Cryptic Taxa Revealed through Combined Analysis of Chromosomes and DNA Barcodes: The Polyommatus ripartii Species Complex in Armenia and NW Iran.

The detection of cryptic species in complexes that have undergone recent speciation is often difficult, since many standard nuclear markers have not yet accumulated differences between closely related taxa, and differences in mitochondrial markers can be leveled out due to mitochondrial introgressions. In these cases, the use of derived chromosomal characters such as non-ancestral chromosomal numbers and/or unusual karyotype features may be a solution to the species delimitation problem. However, non-ancestral but similar karyotypes may arise secondarily as a result of homoplastic evolution, and their interpretation as homologies may lead to incorrect taxonomic conclusions. In our study, we show that the combined use of mitochondrial DNA barcodes and karyotypes helps to solve this problem and identifies cryptic species in situations where each of these markers does not work individually. Using this approach, we show that the fauna of Armenia and adjacent Iran includes the following cryptic taxa of the Polyommatus ripartii species complex (haploid chromosome number, n in parentheses): P. ripartii paralcestis (n = 90), P. ripartii kalashiani, subsp. nov (n close to 90), P. emmeli, sp. nov. (n = 77-79), P. keleybaricus, sp. nov. (n = 86), P. demavendi belovi (n = 73-75), P. demavendi antonius, subsp. nov. (n = 71-73), P. admetus anatoliensis (n = 79) and P. eriwanensis (n = 29-34). Polyommatus admetus yeranyani is synonymized with P. admetus anatoliensis.

Taxonomic Structure and Wing Pattern Evolution in the Parnassius mnemosyne Species Complex (Lepidoptera, Papilionidae).

In our study, using the analysis of DNA barcodes and morphology (wing color, male genitalia, and female sphragis shape), we show that the group of species close to P. mnemosyne comprises the western and eastern phylogenetic lineages. The eastern lineage includes P. stubbendorfii, P. glacialis, and P. hoenei. The western lineage includes three morphologically similar species: P. mnemosyne (Western Eurasia), P. turatii (southwestern Europe), and P. nubilosusstat. nov. (Turkmenistan and NE Iran), as well as the morphologically differentiated P. ariadne (Altai). The latter species differs from the rest of the group in the presence of red spots on the wings. Parnassius mnemosyne s.s. is represented by four differentiated mitochondrial clusters that show clear association with specific geographic regions. We propose to interpret them as subspecies: P. mnemosyne mnemosyne (Central and Eastern Europe, N Caucasus, N Turkey), P. mnemosyne adolphi (the Middle East), P. mnemosyne falsa (Tian Shan), and P. mnemosyne gigantea (Gissar-Alai in Central Asia). We demonstrate that in P. ariadne, the red spots on the wing evolved as a reversion to the ancestral wing pattern. This reversion is observed in Altai, where the distribution areas of the western lineage, represented by P. ariadne, and the eastern lineage, represented by P. stubbendorfii, overlap. These two species hybridize in Altai, and we hypothesize that the color change in P. ariadne is the result of reinforcement of prezygotic isolation in the contact zone. The lectotype of Parnassius mnemosyne var. nubilosus Christoph, 1873, is designated.

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.

Investigations into the Melitaea ornata species complex in the Levant: M. telona and the newly erected species Melitaea klili Benyamini, 2021 (Lepidoptera: Nymphalidae).

Melitaea klili Benyamini, 2021 was recently described from Israel as a species closely related to M. telona Fruhstorfer, 1908, but different in phenology, ecological preferences and with an allopatric distribution. Here, based on comparative examinations of mitochondrial DNA-barcodes, male genitalia and larval behaviour under laboratory conditions, we synonymize M. klili with M. telona. The COI barcodes of M. klili were found to be identical to those of M. telona. Analysis of 658 bp fragment of the mitochondrial gene COI demonstrated that the minimum uncorrected p-distance between M. ornata and M. telona was 1.98%. This value is remarkably less than the 3% threshold traditionally accepted as a species boundary in DNA barcoding studies. The morphological differences between these taxa are minimal. In fact, M. ornata and M. telona represent two phylogenetic lineages, the taxonomic status of which (separate species or subspecies of the same species) is intermediate and debatable.

