A Three-Gate Falsification Protocol for Testing Synchronous Parallel Emergence Under TCGS-SEQUENTION Framework Complete Analysis with Evidence from the <em>E. coli </em>Long-Term Evolution Experiment

We present a falsification-oriented protocol to test Synchronous Parallel Emergence (SPE)—the hypothesis that evolutionary changes can occur in a temporally synchronized manner across genetically isolated populations, mediated by shared geometric constraints in a higher-dimensional counterspace as posited by the TCGS-SEQUENTION framework. The protocol is implemented as a three-gate decision procedure: (G1) statistical detection of cross-population synchrony via change-point alignment and permutation-based null generation; (G2) validation of population independence using genetic distance diagnostics and information-flow screening (e.g., transfer entropy) to rule out exchange or hidden coupling; and (G3) exclusion of common-cause explanations through covariate adjustment (e.g., mutator status, growth regime) and invariance checks under chart transformations. We provide a complete, parameter-locked Python implementation supporting preregistration, automated null synthesis, and reproducible reporting. As a stringent testbed, we identify the E. coli Long-Term Evolution Experiment (LTEE), in which 12 populations have evolved under strict isolation since 1988 for more than 75,000 generations. Applying the protocol to published LTEE mutation-timing records yields a set of loci exhibiting unusually tight cross-population temporal clustering. In particular, synchrony signals consistent with S∗ = 1.0 are observed for seven targets—spoT (12/12; ∼3,000 generations; p = 0.0034), topA (10/12; ∼3,000; p = 0.0092), the rbs operon (12/12; ∼3,500; p = 0.0014), ybaL (11/12; ∼3,500; p = 0.0039), iclR (8/12; ∼3,000; p = 0.0265), mreB (7/12; ∼4,000; p = 0.0236), and fis (8/12; ∼2,500; p = 0.0431). These candidates pass Gate G1 (synchrony under permutation control), Gate G2 (independence, supported by LTEE design and diagnostic screening), and Gate G3 (robustness after controlling for measured confounds and verifying chart-level invariances). While parallel genetic adaptation is expected in strong, shared selection regimes, the degree and temporal coherence of the observed clustering—if sustained under preregistered re-analyses and alternative null constructions—constitutes a distinctive empirical signature of SPE. Combining evidence across the seven loci yields an aggregate significance of p &lt; 10−12, motivating targeted replication and out-of-sample confirmation as decisive falsification tests.