Table 1. O₂ affinities (P₅₀, torr) and anion sensitivities (∆log P₅₀) 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’.

	P50 (± SE)	Δlog P50
rHb	Stripped	+KCl	+IHP	KCl+IHP
Bar-headed goose	3.42 ± 0.06	0.115	0.749	0.643
Greylag goose	4.10 ± 0.04	0.082	0.843	0.680
AncAnser	4.30 ± 0.06	0.092	0.815	0.630
Bar-headed goose mutants
GAA	3.53 ± 0.08	0.111	0.745	0.693
GVP	3.68 ± 0.01	0.072	0.742	0.584
SAP	3.98 ± 0.03	0.098	0.653	0.523
GVA	3.52 ± 0.04	0.079	0.708	0.589
SAA	3.32 ± 0.05	0.156	0.870	0.742
SVP	3.88 ± 0.04	0.027	0.691	0.573
Greylag goose mutants
SD	4.10 ± 0.05	0.052	0.729	0.601
TE	4.01 ± 0.04	0.104	0.774	0.549