More hidden diversity in a cryptic species complex: a new subspecies of Leptideasinapis (Lepidoptera, Pieridae) from Northern Iran.

A new subspecies of Leptideasinapis from Northern Iran, discovered by means of DNA barcoding, is described as Leptideasinapistabarestanassp. nov. The new subspecies is allopatric with respect to other populations of L.sinapis and is genetically distinct, appearing as a well-supported sister clade to all other populations in COI-based phylogenetic reconstructions. Details on karyotype, genitalia, ecology and behaviour for the new subspecies are given and a biogeographical speciation scenario is proposed.

Chromosomal conservatism vs chromosomal megaevolution: enigma of karyotypic evolution in Lepidoptera.

In the evolution of many organisms, periods of slow genome reorganization (= chromosomal conservatism) are interrupted by bursts of numerous chromosomal changes (= chromosomal megaevolution). Using comparative analysis of chromosome-level genome assemblies, we investigated these processes in blue butterflies (Lycaenidae). We demonstrate that the phase of chromosome number conservatism is characterized by the stability of most autosomes and dynamic evolution of the sex chromosome Z, resulting in multiple variants of NeoZ chromosomes due to autosome-sex chromosome fusions. In contrast during the phase of rapid chromosomal evolution, the explosive increase in chromosome number occurs mainly through simple chromosomal fissions. We show that chromosomal megaevolution is a highly non-random canalized process, and in two phylogenetically independent Lysandra lineages, the drastic parallel increase in number of fragmented chromosomes was achieved, at least partially, through reuse of the same ancestral chromosomal breakpoints. In species showing chromosome number doubling, we found no blocks of duplicated sequences or duplicated chromosomes, thus refuting the hypothesis of polyploidy. In the studied taxa, long blocks of interstitial telomere sequences (ITSs) consist of (TTAGG)n arrays interspersed with telomere-specific retrotransposons. ITSs are sporadically present in rapidly evolving Lysandra karyotypes, but not in the species with ancestral chromosome number. Therefore, we hypothesize that the transposition of telomeric sequences may be triggers of the rapid chromosome number increase. Finally, we discuss the hypothetical genomic and population mechanisms of chromosomal megaevolution and argue that the disproportionally high evolutionary role of the Z sex chromosome can be additionally reinforced by sex chromosome-autosome fusions and Z-chromosome inversions.

Whole-Genome Analysis Reveals the Dynamic Evolution of Holocentric Chromosomes in Satyrine Butterflies.

Butterfly chromosomes are holocentric, i.e., lacking a localized centromere. Potentially, this can lead to rapid karyotypic evolution through chromosome fissions and fusions, since fragmented chromosomes retain kinetic activity, while fused chromosomes are not dicentric. However, the actual mechanisms of butterfly genome evolution are poorly understood. Here, we analyzed chromosome-scale genome assemblies to identify structural rearrangements between karyotypes of satyrine butterfly species. For the species pair Erebia ligea-Maniola jurtina, sharing the ancestral diploid karyotype 2n = 56 + ZW, we demonstrate a high level of chromosomal macrosynteny and nine inversions separating these species. We show that the formation of a karyotype with a low number of chromosomes (2n = 36 + ZW) in Erebia aethiops was based on ten fusions, including one autosome-sex chromosome fusion, resulting in a neo-Z chromosome. We also detected inversions on the Z sex chromosome that were differentially fixed between the species. We conclude that chromosomal evolution is dynamic in the satyrines, even in the lineage that preserves the ancestral chromosome number. We hypothesize that the exceptional role of Z chromosomes in speciation may be further enhanced by inversions and sex chromosome-autosome fusions. We argue that not only fusions/fissions but also inversions are drivers of the holocentromere-mediated mode of chromosomal speciation.

Molecular Phylogeny and Taxonomy of the Butterfly Subtribe Scolitantidina with Special Focus on the Genera Pseudophilotes, Glaucopsyche and Iolana (Lepidoptera, Lycaenidae).

