Comparative genomics of parasitic and symbiotic microeukaryotes: Phylogenomic insights into lifestyle transitions and...

Host-associated lifestyles are widespread among microbial eukaryotes, yet their evolutionary origins remain poorly resolved in unculturable lineages. Ciliates are complex unicellular eukaryotes characterized by nuclear dimorphism and extensive genome plasticity. They exhibit diverse host interactions, including ectoparasitism by Trichodina on fishes, opportunistic pathogenesis by Balantidium in mammals, and obligate anaerobic endosymbiosis by Nyctotheroides in amphibians. However, the evolutionary relationships among these host-associated forms have been obscured by a lack of genomic data. Here, we overcome this limitation by applying single-cell whole-genome sequencing to directly recover draft genomes from five ciliate species isolated from vertebrate hosts. Phylogenomic analyses reveal that these host-associated ciliates are distributed across highly divergent clades within the phylum Ciliophora, with no congruence observed between ciliate phylogeny and host phylogeny. This decoupling demonstrates that host association has evolved independently in distantly related lineages without long-term co-diversification. Consequently, functional convergence in key gene categories suggests a shared genetic toolkit for adapting to diverse host environments. Furthermore, comparative functional profiling identifies convergent enrichment in genes involved in environmental sensing, transmembrane transport, and protein complex assembly, despite stark ecological differences among parasitic and symbiotic lifestyles. These shared molecular features suggest that successful colonization of animal hosts may be constrained by common physiological requirements rather than lineage-specific adaptations. By leveraging single-cell genomics, our study establishes a genomic framework for unculturable host-associated ciliates, providing access to previously elusive symbiotic and parasitic lineages and significantly advancing our understanding of the evolution of host dependence across microbial eukaryotes.