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The visual ability and associated photic niche of early mammals is debated. The theory that ancestral mammals were nocturnal is supported by diverse adaptations. However, others argue that photopigment repertoires of early mammals are more consistent with a crepuscular niche, and support for this also comes from inferred spectral tuning of middle/long wavelength-sensitive (M/LWS) opsin sequences. Functional studies have suggested that the M/LWS pigment in the ancestor of Mammalia was either red- or green-sensitive; however, these were based on outdated phylogenies with key lineages omitted. By performing the most detailed study to date of middle/long-wave mammalian color vision, we provide the first experimental evidence that the M/LWS pigment of amniotes underwent a 9-nm spectral shift towards shorter wavelengths in the Mammalia ancestor, exceeding predictions from known critical sites. Our results suggest early mammals were yellow-sensitive, possibly representing an adaptive trade-off for both crepuscular (twilight) and nocturnal (moonlight) niches.

Molecular Data Support an Early Shift to an Intermediate-Light Niche in the Evolution of Mammals