The Palearctic blue butterfly genus Pseudophilotes Beuret, 1958 is not homogenous regarding the morphology of its genital structures. For this reason, some of its species have been considered to be representatives of other genera of the subtribe Scolitantidina (subfamily Polyommatinae). Here, we address these taxonomic problems by analyzing the phylogenetic relationships between the genera, subgenera, and species of this subtribe inferred via the analysis of five nuclear and two mitochondrial DNA sequences. We demonstrate that the enigmatic Asian species P. panope (Eversmann, 1851) belongs to the genus Pseudophilotes but not to Praephilotes Forster, 1938 or Palaeophilotes Forster, 1938 and does not represent the independent genus Inderskia Korshunov, 2000, as hypothesized previously. We synonymize P. svetlana Yakovlev, 2003 (syn. nov.) and P. marina Zhdanko, 2004 (syn. nov.) with P. panope. We demonstrate a deep genetic divergence between lineages that were previously considered as subspecies of the single species Iolana iolas (Ochsenheimer, 1816). As a result, we confirm the multispecies concept of the genus Iolana Bethune-Baker, 1914. We show that the Holarctic genus Glaucopsyche can be divided into four subgenera: Glaucopsyche Scudder, 1872 (=Shijimiaeoides Beuret, 1958), Apelles Hemming, 1931, Bajluana Korshunov and Ivonin, 1990, and Phaedrotes Scudder, 1876.

Reassessment of the status of some European and Asian Melitaea taxa described as subspecies of Melitaea phoebe ([Denis amp; Schiffermller], 1775), with designations of lectotypes where appropriate (Lepidoptera: Nymphalidae).

Morphological characters of value in distinguishing Melitaea phoebe from M. ornata are exemplified from photographs of specimens from sympatric and partially synchronic populations in the Russian Federation and Slovenia. Type material of a number of subspecies classified as M. phoebe have been examined and confirmed as being subspecies of M. phoebe or as subspecies of either M. ornata or M. telona. Larval and adult morphology of M. telona is very similar to those of M. ornata, making morphological separation difficult. Distribution of M. telona forms a major criterion for separation. Lectotypes are designated for amanica Rebel, 1917 and dorae Graves, 1925. The name enoch Higgins, 1941 is recognised as a distinct species and the names abbas Gross Ebert, 1975 and zagrosi Tth Varga, 2011 synonymised with Melitaea enoch.

DNA Barcodes Combined with Multilocus Data of Representative Taxa Can Generate Reliable Higher-Level Phylogenies.

Taxa are frequently labeled incertae sedis when their placement is debated at ranks above the species level, such as their subgeneric, generic, or subtribal placement. This is a pervasive problem in groups with complex systematics due to difficulties in identifying suitable synapomorphies. In this study, we propose combining DNA barcodes with a multilocus backbone phylogeny in order to assign taxa to genus or other higher-level categories. This sampling strategy generates molecular matrices containing large amounts of missing data that are not distributed randomly: barcodes are sampled for all representatives, and additional markers are sampled only for a small percentage. We investigate the effects of the degree and randomness of missing data on phylogenetic accuracy using simulations for up to 100 markers in 1000-tips trees, as well as a real case: the subtribe Polyommatina (Lepidoptera: Lycaenidae), a large group including numerous species with unresolved taxonomy. Our simulation tests show that when a strategic and representative selection of species for higher-level categories has been made for multigene sequencing (approximately one per simulated genus), the addition of this multigene backbone DNA data for as few as 5-10% of the specimens in the total data set can produce high-quality phylogenies, comparable to those resulting from 100% multigene sampling. In contrast, trees based exclusively on barcodes performed poorly. This approach was applied to a 1365-specimen data set of Polyommatina (including ca. 80% of described species), with nearly 8% of representative species included in the multigene backbone and the remaining 92% included only by mitochondrial COI barcodes, a phylogeny was generated that highlighted potential misplacements, unrecognized major clades, and placement for incertae sedis taxa. We use this information to make systematic rearrangements within Polyommatina, and to describe two new genera. Finally, we propose a systematic workflow to assess higher-level taxonomy in hyperdiverse groups. This research identifies an additional, enhanced value of DNA barcodes for improvements in higher-level systematics using large data sets. [Birabiro; DNA barcoding; incertae sedis; Kipepeo; Lycaenidae; missing data; phylogenomic; phylogeny; Polyommatina; supermatrix; systematics; taxonomy].

