Graduate school presentation for Advanced Genetics course.

1. The Role of Subspecies in
the Field of Big Cat
Conservation
Courtney Dunn
Graduate Student
University of
Central Arkansas

2. Luo S-J, Kim J-H, Johnson WE, Walt Jvd, Martenson J, et al. (2004)
Phylogeography and Genetic Ancestry of Tigers (Panthera tigris).
PLoS Biol 2(12): e442.

3. A Disappearing Species
▪ Over a 90% decrease in
population in just a
century
▪ 3,200 individuals remain
in the wild

4. Threats to Tiger Populations

6. What is a subspecies?
▪ “ populations below the species level that share a
distinct geographic distribution, a group of
phylogenetically concordant characters, and a
unique natural history relative to other subdivisions
of the species.”
Avise and Ball (1990) and O'Brien and Mayr (1991),

7. Amur Tiger (Panthera tigris altaica)

8. Bengal Tiger (Panthera tigris tigris)

9. Malaysian Tiger (Panthera tigris jacksoni)

10. Sumatran Tiger (Panthera tigris sumatrae)

11. Indochinese Tiger (Panthera tigris corbetti)

12. South China Tiger (Panthera tigris amoyensis)

13. The Problem with Subspecies
▪ Historically determined by morphological
characteristics (Mazak 1981; Herrington
1987)
▪ Few specimens documented officially
▪ Previous studies have revealed little
genetic variation (Wentzel et al. 1999)
▪ Few to no geographical boundaries
between subspecies (Kitchener and
Dugmore 2000)
▪ Is it all human caused?

14. Save the species or focus on most critical
subspecies?
▪ A Recent ecology-based approach suggests protection of 160
continuous habitat patches (Dinerstein, et al. 1997)
▪ Reintroduction programs have been discussed (Tilson, et al. 2001)
▪ Zoos focus on subspecies level breeding programs
“To this end, an assessment of population genetic structure of living tigers
interpreted in the context of traditional intraspecific taxonomy and the species'
evolutionary history would benefit both in situ and ex situ conservation
management design.”

15. Assessing Past Difficulties
▪ Low “voucher specimen” availability
▪ Pseudogene insertion of cytoplasmic mitochondrial DNA (mtDNA)
known as Numt
▪ Overall scarcity of genetic diversity in tigers

16. Experimental Set-Up
▪ 134 tigers of known geographical location
▪ Analyzed for three genetic markers
▪ “4 kb of mtDNA sequence derived from primer
pairs that excluded Numt amplification
▪ Allele variation in the major histocompatibility
complex (MHC) DRB
▪ Allele size variation of 30 hypervariable short
tandem repeat loci or microsatellites.”
(Shu-Jin Luo, et al. 2004)
WCS Russia – SiberianTiger Project

17. Table 3.Tigers used in study.

19. DNA Isolation Process
▪ Genomic DNA (Sambrook et al.
1989).
▪ Isolated via a standard proteinanse K
digestion
▪ Phenol-chloroform extraction
▪ Dry Skin and Hair Samples (Boom et
al. 1990) (Hoss and Paabo 1993).
▪ Guanidine thiocyanate
▪ Silica-based purificationWCS Russia – SiberianTiger Project

20. Mitochondrial DNA Analysis
▪ Complicated by the presence of
a large 12.8 kb nuclear mtDNA
fragment in proximity to
chromosome F2 in an ancestral
Panthera species (3 million years
ago).
▪ 15 Cymt-specific primer sets
which had sequence differences
from Numt and the 12.8 kb
nuclear mtDNA were chosen.

21. Table 1. PCR Primers Specific for Cytoplasmic
Mitochondrial DNA Sequences

22. Microsatellite Analysis
▪ 30 microsatellite loci analyzed for the domestic cat
were amplified by PCR using fluorescent markers.
▪ 113 of the total 134 tigers were used in this analysis

23. Phylogenetic Analysis of mtDNA and
Microsatellites
▪ 54 variable sites were specified which defined 25
haplotypes
▪ 30 polymorphisms were phylogenetically
informative due to being observed in more than one
individual

24. Table 2. Haplotypes andVariable Sites in Combined Analysis of 4,078 bp ofTiger (P.tigris) mtDNA Sequences

25. Phylogenetic Analysis of mtDNA and
Microsatellites
▪ Phylogenetic analysis consisted of –
▪ Maxiumum parsimony (MP)
▪ Minimum evolution (ME)
▪ Maximum likelihood (ML)
▪ P. t. sumatrae 80% MP, 70% ME, 66% ML
▪ P. t. tigris 93% MP, 82% ME, 90% ML
▪ Asian Haplotypes grouped together
▪ P. t. altaica, P. t. corbetti, and P. t. jacksoni
Figure 3a. Phylogenetic Relationships amongTigers from mtDNA Haplotypes

26. Figure 3b. Phylogenetic Relationships among Tigers from mtDNA
Haplotypes

27. Figure 4. Phylogenetic Relationships among the Individual Tigers from
Composite Microsatellite Genotypes of 30 Loci

28. Table 2. Haplotypes and Variable Sites in Combined Analysis of 4,078
bp of Tiger (P.tigris) mtDNA Sequences

29. Population Subdivision Analysis
▪ Evaluated four different geographic scenarios and compared on the
basis of molecular variance (AMOVA)
▪ Fst defined as the total variation that is related to genetic differences
between populations.
Table 4. Measures of Geographic Subdivision Based on AMOVA with MtDNA and Microsatellite Data

30. Figure S2. Bayesian Population Structure
Analysis of 111 Tigers
▪ Each individual is represented by a vertical bar.

31. Table 8. Diagnostic
Characters and Habitat of
the Six Phylogeographic
Tiger Groups or Subspecies

32. Discussion
▪ Data reflect the distinction of at least five unique subspecies with the
possibility of a sixth (later confirmed)
▪ Separation of the tiger species into subspecies reflects strong
geographical partitioning of the mitochondrial lineages
▪ Adaptation to rapidly changing habitats and genetic drift may have
resulted in isolated populations during the Holocene (Lister 2004)
▪ Groupings of the Amur, South China, and IndochineseTigers could be
due to the recent extinction of intermediate subspecies

33. Questions?