Hidden genetic diversity of rumen protozoa associated methanogens unraveled by a single cell metagenomic approach

Rumen ciliate protozoa are key components of the ruminal microbiome and are considered important contributors to methane production due to their associations with methanogenic archaea. However, the diversity, evolutionary relationships, and metabolic potential of these protozoa-Archaea associations remain poorly understood. Here, we investigated the diversity and methanogenic potential of archaeal communities associated with rumen ciliates using a single-cell metagenomic approach. We analyzed genomic datasets from 22 ciliate morphospecies, comprising nine newly generated and 13 publicly available datasets, combining ribosomal marker screening, metagenomic taxonomic profiling, phylogenetic reconstruction, and functional annotation. We recovered 49 archaeal ribosomal lineages associated with 11 ciliate species, revealing considerable diversity across multiple methanogenic groups, particularly Methanobacteriales and Methanomassiliicoccales. Phylogenetic analyses revealed lineages closely related to previously characterized methanogens, as well as divergent lineages potentially representing previously uncharacterized archaeal diversity. Metagenomic profiling further expanded the detected archaeal diversity and revealed differences in archaeal community composition among ciliate hosts. Functional reconstruction of KEGG metabolic modules revealed pronounced heterogeneity in methanogenic potential, particularly in pathways associated with CO 2 -dependent and methylotrophic methanogenesis. While some ciliate-associated archaeal fractions exhibited extensive recovery of methanogenesis-related modules, others showed limited representation of these functions, indicating differences in the recovered metabolic repertoires among hosts. These results reveal substantial taxonomic and functional heterogeneity among rumen ciliate-associated archaeal communities and highlight the complexity of their potential contributions to ruminal methane metabolism.