Phylogenetic Structure Revealed through Combining DNA Barcodes with Multi-Gene Data for Agrodiaetus Blue Butterflies (Lepidoptera, Lycaenidae).

The need for multi-gene analysis in evolutionary and taxonomic studies is generally accepted. However, the sequencing of multiple genes is not always possible. For various reasons, short mitochondrial DNA barcodes are the only source of molecular information for some species in many genera, although multi-locus data are available for other species of the same genera. In particular, such situation exists in the species-rich butterfly subgenus Polyommatus (Agrodiaetus). Here, we analyzed the partitioning of this subgenus into species groups by using three data sets. The first data set was represented by short mitochondrial DNA barcodes for all analyzed samples. The second and third data sets were represented by a combination of short mitochondrial DNA barcodes for part of the taxa with longer mitochondrial sequences COI + tRNA-Leu + COII (data set 2) and with longer mitochondrial COI + tRNA-Leu + COII and nuclear 5.8S rDNA + ITS2 + 28S rDNA sequences (data set 3) for the remaining species. We showed that the DNA barcoding approach (data set 1) failed to reveal the phylogenetic structure, resulting in numerous polytomies in the tree obtained. Combined analysis of the mitochondrial and nuclear sequences (data sets 2 and 3) revealed the species groups and the position within these species groups, even for the taxa for which only short DNA barcodes were available.

Chromosomal and DNA barcode analysis of the Polyommatus (Agrodiaetus) damone (Eversmann, 1841) species complex (Lepidoptera, Lycaenidae).

The Polyommatus (Agrodiaetus) damone (Eversmann, 1841) species complex comprises from 5 to 8 species distributed in southeastern Europe and southern Siberia. Here we used chromosomal and DNA-barcode markers in order to test the taxonomic hypotheses previously suggested for this complex. We revealed that all taxa within this group demonstrate chromosomal stasis and share the same or very similar haploid chromosome number (n = 66 or n = 67). This finding is unexpected since the karyotypes are known to be very diverse and species-specific within the other taxa of the subgenus Agrodiaetus Hübner, 1822. Analysis of the mitochondrial gene COI revealed six diverged clusters of individuals within the complex. Each cluster has a specific geographic distribution and is characterized by distinct morphological features in the wing pattern. The clusters mostly (but not always) correlate with traditionally recognized species. As a result of our study, we describe a new subspecies P. (A.) iphigenides zarmitanussubsp. nov. from Uzbekistan and Tajikistan and show that the taxon originally described as Lycaena kindermanni var. melania Staudinger, 1886 represents a subspecies P. (A.) iphigenides melanius (Staudinger, 1886). Polyommatus (A.) samusi Korb, 2017 (syn. nov.) and P. (A.) melanius komarovi Korb, 2017 (syn. nov.) are considered here as junior subjective synonyms of P. (A.) iphigenides iphigenides (Staudinger, 1886).

Karyotype and DNA barcode of Polyommatus (Agrodiaetus) cyaneus (Staudinger, 1899) from its type locality: implication for taxonomic and evolutionary research in Polyommatus blue butterflies (Lepidoptera, Lycaenidae).

Chromosomal and molecular analyses of rapidly evolving organisms such as Polyommatus Latreille, 1804 blue butterflies are essential for understanding their taxonomy and evolutionary history, and the studies of populations from their type localities are crucially important for resolving problems of nomenclature and species identity. Here we present data on the topotypical population of the blue butterfly species described as Lycaena damone var. cyanea Staudinger, 1899. This taxon was described from Khankendi (Nagorno-Karabakh, Caucasus), and rediscovered at the type locality for the first time since it was collected there in 1869. The specimens were found on dry stony meadows with a predominance of Onobrychis radiata Bieberstein, 1810, on upper border of oak forests. Their haploid chromosome number (n) was established as n = 17. Chromosomal and mitochondrial DNA barcode analyses of the studied samples from type-locality provided an opportunity for the critical taxonomic re-examination of Caucasian species of the subgenus Agrodiaetus Hübner, 1822 of the genus Polyommatus Latreille, 1804. The obtained data support the interpretation of the P. (A.) cyaneus (Staudinger, 1899) and P. (A.) carmon (Herrich-Schäffer, 1851) as two different, not closely related species complexes as previously hypothesized by Hugo de Lesse. On the contrary, the treatment by Walter Forster who considered these taxa as two groups of conspecific populations was not supported by our data.

Karyotype of Polyommatus (Agrodiaetus) eriwanensis Forster, 1960 and taxonomic position of P. (A.) interjectus de Lesse, 1960 (Lepidoptera, Lycaenidae).

