Refining Heterolobosean Taxonomy with 18S rDNA Divergence Analysis: Discovery of a Novel Genus from the Coast of Mombasa, Kenya

The class Heterolobosea comprises a morphologically diverse and ecologically versatile assemblage of free-living microbial eukaryotes, yet species- and genus-level boundaries remain difficult to define due to limited diagnostic characters and extensive phenotypic plasticity. Here we apply a reproducible 18S rDNA divergence framework to evaluate taxonomic limits across the group and to determine the placement of a newly discovered heterolobosean isolate collected from coastal sediments of Mombasa, Kenya. Microscopic observations reveal a highly plastic amoeba exhibiting monopodial limax-type locomotion, episodic eruptive hyaline 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 the environmental clone DQ504339.1 from the Lost City hydrothermal field. Pairwise 18S rDNA p-distance analyses show clear bimodality between intragenus and intergenus comparisons, and divergence values among the three Orodruina -associated sequences consistently exceed empirical intrageneric thresholds under all analytical frameworks. Substitution saturation diagnostics confirm that these divergences occur within the linear, phylogenetically informative portion of the SSU gene. Together, the molecular, morphological, and ecological evidence support recognition of the Mombasa lineage as a distinct genus and species, Mombasina parasexualis gen. et sp. nov., within Orodruinidae. This work highlights the deep, underexplored diversity within Tetramitia and underscores the value of standardized divergence-based frameworks for resolving taxonomic boundaries in microbial eukaryotes.