Di-, tri-, and tetranucleotide frequencies covary with lifespan and genome size across protostome invertebrates

Animal lifespans span orders of magnitude, yet how genome sequence covaries with lifespan remains poorly characterized outside vertebrates. Although promoter CpG density has been linked to vertebrate longevity due to its gene-regulatory function through DNA methylation, it is unclear whether such patterns are promoter- and CpG-specific, or if they reflect broader sequence evolution. We curated maximum lifespan estimates for 466 protostome species spanning eight phyla with available genome assemblies and quantified mono-, di-, tri-, and tetranucleotide composition across whole genomes, intergenic regions, and six gene-associated regions (two upstream regions, exons, introns, and two downstream regions) defined using Benchmarking Universal Single-Copy Orthologs. Dinucleotide observed/expected ratios showed significant associations with lifespan and genome size in different ways. Lifespan-associated motifs were most pronounced in gene-associated non-coding regions, especially in introns and downstream regions, whereas genome-size effects were strongest in whole-genome and intergenic sequence. Tri- and tetranucleotide observed/expected ratios broadly recapitulated this regional organization. In contrast, GC content was not associated with lifespan across regions, indicating that the observed signals are not explained by mononucleotide composition but instead by how those nucleotides are arranged into short sequence motifs. These results suggest that lifespan and genome size show distinct but overlapping associations with regional sequence composition across invertebrate species and that lifespan-associated motif evolution extends beyond vertebrate promoter methylation architectures.