However, since the credibility interval puma creeper 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 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.
In a thorough study of puma phylogeography, Culver et al. (2000) assessed the current and historical genetic diversity present in this species, based on a large sampling of individuals from across its range. That study indicated that most of the 32 classical puma rihanna fenty puma subspecies did not correspond to definable genetic units and reduced the number of recognized subspecies to six. Four of these subspecies were distributed in South America.
In the present study we employ this longer ND5 segment to investigate the rihanna puma shoes 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.
Culver et al. , 2000 ; puma by rihanna creeper Castilho et al. , 2011 ; Miotto et al. , 2011 , 2012 ). Therefore, as a whole we collected novel data from a total of 186 individuals. These samples were originated from a diverse array of geographic regions throughout most of the puma range, with greater emphasis on South America (see Table S1 ). Two samples of Puma yagouaroundi were also included to be used as outgroups in some analyses.To select a marker that would provide suitable information levels.
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 fenty puma creepers 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.
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.