The karyotype of Polyommatus (Agrodiaetus) eriwanensis Forster, 1960 from the type locality ("Eriwan" [Yerevan, Armenia]) and other localities in Armenia was investigated. The number of chromosomal elements (bivalents+ multivalents) observed in male meiosis I was found to vary from 29 to 34. In individuals with n = 34, all observed elements were represented by bivalents. In other specimens, heterozygosity for different number of chromosomal fusions resulted in multivalent formation at MI stage and consequently in a lower number of recognizable chromosomal elements. We show that all karyotype peculiarities of P. (A.) interjectus de Lesse, 1960 (n = 29-32) from Turkey are similar to those in A. eriwanensis. The butterflies of these taxa have allopatric distribution and can be considered as conspecific.

Karyotype reinvestigation does not confirm the presence of two cryptic species and interspecific hybridization in the Polyommatus (Agrodiaetus) damocles complex in the Crimea (Lepidoptera, Lycaenidae).

The karyotype of the blue butterflies from the Angarskiy Pass (Crimea), previously attributed to Polyommatus (Agrodiaetus) poseidon (Herrich-Schäffer, 1851), was re-examined. In all 19 studied individuals, we found the haploid chromosome number n = 26, including 7 pairs of relatively large and 19 pairs of relatively small chromosomes. According to the chromosome number and karyotype structure, the studied population does not differ from P. (A.) damocles krymaeus (Sheljuzhko, 1928) from the eastern part of the Crimean Mountains. This result does not confirm the previously formulated hypotheses, according to which (1) two morphologically similar but karyologically different species, P. (A.) poseidon and P. (A.) damocles krymaeus, occur sympatrically in the Crimea and (2) there is hybridization between these taxa on the Angarskiy Pass. Thus, only three species of the subgenus Agrodiaetus Hübner, 1822 have been reliably established for the Crimea: P. (A.) damone pljushtchi Lukhtanov & Budashkin, 1993, P. (A.) damocles krymaeus (Sheljuzhko, 1928) and P. (A.) ripartii budashkini Kolev & de Prins, 1995.

A new butterfly species from south Russia revealed through chromosomal and molecular analysis of the Polyommatus (Agrodiaetus) damonides complex (Lepidoptera, Lycaenidae).

Finding a new species is a rare event in easy-to-see and well-studied organisms like butterflies, especially if they inhabit well-explored areas such as the Western Palaearctic. However, even in this region, gaps in taxonomic knowledge still exist and here we report such a discovery. Using a combined analysis of chromosomal and molecular markers we demonstrate that Polyommatus blue populations from Daghestan (South Russia), previously identified as P. aserbeidschanus, represent in fact a new species which is described here as P. australorossicussp. n. We also show that the enigmatic Polyommatus damonides described as a form of Polyommatus damone and later considered as an entity similar to P. poseidon or P. ninae is conspecific with a taxon previously known as P. elbursicus. As a result of our study, we propose several taxonomic changes within the P. damonides species complex and suggest the following new combinations: P. damonides elbursicus Forster, 1956, comb. n. and P. damonides gilanensis Eckweiler, 2002, comb. n.

Taxonomic position of several enigmatic Polyommatus (Agrodiaetus) species (Lepidoptera, Lycaenidae) from Central and Eastern Iran: insights from molecular and chromosomal data.

The species-rich subgenus Polyommatus (Agrodiaetus) has become one of the best studied groups of Palearctic blue butterflies (Lepidoptera, Lycaenidae). However, the identity and phylogenetic position of some rare taxa from Iran have remained unclear. An enigmatic, recently described Central Iranian species Polyommatus (Agrodiaetus) shirkuhensis ten Hagen et Eckweiler, 2001 has been considered as a taxon closely related either to Polyommatus (Agrodiaetus) eckweileri ten Hagen, 1998 or to Polyommatus (Agrodiaetus) baltazardi (de Lesse, 1962). Polyommatus (Agrodiaetus) baltazardi, in its turn, was treated as a taxon close to Iranian-Pakistani Polyommatus (Agrodiaetus) bogra Evans, 1932. Here we used a combination of molecular and chromosomal markers to show that none of these hypotheses was true. Instead, Polyommatus (Agrodiaetus) shirkuhensis was recovered as a member of a species group close to Polyommatus (Agrodiaetus) cyaneus (Staudinger, 1899). From genetically closest species, Polyommatus (Agrodiaetus) kermansis (de Lesse, 1962), Polyommatus (Agrodiaetus) cyaneus and Polyommatus (Agrodiaetus) sennanensis (de Lesse, 1959), it differs by the wing coloration. From morphologically similar Polyommatus (Agrodiaetus) mofidii (de Lesse, 1963) and Polyommatus (Agrodiaetus) sorkhensis Eckweiler, 2003, it differs by its chromosome number, n=21. Polyommatus (Agrodiaetus) bogra and Polyommatus (Agrodiaetus) baltazardi were found to be members of two different species groups and, thus, are not closely related.

Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies.

