Short Communication
Short Communication
Chromosomal identification of cryptic species sharing their DNA barcodes: Polyommatus (Agrodiaetus) antidolus and P. (A.) morgani in Iran (Lepidoptera, Lycaenidae)
expand article infoVladimir A. Lukhtanov, Nazar A. Shapoval§
‡ Russian Academy of Sciences, St. Petersburg, Russia
§ St. Petersburg State University, St. Petersburg, Russia
Open Access


DNA barcoding has been suggested as a universal tool for molecular species identification; however, it cannot be applied in cases when morphologically similar species share their DNA barcodes due to the common ancestry or mitochondrial introgression. Here we analyze the karyotype of Polyommatus (Agrodiaetus) morgani (Le Cerf, 1909) from the region of its type locality in the southern Zagros Mountains in Iran, provide first chromosomal evidence for P. (A.) antidolus (Rebel, 1901) in Iran and demonstrate that these two species can be easily identified through analysis of their karyotypes whereas they share their mitochondrial barcodes.


Ancestral polymorphism, biodiversity, chromosomes, chromosomal fusion/fission, cryptic species, cytogenetics, DNA barcoding, incomplete lineage sorting, karyosystematics, mitochondrial introgression, nomenclature, species identification, taxonomy


Cryptic species, morphologically indistinguishable or highly similar biological entities, represent a substantial portion of plant and animal diversity, and therefore the search for these species is important for taxonomic, ecological and evolutionary studies (Beheregaray and Caccone 2007, Pfenninger and Schwenk 2007, Dincă et al. 2013, Vodă et al. 2015). Cryptic species can usually be identified through analysis of molecular markers (Vodă et al. 2015), e.g. through analysis of the so-called DNA barcodes, short genetic sequences from a standard part of the genome (Hebert et al. 2003). However, the use of the standard DNA barcodes such as short fragments of the mitochondrial gene COI and the non-coding nuclear sequence, internal transcribed spacer 2 (ITS2), is sometimes insufficient to distinguish between evolutionarily young sister species, either because they can be weakly differentiated regarding these markers or because they are too polymorphic (Avise 2000, Lukhtanov et al. 2015a, b, 2016). The absence of lineage sorting among species can often pose a problem for the use of molecular markers in rapidly evolving taxa because the time to coalescence for alleles within lineages can be greater than the time required for speciation (Avise 2000, Kandul et al. 2004). Chromosomal characters in many groups can evolve more rapidly (Lukhtanov 2015, Vershinina and Lukhtanov 2017), and because they are often present as fixed differences, these characters could serve as applicable markers for recently evolved taxa (King 1993, Dobigny et al. 2005, Lukhtanov et al. 2015a, Vishnevskaya et al. 2016).

Polyommatus (Agrodiaetus) antidolus (Rebel, 1901), P. (A.) kurdistanicus (Forster, 1961) and P. (A.) morgani (Le Cerf, 1909), a complex of three closely related allopatric species distributed in east Turkey as well as in west and central Iran (Fig. 1) (Eckweiler and Bozano 2016), represent a good example of such situation. Despite morphological similarity (Fig. 2) and identity of COI barcodes in the majority of the studied populations (see Table 2 and sequences published in Wiemers 2003, Wiemers and Fiedler 2007, Kandul et al. 2004, 2007, Lukhtanov et al. 2015b and see Lukhtanov et al. 2015b for the exceptions), they can be easily identified by their chromosome numbers. Haploid chromosome numbers (n) were found to be n=25-27 in P. (A.) morgani, n=39-42 in P. (A.) antidolus and n=61-62 in P. (A.) kurdistanicus (de Lesse 1960, 1961, Lukhtanov et al. 1998, 2005, 2015b). However, the karyotype has never been studied in Iranian populations from the southern and northern Zagros Mountains including the region of the type locality of P. (A.) morgani (locality 1 in Fig. 1), and this negatively affects the identification and taxonomic interpretation of all known populations. Here we provide first chromosomal data for populations of the complex from the southern and northern Zagros Mountains.

Figure 1.