Integrative analysis reveals cryptic speciation linked to habitat differentiation within Albanian populations of the anomalous blues (Lepidoptera, Lycaenidae, Polyommatus Latreille, 1804).

The Balkan Peninsula is one of the greatest hotspots for biodiversity in Europe. While the region has been investigated thoroughly, some parts remain understudied and may still harbour undiscovered diversity, even in well-studied organisms such as Lepidoptera. Here we investigated the group of the so-called anomalous blue butterflies, also known as 'brown complex' of the subgenus Agrodiaetus Hübner, 1822 including the taxa of the entire Polyommatusaroaniensis (Brown, 1976) species complex. This species complex is distributed in the southern part of the Balkan Peninsula and known to be represented by three closely related allopatric species, differentiated by their chromosome numbers (n) and mitochondrial (mt) DNA. These are P.aroaniensis sensu stricto (Southern Greece, Peloponnese, n=47-48; mt haplogroup aroa1), P.timfristos Lukhtanov, Vishnevskaya et Shapoval, 2016 (Central Greece, Attika, n=38, aroa2) and P.orphicus Kolev, 2005 (North-Eastern Greece, Southern Bulgaria, n=41-42, orph1). Based on an analysis of chromosomal, molecular and morphological markers, we demonstrate that a fourth taxon of this species complex exists in Albania. This taxon possesses the mt haplogroup aroa3, which is the most differentiated within the entire P.aroaniensis species complex, and the karyotype (n=42-43), which differs by one fixed chromosome fission from P.orphicus. The Albanian taxon seems to be ecologically specialised (habitat on dark-coloured, ophiolitic substrate soils) and differs in colouration (wing reflectance) from the others taxa of the P.aroaniensis species group. Based on the evidence here presented and following the current view of the taxonomy of the group, we propose considering the Albanian taxon as a new species, here described as Polyommatuslurae sp. nov. At the contact zone between the new species and P.orphicus, in addition to typical ones, we detected specimens with haplogroup orph2, karyotype n=43 and intermediate morphology, which seem to represent P.lurae × P.orphicus hybrids.

Identification of Natural Hybrids between Ahlbergia frivaldszkyi (Lederer, 1853) and Callophrys rubi (Linnaeus, 1758) (Lepidoptera, Lycaenidae) Using Mitochondrial and Nuclear Markers.

Natural hybridization is rather widespread and common in animals and can have important evolutionary consequences. In terms of taxonomy, exploring hybridization and introgression is crucial in defining species boundaries and testing taxonomic hypotheses. In the present paper, we report on natural hybrid specimens between Ahlbergia frivaldszkyi (Lederer, 1853) and Callophrys rubi (Linnaeus, 1758). To test the hypothesis of their hybrid origin, we employed the molecular mitochondrial (COI gene) and nuclear (wingless, RPS5, and Ca-ATPase genes) markers commonly used in phylogenetic studies and explored the morphology of the specimens. Our analysis revealed that hybrids bear mitochondrial haplotypes of C. rubi, while nuclear fragments are heterozygous, sharing a combination of A. frivaldszkyi and C. rubi lineages. The hybrid specimens combine morphological characters of both genera. Our results for the first time empirically demonstrate the possibility of genetic introgression between these species and between the genera Callophrys and Ahlbergia on the whole.

Chromosomal and DNA barcode analysis of the Melitaea ala Staudinger, 1881 species complex (Lepidoptera, Nymphalidae).

The species of the Melitaea ala Staudinger, 1881 complex are distributed in Central Asia. Here we show that this complex is a monophyletic group including the species, M. ala, M. kotshubeji Sheljuzhko, 1929 and M. enarea Fruhstorfer, 1917. The haploid chromosome number n=29 is found in M. ala and M. kotshubeji and is, most likely, a symplesiomorphy of the M. ala complex. We show that M. ala consists of four subspecies: M. ala zaisana Lukhtanov, 1999 (=M. ala irtyshica Lukhtanov, 1999, syn. nov.) (South Altai, Zaisan Lake valley), M. ala ala (Dzhungarian Alatau), M. ala bicolor Seitz, 1908 (North, East, Central and West Tian-Shan) and M. ala determinata Bryk, 1940 (described from "Fu-Shu-Shi", China). We demonstrate that M. kotshubeji kotshubeji (Peter the Great Mts in Tajikistan) and M. kotshubeji bundeli Kolesnichenko, 1999 (Alai Mts in Tajikistan and Kyrgyzstan) are distinct taxa despite their geographic proximity in East Tajikistan. Melitaea enarea is widely distributed in the southern part of Central Asia and is sympatric with M. kotshubeji.

