The complete primary structures of α^D-2- and β-globin of hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, have been constructed by amino acid sequencing analysis in assistance with nucleotide sequencing analysis of PCR fragments amplified using degenerate oligo-nucleotide primers. Using computer-assisted sequence comparisons, the α^D-2-globin shared a 92.0% sequence identity versus α^D-globin of Geochelone carbonaria, a 75.2% versus α^D-globin of Aves (Rhea americana) and a 62.4% versus α^A-globin of Hb A expressed in adult red blood cells of Geochelone gigantea. Additionally, judging from their primary structures, an identical β-globin was common to the two hemoglobin components, Hb A and Hb D. The α^D-2- and β-globin genes contained the three-exon and twointron configurations and showed the characteristic of all functional vertebrate hemoglobin genes except an abnormal GC dinucleotide instead of the invariant GT at the 5′ end of the second intron sequence. The introns of α^D-2-globin gene were both small (224-bp/first intron, 227-bp/second intron) such that they were quite similar to those of adult α-type globins; the β-globin gene has one small intron (approximately 130-bp) and one large intron (approximately 1590-bp).

A phylogenetic tree constructed on primary structures of 7 α^D-globins from Reptilia (4 species of turtles, 2 species of squamates, and 1 species of sphenodontids) and two embryonic α-like globins from Aves (Gullus gullus) and Mammals (Homo sapiens) showed the following results: (1) α^D-globins except those of squamates were clustered, in which Sphenodon punctatus was a closer species to birds than turtles; (2) separation of the α^A- and α^D-globin genes occurred approximately 250 million years ago after the embryonic α-type globin-genes (π' and ζ) first split off from the ancestor of α-type globin gene family.