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In North America, an unusual pattern was observed in an individual from Florida (USA) that carried haplotype H24, which is shared with animals from southern Central America (Panama and Costa Rica) and is genetically distant from the other lineages found throughout North America. Given the record of human-induced introgression of puma mtDNA lineages from Central America to Florida in the 1960s,it is possible fenty puma slides that this individual descends from a female translocated during that period.

Central America and northern South America (NSA) did not share haplotypes, implying no evidence of historical matrilineal gene flow between these regions. Possible barriers include the northernmost Andean Cordillera or the Darien Straits in Panama, as suggested for jaguars by Eizirik et al. (2001) (but see Ruiz-Garcia et al. , 2013 for a different view). Detailed sampling in that region would be required to distinguish between these two hypotheses.

Both hypotheses remain viable and should be the focus of additional scrutiny. Moreover, northwestern South America has been reported to harbor high levels of genetic diversity and a complex evolutionary history for several mammalian taxa ( e.g. Eizirik et al. , 1998 ; Ruiz-García et al. , 2009 , 2010 , rihanna puma 2012 ; Thoisy et al. , 2010 ), highlighting the need to further investigate pumas from this portion of their range to assess their role in the overall phylogeographic structure of the species.

The network root based on the outgroup Puma yagouaroundi indicates that the most ancestral lineage of current pumas is located primarily in central South America. When we considered M. trumani as the outgroup, once again the rooting was placed primarily in South America, reasserting the area of occurrence of the earliest ancestor of the current lineages and helping reveal the direction of evolution of pumas throughout their distribution.