Butterflies for this study were collected in 2008 in the Balkan Peninsula by V.A. Lukhtanov, N.A. Shapoval and L. Rieppel, in 2016 in Hvoyna village (Bulgaria) by E.A. Pazhenkova and in the Tigirekskiy Reservation (the Altai Mountains, Russia) by M.S.Vishnevskaya in 2007 (Fig. 1, Table 1). We paid special attention to collecting the taxa in their type localities: mount Chelmos (Greece: Peloponnese) (type locality of Agrodiaetus alcestis aroaniensis Brown, 1976), mount Falakró near Granítis (Greece, Makedonía, Dráma district) (type locality of Agrodiaetus eleniae Coutsis & De Prins, 2005) and Hvoyna (south Bulgaria, the Rhodopi mts) (type locality of Polyommatus dantchenkoi orphicus). Unfortunately, in our research we did not have an opportunity to study the holotypes of these taxa. Taking into account a possibility of multiple cryptic species within a local area even in well-studied European butterflies (Dincă et al. 2011, 2013b), in each place we managed to collect (and then to study) as many individuals as possible paying special attention to the specimens with unusual or intermediate morphology.

Figure 1.

Localities of the species collected for the study (the species list is presented in Table 1). 1 Bulgaria: Dragoman (P. admetus) 2 Bulgaria: Hvoyna (P. ripartii pelopi, P. orphicus orphicus) 3 Greece: Granitis (P. nephohiptamenos, P. orphicus eleniae) 4 Greece: Smolikas (P. admetus) 5 Greece: Timfristos Mt (P. ripartii pelopi, P. timfristos) 6 Greece: Parnassos Mt (P. timfristos) 7 Greece: Kalavrita (P. admetus, P. aroaniensis, P. ripartii pelopi). Colored circles match different taxa. Blue circle: P. admetus. Red circle: P. ripartii. Brown circle: P. orphicus orphicus. Lavender circle: P. orphicus eleniae. Yellow circle: P. timfristos sp. n. Grey circle: P. aroaniensis.

Before processing butterflies were put in glassine envelopes and kept alive for less than one hour. Testes were removed and put into a vial with a fresh fixative (3:1, 96% ethanol: glacial acetic acid). The wings were removed and put into a glassine envelope, and the body was placed into a vial with 96% ethanol for further molecular analysis. All chromosome preparations, butterfly bodies in ethanol and wings in glassine envelopes are stored in the Department of Karyosystematics (Zoological Institute of the Russian Academy of Sciences, St. Petersburg).

Testes were stored in the 3:1 fixative for several months at +4 °C and then stained with 2% acetic orcein for 30 days at 20 °C. We used a two-phase method of chromosome analysis following Lukhtanov and Dantchenko (2002b). In the first phase, stained testes were placed into a drop of 40% lactic acid on a slide where spermatocysts were dissected from testis membranes using entomological pins. Intact spermatocytes were transferred into a new drop of 40% lactic acid and covered with a coverslip. A Carl Zeiss Amplival light microscope was used for cytogenetic analysis. During the metaphase I stage, each spermatocyst was observed as a regular sphere consisting of 64 spermatocytes. In the second phase, different degrees of chromosome spreading were observed by gradually increasing pressure on the coverslip. The second phase was useful for studying the bivalent structure and counting the bivalent number. By scaling up the pressure on the coverslip, we were able to manipulate chromosomes, e.g. change their position and orientation on the slide, and consequently to resolve controversial cases of contacting or overlapping bivalents. Haploid chromosome numbers were counted in metaphase I (MI) and/or metaphase II (MII) of meiosis.

We used a 657-bp fragment within the mitochondrial COI gene and a 440-bp fragment within the ITS2 region. DNA was extracted using phenol-chloroform method according to the standard protocol (Sambrook and Russel 2006). The first two abdominal segments were homogenized in lysis buffer [25 mM EDTA, 75 mM NaCl, 10 mM Tris (pH 7.5)]. Then proteinase K (20 mg/ml) and 10% SDS were added and the samples were incubated for 2 h at 60 °C. DNA was extracted from lysate first with phenol/chloroform (1:1) and then with chloroform to remove any remaining phenol. DNA was precipitated with isopropyl alcohol in the presence of 0.1 M NaCl and pelleted by centrifugation. The pellets were washed with 70% ethanol, dried and dissolved in ddH2O. The extracted DNA was stored at -20 °C.

For COI amplification we used the self-designed primers COIF1 (5’-CCACAAATCATAAAGATATTGGAAC-3’) and COIR1 (5’-TGATGAGCTCATACAATAAATCCTA-3’). For ITS2 amplification we used the self-designed primers ITS2F (5’-CATATGCCACACTGTTCGTCTG-3’) and ITS2R (5’-GATATCCGTCAGCGCAACG-3’).