Map of Iran showing the type locality of Polyommatus (Agrodiaetus) morgani and the localities of the analyzed specimens of P. (A.) morgani and P. (A.) antidolus. 1 type locality of P. (A.) morgani, “Deh-Tcheshma” (Deh Cheshme near Farsan, Chaharmahal and Bakhtiari Province) 2P. (A.) morgani, n=27, vic. Sibak, Esfahan Province 3P. (A.) morgani, n=27-29, 25 km E of Mahabad, W. Azerbaijan Province 4P. (A.) morgani, n=27-29, 15 km W of Mahabad, W. Azerbaijan Province 5P. (A.) antidolus, n=39-41, Seir, 4 km S of Urmia, W. Azerbaijan Province.

Material and methods

The butterflies were collected in 2016 in north-west and central Iran: in a mountain valley between Fereydunshahr and Sibak (locality 2), in the vicinity of Darman (25 km E of Mahabad) (locality 3), in the vicinity of Khalifen (15 km W of Mahabad) (locality 4) and in Seir (near Urmia) (locality 5) (Fig. 1). We also included sequences of karyotyped P. (A.) kurdistanicus and P. (A.) antidolus specimens available from GenBank (Wiemers 2003, Lukhtanov et al. 2005) in our analysis. A complete list of specimens included in this study and information about sampling localities are given in Table 1. Karyotypes (Figs 3 and 4) and COI-barcodes (Table 1 and 2) were analyzed using approaches described previously (Lukhtanov et al. 2014, Przybyłowicz et al. 2014). We use the following abbreviations: MI for metaphase I of meiosis and MII for metaphase II of meiosis. Divergences between COI sequences were computed using MEGA6 software (Tamura et al. 2013).

Table 1.

List of studied material (17 specimens). Asterisks indicate unsequenced specimens. Collectors: E. Pazhenkova (EP), N. Shapoval (NS), V. Lukhtanov (VL).

Field Code GenBank number Taxon Chromosome number (n) Locality Altitude Date Collectors/
Q055* morgani n=27 Iran, Esfahan Prov., Sibak (N32°55'; E50°04') 2700 m 02.08.2017 EP, NS, VL
Q060* morgani n=27 Iran, Esfahan Prov., Sibak (N32°55'; E50°04') 2700 m 02.08.2017 EP, NS, VL
Q150 MG457163 morgani n=28-29 Iran, W. Azerbaijan Prov., vic. Darman, 25 km E of Mahabad (N36°45'; E45°52') 1900–2000 m 10.08.2017 EP, NS, VL
Q170 MG457164 morgani n=27 Iran, W. Azerbaijan Prov., vic. Darman, 25 km E of Mahabad (N36°45'; E45°52') 1900–2000 m 10.08.2017 EP, NS, VL
Q171 MG457165 morgani n=27 Iran, W. Azerbaijan Prov., vic. Darman, 25 km E of Mahabad (N36°45'; E45°52') 1900–2000 m 10.08.2017 EP, NS, VL
Q181 MG457166 morgani n=28 Iran, W. Azerbaijan Prov., vic. Darman, 25 km E of Mahabad (N36°45'; E45°52') 1900–2000 m 10.08.2017 EP, NS, VL
Q196 MG457167 morgani n=27-28 Iran, W. Azerbaijan Prov., vic. Khalifen, 15 km W of Mahabad (N36°45'; E45°32') 2100–2200 m 11.08.2017 EP, NS, VL
Q197 MG457168 morgani n=27 Iran, W. Azerbaijan Prov., vic. Khalifen, 15 km W of Mahabad (N36°45'; E45°32') 2100–2200 m 11.08.2017 EP, NS, VL
Q198 MG457169 morgani n=28-29 Iran, W. Azerbaijan Prov., vic. Khalifen, 15 km W of Mahabad (N36°45'; E45°32') 2100–2200 m 11.08.2017 EP, NS, VL
Q237 MG457170 antidolus n=40-41 Iran, W. Azerbaijan Prov., vic. Seir, Urmia (N37°28'; E45°02') 1750 m 14.08.2017 EP, NS, VL
Q238 MG457171 antidolus n=39-40 Iran, W. Azerbaijan Prov., vic. Seir, Urmia (N37°28'; E45°02') 1750 m 14.08.2017 EP, NS, VL
Q239 MG457172 antidolus n=39 Iran, W. Azerbaijan Prov., vic. Seir, Urmia (N37°28'; E45°02') 1750 m 14.08.2017 EP, NS, VL
AY557093 antidolus n=42 Turkey, Hakkari Prov., Dez Çay 1500 m 22.07.1999 Wiemers 2003
AY557095 antidolus n=ca44 Turkey, Hakkari Prov., Haruna Geçidi, SE Yüksekova 2000 m 21.07.1999 Wiemers 2003
AY557108 kurdistanicus n=ca>55 Turkey, Van Prov., Erek Dagi 2200 m 25.07.1999 Wiemers 2003
AY557074 kurdistanicus n=ca54-56 Turkey, Van Prov., Çatak 1600–1900 m 25.07.1999 Wiemers 2003
AY496762 kurdistanicus n=62 Turkey, Van Prov., Çatak July 2001 Lukhtanov et al. 2005
Table 2.

