Ralbovszki, D. M.; Westfall, J. J.; Mori, Y.; Tahas, S. A.; Bertelsen, M. F.; Korshunova, I.; Skov, J.; Gorodkin, J.; Hemberg, M.; Seemann, E. S.; Hall, V.; Witter, M.; Khodosevich, K.

Spatial navigation is a fundamental mammalian ability, supported by the entorhinal cortex (EC), a structurally conserved yet functionally diverse region across mammalian species. However, how molecular signaling underlies both shared and species-specific navigational strategies remains unclear. Here, we present a cross-species single-cell atlas of the EC from human, Hamadryas baboon, mouse, and Egyptian fruit bat - species spanning distinct evolutionary lineages and navigational demands, including true 3D navigation in bats. Using this resource, we identify conserved principal neuron populations as well as species-specific innovations, including mixed-layer or functional identities and fruit bat-specific subtypes. GABAergic interneurons neurons show strong conservation of somatostatin (SST) and parvalbumin (PV) families, while VIP GABAergic neurons exhibit pronounced species-specific divergence, with an expanded repertoire in primates. Integration with whole-brain diffusion tensor imaging reveals conserved and species-specific connectivity between the EC, hippocampus, and sensory cortices. Major species-specific cellular innovations were further validated using orthogonal histological approaches, confirming their anatomical and laminar organization. Overall, this atlas provides a comparative framework available for the research community to dissect the molecular, cellular, and circuit principles underlying conserved and specialized spatial navigation across mammals.