Кавказский энтомологический бюллетень 15(1): 203–205 © Caucasian Entomological Bulletin 2019

Some nuances of molecular phylogeny

of the genus Satyrium Scudder, 1876 (Lepidoptera: Lycaenidae)

Некоторые нюансы молекулярной филогении рода Satyrium Scudder, 1876 (Lepidoptera: Lycaenidae)

B.V. Stradomsky

© 1, E.S. Fomina2

© Б.В. Страдомский 1, Е.С. Фомина2

1Rostov Branch of the Russian Entomological Society, PO Box 3318, Rostov-on-Don 344092 Russia. E-mail: bvstr@yandex.ru

2Federal Research Centre the Southern Scientific Centre of the Russian Academy of Sciences, Chekhov str., 41, Rostov-on-Don 344006 Russia

1Ростовское отделение Русского энтомологического общества, а/я 3318, Ростов-на-Дону 344092 Россия, E-mail: bvstr@yandex.ru 2Федеральный исследовательский центр Южный научный центр Российской академии наук, пр. Чехова, 41, Ростов-на-Дону 344006 Россия

Key words: Lepidoptera, Lycaenidae, Satyrium, Fixsenia pruni, molecular markers, developmental stages.

Ключевые слова: Lepidoptera, Lycaenidae, Satyrium, Fixsenia pruni, молекулярные маркеры, стадии развития.

Abstract. Molecular studies (based on COI of mDNA and nuclear ribosomal ITS2 region) demonstrated that certain concepts of Satyrium Scudder, 1876 reflected by recent literature are not monophyletic. Two species are transferred to the genus Fixsenia Tutt, 1907: Fixenia pruni (Linnaeus, 1758), comb. resurr. (from Satyrium), Fixenia herzi (Fixsen, 1887), comb. resurr. (from Satyrium). This opinion is supported by differences between preimaginal stages of pruni and species of the genus Satyrium. The unusual structure of female genitalia of pruni should be also noted. The taxon Fixsenia Tutt, 1907, sensu nova (F. pruni and its closest relatives) is interpretated here as a genus to improve the classification.

Резюме. Молекулярные исследования на основе COI мДНК и ядерной рибосомальной области ITS2 показали, что таксон Satyrium Scudder, 1876, широко интерпретируемый в современной литературе, не является монофилетическим. Два вида перенесены в род Fixsenia Tutt, 1907: Fixenia pruni (Linnaeus, 1758), comb. resurr. (из Satyrium) и Fixenia herzi (Fixsen, 1887), comb. resurr. (из Satyrium). В пользу этого свидетельствуют различия преимагинальных стадий pruni и представителей рода Satyrium. Также необходимо отметить оригинальность строения гениталий самок pruni. Таксон Fixsenia Tutt, 1907, sensu nova (включающий F. pruni и наиболее близкие к нему виды) интерпретируется как род в целях усовершенствования классификации.

Currently, interpretation of taxonomic position of Palaearctic species of the genus Satyrium Scudder, 1876 is quite ambiguous. For one, many authors assign all these specimens directly to this genus [Gorbunov, 2001; Kudrna, 2002; Settele et al., 2008; Lafranchis et al., 2015]. At the same time there is a division of the genus Satyrium into Nordmannia Tutt, 1907 and Strymonidia

Research Article / Научная статья

DOI: 10.23885/181433262019151-203205

Tutt, 1908 [Higgins, Riley, 1978], or into Armenia Dubatolov et Korshunov, 1984, Superflua Strand, 1910 and Nordmannia [Tuzov et al., 2000], or into Nordmannia and Fixsenia Tutt, 1907 [Inomata, 1990, Korshunov, 2002], or Noredmannia and Strymonidia [Min, Xiaoling, 2002]. To clarify taxonomic position of Palaearctic specimens of the genus Satyrium, the authors performed their molecular genetic analysis with Nearctic specimens Satyrium s. str. involved. The authors examined mitochondrial and nuclear DNA sequences and structures of female genitalia and developmental stages.

