Multilayer analysis reveals extensive hidden population complexity in a dairy starter microbiome comprising few species

The genomic and epigenomic complexity of microbiomes with low species richness remains poorly characterized. Here, we integrated 111 non-redundant isolate genomes with deep long-read metagenomics to investigate an undefined mesophilic dairy starter. Closely related isolates harboured distinct combinations of features associated with fermentation traits and phage defence, including surface polysaccharide genotypes, plasmids, prophages, defence systems, and methylation profiles. Metagenomics revealed additional elements and inferred host associations. However, the limited overlap between approaches across core-genome lineages and accessory repertoires underscored complementary but incomplete recovery, to the extent that the dominant metagenomic cell wall polysaccharide genotype (29.6% abundance) was absent from isolates. Metagenomic methylation signatures supported 36 species-level plasmid-host assignments, corroborated by isolate genomes in nine of ten testable cases. Our findings revealed extensive combinatorial diversity hidden from either approach alone, providing a plausible molecular basis for phage resilience, while likely representing only a lower bound on population complexity.