Quantifying genus-level divergence using 18S rDNA and its application to heterolobosea with discovery of a novel...

The phylum Heterolobosea comprises a morphologically diverse and ecologically versatile assemblage of free-living microbial eukaryotes, yet genus-level boundaries remain difficult to resolve due to limited diagnostic characters and extensive phenotypic plasticity. Here, we apply a reproducible 18S rDNA divergence framework to quantify genus-level divergence in Heterolobosea and to evaluate taxonomic placement of a newly discovered isolate from coastal sediments of Mombasa, Kenya. Microscopic observations reveal a highly plastic amoeba exhibiting monopodial limax-type locomotion, episodic eruptive activity, and the formation of large multinucleate and polyploid stages that fragment into smaller cells, suggesting an unusual parasexual-like life cycle. Phylogenetic analyses recover the isolate in a strongly supported clade with Orodruina flavescens and an uncultured environmental lineage from the Lost City hydrothermal field. Pairwise 18S rDNA distance analyses show a clear bimodal separation between intragenus and intergenus comparisons with divergence values among these lineages consistently exceed empirical intrageneric thresholds across multiple analytical frameworks. Substitution saturation diagnostics confirm that these divergences occur within the phylogenetically informative region of the SSU rDNA gene. Together, the molecular, morphological, and ecological evidence support recognition of the Mombasa lineage as a distinct genus and species, Mombasina parasexualis gen. nov. et sp. nov., within Orodruinidae. This study reveals previously underappreciated diversity within Tetramitia and demonstrates the broader utility of quantitative divergence-based frameworks for resolving genus-level boundaries in microbial eukaryotes.