The Role of Subspecies in the Field of Big Cat Conservation

Courtney DunnGraduate StudentUniversity of Central Arkansas

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. 

A Disappearing Species

▪Over a 90% decrease in population in just a century

▪ 3,200 individuals remain in the wild

Threats to Tiger Populations

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),

Amur Tiger (Panthera tigris altaica)

Bengal Tiger (Panthera tigris tigris)

Malaysian Tiger (Panthera tigris jacksoni)

Sumatran Tiger (Panthera tigris sumatrae)

Indochinese Tiger (Panthera tigris corbetti)

South China Tiger (Panthera tigris amoyensis)

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?

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.”

Assessing Past Difficulties

▪ Low “voucher specimen” availability ▪ Pseudogene insertion of cytoplasmic mitochondrial DNA

(mtDNA) known as Numt▪ Overall scarcity of genetic diversity in tigers

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 – Siberian Tiger Project

Table 3. Tigers used in study.

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 purification WCS Russia – Siberian Tiger Project

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.

Table 1. PCR Primers Specific for Cytoplasmic Mitochondrial DNA Sequences

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

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

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

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 among Tigers from mtDNA Haplotypes

Figure 3b. Phylogenetic Relationships among Tigers from mtDNA Haplotypes

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

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

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

Figure S2. Bayesian Population Structure Analysis of 111 Tigers

▪ Each individual is represented by a vertical bar.

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

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 Indochinese Tigers could be due to the recent extinction of intermediate subspecies

Questions?