Antarctic fish hemoglobins: evidence for adaptive evolution at subzero temperature

Abstract

Notothenioids represent a large group of marine teleosts that are mostly endemic to the Antarctic Ocean. In this environment, the low metabolic demand and the high oxygen concentration reduce the need for hemoglobin(s) [Hb(s)]. The extreme condition is represented by the icefish (Channichthyidae, Notothenioidei), the only vertebrates that lack Hb. We obtained the nucleotide sequence coding for the beta-globin chain of the single major Hb form in six red-blooded notothenioids. These included Gymnodraco acuticeps, one of the closest species to the Hb-less icefish, which is also the only known fish having a single Hb without Bohr effect. This species shows a higher rate of nonsynonymous substitutions (KA), in contrast with the homogeneity of synonymous substitution (KS) rates, and KA/KS ratios significantly greater than one in the majority of comparisons. These results are suggestive of positive selection, diversifying the single major Hb toward specialized functions. A single Hb that is free to diversify means that its role in routine oxygen transport can be reduced in the presence of a combination of physiological, ecological, and environmental factors. Although a reduced "routine" function for Hb, as is apparent in G. acuticeps, might, indeed, evoke the lack of Hb in icefish, evidence of diversifying selection reported here is at variance with the hypothesis of a simple trend from a single Hb toward the Hb-less condition.

Figure 1

(A) Phylogenetic relationships among the studied species, based on mitochondrial DNA sequences (12). Two species (Chionodraco hamatus and Cryodraco antarcticus) belonging to the Hb-less family Channichthyidae were also included. The systematic position of N. coriiceps was unresolved, as reflected by the basal polytomy. (B) Molecular phylogram of β-globin genes of the notothenioid species included in this study. The tree topology was identical under all the reconstruction methods used. The minimum evolution tree (based on pairwise nucleotide distances estimated with Jukes–Cantor correction for multiple hits) is presented. The tree was rooted with S. salar as outgroup. Bootstrap values higher than 50 are shown above branches.

Figure 2

K_A and K_S values were estimated for each branch of the globin tree with the reconstructed sequences at ancestral nodes. Numbers above lineages indicate the minimum number of amino acid substitutions to explain differences between reconstructed sequences. K_A/K_S ratios are shown below branches. Results from codon analysis with paml (see text) are reported on the left. Different shades on branches (white, black, and gray) refer to the different K_A/K_S values estimated along the tree.

Figure 3

Differences among sites in the rate of amino acid substitutions were examined generating a variability profile along the β-globin sequence. First, we used an MP approach to estimation of the parameter a of the γ distribution (36) with four categories of rate, based on the obtained topology (19). Second, substitution rates per site were estimated with a discrete γ model, as implemented in paml. Evolutionary rates were scaled such that the mean of rates across sites was one. On the horizontal axis are reported the amino acid residues of the major notothenioid Hb (1–146). On the vertical axis are indicated the “relative” values of substitution rate at each site. Gray boxes, protein regions that are essential for proper Hb function.