Xenarthra (sloths, anteaters, and armadillos) are the quintessential South American mammals. Among the xenarthrans, sloths constitute the most diverse paleontologically with almost 100 fossil genera recorded. However, this abundant sloth fauna in the Americas became extinct around 10,000 years ago. Only six species belonging to two genera, Bradypus (four three-toed sloth species) and Choloepus (two two-toed sloth species) are alive today in Central and South America. Bradypus variegatus is the sloth species with the widest geographical distribution in the Neotropics. Some regional population genetics have been reported, especially in Brazil, but with limited sample sizes. Herein, we sequenced 77 samples of Bradypys variegatus (Panama, Colombia, Venezuela, Peru, Bolivia, and Brazil; 65 new samples and 12 from GenBank), plus one B. tridactylus, one B. pygmaeus, and five B. torquatus (one new and four from GenBank) at the mitochondrial (mt) control region. Additionally, 25 of these samples, representing the four species, were sequenced for the entire mitochondrial genome. Our results indicate that there are at least six main genetically different haplogroups of B. variegatus. They are the trans-Andean, western Amazon (with some other internal groups), Tapajos River, Tocantins River, Negro River, and the Brazilian eastern Atlantic forest (with two recognizable sub-groups in northern and southern areas) ones, with the ancestor of the trans-Andean haplogroup the first to diverge. A very strong genetic heterogeneity and a striking spatial pattern were detected among these different geographical areas, with the trans-Andean, western Amazon, and Tocantins populations showing the highest levels of genetic diversity, meanwhile the Tapajos and the Brazilian eastern populations yielding lower levels of genetic diversity. The following conclusions were as follows: 1) B. torquatus should be considered as a different genus (Scaeopus) because of its extreme genetic differences from other Bradypus taxa as was previously claimed by other authors; 2) B. pygmaeus is not a recent species adapted to Holocene island environments from the Central America B. variegatus group; and 3) if we adopt a strict version of the Phylogenetic Species Concept (PSC), the highly significant monophyletic clade of the trans-Andean B. variegatus population should be defined as a valid and differentiated species of three-toed sloth. However, we are reluctant to define this population as a new species until new data shows possible strong karyotype differences and/or pre- or post-zygote reproductive barriers (Biological Species Concept, BSC) between the trans and the cis-Andean populations of the three-toed brown-throated sloth.
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