Retrotransposed gene copies persist under relaxed selection in wild Spodoptera frugiperda.

Copy number variations (CNVs) are major sources of genetic variation, affecting large portions of genomes. Typical CNVs are known to have short evolutionary half-lives due to deleterious effects, with documented events of adaptive evolution. However, it is not known whether the same trends apply to CNVs preserving intact Open Reading Frames (iORF-CNVs), which can encode functional proteins. Here, we investigate the mechanistic origins and evolutionary dynamics of iORF-CNVs in Spodoptera frugiperda, a globally distributed pest species where CNV-mediated adaptive evolution has been reported. Using PacBio HiFi reads, we generated high-quality, individually assembled genomes for 36 field-collected individuals and identified 349 genes with polymorphic iORF-CNVs. These elements are short, intronless, and display molecular signatures of LINE-mediated retrotransposition, including cis- and trans-acting effects. A majority of iORF-CNVs (68.0% to 77.28%) exhibited at least one molecular signature of retrotransposition. iORF-CNVs showed lower divergence at the first and second codon positions than at the third codon positions, but they had higher nonsynonymous-to-synonymous polymorphism ratios than non-iORF-CNV genes, implying purifying selection with relaxed constraint. Orthology analyses indicated that these CNVs originate from genes under weak evolutionary constraint, while transcriptomic and promoter motif analyses revealed that many are transcribed and retain regulatory features. We conclude that observed iORF-CNVs are generated through retrotransposition from weakly constrained genes and that the duplicates are selectively maintained for coding potential, albeit under reduced purifying selection compared with non-iORF-CNV genes. These results imply that pseudogenization is not an inevitable evolutionary fate of retrotransposed gene copies when the open reading frame is preserved.