Development of new barcoding loci in gall-forming aphids (Eriosomatinae: Eriosomatini): Comparing three mitochondrial genes, ATP6, ATP8, and COI

•We screened 542 sequences of 8 genes for developing new molecular markers.•The ATP6 and ATP8 gene was statistically tested in comparison to the COI gene from 25 aphid species.•The ATP8 gene reveals lower intra- and higher inter-specific genetic distances than the COI gene.

The mitochondrial COI gene has often been utilized as a molecular marker for species identification. However, it has sometimes caused misidentification for some pairs of closely related species. For detecting complementary barcoding loci, we first screened candidate genes by calculating genetic distances within and between species based on 542 sequences collected from the Genbank by using aphids of the Eriosomatini as an example. Of eight genes analyzed, we selected the ATP6 and ATP8 genes, which exhibited lower intraspecific and higher interspecific genetic divergences than did the COI gene. Secondly, we tested the usefulness of these genes by calculating genetic distances between all the combinations of 44 individuals of 23 eriosomatine species for each of the ATP6, ATP8, and COI genes. In the ATP8 gene, the average intraspecific divergence was lowest (0.6%) and the average interspecific divergence was highest (14.7%). The ATP8 gene evolved more rapidly than did the COI gene if genetic divergence between individuals was sufficiently large, whereas it evolved more slowly than did COI if genetic divergence was less than a threshold (1% in COI distance). As a result, species with intraspecific variation in COI and ATP6 exhibited no genetic variation in ATP8. The pattern of genetic divergence in ATP8 well accorded with the pattern of species delimitation in the present taxonomic system. Thus, we conclude that the use of the ATP8 gene in DNA barcoding could improve the accuracy of species identification in the Eriosomatini and possibly other insect groups.