Newly Discovered Occurrences and Gene Tree of the Extracellular Globins and Linker Chains from the Giant Hexagonal Bilayer Hemoglobin in Metazoans

Multicellular organisms depend on oxygen-carrying proteins to transport oxygen throughout the body; therefore, proteins such as hemoglobins (Hbs), hemocyanins, and hemerythrins are essential for maintenance of tissues and cellular respiration. Vertebrate Hbs are among the most extensively studied proteins; however, much less is known about invertebrate Hbs. Recent studies of hemocyanins and hemerythrins have demonstrated that they have much wider distributions than previously thought, suggesting that oxygen-binding protein diversity is underestimated across metazoans. Hexagonal bilayer hemoglobin (HBL-Hb), a blood pigment found exclusively in annelids, is a polymer comprised up to 144 extracellular globins and 36 linker chains. To further understand the evolutionary history of this protein complex, we explored the diversity of linkers and extracellular globins from HBL-Hbs using in silico approaches on 319 metazoan and one choanoflagellate transcriptomes. We found 559 extracellular globin and 414 linker genes transcribed in 171 species from ten animal phyla with new records in Echinodermata, Hemichordata, Brachiopoda, Mollusca, Nemertea, Bryozoa, Phoronida, Platyhelminthes, and Priapulida. Contrary to previous suggestions that linkers and extracellular globins emerged in the annelid ancestor, our findings indicate that they have putatively emerged before the protostome–deuterostome split. For the first time, we unveiled the comprehensive evolutionary history of metazoan HBL-Hb components, which consists of multiple episodes of gene gains and losses. Moreover, because our study design surveyed linkers and extracellular globins independently, we were able to cross-validate our results, significantly reducing the rate of false positives. We confirmed that the distribution of HBL-Hb components has until now been underestimated among animals.