Shedding light on Amazonian phylogeographic patterns and evolutionary history of night monkeys (Genus: Aotus ) using reconstructed fecal metagenomic shotgun sequencing

Taxonomic classification, phylogenetics, and evolutionary history of night monkeys ( Aotus ) remain subjects of ongoing debate, particularly regarding species boundaries and the complex interplay of drivers underlying Neotropical primate diversification, including the historical role of the Amazon River as a biogeographic divide, Andean uplift, and Pliocene–Pleistocene climatic fluctuations. This study refines our understanding of Aotus systematics by employing fecal metagenomics to generate complete mitochondrial genomes from strategically collected samples across the lower Amazon Basin. Phylogenetic analyses integrating Maximum Likelihood, Bayesian inference, and species delimitation frameworks revealed several corrections to previously proposed biogeographic ranges and clarified evolutionary relationships among taxa. Phylogenomic reconstruction based on complete mitogenomes supports a reorganized classification into three principal clades—northern, western, and southern— originating in the Early Pliocene. Additionally, an expanded mitochondrial cytochrome c oxidase subunit I (COI) dataset provided greater resolution of haplogroups within multiple species, revealing fine-scale geographic structure corresponding to major river barriers. Divergence time estimates were consistent with earlier studies, indicating a most recent common ancestor of the family Aotidae at approximately 17.2 million years ago and rapid diversification within Aotus beginning around 4.5 million years ago. Collectively, these results refine species distributions, illuminate the evolutionary history of Aotus , and provide an updated phylogeographic framework with direct implications for taxonomy and conservation management across the genus.