Assigning confidence to sequence comparisons for species identification: A detailed comparison of the cytochrome b and cytochrome oxidase subunit I mitochondrial genes

Species identification is a tool used extensively in forensic science; particularly in the investigation of wildlife crime. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b), and identification is generally carried out through the use of DNA sequencing. However, there is currently no standard method to quantify the data from sequence comparisons for presentation in reports and to courts as there have been no detailed studies of the expected levels of inter- and intraspecific variation. For the first time this study provides a detailed comparison of the effectiveness of these two loci. Interspecific and intraspecific variation are assessed and statistical confidence is applied to sequence comparisons. Comparison of 217 different mammalian species revealed that cyt b more accurately reconstructed their phylogeny and known relationships, and gave better resolution when separating species based on sequence data. Intraspecific variation was assessed using three model species and showed variation ranging from 0 to 1.16% (Kimura 2-parameter p-distance (K2P). ×. 100%), indicating that some level of variation should be expected. Interspecific variation was greater in cyt b than in COI. Using a K2P (×100) threshold of 1.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI. This study allows, for the first time, application of statistical confidence to sequences comparisons for species identification.