bioRxiv2026
Identifying discrete microbial assemblages and their environmental drivers across multiple biological fractions simultaneously remains a central challenge in aquatic microbial ecology. We applied an integrated analytical pipeline built around Latent Dirichlet Allocation (LDA) to an eight-year 16S rRNA amplicon time series from the Laurentian Great Lakes, spanning four size-fractionated biological blocks -- free-living prokaryotes, particle-associated prokaryotes, and small and large chloroplast-containing eukaryotes. LDA resolved ecologically coherent subcommunities whose taxonomic identity was consistently defined at the order and class level, with fingerprint taxa confirmed by discriminant analysis. Shannon entropy differences between blocks reflected fundamental differences in dispersal capacity and environmental filtering -- free-living prokaryotes and large eukaryotes showed higher mixing than particle-associated prokaryotes and small eukaryotes. Temperature dominated environmental structuring across all blocks, assessed through Limma and random forest with SHAP, with secondary drivers differing by size fraction. Group Compositional Analysis jointly integrating all four blocks revealed that thermal stratification and lake chemistry organize microbial communities coherently across all size fractions simultaneously. Warm stratified and cold inversely-stratified waters harbored largely non-overlapping assemblages across all four blocks, with cold water specialists -- including chemolithotrophic deep-branching lineages and silica-dependent diatoms -- having no warm water equivalents. ImportanceThe Laurentian Great Lakes are among the fastest warming lakes in the world, yet the microbial communities that drive their biogeochemical cycles remain poorly characterized across size fractions and thermal habitats. Using eight years of samples spanning all five Great Lakes, we show that free-living bacteria, particle-associated bacteria, small phytoplankton, and large phytoplankton all respond coherently to the same master environmental gradients -- thermal stratification and lake chemistry -- despite fundamental differences in organism size, trophic role, and sequencing protocol. Warm stratified and cold water habitats support distinct microbial communities, and the cold water specialists identified here have no warm water equivalents. As the Great Lakes warm and cold water habitats shrink, the microbial communities that depend on them will not simply become less abundant they will be replaced by fundamentally different assemblages.