Controlling for life-history traits in vertebrates reveals that effective population size does not affect mutation...

Why mutation rates (μ) and genome sizes (GS) vary among species remains a central question in evolutionary genetics. Two influential models, the drift-barrier hypothesis (DBH) and the mutational-hazard hypothesis, propose that effective population size (N_e) shapes these traits via the efficiency of selection, predicting higher μ and larger genomes in small populations. A recent comparative analysis of vertebrates reported a significant negative correlation between N_e and μ, interpreted as support for the DBH. Using phylogenetic path analysis, we reanalyze the same dataset of 55 vertebrate species spanning mammals, birds, reptiles, and fishes, in which μ was estimated from high-coverage parent-offspring trios, while explicitly controlling for six life-history traits within a causal framework that tests model-implied conditional independencies. We show that the reported N_e-μ association is entirely mediated by generation time (GT), which independently influences both variables; once this "back-door" path is blocked, N_e has no detectable effect on μ. Once GT is accounted for, mating system shows the largest association with μ. Parallel analyses of GS within the same validated life-history framework reveal that GS is unrelated to N_e, μ, or their interaction and is decoupled from the life-history covariation that strongly structures μ. These results are robust to alternative N_e estimators and causal model formulations. Together, our findings indicate that N_e provides little explanatory power for variation in μ or GS across vertebrates, challenging the presumed universality of drift-limited genome evolution.