Genomic insights into the evolutionary history and cryptic diversity of two-toed sloths (Choloepus) in Amazonia

• Genome-wide data reveal cryptic diversity in two-toed sloths ( Choloepus spp .). • C. didactylus and east-Andean C. hoffmanni form a single monophyletic group. • Nuclear data suggest a more recent split in the C. hoffmanni - C. didactylus complex. • Pleistocene climate changes drove population size oscillations. • Conservation assessments should consider distinct lineages for effective protection. Amazonia has undergone significant geological and climatic changes over millions of years, shaping its rich biodiversity. However, the impact of historical environmental change on driving species diversification remains debated. Two-toed sloths (genus Choloepus ) provide a valuable model for studying this connection due to their wide distribution across Amazonia and their restricted arboreal lifestyle, which makes them especially sensitive to shifts in forest landscapes. Previous studies have highlighted inconsistencies in phylogenetic relationships, karyotypic variation, and subtle morphological differences between the two currently recognized Choloepus species, underscoring the need for a taxonomic revision of the genus. Here, we present the first genome-wide dataset from multiple Choloepus populations, combining nuclear and mitochondrial data to investigate their evolutionary history and phylogenetic relationships in relation to Amazonian biogeography. Our results indicate that C. hoffmanni is paraphyletic, with its east-Andean population grouping with C. didactylus . Divergence time estimates based on nuclear loci suggest a more recent split (∼3.57 mya) between C. hoffmanni and C. didactylus than previously inferred from mitochondrial DNA (∼8 mya). Furthermore, the separation of South and East/Northeast Amazonian lineages coincides with the onset of Pleistocene climatic changes, which likely isolated populations via habitat fragmentation. Demographic reconstructions reveal population size oscillations during glacial periods, with the East Amazonian lineage exhibiting lower population size and genetic diversity, possibly due to the region’s particular climatic instability and recent deforestation. Our findings support the existence of previously unknown Choloepus lineages suggesting that the genus may consist of at least three species and highlighting the need for a taxonomic revision based on a broader geographic sampling. The distinct evolutionary lineages may also require independent conservation assessments amidst the increasing threat of habitat loss.