Proliferating cell nuclear antigen (
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PCNA ) plays critical roles in eukaryoticDNA replication and replication‐associated processes. It is typically encoded by one or two gene copies (pcna ) in eukaryotic genomes. Recently reported higher copy numbers ofpcna in some dinoflagellates raised a question of how this gene has uniquely evolved in this phylum. Through real‐timePCR quantification, we found a wide range ofpcna copy number (2–287 copies) in 11 dinoflagellate species (n = 38), and a strong positive correlation betweenpcna copy number and genome size (log10–log10transformed). Intraspecificpcna diverged up to 21% and are dominated by nonsynonymous substitutions, indicating strong purifying selection pressure on and hence functional necessity of this gene. By surveyingpcna copy numbers in eukaryotes, we observed a genome size threshold at 4 pgDNA , above which more than twopcna copies are found. To examine whether retrotransposition is a mechanism ofpcna duplication, we measured the copy number of retroposedpcna , taking advantage of the 22‐nt dinoflagellate‐specific spliced leader (DinoSL ) capping the 5′ end of dinoflagellate nuclear‐encodedmRNA s, which would exist in the upstream region of a retroposed gene copy. We found that retroposedpcna copy number increased with totalpcna copy number and genome size. These results indicate co‐evolution of dinoflagellatepcna copy number with genome size, and retroposition as a major mechanism ofpcna duplication in dinoflagellates. Furthermore, we posit that the demand of faithful replication and maintenance of the large dinoflagellate genomes might have favored the preservation of the retroposedpcna as functional genes.