Download paper 2

Amur Tiger Population Genetic Structure
R. Scudelari
Biology Department, Colorado State University, Fort Collins, CO 80523, USA
[email protected]
Abstract
The tiger population is at a critical point, it can either become extinct in the next hundred
years, or through conservation methods, can grow and prosper. The Amur tiger, which has faced
drastic geographical reduction, is critically endangered. Populations of the tigers are being
separated by human population growth. In order to ensure that the different subpopulations are
not diverging from one another, the molecular make-up of the species will be examined.
Through this, conservationists will be able to tell whether some of the captive tigers need to be or
can be released into the wild to increase the genetic variation, so that gene fragmentation does
not occur.
Keywords: Panthera tigris Altaic; gene fragmentation; population genetic structure; genetic
variation
Introduction
Panthera tigris (Linnaeus, 1758) the largest cat is endangered throughout its territory.
Of the nine subspecies existing in the early 20th century, three have been eliminated. When all
nine subspecies still existed, tigers covered most of Asia with a population of approximately
100,000 (O’Brien et al. 2005). Tigers now occupy seven percent of their historical range, and in
the past decade, the area occupied by tigers has decreased by as much as 41 percent (Dinerstein
et all. 2007).
Panthera tigris altaica (Temminck, 1844), commonly known as the Siberian or Amur
tiger historically has been found across large portions of northern China, the Korean peninsula,
and the southern region of the Russian far east (Heptner & Sludskii, 1972). In the late 19th
century, due to human population growth which is linked with habitat loss and pouching of tigers
in China, there was a drastic decline in the population. In the early 20th century, a similar
scenario occurred in Russia due to the capture of cubs for international zoos and tiger hunting
that lead to a population of 20-30 tigers in the 1940s (Heptner & Sludskii, 1972). Conservation
measures were immediately started with a ban on hunting and a limit on cub capture. Over the
next 55 years the population of wild Amur tigers rose to 428-502 (Miquelle et al. 2007).
Conservation efforts have not only increased the numbers in their natural habitat, but also
in the captive population as well. There are now 421 Amur tigers in captivity with the help of
The Fish and Wildlife Foundation Save the Tiger organization. The current spread of Amur tiger
populations in the wild is separated by human populations, creating the concern that there is a
restriction in gene flow between the different separated populations of tigers. A restriction in
gene flow could cause severe problems in the recovery of the already fragile population. Small
populations are more susceptible to losing genetic variation through inbreeding, accumulation of
deleterious mutations, and genetic drift (Frankham et al. 2002).
This proposed study will expand on current research by closely examining the genetic
make-up of the different population locations in both wild and captive Amur tigers. All these
results will be compared to make sure that gene fragmentation is not occurring. Comparing the
test results will help ensure that the captive populations’ and wild populations’ genetic and
morphological make up have not altered from the original Amur tigers. The main goal is to
make sure the current Amur population genetic make-up has not significantly changed from each
other and from the original Amur population.
Materials and Methods
This study will require samples to be taken from the separated Amur tigers in the wild.
The subdivisions of the population, due to human population growth, the North American
Species Survival Plan tigers, European Breeding Program tigers, and tigers from the zoos in
Asia. In order to ensure that the same tiger is not tested twice, each tiger will be tagged and
chipped. The purpose of chipping the tiger is to demonstrate if the tigers have crossed over into
the other populations or are staying within the subpopulation they reside in. For tigers outside
the natural range it is important to know what area they are taken from or from where their
ancestors were from.
The skin of the Amur tiger will be sampled in order for this study to be preformed. Three
molecular markers will be examined as studied previously including: the mitochondrial DNA
(mtDNA), allele variation in the nuclear major histocompatibility complex class II DRB gene,
and composite nuclear microsatellite genotype based on 30 loci (Luo et al. 2004). After skin
samples are obtained by use of a punch biopsy, the specimen will be placed into liquid nitrogen,
and later placed in a freezer at a temperature below 32 degrees Fahrenheit. Guanidine
thiocyanate and silica-based purification will be used to obtain the DNA from the dry skin (Luo
et al. 2004).
