РОССИЙСКАЯ АКАДЕМИЯ НАУК Институт аридных зон ЮНЦ RUSSIAN ACADEMY OF SCIENCES Institute of Arid Zones SSC CAUCASIAN ENTOMOLOGICAL BULLETIN Том 12. Вып. 1 Vol. 12. No. 1 Ростов-на-Дону 2016 Кавказский энтомол. бюллетень 12(1): 145–156 © CAUCASIAN ENTOMOLOGICAL BULL. 2016 A molecular phylogeny of the subfamily Polyommatinae (Lepidoptera: Lycaenidae) Молекулярная филогения подсемейства Polyommatinae (Lepidoptera: Lycaenidae) B.V. Stradomsky Б.В. Страдомский Institute of Arid Zones SSC RAS, Chekhov str., 41, Rostov-on-Don 344006 Russia. E-mail: [email protected] Институт аридных зон ЮНЦ РАН, пр. Чехова, 41, Ростов-на-Дону 344006 Россия Key words: Lepidoptera, Lycaenidae, Polyommatinae, phylogeny, molecular genetics, morphological research. Ключевые слова: Lepidoptera, Lycaenidae, Polyommatinae, филогения, молекулярно-биологические и морфологические исследования. Abstract. Molecular and morphological study The author also uses such non-taxonomic concept as a of the subfamily Polyommatinae allows to make the section (Section sensu Eliot). following conclusions: the tribe Candalidini and the genus The leading role in the study of taxonomy and Cupidopsis should be excluded from the subfamily. The systematics of organisms is currently allocated to molecular status of the tribe Niphandini should be reduced to a genetic research. This rule undoubtedly applies to Blue subtribe level. Thus, subfamily Polyommatinae consists of butterflies, and the publication of many articles in the last two tribes: Lycaenesthini and Polyommatini. Elimination decade only emphasizes that [Wiemers, 2003; Kandul et of the non-taxonomic rank “section” and the combined al., 2004; Lukhtanov et al., 2005; Vodolazhsky et al., 2009; morphological and genetic analysis make it possible to Wiemers et al., 2010; Ugelvig et al., 2011; Vila et al., 2011; distinguish 22 subtribes within the tribe Polyommatini, Talavera et al., 2013, 2015; Stradomsky, 2014]. Traditional which meet the requirements of monophyly: Brephidiina, morphological methods can not ensure the construction of Pithecopina, Niphandina, Danina, Azanina, Theclinesthina, a natural system of Lycaenidae. High variability of the wing Lycaenopsina, Jamidina, Cacyreina, Actizerina, pattern, structure of genitalia and other features cannot Uranothaumatina, Lampidina, Zizulina, Catochrysopsina, create an adequate system of Blue butterflies. However, the Scolitantidina, Castaliina, Oboroniina, Leptotina, analysis of some morphological characters can be used as Zizeeriina, Fameganina, Everina, and Polyommatina. an additional criterion for the construction of the system. Резюме. Молекулярно-генетическое и First of all, this holds true for the study of genitalia. морфологическое изучение голубянок подсемейства The goal of this study was an attempt to build a Polyommatinae позволяет прийти к следующим natural phylogeny of one of the largest subfamilies of Blue выводам: триба Candalidini и род Cupidopsis должны butterflies, the Polyommatinae. This subfamily contains быть выведены из состава подсемейства, а триба species with the extremely pronounced heterogeneity Niphandini понижена в статусе до уровня подтрибы. of external morphological characteristics and variability Таким образом, в подсемействе Polyommatinae of the genital apparatus. The specimens of the subfamily остаются две трибы: Lycaenesthini и Polyommatini. Polyommatinae were studied with the use of molecular Упразднение внетаксономического ранга «секция» genetic methods. Specifically, we examined the following и комплексный морфолого-генетический анализ genetic markers: sections of the mitochondrial gene позволяют выделить в составе трибы Polyommatini Cytochrome Oxidase subunit I, the nuclear Elongation следующие 22 подтрибы, отвечающие требованию Factor 1-alpha (the nuclear coding sequence) and the монофилии: Brephidiina, Pithecopina, Niphandina, nuclear noncoding sequence internal transcribed spacer 2 Danina, Azanina, Theclinesthina, Lycaenopsina, Jamidina, (the nuclear noncoding sequence). At the same time the Cacyreina, Actizerina, Uranothaumatina, Lampidina, genital structures of Polyommatinae representatives have Zizulina, Catochrysopsina, Scolitantidina, Castaliina, been investigated. Oboroniina, Leptotina, Zizeeriina, Fameganina, Everina и Polyommatina. Material and methods Currently the most commonly used and adequate All specimens examined in this study are archived system of Lycaenidae is the concept of Eliot [1973]. It is at the museum of the Institute of Arid Zones SSC RAS based solely on an analysis of morphological characters. (Rostov-on-Don, Russia) as voucher specimens. Features However, the author uses unreasonably excessive of studied specimens are presented in the Table 1. Eighty fragmentation of many genera, as well as higher-level taxa. nine species were examined. 