The polymerase chain reaction (PCR) was carried out with Taq-polymerase (Sileks) in 20 µl of PCR buffer containing MgCl₂ [2.5 mM], dNTP [200 mM] and forward and reverse primers [20 pmol each]. Amplification of COI gene fragment was carried out with the following conditions: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 30 sec at 94 °C, 30 sec at 51 °C (the annealing temperature) and 30 sec at 72 °C, and then final elongation 5 min for 72 °C. Amplification of ITS2 region fragment was carried out with the following conditions: initial denaturation at 94 °C for 2 min, followed by 30 cycles of 30 sec at 94 °C, 30 sec at 60 °C (the annealing temperature) and 30 sec at 72 °C, and then final elongation 5 min for 72 °C.

After amplification, PCR mix was loaded in 1% agarose gel and specific product was separated by gel electrophoresis (Fig. 2). Pieces of gel containing the DNA fragment of required length were cut out and then double-stranded DNA was purified using the method of ‘DNA purification from agarose gels with MP@SiO₂ magnetic particles’ according to the manufacturer’s protocol (Sileks). Purified DNA fragments were extracted with ddH₂O from magnetic particles pelleted with a magnetic rack and collected in a fresh tube. The concentration of purified DNA was estimated via gel electrophoresis (by comparing the brightness of the sample fragment to the brightness of the DNA marker (in our case 100 bp DNA Ladder, Thermo Fisher Scientific).

Figure 2.

Gel electrophoresis with COI and ITS2PCR products showing the length of the fragments.

All the preparations for sequencing were held in “The Laboratory of Animal Genetics” of Saint-Petersburg State University and “Chromas” Core Facility, Saint-Petersburg State University Research Park. Sequencing was carried out in the Research Resource Center for Molecular and Cell Technologies. GenBank codes of the studied samples are provided in Tables 1 and 2.

The analysis involved 221 COI sequences (169 GenBank sequences and 52 own material) and 117 ITS2 sequences (66 GenBank and 51 own data).

Sequences of different length (from 415 to 657 bp in case of COI and from 415 to 440 bp in case of ITS2) were included into the final dataset alignment. We used BioEdit 7.2.5 software (Hall 1999) to align the sequences and then edited them manually. The final COI alignment included 657 sites, with 137 variable sites and 112 parsimony-informative sites. The final ITS2 alignment included 440 sites, with 52 variable sites and 22 parsimony-informative sites.

Previously, no significant conflict was detected between the mitochondrial COI and nuclear ITS2Agrodiaetus data sets (Vila et al. 2010). Thus, we combined mitochondrial and nuclear sequences to improve phylogenetic signal. This resulted in a concatenated alignment with a total of 1039 bp.

Phylogenetic relationships were inferred using Bayesian Inference (BI), maximum likelihood (ML) and maximum parsimony (MP) analyses. jModelTest was used to determine optimal substitution models for ML inference (Posada 2008).

Bayesian analyses were conducted using MrBayes, version 3.2 (Ronquist et al. 2012). Datasets were partitioned by codon position. Substitution models used for each partition were chosen according to jModelTest (Posada 2008): nst=2 and rates=invgamma for the first position, nst=2 and rates=gamma for the second position, and nst=6 and rates=gamma for the third position of COI barcodes. Substitution model nst=6 and rates=invgamm was chosen for ITS2. In evolution of ITS2 sequences, the mono, bi- and mullti-nucleotide insertions/deletions are frequent and contain phylogenetically important information. To account for this, each indel event was coded as a binary character (1/0, presence/absence of the gap independently of its length) and then used in the Bayesian analyses of ITS2 and concatenated data sets. Two runs of 10 000 000 generations with four chains (one cold and three heated) were performed. Chains were sampled every 10 000 generations, and burn-in was determined based on inspection of log likelihood over time plots using TRACER, version 1.4 (available from http://beast.bio.ed.ac.uk/Tracer).

The ML trees were inferred using MEGA6 under the GTR+G+I model. MP analysis was performed using a heuristic search as implemented in MEGA6 (Tamura et al. 2013). A heuristic search was carried out using the close-neighbor-interchange algorithm with search level 3 (Nei and Kumar 2000) in which the initial trees were obtained with the random addition of sequences (100 replicates). We used nonparametric bootstrap values (Felsenstein 1985) to estimate branch support for ML and MP trees. The bootstrap consensus tree was inferred from 500 replicates.

Median network was constructed using the program Network 4.6.1.3. (Fluxus Technology, fluxus-engineering.com), with the Median Joining algorithm (Bandelt 1999). The algorithm picks close haplotype groups and finds hypothetical ancestors, to join the haplotypes in a common parsimony network. The program shows each haplotype with a colored circle. When the haplotypes are identical, they are united in one bigger circle under one name. Similar haplotypes then are combined in haplogroups (Table 2). The network was constructed on the base of COI alignment, with 191 sequences. The length of the sequences was 612 bp with 116 parsimony-informative sites. The final alignment included only sequences of equal length. Short and ambiguous sequences were excluded.

