ZHANG, H.; Luo, H.

Flavobacteria are keystone taxa in global carbon cycling, degrading complex glycans in marine and terrestrial ecosystems. In both environments, polysaccharides constitute a major fraction of organic matter but differ in origin: marine glycans primarily derive from micro- and macroalgae, while terrestrial counterparts originate from land plants. Flavobacteria deploy distinct suites of carbohydrate-active enzymes (CAZymes) tailored to these habitat-specific substrates, yet the evolutionary drivers of their diversification remain unresolved. Two competing hypotheses exist: one posits glycan specialization as the primary driver of divergence, while the other implicates extrinsic geological factors in shaping their evolution. By integrating mitochondrial- and plastid-based molecular clocks with eukaryotic fossil calibrations, we infer that flavobacteria emerged between 2.15 and 1.98 billion years ago (Gya), shortly after the Great Oxidation Event (GOE; 2.4-2.32 Gya). Subsequent diversification involved three independent marine-to-non-marine transitions during the Proterozoic Eon (1.98-1.70 Gya, 1.72-1.40 Gya, and 1.28-1.14 Gya), temporally aligned with the formation and the fragmentation of the Columbia supercontinent and preceded the evolution of the major glycan contributors in both marine and non-marine niches. This temporal mismatch disfavors the glycan specialization hypothesis, instead implicating tectonic-driven habitat shifts as the primary driver of lineage diversification. Non-marine flavobacteria exhibited higher turnover but lower net diversification rates than marine counterparts, reflecting the challenges of adapting to fragmented non-marine niches. Genome erosion and deleterious mutation accumulation further constrained reverse transitions, locking lineages into non-marine habitats. Our findings highlight Proterozoic tectonics, rather than substrate-specific CAZyme innovation, as the catalyst for flavobacteria\'s evolutionary success across Earth\'s carbon-rich biomes.