Divergent gene expression levels between diploid and autotetraploid Tolmiea (Saxifragaceae) relative to the total transcriptome, the cell, and biomass

• Premise of the study: Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea, we present an integrated approach for cross-ploidy comparisons that account for differences in transcriptome size and cell density, and make multiple comparisons of transcript abundance.• Methods: We use RNA spike-in standards in concert with cell size and density to identify and correct for differences in transcriptome size, and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass.• Key results: In total, ~17% of all loci were identified as differentially expressed (DEGs) between the diploid and autopolyploid species. The per-transcriptome normalization, the method researchers typically use, captured the fewest DEGs (58% of total DEGs) and failed to detect any DEGs not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEGs were recovered respectively. The discrepancy between per-transcriptome and per-cell recovery of DEGs occurs because per-transcriptome normalizations are concentration-based and therefore blind to differences in transcriptome size.• Conclusions: While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid-like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis-related functions.