The reinforcement model of evolution argues that natural selection enhances pre-zygotic isolation between divergent populations or species by selecting against unfit hybrids or costly interspecific matings. Reinforcement is distinguished from other models that consider the formation of reproductive isolation to be a by-product of divergent evolution. Although theory has shown that reinforcement is a possible mechanism that can lead to speciation, empirical evidence has been sufficiently scarce to raise doubts about the importance of reinforcement in nature. Agrodiaetus butterflies (Lepidoptera: Lycaenidae) exhibit unusual variability in chromosome number. Whereas their genitalia and other morphological characteristics are largely uniform, different species vary considerably in male wing colour, and provide a model system to study the role of reinforcement in speciation. Using comparative phylogenetic methods, we show that the sympatric distribution of 15 relatively young sister taxa of Agrodiaetus strongly correlates with differences in male wing colour, and that this pattern is most likely the result of reinforcement. We find little evidence supporting sympatric speciation: rather, in Agrodiaetus, karyotypic changes accumulate gradually in allopatry, prompting reinforcement when karyotypically divergent races come into contact.

Phylogeny of Agrodiaetus Hübner 1822 (Lepidoptera: Lycaenidae) inferred from mtDNA sequences of COI and COII and nuclear sequences of EF1-alpha: karyotype diversification and species radiation.

Butterflies in the large Palearctic genus Agrodiaetus (Lepidoptera: Lycaenidae) are extremely uniform and exhibit few distinguishing morphological characters. However, these insects are distinctive in one respect: as a group they possess among the greatest interspecific karyotype diversity in the animal kingdom, with chromosome numbers (n) ranging from 10 to 125. The monophyly of Agrodiaetus and its systematic position relative to other groups within the section Polyommatus have been controversial. Characters from the mitochondrial genes for cytochrome oxidases I and II and from the nuclear gene for elongation factor 1 alpha were used to reconstruct the phylogeny of Agrodiaetus using maximum parsimony and Bayesian phylogenetic methods. Ninety-one individuals, encompassing most of the taxonomic diversity of Agrodiaetus, and representatives of 14 related genera were included in this analysis. Our data indicate that Agrodiaetus is monophyletic. Representatives of the genus Polyommatus (sensu stricto) are the closest relatives. The sequences of the Agrodiaetus taxa in this analysis are tentatively arranged into 12 clades, only 1 of which corresponds to a species group traditionally recognized in Agrodiaetus. Heterogeneous substitution rates across a recovered topology were homogenized with a nonparametric rate-smoothing algorithm before the application of a molecular clock. Two published estimates of substitution rates dated the origin of Agrodiaetus between 2.51 and 3.85 million years ago. During this time, there was heterogeneity in the rate and direction of karyotype evolution among lineages within the genus. Karyotype instability has evolved independently three times in the section Polyommatus, within the lineages Agrodiaetus, Lysandra, and Plebicula. Rapid karyotype diversification may have played a significant role in the radiation of the genus Agrodiaetus.

Principles of the highly ordered arrangement of metaphase I bivalents in spermatocytes of Agrodiaetus (Insecta, Lepidoptera).

We have investigated the nature of highly ordered bivalent arrangement in lepidopteran spermatocytes by analysing and comparing the patterns of bivalent distribution in intact metaphase I plates of 24 closely related species of the genus Agrodiaetus (Lycaenidae). The studied species greatly differed in haploid chromosome numbers (from n = 13 to n = 90) and in the structure of their karyotypes. We found that the larger the bivalent, the closer to the centre of the metaphase plate it was situated. In species with a high chromosome number and asymmetrical karyotype structure, the largest bivalent was located in the centre of the circular metaphase plate. Bivalents of equal size were approximately equidistant from the centre of the metaphase plate and formed concentric circles around the largest bivalent. These principles are diametrically different from those known in the majority of other animals and plants, in which the smallest elements of the chromosome set are situated in the centre of metaphase plate. The only exception from the above principles was observed in spermatocytes of A. surakovi which were heterozygous for reciprocal translocation involving two or three chromosome pairs. In addition to one large bivalent, the heterozygous cells had a multivalent, the size of which was comparable to or even exceeded that of the largest bivalentin the karyotype. In spite of thelarge size, the multivalent was always situated at the periphery of metaphase plate. This indicated that the chromosome size itself is not the only factor determining the bivalent position. We also found that the structure of the metaphase plate is fundamentally different in mitotic and meiotic cells of Agrodiaetus. In spermatogonial metaphase, chromosomes were tightly brought together, forming a dense compact disk, whereas during metaphase I of spermatocytes, all bivalents were clearly separated from each other, and the distance between adjacent bivalents varied from 0.4 to 1.5 microm. Based on the above findings, we proposed a model of bivalent distribution in the Lepidoptera. According to the model, during congregation in the prometaphase stage there is a centripetal movement of bivalents made by a force directed to the centre of the metaphase plate transverse to the spindle. This force is proportional to the kinetochore size of a particular bivalent. The Lepidoptera have a special near-holokinetic type of chromosome organisation. Therefore, large bivalents having large kinetochores are situated in the central part of metaphase plate. Another possible factor affecting the bivalent position is the interaction of bivalents with the cisternae of the membrane system compartmentalising the intraspindle space.