Hebert et al. (2003) first proposed the idea that animal species can be distinguished by comparing a 648-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. The length of the fragment is technically limited by the Sanger sequencing method. The COI barcoding can be used for two purposes:

A.)  Indication/discovery/delimitation of new species

B.)  Identification of known species

In both cases, query sequences are compared with sequences stored in reference libraries like GenBank and BOLD (Barcode of Life Data Systems). The reference libraries need to be accurate, comprehensive and taxonomically updated. 

To determine genetic diversity levels between taxa, pairwise genetic distances need to be established. 

COI sequences have been chosen as species markers because it turned out that in vertebrates (including birds) species are separated from close congeneric species by CO1 sequence divergences higher than 2%, while sequence divergences among conspecifics are usually less than 2% (Hebert et al., 2003). The 2%-threshold genetic distance is not a strict biological boundary, but rather a heuristic tool. 

Misleadingly, this phenomenon is referred to as the “barcode gap” (Meyer & Paulay, 2005). 

In a comparative avian mitogenomic study, the CO1 gene proved to be the one with the least amount of rate heterogeneity across avian orders, thus being closest to a “molecular clock” (Pacheco et al., 2011). Thus, the suitability of COI gene sequences as indicators of species limits simply reflects the fact that they serve as a reliable time scale. It should be noted, however, that mitochondrial DNA is inherited maternally only. 

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