Divergence between COI sequences. The numbers of base differences per site between sequences are shown. The shared barcodes (uncorrected COI p-distance = 0) are shown in bold.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
(1) AY557095antidolus
(2) AY557089antidolus 0.0015
(3) AY557108kurdistanicus 0.0015 0
(4) AY557074kurdistanicus 0.0015 0 0
(5) AY496762kurdistanicus 0.0015 0 0 0
(6) Q150 morgani 0.0015 0 0 0 0
(7) Q170 morgani 0.0029 0 0 0 0 0
(8) Q171 morgani 0.0029 0.0015 0.0015 0.0015 0.0015 0.0015 0.0015
(9) Q181 morgani 0.0015 0 0 0 0 0 0 0.0015
(10) Q196 morgani 0.0015 0 0 0 0 0 0 0.0015 0
(11) Q197 morgani 0.0015 0 0 0 0 0 0 0.0015 0 0
(12) Q198 morgani 0.0015 0 0 0 0 0 0 0.0015 0 0 0
(13) Q237 antidolus 0.0015 0 0 0 0 0 0 0.0015 0 0 0 0
(14) Q238 antidolus 0.0015 0 0 0 0 0 0 0.0015 0 0 0 0 0
(15) Q239 antidolus 0.0015 0 0 0 0 0 0 0.0015 0 0 0 0 0 0

Results and discussion

In order to investigate the topotypical population of P. (A.) morgani, we first searched for it in its exact type locality in “Deh Tcheshma” (mountain area near the village Deh Cheshme, close to the city Farsan, Chaharmahal and Bakhtiari Province, Iran) (locality 1 in Fig. 1). Unfortunately, we were unable either to find it there or to locate a biotope suitable for butterflies of the P. (A.) antidolus - P. (A.) kurdistanicus - P. (A.) morgani complex. In our opinion, P. (A.) morgani is extinct in its type locality, probably due to climate change and aridification during the last 100 years. Fortunately, we were able to find typical P. (A.) morgani in a small, relatively humid mountain valley between Fereydunshahr and Sibak, 90 km NW of Farsan (N32°55; E50°04’, Esfahan Province, Iran) (locality 2 in Fig. 1). In two studied specimens from the latter locality, at the MI stage, the haploid chromosome number n = 27 was found (Figs 3a, b). The meiotic karyotype was strongly asymmetric, with a group of larger bivalents (from 6 to 10 in different cells) and a group of smaller bivalents (from 17 to 21 in different cells). The number of bivalents that were classified as “larger” and “smaller” was variable, most likely depending on the bivalent orientation. However, in some metaphase plates, the distinction between the larger and smaller bivalents was unclear, and the bivalents gradually decreased in size, with the largest bivalent approximately 10 times larger than the smallest one. Thus, the results obtained confirm the previous taxonomic interpretations (de Lesse 1960, 1961, Carbonell 2003, Lukhtanov et al. 1998, 2005, 2015b, Eckweiler and Bozano 2016) that considered the populations with n=25–27 as P. (A.) morgani.

Figure 2.

Male wing pattern of P. (A.) morgani, P. (A.) antidolus and P. (A.) kurdistanicus. aP. (A.) morgani, Iran, Kordestan Province, Senandaj, 1800 m, 20 July 2000, leg. P. Hofmann bP. (A.) antidolus Turkey, Hakkari Province, Ogul-Tal, 1500–1900 m, 1 August 1984, leg. Schurian cP. (A.) kurdistanicus Turkey, Van Province, 10 km S of Van, 1900–2100 m, 10 August 1978, leg. Görgner.

