Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified

Abstract

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineagespecific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.cytochrome c oxidase I ͉ Decapoda ͉ Orthoptera

Population genetic structure of Calanoides natalis (Copepoda, Calanoida) in the eastern Atlantic Ocean and Benguela upwelling system

“…They have been found in a wide range of crustacean taxa including calanoid copepods (Bucklin et al, 1999(Bucklin et al, , 2000Machida and Lin, 2017) and diaptomid calanoids (Thum and Harrison, 2009;Marrone et al, 2013). The aberrant sequences found in this study do not contain any stop codons or frame shifts, and therefore do not resemble classical nuclear mitochondrial pseudogenes (so called numts) that often contain InDels (Insertion/Deletions) and mutations at any codon position (Song et al, 2008;Buhay, 2009;Calvignac et al, 2011). Still, the pronounced nucleotide diversity of the sequences may indicate a recent translocation of a COI copy to the nucleus.…”

“…co-amplification of highly divergent paralogous copies can lead to misinterpretation of species diversity (Song et al, 2008). They have been found in a wide range of crustacean taxa including calanoid copepods (Bucklin et al, 1999(Bucklin et al, , 2000Machida and Lin, 2017) and diaptomid calanoids (Thum and Harrison, 2009;Marrone et al, 2013).…”

“…However, recent discoveries of cryptic and pseudo-cryptic species complexes in copepods (Goetze, 2003;Goetze and Ohman, 2010;Laakmann et al, 2012;Andrews et al, 2014;Cornils and Held, 2014) clearly demonstrate that genetic diversity does not necessarily lead to distinguishable morphological features. Additionally, DNA barcoding solely relying on the mitochondrial marker genes such as cytochrome c oxidase subunit I (COI) may lead to the misinterpretation of species diversity (Song et al, 2008).…”

Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead‐end

“…Protein coding sequences that contained stop codons were removed to eliminate potential nuclear mitochondrial genes (Song et al. 2008; Buhay 2009). The rRNA genes were aligned with PASTA version 1.6 (Mirarab et al.…”

Genetic aspects of mitochondrial genome evolution

“…In PCR-based approaches, unrecognised divergent NUMTs can introduce noise and unrecognised biases. In the DNA barcoding approaches, furthermore, they cause a systematic overestimate of the number of species (Song et al, 2008). …”