Two-toed sloth species, Linnaeus's and Hoffmman's, are frequent residents of zoo collections in North America. However, species identification has always been problematic because of their large overlap in external morphology, which represents an obstacle to the captive breeding program. We describe here a PCR-based technique that allows species identification of two-toed sloths without requiring sequencing, by using a mitochondrial marker (COI gene) and restriction enzyme assay. We also report intra- and inter-specific patterns of chromosome variation in captive two-toed sloths. Molecularly, we identified 22 samples of Linnaeus's and Hoffmman's two-toed sloths corresponding to 14 and 8 individuals, respectively. One animal was identified as a hybrid using the nuclear gene Enam having alleles derived from both species. The chromosome number in Hoffman's two-toed sloths showed low variation ranging only between 50 and 51. In contrast, Linnaeus's two-toed sloths appeared to vary widely, with diploid numbers ranging from 53 to 67, suggesting distinct geographic groups. The species identification method presented here represents a low-cost easy-to-use tool that will help to improve management of the captive population of two-toed sloths.
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