Multiple full‐length variants of the mitochondrial COI DNA barcode region are prevalent in north European sawflies

Abstract DNA barcoding, the use of a standard DNA fragment for species identification, has emerged as a major field of biodiversity research. The effectiveness of this approach rests on the premise that much less variation exists within species than between them. While exceptions occur, this has been demonstrated in many animal taxa where the COI gene is effective in species discrimination. Sawflies are an exception to this pattern because DNA barcodes often fail to distinguish congeneric species. Using high‐throughput single‐molecule DNA sequencing to recover COI sequences from thousands of sawflies, we found that single individuals often possess multiple, seemingly functional, full‐length DNA barcodes (i.e., unrecognizable as nuclear pseudogenes)—a phenomenon not documented at similar prevalence in any animal taxon. While the evolutionary causes of multiple variants require further investigation, our observation is remarkable as it violates the one‐barcode‐one‐specimen assumption. The presence of multiple variants of barcodes within individuals does not jeopardize the concept, but it introduces a complexity for species inventories based on metabarcoding. They will overestimate the species count when barcode‐based operational species units are used as species proxies. Similarly, DNA barcode reference libraries must consider how best to deal with the high frequency of multiple intra‐individual variants.