Fig. 3a–c

The haploid chromosome number n=80 was found in MI and MII cells of two studied individuals from South and Central Greece. In two specimens (Greece, Smolikas Mt and Bulgaria) we counted approximately n=ca80 at MI. The last count was performed with an approximation due to the overlapping of some bivalents. The karyotype displayed one larger bivalent in the centre of the MI plate and one larger univalent in the centre of the MII plate.

Figure 3.

Polyommatus (Agrodiaetus) karyotypes. Bar =10 µ. a–b P. admetus, sample LR08D109, Greece, MI, n=80. One large bivalent in the centre of the plate can be seen c P. admetus, sample LR08D109, Greece, MII, n=80. One large chromosome in the centre of the plate can be seen d P. ripartii pelopi, sample LR08D249, Greece, MI, n=90. Two large bivalents in the centre of the plate can be seen e P. ripartii pelopi, sample LR08D144, Greece, MI, n=90. Two large bivalents in the centre of the plate can be seen f P. ripartii pelopi, sample LR08D145, Greece, MI, n=90. Two large bivalents in the centre of the plate can be seen g P. ripartii pelopi, sample LR08D92, Greece, MII, n=90. Two large chromosomes in the centre of the plate can be seen h P. nephohiptamenos, sample LR08D494, Northern Greece, MI, n=90. All the bivalents are situated in a plane with the largest elements in the centre of the circular metaphase plate. Bivalents are clearly separated from each other by gaps. Two bivalents are larger than the rest. i P. aroaniensis, sample LR08D102, Greece, MI, n=47.

Fig. 3d–g

The haploid chromosome number was determined to be n=90 in MI and MII cells of seven studied individuals from different localities (Greece, Bulgaria). At MI, two bivalents were especially large and were situated in the centre of the metaphase plates. Bivalent 1 was 1.4–1.6 times larger than bivalent 2. The sizes of the remaining 88 bivalents decreased more or less linearly. At MII, two univalents were especially large and were situated in the centre of the metaphase plates. Chromosome 1 was 1.4–1.6 times larger than chromosome 2. The sizes of the remaining 88 chromosomes decreased more or less linearly. In three specimens we counted approximately n=ca 90 at MI. The last count was an approximation due to the overlapping of some bivalents. In three specimens, the diploid chromosome number was estimated as 2n=ca180 in male asynaptic meiosis.

Fig. 3h

The haploid chromosome number was determined to be n=90 in MI and MII cells of two studied individuals. At MI, two bivalents (one big and one medium-sized) were larger than the others. At MII, two univalents (one big and one medium-sized) were larger than the rest. The sizes of the remaining 88 bivalents and univalents decreased more or less linearly. In four specimens we counted approximately n=ca90 at MI. The last count was an approximation due to the overlapping of some bivalents.

Fig. 3i

In the single studied specimen collected in the type locality (Greece, Mt. Chelmos) haploid chromosome number n=47 was found in MI cells. Bivalents were fairly well differentiated with respect to their size. However, it was difficult to subdivide them objectively into size groups because the sizes of the 47 bivalents decrease more or less linearly.

Figs 4a–h, 5a–d

The haploid chromosome number was determined to be n=38 in prometaphase, MI and MII cells of the holotype and six studied paratypes. Bivalents at MI and prometaphase and univalents at MII were fairly well differentiated with respect to their size; however, it was difficult to subdivide them objectively into size groups because the sizes of the 47 elements decrease more or less linearly.

Figure 4.

Polyommatus (Agrodiaetus) timfristos karyotypes. Bar = 10 µ. a P. timfristos, sample LR08D205, Central Greece, Parnassos, first prometaphase of meiosis, n=38 b P. timfristos, sample LR08D205, Central Greece, Parnassos, MI, n=38 c P. timfristos, holotype, sample LR08D247, Central Greece, Timfristos, MI, n=38 d P. timfristos, sample LR08D255, Central Greece, Timfristos, MI, n=38 e P. timfristos, sample LR08D258, Central Greece, Timfristos, MI, n=38 f P. timfristos, sample LR08D258, Central Greece, Timfristos, MI, n=38 g P. timfristos, sample LR08D273, Central Greece, Timfristos, MI, n=38 h P. timfristos, sample LR08D274, Central Greece, Timfristos, MII, n=38.

Figure 5.