146 B.V. Stradomsky Table 1. List of material with voucher codes and GenBank accession numbers. Таблица 1. Исследованный материал: музейные номера и присвоенные номера Генбанка. A molecular phylogeny of the subfamily Polyommatinae (Lepidoptera: Lycaenidae) 147 Table 1 (continuation). Таблица 1 (продолжение). Parameters for methods of DNA extraction were 5’-TAG CGA AAA TGA CTT TTT TCT A-3’ (reserve described previously [Vodolazhsky, Stradomsky, 2008]. forward 5’-GGT CAA CAA ATC ATA AAG ATA We amplified DNA 5’ section of the mitochondrial TTG G-3’) with reverse, 5’-TTG CTC CAG CTA ATA gene Cytochrome Oxidase subunit I (COI), the nuclear CAG GTA A-3’ (reserve reverse 5’-TAA ACT TCA GGG Elongation Factor 1-alpha (Ef-1a) and the nuclear TGA CCA AAA AAT CA-3’) were used to amplify COI. noncoding sequence internal transcribed spacer 2 (ITS2) Ef-1a was amplified with forward, 5’-TAC CAT CGA GAA on the Mastercycler gradient (Eppendorf). The following GTT CGA GAA G-3’ (reserve forward 5’-TGA AGG CCG cycling protocols were used: an initial 4 min denaturation AAC GTG AAC GTG G -3’) and reverse, 5’-GCC ACC at 95° C and 40 cycles of 30 s denaturation at 95° C, 30 s CCT TGA ACC AGG GCA T-3’. ITS2 was amplified with annealing at 53° C and 60 s extension at 72° C. forward, 5’-GGG CCG GCT GTA TAA AAT CAT A-3’ We used the following PCR primer pairs: forward, (reserve forward 5’-ACT CCT GTC TGA GGG CCG 148 B.V. Stradomsky GCT G-3’) and reverse, 5’-AAA AAT TGA GGC AGA torsion on the apex and long sickle-shaped branches of CGC GAT A-3’ (reserve reverse 5’-TGA GGC AGA gnathos. CTC GAT ATC CGT C-3’) [Stradomsky, Fomina, 2013; We should also note that the butterflies of the genus Stradomsky, 2014]. Cupidopsis Karsch, 1895 have 10 veins on the forewing, Amplified fragments were separated using an which is characteristic only for some genera of the subfamily automated sequencing machine (Applied Biosystems 3500). Theclinae, including the genus Hypothecla Semper, 1890. The analysis of primary nucleotide sequences was Thus, the results of molecular genetic analysis, as well made with the help of the application BioEdit Sequence as some significant morphological characteristics, indicate Alignment Editor, version 7.0.5.3 [Hall, 1999]. that the species of the genus Cupidopsis (and, consequently, Summary COI-Ef-1a-ITS2 nucleotide sequences Cupidopsis section sensu Eliot), most likely do not belong were treated quantitatively using MEGA5 [Tamura et al., to the subfamily Polyommatinae. 2011] methods Minimum-Evolution (ME) and Maximum All the other taxa of the subfamily Polyommatinae, Likelihood (ML) and were represented as ME- и ML- except those mentioned above, form a monophyletic cladograms. group. The first of the isolated clades of the subfamily includes genera Cupidesthes Aurivillius, 1895 and Anthene Results and discussion Doubleday, 1847, which constitute the tribe Lycaenesthini. Members of the tribe are still close to the species of the This study based on a complex analysis of three outgroup based on morphological features, which is genetic markers of blues butterflies from 4 tribes and especially characteristic for the genus Cupidesthes. The 30 sections (sensu Eliot) of subfamily Polyommatinae, as genitalia of representatives of the genera Cupidesthes well as an outgroup which includes some members of the (subfamily Polyommatinae) and Satyrium Scudder, 1876 subfamilies Lycaeninae, Theclinae and Miletinae. We used (subfamily Theclinae) are very similar in the lateral and nucleotide sequences that are associated with various types in the ventral view (Figs 9–12). They have an expressed of the evolutionary process as a marker: mitochondrial saccus, wide domed dorsal structures, a long thin aedeagus gene COI, the nuclear gene encoding a protein Ef-1a, as with adjacent small elongated valvae, long widely rounded well as nuclear nucleotide sequence noncoding a protein branches of gnathos, and small ventrally oriented lobes of ITS2, which is largely nondependent on external factors uncus. selection. The next three sister clades are two sections and one Obtained ME- and ML-cladograms (Figs 1, 2) tribe sensu Eliot: Brephidium section, Pithecus section have the maximum similarity, except for a insignificant and the tribe Niphandini sensu Eliot. Since all of these displacement of some small clades. It should be noted clades are at least equivalent, it is necessary to set a lower that the obtained dendrograms have significant similarity status for the tribe Niphandini and set it as a subtribe with the ML-phylograms of tribe Polyommatini, which is Niphandina, placed in the tribe Polyommatini. In addition, based on the analysis of species and genes which are in there is a need to clarify a taxonomic status of