O₂ affinities (<i>P</i>₅₀, torr) and anion sensitivities (∆log <i>P</i>₅₀) of rHbs representing bar-headed goose, greylag goose, their reconstructed ancestor (AncAnser), and all possible mutational intermediates connecting AncAnser with each of the two descendant species.

O2 equilibria were measured in 0.1 mM Hepes buffer at pH 7.4 (± 0.01) and 37°C in the absence (stripped) and presence of Cl- ions (0.1 M KCl]) and IHP (at two-fold molar excess over tetrameric Hb). Anion sensitivities are indexed by the difference in log-transformed values of P50 in the presence and absence of Cl- ions (KCl) and IHP. The higher the ∆log P50 value, the higher the sensitivity of Hb-O2 affinity to the presence of a given anion or combination of anions. For the bar-headed goose mutants (all mutational intermediates between wildtype bar-headed goose and AncAnser), three-letter genotype codes denote amino acid states at α18, α63, and α119 (amino acid abbreviations underlined in bold = derived [non-ancestral]). At these same three sites, AncAnser is ‘GAP’ the wildtype genotype of bar-headed goose is ‘SVA’. For the greylag goose mutants (all mutational intermediates between wildtype greylag goose and AncAnser), two-letter genotype codes denote amino acid states at β4 and β125. At these same two sites, AncAnser is ‘TD’ the wildtype genotype of greylag goose is ‘SE’.