Polyommatus (Agrodiaetus) karyotypes. Bar = 10 µ. a P. timfristos, sample LR08D205, Central Greece, Parnassos, MII, n=38 b P. timfristos, sample LR08D205, Central Greece, Parnassos, MII, n=38 c P. timfristos, sample LR08D205, Central Greece, Parnassos, MII, n=38 d P. timfristos, sample LR08D258, Central Greece, Timfristos, MII, n=38 e P. orphicus eleniae, sample LR08D433, Northern Greece, MI, n=41 f P. orphicus eleniae, sample LR08D431, Northern Greece, MI, n=42 g P. orphicus eleniae, sample LR08D431, Northern Greece, MI, n=ca42 h P. orphicus eleniae, sample LR08D437, Northern Greece, MII, n=41 i P. orphicus eleniae, sample LR08D437, Northern Greece, MII, n=41 j P. orphicus eleniae, sample LR08D431, Northern Greece, MII, n=42.

Two different haploid chromosome numbers (n=41 and n=42) were observed in MI and MII cells of the four specimens studied. This variation was most likely caused by polymorphism for one chromosome fussion/fission. This polymorphism resulted in three types of MI karyotype: n=41 (homozygous for chromosomal fusion/fission, one pair of fused chromosomes), n=42 (homozygous for chromosomal fusion/fission, two pairs of unfused chromosomes) and n=41 (heterozygous for chromosomal fusion/fission, 40 bivalents and one trivalent). Bivalents at MI and univalents at MII were fairly well differentiated with respect to their size; however, it was difficult to subdivide them objectively into size groups because the sizes of the elements decrease more or less linearly.

Fig. 5e–j

Chromosome numbers (n=41 and n=42) were observed in MI and MII cells of the four specimens studied. This variation was most likely caused by polymorphism for one chromosome fussion/fission. This polymorphism resulted in three types of MI karyotype: n=41 (homozygous for chromosomal fusion/fission, one pair of fused chromosomes), n=42 (homozygous for chromosomal fusion/fission, two pairs of unfused chromosomes) and n=41 (heterozygous for chromosomal fusion/fission, 40 bivalents and one trivalent). Bivalents and univalents were fairly well differentiated with respect to their size; however, it was difficult to subdivide them objectively into size groups because the sizes of the elements decrease more or less linearly.

The complicated relationships between species of P. admetus and P. dolus groups were also reflected by a haplotype network (Figs 10 and 11) constructed on the base of COI. To construct the network we used 191 specimens that were collapsed in 96 haplotypes representing 26 haplogroups (Table 2): 10 haplogroups for P. admetus group and 16 haplogroups for P. dolus group.

Figure 10.

Haplotype network of P. admetus species group. Colored circles represent different taxa. Each line segment represents a mutation step, and white small circles represent “missing” haplotypes.

Figure 11.

Haplotype network of P. dolus species group. Colored circles represent different taxa. Each line segment represents a mutation step, and white small circles represent “missing” haplotypes.

Polyommatus ripartii was represented by 82 specimens divided in 38 haplotypes and four haplogroups which corresponded completely with the four clades revealed on the Bayesian tree (Fig. 10). Polyommatus admetus sensu auctorum was found to include two haplogroups. One haplogroup was represented by specimens from the Balkan and west Turkey (P. admetus admetus), and the other haplogroup was represented by specimens from Armenia and Azerbaijan (P. admetus yeranyani + P. admetus malievi). These two haplogoups were clearly distinct from one another as can be seen in the number of nucleotide substitutions between them. Polyommatus nephohiptamenos was represented by a distinct haplogroup most close to P. ripartii ripartii haplogroup.

As a by-product of our study, we also discovered that within our samples P. demavendi comprised two haplogroups. One haplogroup was represented by specimens of P. demavendi belovi, whilst the other was represented by P. demavendi lorestanus. Polyommatus pseudorjabovi was represented by a single differentiated haplogroup. A distinct haplogroup represented by a single haplotype was found within P. khorasanensis.

Concerning P. dolus group (Fig. 11) we would like to mention that all recognized species, except for P. fulgens and P. fabressei, were represented by clearly distinct COI haplogroups. Polyommatus fulgens and P. fabressei were closely related and even shared one haplotype, despite clear differences in butterfly wing color and karyotypes.

Haplotypes of our target taxa (P. aroaniensis, P. timfristos sp. n., P. orphicus and P. humedasae) formed together a single cluster. However, all these taxa were distinct, and they did not share any common haplotypes. Therefore, this cluster could be subdivided into four haplogroups: ar (P. aroaniensis), tim (P. timfristos), orph (P. orphicus) and hum (P. humedasae) (Table 2, Fig. 11).

Despite presumed conspecifity (Kolev 2005), Polyommatus orphicus and P. dantchenkoi were found to be in the opposite parts of the recovered net, being separated by a number of other species (P. alcestis, P. violetae, P. aroaniensis, P. timfristos). The chromosomally distinct taxa P. alcestis and P. karacetinae were found to be also distinct with respect to their COI haplotypes. These two taxa were already treated as different species by Wiemers et al. (2009).

