Titi monkeys, subfamily Callicebinae, are a diverse, species-rich group of Neotropical primates with an extensive range across South America. Their distribution in space and time makes them an interesting primate model for addressing questions of Neotropical historical biogeography. Our aim was to reconstruct the biogeographic history of Callicebinae to better understand their diversification patterns and the history of their colonisation of South America since the late Miocene. We reconstructed a time-calibrated phylogeny of 19 titi species under Bayesian inference using two mitochondrial and 11 nuclear loci. Species were assigned across eight Neotropical areas of endemism, and statistical biogeographic methods implemented in BioGeoBEARS were employed to estimate ancestral areas using 12 biogeographic models. Our results indicate that the most recent common ancestor to extant titi monkeys was widespread from the present-day Andean foothills in the Colombian Amazon, through the wet and dry savannas of Bolivia and Brazil, to the southern Atlantic forest of eastern Brazil. Genus-level divergences were characterised by vicariance of ancestral range in the late Miocene. Species-level diversification in Cheracebus and the Plecturocebus moloch group occurred as they spread across the Amazon in the Pleistocene and were largely characterised by a sequential, longdistance “island-hopping” dispersal model of speciation from a narrow area of origin through jump dispersal across rivers. This study comprises the first large-scale investigation of the evolutionary history of titi monkeys in the context of Amazonian and South American historical biogeography and sheds light on the processes that generated the great diversity found among Callicebinae.

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