Uncovering hidden complexity: COI heteroplasmy in Euphausia mucronata G.O. Sars, 1883

Over the decades, the Cytochrome Oxidase I (COI) gene has been widely used as a low-cost standardized identification method for phylogenetic and phylogeographic studies. However, the literature of the COI gene amplification, cloning, and sequencing using traditional methods, such as Sanger sequencing, suggest that individuals from certain species might present multiple COI copies as consequence of contamination, NUMTs and heteroplasmy. Here we investigated whether Euphausia mucronata, a hypoxia-tolerant endemic and abundant krill species from the Humboldt Current System along the western coast of South America, presents multiple COI copies within individuals in order to elucidate the likely effects on phylogenetic and phylogeographic studies. By cloning and sequencing the COI gene of E. mucronata individuals we identified multiple copies within individual samples, with no evidence of contamination or NUMTs, suggesting heteroplasmy as the cause of these copies. Genetic distances between COI copies were small, with intra-individual divergence observed, interpreted as the result of significant variability of the oceanic environment where the species lives. Phylogenetic analyses used to validate the use of COI for the identification of E. mucronata showed that multiple COI copies did not affect species identification. Nevertheless, because COI copies within single individuals were not geographically related, they could influence population studies, as seen in genetic variations among individuals from different sampling sites. Our results demonstrate that the random amplification or sequencing of a single copy of the COI gene in E. mucronata might not pose a phylogenetic concern, but it could have implications for phylogeographic or population studies.