show Abstracthide AbstractOhno (1967) originally proposed that the sex difference in X-linked gene dose caused by the decay of Y-linked genes may impose a "peril of hemizygosity" and that regulatory mechanisms must compensate to make X=XX=AA at the level of expression. Recent evidence suggests that Ohno's paradigm is not universal, but our understanding remains unclear because estimating the ancestral expression of X-linked genes is difficult or impossible in many systems. Many studies assess dosage compensation (DC) by comparing X to Autosome (AA) expression ratios, thereby implicitly assuming that the current average AA expression is a good proxy for the average ancestral expression of X-linked genes. A more appropriate test would be whether X=XX=Ancestral expression, where "Ancestral" is the inferred expression level of each X-linked gene before becoming X-linked. The few studies that have attempted to compare X (or Z) linked gene expression to corresponding ancestral levels have relied on distantly related taxa that include changes in chromosome number and sex-determination system. Here, we study the evolution of dosage compensation by comparing the expression of neo-X chromosome genes in Tribolium confusum to their inferred ancestral, autosomal expression state. The ancestral expression is estimated by analyzing RNA-Seq data across a time-calibrated phylogeny that includes four additional closely related species that all share an ancestral karyotype where the neo-X genes of T. confusum remain autosomal. We find that the neo-X in T. confusum is dosage balanced (X=XX) and dosage compensated (X=Ancestral), suggesting a chromosome-wide dosage compensation mechanism as envisioned by Ohno. Further, we observe that DC in T. castaneum, which was previously contentious, is fully balanced and compensated (X=XX=Ancestral). The conservation of dosage compensation mechanism of flour beetles suggests mammalian-like X chromosome inactivation in somatic tissues, which has important implications in understanding sex chromosome dosage compensation.