Mitochondrial Impostors: Prevalence and Impacts of NUMTs on Genetic and Evolutionary Studies in Carnivora

Nuclear mitochondrial pseudogenes are mitochondria-derived DNA sequences integrated into the nuclear genome, which can introduce errors in species identification, phylogenetic inference, and population genetics. Although nuclear mitochondrial pseudogene contamination has been reported in some Carnivora species, a systematic investigation into the prevalence and impacts of nuclear mitochondrial pseudogenes across an order is still lacking. In this study, 22,102 mitochondrial DNA sequences of 80 Carnivora species from 14 families and 54 genera were retrieved from the public National Center for Biotechnology Information database and further analyzed. Using alignment-based methods, 158 problematic sequences/sequence groups were identified and categorized into four types: nuclear mitochondrial pseudogenes, species misidentification or mislabeling, sequence errors, and anomalous sites. Among families, Felidae exhibited the highest rate of nuclear mitochondrial pseudogene contamination, particularly in species of the genus Panthera. In contrast, no nuclear mitochondrial pseudogene contamination was detected in members of Ursidae and Ailuridae. Phylogenetic analysis revealed multiple independent origins of nuclear mitochondrial pseudogene, with some tracing back to the common ancestor of Carnivora. To mitigate nuclear mitochondrial pseudogene-related errors, rigorous sequence verification strategies, such as sequence alignment and phylogenetic validation, should be implemented. In conclusion, our findings highlight the necessity of nuclear mitochondrial pseudogene awareness in genetic and evolutionary studies of Carnivora and other taxa.