Organisms encounter a diverse array of habitats and adapt to these environments with an equally diverse array of structures and functions. The long-term goal of our studies is to elucidate mechanisms that drive these adaptive changes at the molecular and functional levels. We plan to accomplish this goal using vision as a model system. In the traditional view of photo-transduction, not only are amino acid (AA) sites that are involved in the spectral tuning of visual pigments located only in or near the "retinal binding pocket" but also they modulate the wavelength of maximal absorption (?max) of visual pigments mostly in an additive fashion. It is now clear, however, that neither of these "assumptions" holds in nature. Hence, to identify all critical AA changes and understand their individual and synergistic effects on the ?max-shift, some new approaches must be taken. Only when we establish the fundamental principle of the spectral tuning, the molecular mechanisms of adaptive evolution of visual pigments (and color vision) will be understood fully. [unreadable] Here we propose to clone all opsin genes of visual pigments of five deep-sea fishes lampfish (S. leucepsarus), loosejaw (A. scintillans), scabbardfish (L. fitchf), thornyhead (S. altivelis), and viperfish (C. macouni). We will then study both the molecular bases of spectral tuning and the mechanisms of adaptive evolution of visual pigments in a wide range of vertebrate species. Living at different depths, ranging from 200 to 4,000 m, the deep-sea fishes receive varying levels of sunlight at ~480 nm. In addition, the lampfish and viperfish emit bioluminescence at ~480 and the loosejaw at ~480 and ~700 nm. We plan to explore three features of visual pigments: 1) the molecular and chemical bases of the spectral tuning of visual pigments; 2) statistical and experimental analyses of positively selected AA changes; and 3) co-evolution of paralogous pigments in each of the five deep-sea fish species. Using computational methods in theoretical chemistry, we plan to test four specific hypotheses of spectral tuning and identify the chemical principles by which absorption spectra of visual pigments are determined. To test whether the evolutionary patterns of different paralogous pigments are synchronized in each species according to the light distribution of the habitat, we shall compare the evolutionary rates of nucleotide (or AA) substitution to those of the duplicated a and [unreadable]-globin genes in the same species, which will also be cloned and sequenced. [unreadable] [unreadable]