Кавказский энтомол. бюллетень 14(1): 91–93 © CAUCASIAN ENTOMOLOGICAL BULL. 2018

Molecular-genetic data for the better knowledge

of the identity of Polyommatus elena Stradomsky et Arzanov, 1999 (Lepidoptera: Lycaenidae)

Молекулярно-генетические данные к лучшему познанию идентичности Polyommatus elena Stradomsky et Arzanov, 1999 (Lepidoptera: Lycaenidae)

B.V. Stradomsky, E.S. Fomina Б.В. Страдомский, Е.С. Фомина

Federal Research Center the Southern Scientific Centre of the Russian Academy of Sciences, Chekhov str., 41, Rostov-on-Don 344006 Russia. E-mail: bvstr@yandex.ru

Федеральный исследовательский центр Южный научный центр РАН, пр. Чехова, 41, Ростов-на-Дону 344006 Россия

Key words: Lepidoptera, Lycaenidae, Polyommatus elena, COI, ITS2 DNA sequences.

Ключевые слова: Lepidoptera, Lycaenidae, Polyommatus elena, COI, ITS2-последовательности ДНК.

Abstract. It is demonstrated that specimens of Polyommatus elena Stradomsky et Arzanov, 1999 form separate branches on cladograms of their mitochondrial COI and nuclear ITS2 sequences that are separate from specimens of P. icarus (Rottemburg, 1775) and P. icadius (Grum-Grshimailo, 1890). This may give evidence of species independence of Polyommatus elena.

Резюме. Показано, что экземпляры Polyommatus

elena Stradomsky et Arzanov, 1999 на кладограммах митохондриальной COI и ядерной ITS2- последовательностей ДНК образуют отдельные, независимые ветви от представителей P. icarus (Rottemburg, 1775) и P. icadius (Grum-Grshimailo, 1890), что может свидетельствовать о самостоятельности вида.

Described about 20 years ago Polyommatus elena Stradomsky et Arzanov, 1999 had, until now, only morphological characteristics that determined its species status [Stradomsky, Arzanov, 1999; Polumordvinov et al., 2005; Stradomsky, 2006]. Yet nowadays the full characterization of a taxon should include molecular- genetic data as well. Carrying out a DNA analysis required fresh specimens of P. elena. The type locality of the species was recently completely destroyed by a construction project. However, three specimens which met related morphological criteria of P. elena (i.e. peculiar features of genitalia) were found on a virgin land in Belaya Kalitva District, Rostov Region, Russia (Fig. 1). The authors examined mitochondrial and nuclear DNA sequences, structures of genitalia and wing pattern characteristics of those specimens in comparison with related species P. icarus (Rottemburg, 1775) and P. icadius (Grum-Grshimailo, 1890).

Material and methods

The characteristic data of studied specimens of P. elena

and the compared specimens of P. icarus and P. icadius

DOI: 10.23885/1814-3326-2018-14-1-91-93

(place of collection, vouchers, accession numbers of COI and ITS2 sequences registered in GenBank) are presented in Table 1.

We amplified DNA 5’ section of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and the nuclear noncoding sequence internal transcribed spacer 2 (ITS2) on the Mastercycler gradient (Eppendorf). The following cycling protocols were used: an initial 4 min denaturation at 95 °C and 40 cycles of 30 s denaturation at 95 °C, 30 s annealing at 53 °C and 60 s extension at 72 °C.

We used the following PCR primer pairs: forward, 5’-TAG CGA AAA TGA CTT TTT TCT A-3’ with reverse,

Fig. 1. Localities of Polyommatus elena in Rostov Region, Russia. 1 – type locality; 2 – locality of the new samples.

Рис. 1. Местонахождения Polyommatus elena в Ростовской области.

1 – типовое местонахождение; 2 – местонахождение новых экземпляров.

92 B.V. Stradomsky, E.S. Fomina

Table 1. List of material with voucher codes and GenBank accession numbers.

Таблица 1. Используемый материал с музейными номерами и номерами последовательностей в GenBank.

5’-TTG CTC CAG CTA ATA CAG GTA A-3’ were used

to amplify COI. ITS2 was amplified with forward, 5’-GGG CCG GCT GTA TAA AAT CAT A-3’ and reverse, 5’-AAA AAT TGA GGC AGA CGC GAT A-3’ [Stradomsky, 2016].

Amplified fragments were separated using an automated sequencing machine (Applied Biosystems 3500). The analysis of primary nucleotide sequences was made with the help of the application BioEdit Sequence

Alignment Editor, version 7.0.5.3 [Hall, 1999].

COI and ITS2 nucleotide sequences were treated quantitatively using MEGA5 [Tamura et al., 2011] methods Minimum Evolution (ME) and were represented as ME- cladograms.

Results and discussion

The results of our molecular-genetic studies are presented in form of MP cladograms of DNA sequences for mitochondrial COI gene (Fig. 2) and nuclear nucleotide sequence ITS2 (Fig. 3), not linked together.

The data presented suggest that both mitochondrial and nuclear DNA sequences of P. icarus, P. icadius and P. elena form distinct independent branches in cladograms.

