins extracted from the 2 types
of ferns (labeled CAN and COP) and separated by protein
electrophoresis (different proteins are identified in 1, 2, 3).
b. This gel shows that the protein banding patterns of the 2 types of
ferns are different from each other.
c. Along with other sorts of evidence, these results suggest the 2 ferns
are different enough from each other that they should be recognized
as different species.
Figure 2. Results of a study of 2 “forms” of a fern, CAN and COP using
electrophoresis. Numbers 1-3 refer to different proteins.
B. mtDNA fragment analysis
1. In the 1960's it was discovered that certain enzymes, called restriction
enzymes, could cut specific regions (or sequences) of DNA by
recognizing specific base pair sequences, such as GATATC.
a. Restriction fragment length polymorphism (RFLP) analysis employs
these enzymes to break mtDNA at specific locations in the
nucleotide-pair sequences, resulting in mtDNA fragments of given
lengths.
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b. Because mtDNA mutates 5-10 times more rapidly than nuclear DNA,
genotypic analysis based on it is very sensitive to genetic differences.
2. Figure 3 demonstrates the use of RFLP fragment analysis on mtDNA
taken from black rhinos (Diceros bicornis). In this example, a restriction
enzyme is applied to samples of mtDNA from 3 different rhinos (samples
1, 2, and 3).
Figure 3. A hypothetical example of the use of restriction fragment length
polymorphism (RFLP) analysis of black rhino (Diceros bicornis) mtDNA. From
Ashley, M.V. 1999. American Scientist 87:28-34.
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a. In sample 1, the enzyme finds 2 locations to cut the 16,000-basepair-long mtDNA, resulting in 1 fragment 14,200 base pairs long
(14.2kb) and another 1,800 base pairs long (1.8kb).
b. In sample 2, the mtDNA is cut at 3 locations, producing 1 fragment in
common with sample 1 (the 1.8kb fragment) and 2 fragments
(10.2kb and 4kb) from the remaining 14.2kb.
c. Sample 3 is divided into 4 fragments by the restriction enzyme, with
the 10.2kb fragment seen in sample 2 divided into 8kb- and 2.2kb
fragments. Because the restriction enzyme found more target
sequences in sample 3 than in sample 2 and more in sample 2 than in
sample 1, clear genetic differences are present between the 3
samples of black rhino mtDNA. An electric current passing through
the samples causes the shorter sequences to move toward the
bottom.
C. Polymerase Chain Reaction (PCR)
1. This technique revolutionized modern molecular genetics. It allows the
amplification of fragments of DNA from minute amounts of template.
2. See Box 3.2 on pages 42 and 43 in Mills (2007) for a good description of
PCR.
D. Microsatellite DNA
1. Microsatellites are used today to address many conservation questions.
They are short tandem repeats of nucleotides; the number of times a
nucleotide sequence repeats in a microsatellite can vary (AGCTAGCT vs.
AGCTAGCTAGCTAGCT) and be unique at the individual and/or species
level.
2. This variation in microsatellite length can easily be measured by
amplifying the region with PCR and then sending it through an
electrophoresis gel to show bands.
3. These genetic markers have proven to be useful in wildlife studies to
address such questions as:
a. the estimation of gene flow between populations;
b. to describe the genetic variation in and between populations;
c. to examine the effects of hybridization;
d. in pedigree analysis.
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E. DNA fingerprinting
1. The variation in RFLP band number comes only from mutations at the
restriction site.
2. Other approaches take advantage of the fact that the DNA in between
restriction sites may also differ among individuals and species due to
stretches of DNA being repeated for varying numbers of times.
3. DNA fingerprinting is based on stretches of DNA 10-100 bp long
repeated up to several hundred times; because different animals
typically have different numbers of repeats, the fragments on the gel
appear as 10-30 individual-specific bands.
4. These fingerprints, analogous to a bar code on a grocery product, are
formally called multilocus minisatellite markers. DNA fingerprinting has
been useful for pedigree analysis and in estimating the genetic variation
in populations.
F. DNA Sequencing
1. One challenge for the conservation biologist is to define the
conservation units to be conserved, typically a species or subspecies.
2. Many of the species and subspecies we recognize today were described
in the late 1800's and early 1900's using morphological characters.
Conservation geneticists use molecular methods, such as the sequencing
of mitochondrial and nuclear genes, in order to better identify these
units.
3. Using these tools, the conservation scientist may find undetected
variation that reveals a new species or alternatively, may find that taxa
which are differentiated morphologically are not genetically different.
V. Uses of Genetic Tools
A. Taxonomy and hybridization
1. Genetic research indicates that populations of tuatara (Sphenodon
punctatus) in New Zealand should be managed as multiple taxonomic
groups instead of 1 and seaside sparrows (Ammodramus maritimus)
along the Atlantic seaboard and Gulf of Mexico should be managed as 2
taxonomically important units instead of 9. See Box 3.3, pages 49 and
50, in Mills (2007).
2. Genetic tools are being used to detect hybridization in wildlife
populations too. Hybridization with coyotes is the biggest threat to red
wolf (Canis rufus) persistence in eastern North Carolina. Management
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requires hybrids be identified using molecular methods and eliminated
or sterilized.
3. The red wolf is one of 2 species of wolves in North America, the other
being the gray wolf, Canis lupus.
a. The red wolf is one of the world’s most endangered wild canids.
Once common throughout the southeastern United States, red wolf
populations were decimated by the 1960s due to intensive predator
control programs and loss of habitat.
b. A remnant population of red wolves was found along the Gulf coast
of Texas and Louisiana.
1) After being declared an endangered species in 1973, efforts were
initiated to locate and capture as many wild red wolves as
possible.
2) Of the 17 remaining wolves captured by biologists, 14 became the
founders of a successful captive breeding program.
3) Consequently, the United States Fish and Wildlife Service declared
red wolves extinct in the wild in 1980.
c. By 1987, enough red wolves were bred in captivity to begin a
restoration program on Alligator River National Wildlife Refuge in
northeastern North Carolina. Since then, the experimental
population area has expanded to include three national wildlife
refuges, a Department of Defense bombing range, state-owned
lands, and private property, spanning a total of 1.5 million acres.
d. An estimated 100 red wolves roam the wilds of northeastern North
Carolina and another 150 comprise the captive breeding program,
still an essential element of red wolf recovery. See box 3.5, page 52
in Mills (2007) for more details.
B. Determining species identity, individual identity, and gender
1. Carnivore species identity, gender, and individual identity has been
determined from DNA extracted from hair follicles left on hair-rub pads
or from scats.
2. From saliva left in the puncture wounds of sheep, the individual identity
of sheep-killing coyotes have been determined.
3. Estimation of the abundance of humpback whales in the North Atlantic
and black bears in western Arkansas have been determined from DNA
extracted from “plugs” of whale tissue and the follicles of bear hair.
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C. Forensic applications
1. What species were killed to produce so-called whale meat in
restaurants?
2. Using blood samples taken from a poacher’s gun or a set of antlers to
find out what species have been poached.
D. Abundance estimation using non-invasive genetic sampling
1. Barbed-wire enclosure hair-traps are used to collect hair from bears.
2. The barbed-wire is positioned knee-high above the ground and bait
(rancid fish entrails) is poured on a stack of woody debris placed on the
forest floor in the center of the trap.
3. When bears walk over or under the wire to investigate the lure, they
often leave hair samples on barbs. DNA is extracted from cells adhering
to the follicle of the hair allowing individual and gender identification.
4. The “Capture” data are then used in CMR models to estimate population
size.