From numerous specimens collected in Armenia in 1996, 1997, 2001 and 2007 only 14 males from four different populations showed metaphase cells which were acceptable for karyotype analysis.

The variable number of distinct chromosome elements was found in MI, MII and AI cells of the studied specimens. In six individuals, 34 chromosome elements were counted (Table 1, Figs 1–6); all observed elements were distinct bivalents in MI and AI cells. Thus, the haploid chromosome number was n = 34 and the diploid number may be calculated as 2n = 68 for these six individuals. In the chromosome set with n = 34, the bivalents were oval or dumb-bell shaped. They were differentiated with respect to their size: the area of the first bivalent was 4–5 times as large as that of the last bivalent (Figs 1–4). In two cells the number of chromosome elements was determined as n = ca34 with an approximation due of imperfect spreading of bivalents.

Figures 1–7.

Karyotype in male meiosis of Polyommatus (Agrodiaetus) eriwanensis 1–4 metaphase I of meiosis, squash preparation, n = 34 5 metaphase I of meiosis, intact (not squashed) preparation, n = 34 6 metaphase II of meiosis, intact (not squashed) preparation, n = 34 7 metaphase I of meiosis, intact (not squashed) preparation, n = 29, multivalents are shown by arrows. Scale bar: 10 μm in all figures.

If the number of chromosome elements was lower than 34 (29, ca30, ca31, ca32 or 33), from 1 to 6 V-shaped large elements were observed in MI cells. We treat these V-shaped elements as multivalents. For example, in the specimens KL-1997-6-9, 8 cells showed clearly 32 bivalents + 1 multivalent (possibly a trivalent). A very interesting karyotype was found in the specimen KL-1997-7. In this specimen in all studied MI plates we could find 29 chromosome elements including oval or dumb-bell shaped bivalents and three V-shaped multivalents (Fig. 7). The karyotype contained no exceptionally big or small bivalents or multivalents. We suppose that studied cells were heterozygous for multiple chromosome fusions. Small chromosomes of the standard n = 34 karyotype were probably involved in these rearrangements and were observed as parts of the multivalents. Thus, it is evident that the difference in the number of visible chromatin bodies in MI plates does not reflect the real variation of diploid number. Heterozygosity for chromosome fusion(s) may result in multivalent formation at MI stage and consequently in change of number of recognizable chromosome units.

Thus, the number of chromosome elements (bivalents+multivalents) observed in MI was found to vary from 29 to 34. Similar chromosome numbers were found in the following species of the P. dolus clade: n = 29-32 in P. (A.) interjectus (de Lesse, 1960) (de Lesse 1960), n = 38 in P. (A.) timfristos Lukhtanov, Vishnevskaya & Shapoval, 2016 (Vishnevskaya et al. 2016), n = 39 in P. (A.) humedasae (Toso & Balletto, 1976) (Vila et al. 2010), n = 41-42 in P. (A.) orphicus Kolev, 2005 (Kolev 2005), n = 40–42 in P. (A.) dantchenkoi (Lukhtanov & Wiemers, 2003) (Lukhtanov et al. 2003) and n = 43 in P. (A.) rjabovianus masul Lukhtanov, Dantchenko, Vishnevskaya & Saifitdinova, 2015 (Lukhtanov et al. 2015).

We calculated barcoding gaps between these taxa using published COI sequences (Lukhtanov et al. 2003; Wiemers 2003; Kandul et al. 2004; Vila et al. 2010; Vishnevskaya et al. 2016) deposited in GenBank (Table 2). As one can see, the species pair P. eriwanensis – P. interjectus is characterized by a minimum COI barcoding gap which is much lower than ‘standard’ DNA-barcode species threshold (3%) (Hebert et al. 2003; Lukhtanov et al. 2016). This pair is also characterized by overlapping numbers of chromosomes. In the MI and MII karyotype of P. (A.) interjectus de Lesse (1960) found from 29 up to 32 chromosome entities with variable number of small and large elements. On the pictures in his paper one can see distinct V-shaped large elements, which are probably multivalents. The chromosome elements were strongly differentiated in P. (A.) interjectus with respect to their size: the area of the largest elements was 4–5 times as large as that of the smallest ones (de Lesse 1960: figs 7 and 8). All these peculiarities are similar to those in P. (A.) eriwanensis. These taxa have allopatric distributions (Fig. 8). Therefore, the taxon studied by de Lesse (1960) can be treated as a subspecies P. (A.) eriwanesis interjecus rather than a distinct species.

Figure 8.

Geographic distribution of P. (A.) eriwanensis eriwanensis (red circles) and P. (A.) eriwanensis interjectus (blue circles) based on information from Hesselbarth et al. (1995) and Lukhtanov et al. (2003). Ar is Armenia, Az is Azerbaijan, Ge is Georgia, Ru is Russia.

Polyommatus eriwanensis is found in southern Armenia (nominotypical subspecies) and in Ezincan, Erzurum, Sivas and Tunceli Provinces in Turkey (P. eriwanensis interjectus) (Fig. 8). The species is also reported for southern Georgia and western Azerbaijan (Eckweiler and Bozano 2016), as well as for north-western Iran (Tshikolovets et al. 2014). However, the indications for Georgia, Azerbaijan and Iran have not been confirmed by chromosomal or molecular data, and we consider them doubtful.