Globin Evolution in the Australian Lungfish: An Integrated Bioinformatic, Structural and Functional Study

Abstract

This thesis investigates the globin repertoire of the Australian lungfish (Neoceratodus forsteri), the closest living relative of tetrapods, to gain insight into vertebrate globin evolution during the transition from water to land. The lungfish genome is exceptionally large and highly repetitive, so a major part of the work focused on developing and applying complementary bioinformatic strategies for reliable gene identification and annotation. A diverse globin repertoire was recovered using a lungfish specific repeat-masked genome annotation pipeline combined with sequence similarity-based searches, phylogenetic analyses, transcriptomic validation, and structural prediction. This repertoire includes representatives of the major vertebrate globin families. A total of 103 globin-like candidates were identified. Of these, 18 high-confidence sequences were selected for further analysis. These included multiple copies of myoglobin and globin E, as well as globin X, globin Y, androglobin and hemoglobin α/β components. Importantly, they also included neuroglobin (Ngb) and cytoglobin (Cygb). This study is the first to confirm the presence of Ngb and Cygb in N. forsteri and in lungfishes in general. Both genes were amplified from cDNA and exhibited tissue-specific expression in the brain and heart. They also clustered within the expected vertebrate Ngb and Cygb clades and retained the conserved structural features characteristic of hexacoordinated globins. AlphaFold models and UV-Vis spectroscopy further supported the conservation of the canonical globin fold and heme coordination. In addition to genome-wide and molecular characterization, exploratory cell culture experiments were conducted to investigate the impact of lungfish Ngb and Cygb on cellular stress responses within a heterologous fish cell system. While these assays did not reveal a clear protective effect under the tested conditions, they established a foundation for future functional studies. Together, this thesis shows that lungfish retain a complex and evolutionarily informative globin repertoire and that integrated bioinformatic and experimental approaches are essential for studying genes in exceptionally large, repeat-rich genome.