| As the Southern Ocean cooled to --1.8°C over the past 40 million years, the teleostean clade Notothenioidei became less reliant on hemoglobin and red blood cells, a trend which culminates in the white-blooded, erythrocyte-null Antarctic icefishes. The adult alpha- and beta-globin genes of red-blooded notothenioids are linked in 5'-to-5' orientation such that the intergenic sequences direct divergent transcription of the loci. To gain further insight into their evolution and function, we have compared the intergenic regions of the adult alpha/beta-globin gene complexes from three species of Antarctic rockcods (Nototheniidae), a basal notothenioid group, and from eight species of Antarctic dragonfishes (Bathydraconidae), the red-blooded notothenioids most closely related to the icefishes, as well as the alpha- and beta-globin of 13 species and 10 genera of dragonfish.;The ancestral nototheniid intergene appears to be ∼3 kb in length, although one species contains a duplication of ∼1 kb. The bathydraconid intergenes resolve into three distinct subclasses [long (3.8 kb), intermediate (3.0 kb), and short (1.5-2.3 kb)] that correspond to the subfamilies proposed for the taxon: Gymonodraconinae, Bathydraconinae, and Cygnodraconinae. Using luciferase reporter technology, we assessed the promoter/enhancer activities of the intergenes from the rockcod Notothenia coriiceps and the dragonfishes Akarotaxis nudiceps (Bathydraconinae) and Gerlachea australis (Cygnodraconinae) in the erythropoietic microenvironment of differentiated MEL cells. We found that the N. coriiceps intergene directs high-level transcription in both orientations, the A. nudiceps intergene is active only in alpha orientation, and the short intergene of G. australis supports only weak transcription.;Phylogenetic analysis indicated that dragonfish are paraphyletic. Our alpha, beta-globin, and intergenic data agree with the subfamilies proposed by mitochondrial data but Cygnodraconinae and Gymnodraconinae are more closely related and Bathydraconinae should be split into two subclades. The icefish representative N. ionah was found to have diverged from the dragonfish, after the initial split of the dragonfish family such that N. ionah is more closely related to Cygnodraconinae and Gymnodraconinae. Studies of the beta-globin gene resulted in the discovery of pseudogenes in a number of dragonfish. This raises the question as of multiplicity of beta-globin in dragonfish as well as other notothenioids. Our results show that dragonfish can be resolved into four groups, with icefish more related to a subset of the dragonfish family. Functional and phylogenetic results are consistent with the hypothesis that the notothenioid globin loci are in evolutionary flux, probably due to relaxation of selection pressure for hemoglobin expression. |
