DNA barcoding of the leaf-mining moth subgenus Ectoedemia s. str. (Lepidoptera: Nepticulidae) with COI and EF1- α: two are better than one in recognising cryptic species

We sequenced 665bp of the Cytochrome C Oxidase I (COI)barcoding marker for 257 specimens and 482bp of ElongationFactor 1-α (EF1-α) for 237 specimens belonging to the leafminingsubgenus Ectoedemia (Ectoedemia) in the basal Lepidopteranfamily Nepticulidae. The dataset includes 45 out of 48West Palearctic Ectoedemia s. str. species and several speciesfrom Africa, North America and Asia. Both COI and EF1-αproved reliable as an alternative to conventional species identificationfor the majority of species and the combination of bothmarkers can aid in species validation. A clear barcode gap is notpresent, and in some species large K2P intraspecific pairwisedifferences are found, up to 6.85% in COI and 2.9% in EF1-α.In the Ectoedemia rubivora species complex, the species E. rubivora,E. arcuatella and E. atricollis share COI barcodes andcould only be distinguished by EF1-α. Diagnostic base positions,usually third codon positions, are in this and other casesa useful addition to species delimitation, in addition to distancemethods. Ectoedemia albifasciella COI barcodes fall into twodistinct clusters not related to other characters, whereas theseclusters are absent in EF1-α, possibly caused by mtDNA anomaliesor hybridisation. In the Ectoedemia subbimaculella complex,both sequences fail to unequivocally distinguish the speciesE. heringi, E. liechtensteini, E. phyllotomella and onepopulation of E. subbimaculella. DNA barcodes confirm thatNorth American Ectoedemia argyropeza are derived from aEuropean introduction. We strongly advocate the use of a nuclearmarker in addition to the universal COI barcode markerfor better identifying species, including cryptic ones.