First results of Central Asiatic Euchalcia DNA- studies: comparison of nucleotide sequence differences in COI between Euchalcia herrichi and Euchalcia gyulai (Lepidoptera: Noctuidae)

We submitted first results of the DNA studies of the Central Asiatic owlet moths of the genus Euchalcia. Standard cytochrome C oxidase subunit I (COI) gene fragments were sequenced for DNA barcoding of six specimens belonging to Euchalcia herrichi and Euchalcia gyulai. We compared the received sequences between discussed species and with two European Euchalcia species (E. variabilis and E. consona). We found no variability within the COI sequences of the samples collected in the same locality (Alai Mts., Kyrgyzstan), whereas the difference in COI sequences between two populations (Ketmen Mts., Kazakhstan and Alai Mts., Kyrgyzstan) was 0.005.

In 2017-2019, the expeditions of the Russian Entomological Society (Nizhny Novgorod, Volgograd and Saratov Divisions) to Kyrgyzstan found a local population of recently described species, E. gyulai, in the Alai mountain range, Taldyk Pass (Korb et al. 2016). Moths were flying together with typical Central Asiatic species E. herrichi (Staudinger, 1861). Both species were used in DNA studies. Herein I report their results.
E gyulai -three specimens collected together with E. herrichi in Kyrgyzstan, Alai Mts., Taldyk Pass, 3700 m, using the same automatic autonomous light trap.
The material was sequenced by the BOLD project at the University of Guelph (Ratnasingham and Hebert 2007). All sequence operations were conducted using the MEGA v. 7.0 (Kumar et al. 2016) and BioEdit v. 7.2.5 (Hall 1999).

Results
We did not discover any variability in three COI sequences of E. gyulai. The COI sequences of E. herrichi from Alai Mts. were also identical. The sequences of the latter species from Alai Mts. and from Ketmen Mts. had a small difference (p-distance 0.005). The differences between COI sequences of E. gyulai and E. herrichi from the same locality (Altai Mts.) are presented below (p-distance 0.139, Fig.1).

Discussion
The COI differences between European Euchalcia species are not as high as between high-mountainous Central Asiatic species. E. variabilis from Finland and E. consona from Germany had 48 different nucleotide pairs (p-distance was 0.078), while E. herrichi and E. gyulai from Altai Mts. had 88 different nucleotide pairs (p-distance was 0.139).
We registered that there was no variability within three samples of E. gyulai: all three received sequences were identical. The COI distances between two closely related species (E. herrichi and E. gyulai are very close habitually and inhabited the same biotopes) were relatively high: 0.139 for species collected from Altai Mts. and 0.135 between the samples of E. gyulai from Altai Mts. and E. herrichi from the Ketmen mountain ridge. The questions of this 'magic number' (what COI sequence p-distance is enough for the species delimination) is still open since the molecular methods were applied for taxonomy. Many scientists concluded, that there is no exact answer to this question. This should be specific for every group of organisms: in some genera the species can be separated by the difference in only 1-3 nucleotides (Burns et al. 2007); in some other groups, like discussed in this paper species from genus Euchalcia, the interspecific differences can widely vary and could reach the p-distance of 0.139. So, the 'universal' p-distance of 0.02, calculated by Huemer et al. (2014), is not an universal number in many cases. It must be calculated independently for every group of organisms basing on the sequencing statistics, comparative morphology, and ecology.