One of the main characteristic features of the anomalous blue butterflies is the upperside wing color. All males and females have brown upper side of the wings, and therefore the group is also called “brown” complex. As for the underside (Fig. 12), there are some differentiated characters of the wing pattern that allow the defining of seven morphological types.

1. Polyommatus ripartii type: hindwing underside with well-developed white streak (character 2 in Fig. 12), spots are small or medium-sized, marginal marking is reduced. This type is found in different species of both P. admetus and P. dolus complexes, e.g. in P. orphicus orphicus (Fig. 13g), P. orphicus eleniae (Fig. 13j), P. nephohiptamenos (Fig. 14e), P. ripartii pelopi (Figs 15a, f, g, h, j, k, 16a, b, c) and P. timfristos (Fig. 16e, h).

2. Polyommatus valiabadi type: the wing underside with exaggerated spots, white streak on the hindwing underside is clearly visible and sharp. This type is found in P. valiabadi, P. rjabovianus and P. pseudorjabovi from Iran and Azerbaijan (Lukhtanov et al. 2015). This type is not found in European Agrodiaetus species.

3. Polyommatus admetus type: the hindwing has no white streak, marginal marking is very well pronounced. This type is found in P. admetus (Fig. 13a, b, c, d).

4. Polyommatus nephohiptamenos type: White streak is well pronounced and very broad on the hindwing, consisting of the main streak and an additional short streak between postdiscal and submarginal areas, just under the main streak. This type is common in P. nephohiptamenos (Fig. 14c, d, f, g, h), not rare in P. ripartii (Fig. 15b,d,i) and also found in P. orphicus orphicus (Fig. 14a) and P. timfristos (Fig. 16g).

5. Polyommatus humedasae type: no white streak on the hindwing, marginal marking is pale. This type is quite common in P. aroaniensis, P. timfristos (Fig. 16f) and P. orphicus (Fig. 14b). It is typical for some populations of P. ripartii from West Europe (Vila et al. 2010) (but not from the Balkan Peninsula).

6. Polyommatus aroaniensis type: the white streak on the hindwing underside demonstrates different level of reduction. This type is found in P. aroaniensis (Fig. 16j), P. timfristos (Fig. 16d, e, i), P. orphicus orphicus (Fig. 14h, i) and P. orphicus eleniae (Fig. 13e, g). It is also found in the population of P. ripartii from the Crimea (Vila et al. 2010) (but not from the Balkan Peninsula).

7. Polyommatus orphicus type: forewing underside with clear white postdiscal streak between discal spot and submarginal marking, white streak on hindwing underside is prominent, often with additional small white streak (Fig. 12). This type is common in P. orphicus orphicus (Fig. 14a); nevertheless, the most characteristic feature (the white postdiscal streak between discal spot and submarginal marking on the forewing underside) can be found in other species, e.g. P. aroaniensis (Fig. 16j) and P. nephohiptamenos (Fig. 14h).

Figure 12.

Polyommatus orphicus orphicus collected in the type locality (Bulgaria, Hvoyna, 3 July 2016). Photo by E. Pazhenkova. White postdiscal streak between discal spot and submarginal marking on the forewing underside (character 1), prominent white streak on the hindwing underside (character 2) and additional white short streak between postdiscal and submarginal areas of the hind wing underside (character 3) are shown.

Figure 13.

The coloration and wing pattern of P. admetus, P. orphicus eleniae and P. orphicus orphicus. The letters correspond to the following sample numbers: a LR-08-D109 upperside and underside b LR-08-386 c LR-08-655 d LR-08-211 e LR-08-433 upperside and underside f LR-08-434 g LR-08-437 h LR-08-545 i LR-08-546 j LR-08-560. Scale bar corresponds to 10 mm in all figures.

Figure 14.

The coloration and wing pattern of P. orphicus orphicus, P. orphicus eleniae and P. nephohiptamenos. The letters correspond to the following sample numbers: a LR-08-D561 b LR-08-431 c LR-08-483 upperside and underside d LR-08-496 e LR-08-498 f LR-08-499 upperside and underside g LR-08-485 upperside and underside h LR-08-494 upperside and underside, white postdiscal streak between discal spot and submarginal marking on the forewing underside is shown by arrow. Bar = 10 mm.

Figure 15.

The coloration and wing pattern of P. ripartii pelopi. The letters correspond to the following sample numbers: a LR-08-D257 upperside and underside b LR-08-471 c LR-08-085 d LR-08-092 e LR-08-120 f LR-08-144 g LR-08-145 h LR-08-249 i LR-08-252 j LR-08-260 k LR-08-291. Bar = 10 mm.

Figure 16.

