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Кавказский энтомол. бюллетень 13(2): 247–248 © CAUCASIAN ENTOMOLOGICAL BULL. 2017
1Southern Scientific Centre of the Russian Academy of Sciences, Chekhov str., 41, Rostov-on-Don 344006 Russia. E-mail: bvstr@yandex.ru
2Universität zu Köln, Auf dem Rosenhügel 15, Köln 50997 Germany
3North-Caucasus Federal University, Pushkin str., 1, Stavropol 355009 Russia
1Южный научный центр РАН, пр. Чехова, 41, Ростов-на-Дону 344006 Россия
2Кёльнский университет, Розенхюгель, 15, Кёльн 50997 Германия
3Северо-Кавказский федеральный университет, ул. Пушкина, 1, Ставрополь 355009 Россия
Key words: Lepidoptera, Lycaenidae, enigmatic specimen, Prosotas lutea, ITS2 DNA sequence.
Ключевые слова: Lepidoptera, Lycaenidae, неопознанный экземпляр, Prosotas lutea, ITS2, последовательности ДНК.
Abstract. With the help of DNA analysis, an enigmatic lycaenid butterfly from Sumatra belonging to the genus Prosotas is determined as Prosotas lutea (Martin, 1895).
Резюме. С помощью анализа ДНК неопознанная голубянка из Суматры определена как Prosotas lutea (Martin, 1895).
During an investigation of the entomofauna of Sumatra in 2016, Valentin Tikhonov collected a most unusual lycaenid butterfly (Color plate 7: 1, 2). The specimen is characterized by the complete reduction of its underside pattern, usually consisting of numerous striae, but with the help of genitalic dissection, was determined as belonging to the genus Prosotas Druce, 1891 (Color plate 7: 3).
To obtain a reliable determination, we decided to analyse molecular sequences and compared them with ITS2 sequences (Internal Transcribed Spacer 2) known from various Prosotas species: P. lutea (Martin, 1895) (Color plate 7: 4, 5), P. gracilis (Röber, 1886), P. pia Toxopeus, 1929 and P. dubiosa (Semper, [1879]). We used the ITS2 sequence because of its great variability, providing significant differences at species level.
Material. 1♂, Prosotas sp.: Indonesia, Ketambe vill., Sumatra, 28.02.2016 (V. Tikhonov), (Voucher No. ILL253, GenBank accession numbers KX083692); 1♂, P. lutea: Myanmar, Hsipaw, Tu River, 1.08.2005 (A. Sokolov), (Voucher No. ILL282, GenBank accession numbers MG551471); 1♂, P. gracilis: Indonesia, Ketambe vill., Sumatra, 28.02.2016, (V. Tikhonov), (Voucher No. ILL256, GenBank accession numbers KX083689); 1♂, P. pia: Malaysia, Sabah, Borneo, 14.02.2014 (D. Pozhogin), (Voucher No. ILL197, GenBank accession numbers KJ934122); 1♂,
P. dubiosa: Thailand, Pai, 1.04.2014 (I. Uchevatov), (Voucher No. ILL193, GenBank accession numbers KJ774016).
DOI: 10.23885/1814-3326-2017-13-2-247-248
We amplified DNA 5’ section of the nuclear noncoding sequence 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: with forward, 5’-GGG CCG GCT GTA TAA AAT CAT A-3’ and reverse,
5’-AAA AAT TGA GGC AGA CGC GAT A-3’ were used to
amplify ITS2 [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].
ITS2 nucleotide sequences were treated quantitatively using MEGA5 [Tamura et al., 2011] methods Minimum Evolution (ME) and were represented as ME-cladogram.
As additional source for information and calculation of cladograms, we also used sequences from GenBank No. KJ934120, KJ934121, KM586809, KP900999, KX013516.
Molecular analysis resulted in a cladogram (Color plate 7: 6) showing the enigmatic specimen at the same position and branch where P. lutea is located. ITS2 difference is less than 1% between both and thus confirms a determination as P. lutea.
Aberrations are not uncommon among lycaenid butterflies and this example shows again that molecular analysis is a powerful tool to determine problematic specimens correctly.
248 B.V. Stradomsky, S. Schröder, V.V. Tikhonov
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.
Stradomsky B.V. 2016. A molecular phylogeny of the subfamily Polyommatinae (Lepidoptera: Lycaenidae). Caucasian Entomological Bulletin. 12(1): 145‒156.
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: 30.11.2017 Accepted / Принята: 10.12.2017
Identification of an enigmatic lycaenid specimen (Lepidoptera: Lycaenidae) Color plate 7
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Figs 1–6. Prosotas spp., morphology of imago and result of molecular-genetic analysis.
1–3 – Prosotas sp. (Voucher No. ILL253): 1 – upperside, 2 – underside, 3 – genitalia; 4–5 – Prosotas lutea (Voucher No. ILL282): 4 – upperside, 5 – underside; 6 – ME-cladograms for ITS2 DNA sequences.
Рис. 1–6. Prosotas spp., морфология имаго и результаты молекулярно-генетического анализа.
1–3 – Prosotas sp. (музейный номер ILL253): 1 – верх, 2 – испод, 3 – гениталии; 4–5 – Prosotas lutea (музейный номер ILL282): 4 – верх, 5 – испод; 6 – ME-кладограмма для ITS2-последовательностей ДНК.