Material and methods

We amplified mDNA 5’ section of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and the nuclear non-coding 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’-GTC AAC AAT CAT AAA ATA TTG G-3’ (reserve forward 5’- TAG CGA AAA TGA CTT TTT TCT-3’) with reverse, 5’-TTG CTC CAG CTA ATA CAG GTA A-3’

(reserve reverse 5’- AAG AAT GAG GTA TTG AGG TTT C-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’

[Wiemers et al., 2010; 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].

204 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.

COI and ITS2 nucleotide sequences were treated quantitatively using MEGA6 [Tamura et al., 2013] methods Minimum-Evilution (ME) and were represented as ME- cladograms.

Network for COI sequences constructed with the NETWORK: Version 4.6.1.6 program.

In order to elaborate the system of the genus Satyrium the authors used also some sequences from GenBank for comparision, namely COI sequences of S. acaudata (Staudinger, 1901) (accession No GenBank FJ664041) and

S. herzi (Fixsen, 1887) (accession No GenBank GU372539). Other material is listed in the Table 1.

Thecla betulae, Quercusia quercus, Neolycaena rhymnus and N. eckweileri were used as an outgroup to root the tree.

Results and discussion

The results of molecular genetic studies (Color plate : 1, 2) suggest that while members of the outgroup to the genus Neolycaena de Nicéville in Marshall et de Nicéville, 1890 are not an outgroup to specimens of the taxon S. pruni, they meet the criteria for an outgroup for the other specimens of the genus Satyrium, including the Nearctic species S. titus (Color plate : 1). This pattern is specific for both COI gene and ITS2 sequence, which is unlinked to COI gene (Color plate : 2).

In order to preserve the principle of monophyly in this case there is the only appropriate solution: to recognize the taxon pruni as not belonging to the genus Satyrium. The another solution is to unite all the genera involved under Satyrium. But that not reflects the reality mirrored by genitalia and early stage characters.

The cladogram on Color plate : 3 demonstrates that the taxon herzi, the type species of the genus Fixsenia, is a sister taxon of pruni. Therefore, both taxa have to be recognized as valid: Fixsenia herzi, and F. pruni. Higgins et al. [1991] included Satyrium pruni to the genus Fixsenia.

Gorbunov [2001] placed Fixsenia as a subgenus of the genus Satyrium. In his view morphological similarity between both S. (F.) herzi and S. (F.) pruni and other specimens of the genus Satyrium based on the structure of genitalia: “Ventral cornutus in vesica reduced, short” [Gorbunov, 2001]. Korshunov [2002] listed the genus Fixsenia with species F. herzi and F. pruni, however without any substantial explanations.

The distinctness of F. pruni is highlighted also by specific characters of its developmental stages which are different from those of species placed in the genus Satyrium: final instar larvae of the F. pruni have yellowish- green heads (Color plate : 4, 5), while those of the genus Satyrium have black heads (Color plate : 6–8). More significant differences are found between pupae. The pupa of F. pruni is black, with very large white spots; abdominal region very convex, with conical lumps on dorsal and lateral sides (Color plate : 9, 10). Pupae of the genus Satyrium are uniformly convex, with smooth, brownish surface with numerous small dark spots (Color plate : 11–16).

There are also differences in structure of female genitalia. Lamella postvaginalis of F. pruni (Color plate : 17) is broadened apically, while the lamella of specimens representing the genus Satyrium is smoothly narrowed apically (Color plate : 18–21).

Consequently, on the basis of combination of morphological and molecular genetic characteristics the taxon pruni and its relatives should be considered as not belonging to the genus of Satyrium, but representing the genus Fixsenia: genus Fixsenia Tutt, 1907, sensu nova (not as a subgenus of the genus Satyrium), Fixenia pruni (Linnaeus, 1758), comb. resurr. (from Satyrium), Fixenia herzi (Fixsen, 1887), comb. resurr. (from Satyrium).

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Received / Поступила: 8.02.2019 Accepted / Принята: 28.02.2019