The coloration and wing pattern of P. ripartii pelopi, P. timfristos sp. n. and P. aroaniensis. The letters correspond to the following sample numbers: a LR-08-D549 b LR-08-551 c LR-08-571 d LR-08-273 e LR-08-205 f LR-08-274 upperside and underside g LR-08-258 h LR-08-247 (Holotype) i LR-08-255 j LR-08-102 upperside and underside. White postdiscal streak between discal spot and submarginal marking on the forewing underside is shown by arrow. Bar = 10 mm.

The chromosome number of P. admetus was first established by H. de Lesse who discovered n=80 in populations from Bulgaria (Kalotina) and W Turkey, and n=78-80 (with predominance of n=79) in populations from the eastern part of Turkey (de Lesse 1960a,b). The last count (n=78-80 with predominance of n=79) was later confirmed for populations from Armenia (Lukhtanov and Dantchenko 2002a), Turkey and Azerbaijan (Dantchenko and Lukhtanov 2005, Lukhtanov et al. 2015a). Here we confirm the haploid chromosome number n=80 for Dragoman near Kalotina (Bulgaria) and demonstrate that this karyotype occurs in other localities in Greece. The karyotype of the European samples (with predominance of n=80) seems to be similar, but not completely identical to the karyotype of samples from east Turkey, Armenia and Azerbaijan (with predominance of n=79).

This transpalearctic species has been known to have a stable karyotype (n=90, including one large, one medium and 88 small elements) throughout its whole distribution range from Spain in the west to the Altai in the east (de Lesse 1960a,b, Kandul 1997, Lukhtanov and Datchenko 2002, Vila et al. 2010, Vershinina and Lukhtanov 2010, Przybyłowicz et al. 2014). The number n=90 was also found in P. ripartii pelopi (Coutsis et al. 1999), and we confirmed this count for samples from South and Central Greece and from Bulgaria.

The haploid chromosome number was erroneously given for this taxon as n=8-11 by Brown and Coutsis (1978), and later corrected by Coutsis and De Prins (2007) who established the chromosome number with an approximation due bivalents overlaps as n=ca84-88. Here we were able to make a precise count of chromosome elements in this taxon and to demonstrate that n=90, exactly as in P. ripartii. We do not confirm the proposed difference between P. nephohiptamenos and P. ripartii in number of large chromosomes (Coutsis and De Prins 2007). In our squash preparations, both species demonstrate one big and one medium-sized element in the haploid chromosome set.

The haploid chromosome number for this taxon was erroneously given as n=15-16 by Brown (1976a), and later corrected to be n=48 in few studied metaphase plates by Coutsis et al. (1999). In the single studied sample we were able to make a precise count of chromosome elements and found the haploid chromosome number to be n=47. Both counts (previous n=48 and n=47 in this study), are essentially different from those found in closely related P. timfristos and P. orphicus (Kolev 2005, this work) and P. humedasae (Troiano et al. 1979, Vila et al. 2010).

The chromosome number of P. orphicus was first established by Kolev (2005) who discovered n=41-42 in population from Hvoyna (Bulgaria), thus, similar to the karyotype found in P. dantchenkoi from remote east Turkey (Lukhtanov et al. 2003).

The chromosome number of P. eleniae was established first by Coutsis and De Prins (2005) who discovered n=41 in population from Falakro Mt near Granitis (Greece). Coutsis and De Prins reported that despite identical chromosome number, karyotypes of P. orphicus and P. eleniae were different in respect to their structure. Karyotype of P. eleniae was reported to be more asymmetrical than karyotype of P. orphicus (that is, the chromosomes were more differentiated with respect to their size).

Here we reinvestigated the karyotypes of P. orphicus and P. eleniae originating directly from their type-localities. Our data confirm previous chromosome number counts, but do not confirm the differences in karyotype structures. In our opinion, the presumed differences could appear because of differences in staining techniques used by Kolev (2005) for P. orphicus and Coutsis and De Prins (2005) for P. eleniae (see Wiemers and De Prins 2004). In our study, we used the same technique for both taxa, and we did not find any differences in the karyotype structure.

The haploid chromosome number of this taxon is established first here as n=38 and thus differs by at least three fixed chromosome fussions/fixions from P. orphicus orphicus and P. orphicus eleniae (n=41-42). This number is similar (but not identical) to that found in P. humedasae (n=39, Vila et al. 2010). We are not sure that the karyotypes of P. timfristos and P. humedasae are related in their origin because they are not found in proximity and separated by an area where P. orphicus with n=41–42 is distributed.

