The evolution of gene functional repertoire in Amorphea: Divergent strategies across Amoebozoa, Fungi, and Metazoa.

Abstract

Metazoa and Fungi have been extensively studied to reconstruct the trajectory of Opisthokont evolution. Their sister group, Amoebozoa, provides additional potential to generate valuable insights into the origins of Opisthokont lineages. Amoebozoa represent a diverse group of amoeboid organisms, which have adapted to a wide range of environments and ecological niches. Studying Amoebozoa not only helps to illuminate Opisthokont evolution but also reveals the mechanisms that have driven their ecological success. Here we report the discovery of Apostamoeba explorator strain BEAP0066, representing a novel lineage within Amoebozoa with intriguing behaviors like the "double-amoeba", a behavior characterized by the bipolarization of a cell into two poles that coexist and act as two semi-independent cells; and the "colonizing rings", the generation of a front of amoebae advancing together and grazing on bacterial mats. By analyzing the gene content of A. explorator and diverse amoebozoans with ancestral gene content reconstructions, correspondence analyses of Clusters of Orthologous Groups (COG) category composition and Pfam clan clustering, we revealed distinct evolutionary trajectories for Amoebozoa, Metazoa, and Fungi. Amoebozoa retained an ancestral Amorphea-like state, characterized by an enrichment of genes related to motility, phagocytosis, and environmental adaptability; while Metazoa specialized in multicellularity-related genes and Fungi in metabolism and transport. These findings suggest that retention of gene function composition, rather than gene loss, played a key role in shaping Amoebozoa evolution. SIGNIFICANCE STATEMENTAmoebae are cells with no defined shape that move by extending temporary cell membrane projections, usually composed of actin, called pseudopodia. Amoeboid cells are present in all major eukaryotic lineages, from the well-known Amoeba proteus in Amoebozoa to human macrophages in Metazoa, Salpingoeca rosetta in Choanoflagellatea, Vampyrella lateritia in Rhizaria, and other examples such as Naegleria fowleri in Heterolobosea. The prevalence of this lifestyle highlights the evolutionary success of the amoeboid form and its ability to adapt to diverse environmental conditions and ecological roles. The study of amoebae and amoeboid cell types is crucial for advancing research in medicine, ecology, and evolution. Understanding the phylogeny of Amoebozoa remains a key focus in phylogenomics, as deep divergences within the group complicate the taxonomic placement of certain taxa and have implications for character evolution. Thus, the discovery, identification, and characterization of new Amoebozoa species can help resolve current uncertainties. Recent studies have identified divergent trajectories in gene content composition within one lineage of Amorphea, the Opisthokonts. Fungi evolved through an expansion of metabolic genes, whereas Metazoa accumulated genes associated with multicellularity. Inspired by these findings, we analyzed the proteomes of Amoebozoa, Metazoa, and Fungi. Using correspondence analysis of the relative composition of Clusters of Orthologous Groups (COG), ancestral reconstruction and the analysis of Pfam domain clan presence in supergroup-specific gene clusters, we aimed to determine whether the three supergroups within Amorphea exhibit distinct clustering patterns. Our results provide evidence of divergent functional evolution in Amoebozoa, Fungi, and Metazoa. Amoebozoa retained an ancestral Amorphea-like state, characterized by an enrichment of genes related to motility, phagocytosis, and environmental adaptability; while Metazoa specialized in multicellularity-related genes and Fungi in metabolism and transport. These findings suggest that retention of gene function composition, rather than gene loss, played a key role in shaping Amoebozoa evolution.