Tshikolovets et al. (2014) and Eckweiler and Bozano (2016) identified the population of the P. (A.) antidolus - P. (A.) kurdistanicus - P. (A.) morgani complex from the vicinity of Mahabad (West Azerbaijan Province) (localities 3 and 4 in Fig. 1) as P. (A.) antidolus; however, they did not provide any chromosomal data to confirm this conclusion. We analyzed seven specimens from two localities close to Mahabad (localities 3 and 4 in Fig. 1). At the prometaphase I, MI and MII stages, n=27 was determined as the basic number in four specimens (Fig. 3c–g), not n=39-42 as expected for P. (A.) antidolus. The number of elements within the karyotype was unstable, varying from n=27 to n=29, most likely due to the presence of two chromosomal fusions/fissions (Figs 3h, i, 4a, b). With respect to the karyotype structure (size and proportion of larger vs. smaller chromosomal elements) the specimens from Mahabad were indistinguishable from the typical P. (A.) morgani described above. The chromosome numbers n=28 and n=29 were not previously reported for P. (A.) morgani (de Lesse 1960, 1961, Lukhtanov et al. 1998, 2005, 2015b). However, since there is no fixed chromosomal difference between the populations from Sibak and Mahabad, we do not see the need for a description of a new taxon from Mahabad, and therefore identify the populations from Mahabad as P. (A.) morgani.

Figure 3.

Karyotype of P. (A.) morgania Q060, MI, n=27 b Q055, MI, n=27 c Q170, prometaphase I, n=27 d Q171, MI, n=27 e Q197, prometaphase I, n=27 f Q196, MI, n=27 g Q196, MII, n=27 h Q196, MII, n=28 i Q196, MII, n=28. Bar = 10 μ.

Finally, in three specimens collected in Seir (near Urmia, locality 5 in Fig. 1) at the MI/MII stages, we found that the number of chromosomal elements varied from 39 to 41. The chromosomes ranged in size from very small to large (Fig. 4c–f). This karyotype (n=39-41) seems to be identical to that found in P. (A.) antidolus in the neighboring Province Hakkari in south-east Turkey, thus providing first chromosomal evidence for P. (A.) antidolus in Iran.

Figure 4.

Karyotypes of P. (A.) morgani and P. (A.) antidolus. aP. (A.) morgani, Q150, MII, n=28 bP. (A.) morgani, Q150, MII, n=29 cP. (A.) antidolus, Q239, MI, n=39, squash preparation dP. (A.) antidolus, Q239, MI, n=39, intact metaphase plate eP. (A.) antidolus, Q237, MII, n=40 fP. (A.) antidolus, Q237, MII, n=41. Scale bar = 10 μ.


We are grateful to Wolfgang ten Hagen for providing information about sampling localities of P. (A.) morgani in Iran. The complete financial support for this study was provided by the grant from the Russian Science Foundation N 14-14-00541 to The Zoological Institute of the Russian Academy of Sciences. The work was partially performed using equipment of the Centre for Molecular and Cell Technologies and Department of Entomology of St. Petersburg State University. A part of this equipment was purchased with support of the St. Petersburg University grant 1.40.490.2017.