The Balkan and west Turkish populations of Polyommatus admetus have a unique hindwing underside pattern (Polyommatus admetus type, Fig. 13a, b, c, d) and can be easily separated on the basis of morphology from other species. However, some taxonomic and identification problems appear if oriental populations of P. admetus sensu lato are considered. In 2004, P. admetus yeranyani from Armenia and P. admetus malievi from Azerbaijan were described (Dantchenko and Lukhtanov 2005). The two last taxa differ from the nominative subspecies morphologically. They usually have a distinct white streak on the underside of the hindwing, and the marginal pattern of the wing underside is not as prominent as in P. admetus admetus. In fact, P. admetus yeranyani and P. admetus malievi are phenotypically similar to P. ripartii and P. demavendi, and their identification is not always easy. Karyological analysis revealed a minor difference between the western and oriental forms (see above), and molecular analysis demonstrated that they were differentiated with respect to COI barcodes and did not constitute together a monophyletic entity. This barcode distinctness is especially clearly expressed in the haplotype network (Fig. 10). Therefore, in accordance with the criterion of avoiding non-monophyletic groups in taxonomy (Vila et al. 2013), they should be treated as distinct species P. admetus and P. yeranyani.

The distribution of COI haplotypes in P. ripartii demonstrates a very complex picture. This taxon is represented by several clades on the phylogenetic reconstructions. The West-European clade includes butterflies from France, Italy and Spain. Another clade (a “mixed”, or Eurasian clade) includes butterflies from the whole Western Palaearctic region from Spain to Mongolia. Eastern Turkish-Caucasian clade (P. ripartii paralcestis) is strongly differentiated and appears as a group close to P. demavendi. Complicated taxonomy and phylogeography of P. ripartii have recently been topics of several specific studies and publications (Vila et al. 2010, Vodolazhsky et al. 2011, Dincă et al. 2013a, Przybyłowicz et al. 2014) and are out of the focus of the present paper. The sequences obtained in our study confirm that Balkan samples represent one of the major clades within P. ripartii populations, thus P. ripartii pelopi is confirmed as a valid subspecies.

Taxonomic interpretation of this local Balkan endemic is difficult since it is morphologically very similar and chromosomally seems to be identical to the close species P. ripartii. However, distinct COI barcodes in combination with ecological differentiation (P. nephohiptamenos is a high altitude species, whereas P. ripartii pelopi can be found usually at middle and low elevations) do not allow us to reject the pre-existing taxonomic hypotheis that P. nephohiptamenos represents a distinct taxonomic entity. The fact that P. nephohiptamenos retains its homogeneity with respect to COI being surrounded by closely related P. ripartii is additional indirect evidence for a presence of genetic boundaries between them. Further molecular and genetic studies are required to understand the real taxonomic status of P. nephohiptamenos.

Polyommatus dantchenkoi orphicus was described (Kolev 2005) and later considered (Tshikolovets 2011, Eckweiler and Bozano 2016) as a subspecies of P. dantchenkoi, a species known from east Turkey, because P. d. dantchenkoi and P. d. orphicus shared a similar phenotype and number of chromosomes (Lukhtanov et al. 2003, Kolev 2005). At times, P. orphicus has been considered as a distinct species (e.g. Van Swaay et al. 2010); however, its species level status was not justified.

Our molecular data demonstrate that, despite similarity in number of chromosomes, P. d. dantchenkoi and P. d. orphicus are not closely related as was previously thought. In the haplotype network, these taxa were found to be placed in the opposite parts of the recovered net, being separated by a number of other species (Fig. 11). Their merging would result in a polyphyletic assemblage (Fig. 8). Avoiding non-monophyletic groups is a preferable option in practical taxonomy (Talavera et al. 2013a). Therefore, P. dantchenkoi and P. orphicus should be considered as two distinct species. We should also note that the COI barcodes alone (as in our study) can provide weak evidence for monophyly or non-monophyly of taxa since trees inferred from single markers sometimes display relationships that reflect the evolutionary histories of individual genes rather than of the species being studied. In case of Agrodiaetus, COI barcodes showing such a discrepancy between species and gene trees may be a result of interspecific mitochondrial introgression (Lukhtanov et al. 2008, 2015b). Despite this limitation, we argue that monophyletic clusters resulting from the DNA barcode analysis are better primary taxonomic hypotheses than para- or polyphyletic ones (Lukhtanov et al. 2016).

Polyommatus eleniae was described from a place located 80 km south-west from the type locality of P. orphicus. Polyommatus orphicus and P. eleniae have the same number of chromosomes, but it was supposed that they were different in karyotype structure (Coutsis and De Prins 2005). Additionally, it was supposed that P. eleniae differed from P. orphicus by the constant lack of a white postdiscal streak on the forewing underside (character 1 on Fig. 12) and by strong reduction or total lacking of a white streak on the hindwing underside (character 2 on Fig. 12) (Coutsis and De Prins 2005). In P. orphicus these streaks are supposed to be always sharply defined (Kolev 2005). Our study does not support the difference in karyotypes (see above). Our analyses showed that the supposed differences in morphology disappeared if individual variations were taken into account. Although the “typical” phenotype of P. orphicus (Figs 12 and 14a) often present in in Hvoyna, the individuals with different level of reduction of white streak on both fore- and hindwing underside are very common (Fig. 13h, i, j). These individuals with confidence can be identified as P. orphicus as they have the same karyotype and do not differ in mitochondrial haplotypes. Thus, the morphological difference between individuals from Hvoyna (P. orphicus) and Falakro Mt (P. eleniae) is not clear and is not based on fixed characters. The difference in karyotypes was also not confirmed in our analysis (see the section Chromosomal diversity above). Therefore, we conclude that the population from Falakro Mt is most probably conspecific with P. orphicus and can be treated as a subspecies P. orphicus eleniae.

