Interspecies variation of 45S ribosomal DNA in vertebrates

Ribosomal DNA (rDNA) remains one of the least resolved components of complex genomes despite its central role in cellular function and evolutionary inference. Using chromosome-scale assemblies from the Vertebrate Genomes Project, we present a comparative analysis of the 45S rDNA locus across vertebrates. rDNA organization varies widely among clades, with lineage-specific differences in chromosomal distribution, copy number, and positional bias. Mammals and birds typically restrict rDNA to few, often terminal loci, whereas amphibians and several fish lineages exhibit highly dispersed architectures, including extreme cases spanning dozens of chromosomes. Copy number varies by more than an order of magnitude and does not scale simply with repeat-unit size or predicted functional demand, consistent with rDNA functioning as a flexible genomic reservoir rather than a direct proxy for ribosome production. At the sequence level, the 45S unit resolves into distinct evolutionary regimes. Genes encoding rRNAs (18S, 5.8S, 28S) remain highly conserved and retain strong phylogenetic signal, whereas internal transcribed spacers show rapid divergence, lineage-specific expansion, and pronounced asymmetry between intergenic spacers ITS1 and ITS2. Sequence-structure analyses reveal a gradient of constraint across the locus, from tightly coupled evolution in rRNA-encoding regions to increasingly permissive regimes in spacers. Structure-informed phylogenetic inference yields modest but consistent improvements under high divergence, highlighting its value when sequence signal degrades. Together, these results establish the 45S rDNA locus as a multi-layered evolutionary system integrating deep conservation with structural plasticity across vertebrates.