However, since the credibility interval of this estimate puma shoes slightly overlaps the timing of the GABI, this issue is still not fully settled. Interestingly, Barnett et al. (2005) provided molecular evidence indicating that the extinct North American felid Miracinonyx trumani is the puma's closest relative, with a divergence time estimated at 3.19 MYA. This finding would support the hypothesis of a North American origin for the puma, with subsequent colonization of South America by this species.

Indicating that most of the species' historical subdivision occurred in that subcontinent. In addition, the genetic diversity within South America was found to be larger than in Central and North America, suggesting that pumas from the latter subcontinent actually derive from a recent re-colonization event, following extinction puma trainers in North America in the Late Pleistocene (ca. 10,000 12,000 years ago).Within the mtDNA, the NADH dehydrogenase subunit 5 ( ND5 ) gene has been successfully used in phylogenetic and phylogeographic studies of felids and other carnivores ( e.g. Culver et puma suede al.

2000 ; Trinca et al. , 2012 ). In a previous study focusing on pumas ( Culver et al. , 2000 ), three mtDNA segments were employed ( ND5 , 16S and ATP8 ). Of these, ND5 showed the highest polymorphic content in this species, based on a segment spanning 318 bp. A new primer set for this gene was designed specifically for carnivores ( Trigo et al. , 2008 ), amplifying a longer fragment ( ca. 750 bp) puma sliders and exhibiting successful amplification across several families .

In the present study we employ this longer ND5 segment to investigate the evolutionary history of P. concolor , with emphasis on South American populations, which were previously found to harbor high levels of diversity and inferred to have played a key role in the historical demography of this species ( Culver et al. , 2000 ). Given that the geographic sampling of South American pumas was limited in that first study, we aimed here to expand the representation of the various regions of this sub-continent.

We obtained blood and tissue samples from 77 pumas including wild individuals captured during field-ecology projects, caught by farmers or road-killed, as well as captive animals with known geographic origin ( Table S1 ). In addition, we also collected data from 109 additional individuals whose DNA was already available in puma uk the participant laboratories, some of which had been used in earlier genetic studies employing different markers .

We initially examined the mtDNA fragments used in previous studies, especially those involving Neotropical felids ( e.g. Eizirik et al. , 1998 , 2001 ; Johnson et al. , 1998 , 1999 ; Culver et al. , 2000 ). We selected the ca. 750 bp-long fragment of the ND5 gene reported by Trigo et al. (2008) , thus considerably increasing the information content derived from this marker relative to the previous phylogeographic study of the puma ( Culver et al. , 2000 ). Finally.