Genomic introgression from a distant congener in the Levant fritillary butterfly, Melitaea acentria.

Introgressive hybridization is more common in nature than previously thought, and its role and creative power in evolution is hotly discussed but not completely understood. Introgression occurs more frequently in sympatry between recently diverged taxa, or when the speciation process has not yet been completed. However, there are relatively few documented cases of hybridization that erodes reproductive barriers between distantly related species. Here, we use whole genome and mitochondrial data to examine how introgression from a distant congener affects pattern of genetic differentiation in the Levant fritillary butterfly Melitaea acentria. We show that this local taxon has evolved as a peripatric geographic isolate of the widespread Melitaea persea, and that there has been significant unidirectional gene flow from the sympatric, nonclosely related Melitaea didyma to M. acentria. We found direct evidence of ongoing sporadic hybridization between M. didyma and M. acentria, which are separated by at least 5 million years of independent evolution. Elevated differentiation and lower level of introgression on the sex Z chromosome compared to autosomes suggest that the Z chromosome has accumulated loci acting as intrinsic postzygotic barriers. Our results show that introgression from M. didyma has been an additional source of nucleotide diversity in the M. acentria population, providing material for drift and selection.

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).

Incomplete Sterility of Chromosomal Hybrids: Implications for Karyotype Evolution and Homoploid Hybrid Speciation.

Heterozygotes for major chromosomal rearrangements such as fusions and fissions are expected to display a high level of sterility due to problems during meiosis. However, some species, especially plants and animals with holocentric chromosomes, are known to tolerate chromosomal heterozygosity even for multiple rearrangements. Here, we studied male meiotic chromosome behavior in four hybrid generations (F1-F4) between two chromosomal races of the Wood White butterfly Leptidea sinapis differentiated by at least 24 chromosomal fusions/fissions. Previous work showed that these hybrids were fertile, although their fertility was reduced as compared to crosses within chromosomal races. We demonstrate that (i) F1 hybrids are highly heterozygous with nearly all chromosomes participating in the formation of trivalents at the first meiotic division, and (ii) that from F1 to F4 the number of trivalents decreases and the number of bivalents increases. We argue that the observed process of chromosome sorting would, if continued, result in a new homozygous chromosomal race, i.e., in a new karyotype with intermediate chromosome number and, possibly, in a new incipient homoploid hybrid species. We also discuss the segregational model of karyotype evolution and the chromosomal model of homoploid hybrid speciation.

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.

Linking karyotypes with DNA barcodes: proposal for a new standard in chromosomal analysis with an example based on the study of Neotropical Nymphalidae (Lepidoptera).

Chromosomal data are important for taxonomists, cytogeneticists and evolutionary biologists; however, the value of these data decreases sharply if they are obtained for individuals with inaccurate species identification or unclear species identity. To avoid this problem, here we suggest linking each karyotyped sample with its DNA barcode, photograph and precise geographic data, providing an opportunity for unambiguous identification of described taxa and for delimitation of undescribed species. Using this approach, we present new data on chromosome number diversity in neotropical butterflies of the subfamily Biblidinae (genus Vila Kirby, 1871) and the tribe Ithomiini (genera Oleria Hübner, 1816, Ithomia Hübner, 1816, Godyris Boisduval, 1870, Hypothyris Hübner, 1821, Napeogenes Bates, 1862, Pseudoscada Godman et Salvin, 1879 and Hyposcada Godman et Salvin, 1879). Combining new and previously published data we show that the species complex Oleria onega (Hewitson, [1852]) includes three discrete chromosomal clusters (with haploid chromosome numbers n = 15, n = 22 and n = 30) and at least four DNA barcode clusters. Then we discuss how the incomplete connection between these chromosomal and molecular data (karyotypes and DNA barcodes were obtained for different sets of individuals) complicates the taxonomic interpretation of the discovered clusters.

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.