  • Avise JC (2000) Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, Massachusetts, 464 pp.
  • Carbonell F (2003) Agrodiaetus antidolus pertekensis n. ssp., de Turquie orientale, et A. morgani sanandajensis n. ssp., de l’ouest de l’Iran (Lep., Lycaenidae). Bulletin de la Societe Entomologique de France 108(5): 445–446.
  • de Lesse H (1960) Spéciation et variation chromosomique chez les Lépidoptères Rhopalocères. Annales des Sciences Naturelles Zoologie et Biologie Animale, 12e série.2: 1–223.
  • de Lesse H (1961) Les nombres de chromosomes chez Agrodiaetus dolus Hübner et les espèces voisines (Lycaenidae). Alexanor 2(2): 57–63.
  • Dincă V, Wiklund C, Lukhtanov VA, Kodandaramaiah U, Norén N, Dapporto L, Wahlberg N, Vila R, Friberg M (2013) Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex. Journal of Evolutionary Biology 26(10): 2095–2106.
  • Dobigny G, Aniskin V, Granjon L, Cornette R, Volobouev V (2005) Recent radiation in West African Taterillus (Rodentia, Gerbillinae): the concerted role of chromosome and climatic changes. Heredity 95(5): 358–368.
  • Eckweiler W, Bozano GC (2016) Guide to the butterflies of the Palearctic region. Lycaenidae part IV. Omnes artes, Milano, 132 pp.
  • Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270(1512): 313–321.
  • Kandul NP, Lukhtanov VA, Dantchenko AV, Coleman JWS, Sekercioglu CH, Haig D, Pierce NE (2004) Phylogeny of Agrodiaetus Hübner 1822 (Lepidoptera: Lycaenidae) inferred from mtDNA sequences of COI and COII and nuclear sequences of EF1–α: Karyotype diversification and species radiation. Systematic Biology 53(2): 278–298.
  • King M (1993) Species evolution: the role of chromosomal change. Cambridge University Press, Cambridge, 360 pp.
  • Lukhtanov VA (2015) The blue butterfly Polyommatus (Plebicula) atlanticus (Lepidoptera, Lycaenidae) holds the record of the highest number of chromosomes in the non-polyploid eukaryotic organisms. Comparative Cytogenetics 9(4): 683–690.
  • Lukhtanov VA, Shapoval NA, Dantchenko AV (2014) Taxonomic position of several enigmatic Polyommatus (Agrodiaetus) species (Lepidoptera, Lycaenidae) from Central and Eastern Iran: insights from molecular and chromosomal data. Comparative Cytogenetics 8(4): 313–322.
  • Lukhtanov VA, Kandul NP, Plotkin JB, Dantchenko AV, Haig D, Pierce NE (2005) Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies. Nature 436(7049): 385–389.
  • Lukhtanov VA, Kandul NP, De Prins WO, van der Poorten D (1998) Karyology of species of Polyommatus (Agrodiaetus) from Turkey: new data and their taxonomic consequences (Lepidoptera: Lycaenidae). Holarctic Lepidoptera 5(1): 1–8.
  • Lukhtanov VA, Sourakov A, Zakharov EV (2016) DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera, Papilionidae). Systematics and Biodiversity 14(6): 599–613.
  • Lukhtanov VA, Dantchenko AV, Vishnevskaya MS, Saifitdinova AF (2015a) Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae). Biological Journal of the Linnean Society 116(2): 468–485.
  • Lukhtanov VA, Shapoval NA, Anokhin BA, Saifitdinova AF, Kuznetsova VG (2015b) Homoploid hybrid speciation and genome evolution via chromosome sorting. Proceedings of the Royal Society B: Biological Sciences 282(1807): 20150157.
  • Pfenninger M, Schwenk K (2007) Cryptic animal species are homogeneously distributed among taxa and biogeographical regions. BMC Evolutionary Biology 7: 121.
  • Przybyłowicz Ł, Lukhtanov V, Lachowska-Cierlik D (2014) Towards the understanding of the origin of the Polish remote population of Polyommatus (Agrodiaetus) ripartii (Lepidoptera: Lycaenidae) based on karyology and molecular phylogeny. Journal of Zoological Systematics and Evolutionary Research 52(1): 44–51.
  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30(12): 2725–2729.
  • Tshikolovets VV, Naderi A, Eckweiler W (2014) The butterflies of Iran and Iraq. Tshikolovets Publications, Pardubice, 366 pp.
  • Vishnevskaya MS, Saifitdinova AF, Lukhtanov VA (2016) Karyosystematics and molecular taxonomy of the anomalous blue butterflies (Lepidoptera, Lycaenidae) from the Balkan Peninsula. Comparative Cytogenetics 10(5): 1–85.
  • Vodă R, Dapporto L, Dincă V, Vila R (2015) Cryptic matters: overlooked species generate most butterfly beta-diversity. Ecography 38(4): 405–409.
  • Wiemers M (2003) Chromosome differentiation and the radiation of the butterfly subgenus Agrodiaetus (Lepidoptera: Lycaenidae: Polyommatus) a molecular phylogenetic approach. Ph.D. Dissertation, University of Bonn, Bonn, Germany, 203 pp.
  • Wiemers M, Fiedler K (2007) Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae). Frontiers in Zoology 4: 8. 9994-4-8
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