A Divergence-Calibrated Framework for Robust Cross-Species Comparison of Effective Population Size Ratios

1. Comparing effective population size (Nₑ) ratios among autosomes, the X, Y, and mitochondrial genomes is a powerful approach for inferring sex-specific demography and selection; however, chromosome- and lineage-specific mutation-rate differences have prevented reliable cross-species comparisons. 2. We introduce a phylogenetically informed framework that uses interspecific divergence to calibrate chromosome-specific mutation rates, yielding unbiased Nₑ ratios that are free of external priors and directly comparable across diverse taxa. 3. For each genomic compartment, we combine genome-wide nucleotide diversity (π) with divergence (D) from a high-quality outgroup. Because neutral mutation rate (μ) and divergence time cancel in the π/D ratio, this approach produces internally calibrated, chromosome-specific Nₑ ratios and is implemented as an open-source, automated pipeline. 4. Applying this framework to hominids and three ant species with a Y-like chromosome reveals Nₑ-ratio patterns that are consistent with known biology, demonstrating the method’s ability to recover species-specific signals of reproductive strategy and population history. We provide a fully implemented and well-documented computational tool to facilitate broad adoption. Together, this work establishes a generalizable and robust basis for inferring reproductive strategies and demographic history through comparative analyses of Nₑ ratios.