Population samples were selected to exclude known related individuals, or in the case of Florida, known hybrids (O'Brien et al. Samples of cheetah, jaguarundi, Geoffroy's cat (Oncifelis geoffroyi), and domestic cat (Felis catus) were included as outgroup species. Products for both single-strand conformation polymorphism (SSCP) and sequencing of mt DNA regions were obtained by PCR amplification of genomic DNA (Saiki et al.
DNA was extracted from white blood cells, whole blood, primary fibroblast cultures from skin biopsies, or tissues following a phenol-chloroform protocol (Sambrook et al. DNA from hide, hair, and bone samples was extracted using a silica based, guanidium extraction method (Hoss and Paabo 1993; Johnson et al. 1985) using primers that amplify portions of the 16S r RNA (76S), NADH-5 (ND5), and ATPase-8 (ATP8) genes (Johnson et al. PCR amplification of museum samples was performed with two pairs of hemi-nested primers for 16S and ND5 (Johnson et al.
1998) except 16S-3F-5'GA-GACCCATTAATTTCAACCG-3' is substituted for 16S-1NF.
PCR reactions were performed using 20 ng of genomic DNA in the presence of 10 m M Tris-HCl (p H 8.3), 50 m M KC1, 1.5 m M Mg Cl2, 200 |x M each of d ATP, d CTP, d GTP, d TTP, 0.16 mg/ml BSA, 1 |jl M of each primer, and 1 unit Taq poly-merase enzyme in a volume of 10 jjl I (2.5 units in 25 jjl I for museum specimens).
The marked uniformity of mt DNA and a reduction in microsatellite allele size expansion indicates that North American pumas derive from a recent (late Pleistocene circa 10,000 years ago) replacement and recolonization by a small number of founders who themselves originated from a centrum of puma genetic diversity in eastern South America 200,000-300,000 years ago.
The recolonization of North American pumas was coincident with a massive late Pleistocene extinction event that eliminated 80% of large vertebrates in North America and may have extirpated pumas from that continent as well.
Some 32 separate geographic subspecies of puma have been described based on geographic and morphometric criteria (Figure 1A) (Neff 1983; Young and Goldman 1946). Captive animals were wild born and of known geographic origin.
Phylogenetic analyses of mt DNA variation were conducted for samples which successfully amplified for all three gene segments (n = 286).1982; Stehli and Webb 1985; Webb 1978; Webb and Marshall 1981; Webb and Rancy 1996). Four of the six recognized puma OTUs reflect geographic (potential faunal) boundaries, while the other two are likely hybrid (or intergrade) zones from geographically adjacent subspecies.Although the event would suggest that modern pumas originated from a North American ancestry, the puma fossil record in South America is so poor that a more recent neotropical origin and northward dispersal cannot be ruled out (Kurten 1976; Werdelin 1989). schorgeri) are presumed extinct, and three subspecies (P. Consistency of the genetic/phylogenetic results with each category of genetic markers offers confidence in subspecies classification and has implications for natural history and present conservation management of the puma.The peopling of North and South America clearly diminished puma populations in recent centuries, reducing the range by two-thirds in North America as a consequence of habitat destruction and human depredation (Figure 1A). Biological samples were collected from 315 pumas and were obtained from the wild (n = 148), captive facilities (n = 113), and museums (n = 54) (Figure 1A).Except for the small endangered population of Florida panther (Puma concolor coryi) living in cypress swamps of south Florida, pumas are extinct east of the Mississippi (Hansen 1992; Maehr 1998; Roelke et al. Nonetheless, elimination of bounties and recent legislative protection has led to an increase in puma numbers in many areas since 1900. Samples included 1-35 animals from each of 32 subspecies.