This taxon was first described by Brown (1976a) as a subspecies of P. alcestis and two years later was raised to species rank (Brown and Coutsis 1978). Despite its similarity to other taxa of the brown complex, especially with P. humedasae, P. orphicus orphicus and P. o. eleniae, it differs by its karyotype and COI barcodes. Its species distinctness confirmed by chromosomal analysis (Coutsis et al. 1999) has never been questioned. Thus, there has been no problem with treatment of P. aroanisnsis as a separate species. However, there are numerous identification problems associated with P. aroaniensis because several populations from Central and Northern Greece, as well as from other countries of the Balkan Peninsula were identified as P. aroaniensis (see the section Distribution areas below), but their karyotypes were not studied. In our work, we discovered that two of these populations (from Timfristos Mt and Parnassos Mt) represented a previously unrecognized species. Below we name it and provide its formal description.

Figs 24–26

This species is widespread in the Balkan Peninsula. It is local in the northern part of Hungary (Ilonczai and Bálint 2001, Bálint et al. 2006) and recorded in Slovakia (Kulfan and Kulfan 1992, Eckweiler and Bozano 2016). It has been shown to be widely distributed in the western part of Romania, but no exact localities were provided (Eckweiler and Bozano 2016). It is common in Greece and found in Croatia, Bosnia and Herzegovina, Montenegro, Serbia, Bulgaria, The Republic of Macedonia, Albania and European Turkey (Sijaric and Mihljevic 1972, Hesselbarth et al. 1995, Abadjiev 2001, Tolman and Lewington 2008, Eckweiler and Bozano 2016).

Fig. 27

Polyommatus ripartii is widespread in the southern part of the Balkan Peninsula (Greece and Bulgaria); however, it is more local in the north. It is known from Albania, the Republic of Macedonia, south Serbia (Kudrna et al. 2011, Eckweiler and Bozano 2016), Bosnia and Herzegovina (Koren 2010). It was mentioned for European Turkey (Hesselbarth et al. 1995, Tolman and Lewington 2008) and recently found in Croatia (Koren 2010, Dincă et al. 2013a).

Figs 28–30

Polyommatus nephohiptamenos has a dot-like distribution area and is known from the high altitudes of north-east Greece (Mt Pangeon, Mt Phalakro and Mt Orvilos) and south-west Bulgaria (Mt Orvilos, also known as Mt Slavyanka, Mt Alibotush and Kitka Planina) (Kolev 1994, Tolman and Lewington 2008, Eckweiler and Bozano 2016).

Fig. 31

Polyommatus aroaniensis has been considered as a relatively widespread species (Kolev and van der Poorten 1997). Apart from its type-locality (South Greece, Peloponnese), it has been recorded in different parts of Central and Northern Greece (Brown 1976a, Wakeham-Dawson and Spurdens 1994, Wakeham-Dawson 1998, Pamperis 2009), from a few areas in south Macedonia (Kolev and van der Poorten 1997, Melovski and Bozhinovska 2014) and from some localities in south-west Bulgaria and one isolated place in the central part of the country (Abadjiev 2001, Kolev 1994, Kolev and van der Poorten 1997). Tshikolovets (2011) and Eckweiler and Bozano (2016) show its distribution extending into Albania, although the species has not been recorded from this country in recent surveys (Verovnik and Popović 2013). Verovnik et al. (2015) recorded it in Bosnia and Herzegovina. Koren and Laus (2015) recorded it in Croatia; however, this record was not confirmed by molecular data (Lovrenčić et al. 2016).

Our chromosomal data confirm P. aroaniensis in South Greece (Peloponnese), but cannot confirm it in Central and Northern Greece and in Bulgaria where it is replaced by the closely related allopatric species P. timfritos and P. orphicus. In the light of the data obtained, the occurrence of P. aroaniensis in Bulgaria, Albania, Macedonia and Bosnia and Herzegovina seems to be doubtful and requires a confirmation based on chromosomal analysis. We cannot exclude that the populations from Albania, Macedonia and Bosnia and Herzegovina could represent P. orphicus or even undescribed taxa of the subgenus Agrodiaetus.

Figs 32–35

This species is known from Timfristos and Parnassos Mts in Central Greece only.

Figs 36–39

This species is known from South Bulgaria and Northern Greece only. However, its occurrence in other countries in the northern Balkan is theoretically possible (see above).