Comprehensive computational insights into Plasmodium retrocopies reveals potential roles in virulence-associated functions.

Retrocopies are processed copies of other genes that originate through reverse transcription and are randomly integrated into the genome. Once considered genomic "junk," emerging evidence shows they contribute to novel gene evolution and may aid adaptation to environmental and lifestyle changes. However, the role of retrocopies in the emergence of virulence in Plasmodium parasites remains largely unexplored. This study systematically characterizes retrocopies across 23 Plasmodium pathogenic genomes to elucidate their evolutionary origins, functional relevance, and potential contributions to the emergence of virulence. Using a stringent computational framework, we identified 724 high-confidence retrocopies exhibiting canonical signatures of retroposition. Retrocopies are frequently intact, under purifying selection, and often display detectable apparent transcription during intraerythrocytic stages, with a subset showing FPKM values from RNA-seq data comparable to or exceeding those of their parental genes, particularly in PPIase, PIR, and actin-related families linked to host interaction and immune evasion. Domain, GO, and KEGG enrichment analyses indicate that retrocopies are preferentially associated with processes such as cytoadhesion, chromatin remodeling, stress response, and cytoskeletal organization, and that retroposition has contributed to the expansion and diversification of pathogenic gene families in a lineage and host-specific manner. Ancestral state reconstruction indicates episodic gains and losses of retrocopies, with notable expansions in primate-infecting lineages. The findings show retrocopies in Plasmodium are widespread, evolutionarily dynamic, and functionally associated with key virulence-associated pathways rather than representing inert genomic byproducts. This study highlights retroposition as an underappreciated mechanism contributing to genomic plasticity in malaria parasites and provides a framework for future functional investigations.