The results of molecular and genetic analysis correlate well with the results of comparison of genitalia structure characteristics. In lateral projection, unci of P. icarus and

P. icadius have an expanding at their basal part (Color plate 8: 6, 14), while uncus of P. elena is narrowed along the entire length (Colorplate 8: 10). In ventral projection, uncus lobes of P. icarus and P. icadius have a conical form (Color plate 8: 7, 15), while uncus lobes of P. elena have a blade form (Color plate 8: 11). A distinctive feature of the structure of female genitalia of P elena is a sclerotized part at the top of lamellae postvaginalis (Color plate 8: 21), which is absent in P. icarus (Color plate 8: 18). Also the habitus of imago of

P. elena (Color plate 8: 8, 9, 19, 20) has very characteristic features in comparison with specimens of P. icarus and

P. icadius (Color plate 8: 4, 5, 12, 13, 16, 17). It should be taken into account that the specimens of P. icarus

2 3

Figs 2–3. Polyommatus spp., ME-cladograms.

2 – for COI DNA sequences; 3 – for ITS2 DNA sequences. Рис. 2–3. Polyommatus spp., MЕ-кладограммы.

2 – для COI-последовательностей ДНК; 3 – для ITS2-последовательностей ДНК.

Color plate 8 Molecular-genetic data for the better knowledge of the identity of Polyommatus elena

Figs 4–15. Polyommatus spp., males.

4–7 – P. icarus (Voucher No ILL041); 8–11 – P. elena (Voucher No ILL075); 12–15 – P. icadius (Voucher No ILL022). 4, 8, 12 – upperside; 5, 9, 13 – underside; 6, 10, 14 – male genitalia, lateral view; 7, 11, 15 – uncus and gnathos, ventral view.

Рис. 4–15. Polyommatus spp., самцы.

4–7 – P. icarus (музейный № ILL041); 8–11 – P. elena (музейный № ILL075); 12–15 – P. icadius (музейный № ILL022). 4, 8, 12 – верх; 5, 9, 13 – испод; 6, 10, 14 – гениталии самца в латеральной проекции; 7, 11, 15 – ункус и гнатос в вентральной проекции.

Figs 16–21. Polyommatus spp., females.

16–18 – P. icarus (Voucher No ILL283); 19–21 – P. elena (Voucher No ILL284). 16, 19 – upperside; 17, 20 – underside; 18, 21 – lamellae postvaginalis.

Scale bars 1 mm.

Рис. 16–21. Polyommatus spp., самки.

16–18 – P. icarus (музейный № ILL283); 19–21 – P. elena (музейный № ILL284). 16, 19 – верх; 17, 20 – испод; 18, 21 – поствагинальная пластинка.

Масштабные линейки – 1 мм.

Molecular-genetic data for the better knowledge of the identity of Polyommatus elena 93

(Vouchers Nos. ILL041, ILL283) and P. elena (Vouchers Nos. ILL075, ILL284) both originate from Rostov Region,

i.e. the specimens are in sympatry.

It can be concluded that specimens, which by their morphological characters are categorized as P. elena, also have segments of unlinked mitochondrial and nuclear DNA sequences different from those of related species. It could be assumed that this is a specific feature of a particular population of P. icarus, but then it would be difficult to explain sympatric habitation of populations which have different sets of morphological characters as well as specific structure of both mitochondrial and nuclear markers. According to an expert’s opinion, “combined analysis of mitochondrial and nuclear markers is a simple and efficient way to identify the cryptic species in sympatry” [Lukhtanov, Shapoval, 2008: 436].

It would be logical to assume, therefore, that P. elena is a very young species in a state of formation.

Acknowledgements

The authors are grateful to Dr. V.A. Lukhtanov (Saint Petersburg State University, St. Petersburg, Russia) for fruitful discussion and valuable advice.

References

Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 41: 95–98.

Lukhtanov V.A., Shapoval N.A. 2008. Detection of cryptic species in sympatry using population analysis of unlinked genetic markers: a study of the Agrodiaetus kendevani species complex (Lepidoptera: Lycaenidae). Doklady Biological Sciences. 423(1): 432–436.

Polumordvinov O.A., Stradomsky B.V., Arzanov Yu.G. 2005. Identification and occurence of Polyommatus elena Stradomsky et Arzanov, 1999 (Lepidoptera: Lycaenidae). Caucasian Entomological Bulletin. 1(1): 87–88 (in Russian).

Stradomsky B.V. 2006. Characteristics of taxon Polyommatus elena Stradomsky et Arzanov, 1999 (Lepidoptera: Lycaenidae). Eversmannia. 5: 15–19 (in Russian).

Stradomsky B.V. 2016. A molecular phylogeny of the subfamily Polyommatinae (Lepidoptera: Lycaenidae). Caucasian Entomological Bulletin. 12(1): 145–156.

Stradomsky B.V., Arzanov Yu.G. 1999. Polyommatus elena sp. n. and Polyommatus neglectus sp. n. – new taxones of the family Lycaenidae (Lepidoptera). Izvestiya Kharkovskogo entomologicheskogo obshchestva. 7(2): 17–21 (in Russian).

Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology end Evolution. 28(10): 2731–2739.

Received / Поступила: 12.10.2017 Accepted / Принята: 6.03.2018