In parallel, molecular data ( Johnson et al. , 2006 ) puma creeper have led to an estimate of its divergence from the sister-species P. yagouaroundi of 4.17 MYA (C.I.: 3.16 6.01MYA), suggesting a much longer history as a distinct evolutionary lineage. The speciation event that separated these lineages may have occurred in North or South America, with the molecular dating estimate supporting the former, as it tends to predate the Great American Biotic Interchange (GABI) (ca. 2.5 3.5 MYA).
However, since the credibility interval of this estimate slightly overlaps the timing of the GABI, this issue is still not fully settled. Interestingly, Barnett et al. (2005) provided molecular evidence indicating that rihanna fenty puma 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 rihanna puma shoes colonization of South America by this species.
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 puma by rihanna creeper et al. , 2008 ), amplifying a longer fragment ( ca. 750 bp) and exhibiting successful amplification across several families .
So as to allow refined inferences of population structure, maternal gene flow and demographic history. In addition to expanding the geographic coverage of South American regions to refine inferences on patterns of matrilineal subdivision, we have performed novel analyses on puma demographic history, which revealed consistent evidence of a recent population expansion in South America, prior to re-colonization of North America.
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, fenty puma creepers we also collected data from 109 additional individuals whose DNA was already available in the participant laboratories, some of which had been used in earlier genetic studies employing different markers .
Sequence electropherograms were visually inspected and edited using Chromas Lite 2.01 or FinchTV 1.4.0. Sequences were aligned with the CLUSTALW algorithm ( Higgings et al. , 1996 ) implemented in MEGA 4 ( Tamura et al. , 2007 )To investigate the evolutionary relationships among puma mtDNA lineages, haplotype networks were built using the median-joining approach ( Bandelt et al. , 
1999 ), as implemented in the software Network 4.5.1.6.
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