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The effects of all tuning site substitutions found in this study are known [ 22, 23], with one exception: a leucine residue in the spectral tuning site 93 of the Rifleman Acanthisitta chloris.
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We assumed the effects of the key tuning sites to be additive.
Reconstructed amino acid substitutions at major spectral tuning sites were mapped on a landbird phylogeny.
Several LWS opsin spectral tuning sites have been identified through comparative sequence analysis and site-directed mutagenesis studies [ 6, 45- 47].
This assumption should provide reasonable approximations of λmax [ 51], although it disregards potential interactions between the tuning sites (see [ 52]).
We translated our DNA sequences into aa's to identify the spectral tuning sites 86, 90, and 93 [ 5, 10].
Amino acid translations showing the spectral tuning sites 86, 90 and 93 [ 5] are presented in Table 1.
Then we calculated λmax from the tuning sites following in vitro changes in λmax reported by Wilkie et al. [ 22].
The ancestral aa residues cannot be reconstructed for the two most important spectral tuning sites of the avian SWS1 opsin.
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