Multiple variants of the mitochondrial COI DNA barcode region are prevalent in North European sawflies

Abstract

DNA barcoding, the use of standard segments of DNA to assign specimens to a species, has emerged as a major field of biodiversity research over the last 20 years. Large-scale global initiatives are building DNA barcode reference libraries for animals, fungi, and plants, while pipelines are being developed for metabarcoding-based biomonitoring. The effectiveness of these approaches rests on the premise that much less variation exists within species than between them. While exceptions occur, this principle has been demonstrated to apply in the many animal taxa where the barcode region of the COI gene is effective in species discrimination. Sawflies are an exception to this general pattern because DNA barcodes often fail to distinguish congeneric species, an observation which prompted us to search for an explanation. 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 - 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 its occurrence does introduce 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, reference libraries must consider how best to deal with the high frequency of multiple variants in sawflies and any other groups of organisms. Significance StatementDNA barcoding is revolutionizing biodiversity science by enabling the accurate identification of organisms, accelerating taxonomic workflows, and permitting DNA-based biomonitoring. The DNA barcode region for the animal kingdom, mitochondrial COI, is highly effective in discriminating species in almost all studied animal groups. However, the use DNA barcoding is sometimes complicated by the presence of nuclear pseudogenes (NUMTs) or by variants of the mitogenome itself (heteroplasmy) within individuals. By using high-throughput sequencing (HTS) to analyze thou-sands of specimens, we demonstrate that multiple, seemingly functional, full-length variants of the COI barcode region are frequent in North European sawflies. Since these variants are sometimes very divergent, it is important to consider the impact of this within-individual variability on studies based on DNA barcodes.