Possibility of numt co-amplification from gigantic genome of Orthoptera: testing efficiency of standard PCR protocol in producing orthologous COI sequences

Mitochondrial DNA has been the preferential genome biodiversity studies. However, several factors contribute to its inadequacy. Numts constitute one of the main complications that prevent obtaining orthologous mitochondrial sequences. Orthoptera have been a model group in numt studies because of their huge genome size. In this study we aimed to; (i) test efficiency of standard PCR protocol in producing orthologous sequences of cytochrome C oxidase, (ii) study presence/absence of numts in several unstudied Orthoptera species, (iii) test if there is a threshold between the length of mtDNA targeted for amplification and possibility of encountering numts, and (iv) estimate reliability of the sequences in databases in light of these findings. For these aims we studied 38 species of Orthoptera representing different sublineages and genome sizes. DNA extracted from each sample was used to amplify five different fragments of COI region by standard PCR protocol. Sequenced PCR amplicons were checked for numt possibility by several different numt criteria. No sequences without numt signs were obtained for the first fragment. The number of samples with numt signs for the other four fragments differed between the suborders Ensifera and Caelifera. The percentage of samples with numt signs was higher in Caelifera than Ensifera for all fragments. The numt percentage considerably decreased for the longest two fragments. Numts are more prevalent in families with larger genome size. We arrived at the following conclusions: (i) numts are common in all members of Orthoptera, but, their prevalence differs among intra-lineages, especially more prevalent in Caelifera, (ii) there seems a correlation between numt rate and genome size, (iii) there is no threshold to avoid numt co-amplification, but, a 1,000 bp length may be a threshold for Ensifera, (iv) Folmer region of COI doesn't seem an appropriate marker for animal barcoding. Additionally, a phylogenetic tree produced from the numt sequences of fragment four detected in genus Anterastes suggested a paleonumt gained in generic ancestor a 3.5–4 times slower divergence rate for numt sequences