Hepcidin is a small bioactive peptide with dual roles as an antimicrobial peptide and as the principal hormonal regulator of iron homeostasis in human and mouse. Hepcidin homologs of very similar structures are found in lower vertebrates, all comprise 20-25 amino acids with eight highly conserved cysteines forming four intramolecular disulfide bonds, giving hepcidin a hairpin structure. Hepcidins are particularly diverse in teleost fishes, which may be related to the diversity of aquatic environments with varying degree of pathogen challenge, oxygenation, and iron concentration, factors known to alter hepcidin expression in mammals. We characterized the diversity of hepcidin genes of the Antarctic notothenioid fishes that are endemic to the world's coldest and most oxygen-rich marine water. Notothenioid fishes have at least four hepcidin variants, in two distinctive structural types. Type I hepcidins comprise three distinct variants that are homologs of the widespread 8-cysteine hepcidins. Type II is a novel 4-cysteine variant and therefore only two possible disulfide bonds, highly expressed in hematopoietic tissues. Analyses of dN/dS substitution rate ratios and Likelihood Ratio Test under site-specific models detected significant signal of positive Darwinian selection on the mature hepcidin coding sequence, suggesting adaptive evolution of notothenioid hepcidins. Genomic PCR and Southern hybridization showed that the novel type II hepcidin occurs exclusively in lineages of the Antarctic notothenioid radiation but not in the basal non-Antarctic taxa, and lineage-specific positive selection was detected on the branch leading to the type II hepcidin clade under Branch-site models, suggesting adaptive evolution of the reduced-cysteine variant in response to the polar environment. We also isolated a structurally distinct 4-cysteine hepcidin from an Antarctic eelpout that is unrelated to the notothenioids but inhabits the same freezing water. Neighbor-Joining analyses of teleost hepcidins showed the eelpout 4-cysteine variant arose independently from the notothenioid version, which lends support to adaptive evolution of reduced cysteine hepcidin variants on cold selection. The NJ tree also showed taxonomic-specific expansions of hepcidin variants, indicating duplication and diversification of hepcidin genes play important roles in evolutionary response to diverse ecological conditions.

Key Words: hepcidin ？ iron regulation ？ cold adaptation ？ positive selection ？ gene duplication