When testing Panthera species mtDNA, it was discovered that it contains a large 12.8 Kb
nuclear mtDNA fragment on chromosome F2. It has existed there for three million years and
been confirmed in ancestral species (Johnson et al. 1996). To overcome this issue, the use of
Cymt-specific primer set will be used to complete the tiger Numt and homologous 12.8 kb Cymt
sequence (Luo et al. 2004). The genes will then be put through PCR, results will be transferred
to a computer system, and then the phylogenic relationship among the mtDNA will be observed.
For the microsatellite analysis, 30 loci will be viewed by running the sample through PCR with
fluorescent primers. The results be entered into computers and patterns will be tracked. All of
the results will be grouped based off of where the tiger was found and compared to all other
locations, as well as the data from an ancestral Amur tiger.
Expected Results
After running these three molecular marker tests, it should be clear whether there has
been a divergence from the original genes, as well as whether the different subpopulations, both
captive and non-captive, have the same genetic make-up. Most if not all of the genes should be
seen as not having differed from each other’s subpopulation. It should be expected that the tigers
in captivity have some differing genes from wild tigers. However, there should be a huge
portion that overlaps if the conservation measures are being properly conducted. The living
tigers of today will most likely have differing genes from the ancestral Amur tiger. However,
which habitat differs most from the ancestral one (captive or wild) can only be determined by
studying the data.
Discussion
This study will be conducted in order to determine the level of genetic variation within
Amur tiger populations. Due to historical events in the population, it is important to keep in
mind that the Amur tiger population is already known for having a low genetic variation in
comparison to the other subspecies of tigers. It will be important to note where the difference are
in the genes if any. If there are differences, it will be necessary to examine if this is a new
finding or if this was seen in previous studies. If there are new differences within the
subpopulations, it can be assumed it is due to the human population growth into their habitat,
splitting the population and/or the population adapting to a different environment. If the genetic
variation is limited in the wild, the release of captive tigers into the population of wild tigers is
essential to the survival of the Amur tiger in the wild. With the use of trackers in the animals, it
will be important for conservationists to watch the movement of the wild populations to make
sure they are indeed breeding with one another. If there is no breeding between populations, the
possibility of a weakened gene pool and mutations exists. Bottlenecking events lead to a low
genetic diversity, which leads to adverse effects (inbreeding), and will then lead to a heightened
risk of extinction.
Some possible errors that could occur in the study would include improper scientific
technique, which could include errors related to the chemicals or reagents. Improper room
temperature could also cause the results to be incorrect. Poor or insufficient samples of skin
could also cause problems in the study. The temperature that the skin samples are stored is
important also; if not cold enough, the skin cells will die. Should not enough DNA be added to
run the PCR, the PCR will not properly operate. Cross contamination on improperly cleaned
study devices can also read to invalid results.
References
Black, W. (2010, March). Molecular Taxonomy. [Lecture notes]. Presented to the class BZ 424:
Principles of Systematic Biology, Fort Collins, CO.
Dinerstein E, Loucks C, Wikramanayake E et al. (2007) The fate of wild tigers. BioScience, 57,
508–514
Frankham R, Ballou J, Briscoe D (2002) Introduction to Conservation Genetics. Cambridge
University Press, Cambridge, UK
Heptner V, Sludskii A (1972) Carnivora (hyaenas and cats). In: Mammals of the Soviet Union
(eds Heptner V, Naumov N), pp. 95–193. Amerind Publishing Co., New Dehli.
Kerley, L., Goodrich, J., Miquelle, D., Smirnov, E., Quigley, H., & Hornocker, M. (2002).
Effects of Roads and Human Disturbance on Amur Tigers. Conservation Biology, 16(1),
97-108. doi:10.1046/j.1523-1739.2002.99290.x.
Kondratieff, B. (2010, February)Species Concept. [Lecture notes]. Presented to the class BZ 424:
Principles of Systematic Biology, Fort Collins, CO.
Luo S, Kim J, Johnson W, et al. (2004) Phylogeography and Genetic Ancestry of Tigers
(Panthera tigris). PLoS Biology, 2(12), 2275-2293.
Miquelle D, Pikunov D, Dunishenko Y et al. (2007) 2005 Amur Tiger Census. Cat News, 46,
14–16.