We show how combination of chromosomal and molecular markers can be applied for proper species identification in Agrodiaetus Hübner, 1822 blue butterflies. Using this approach we provide first evidence for presence of P. (A.) poseidon (Herrich-Schäffer, [1851]) in Georgia.

Agrodiaetus , chromosome number, COI , karyotype, Lycaenidae , Polyommatus

The blue butterfly subgenus Agrodiaetus Hübner, 1822 belongs to the genus Polyommatus Latreille, 1804 (Talavera et al. 2013). In the last years, this group become a model system for study of speciation and chromosome evolution (Lukhtanov et al. 2015, Vershinina et al. 2015). Despite this, its taxonomy is still poorly elaborated and identification of individual species is difficult due to their morphological similarity. Species within the subgenus are mostly uniform and exhibit few differences in characters traditionally used in classification, such as wing pattern and/or aspects of the male and female genitalia (Lukhtanov et al. 2006, Vila et al. 2010). The genus was estimated to have originated very recently (Kandul et al. 2004) and, thus, many Agrodiaetus species may have not had sufficient time to acquire extensive genetic differences. In particular, COI barcode gap is low or even absent between numerous closely related species of Polyommatus (Agrodiaetus) (Wiemers and Fiedler 2007). In opposite to majority of other butterflies and moths (Lukhtanov 2014), many Agrodiaetus species have evolved distinctive karyotypes. They show one of the highest interspecific karyotypic diversities known in the animal kingdom with haploid chromosome numbers ranging from n = 10 to n = 134 (Lukhtanov et al. 2005). Therefore, karyotypic features provide important identification characters for many described species that are virtually indistinguishable by their morphology. However, it should be noted that in few cases the chromosome number may be identical in different species (see Results and Discussion).

Here we show how combination of chromosomal and molecular markers can be applied for proper species identification in Agrodiaetus. Using this approach we provide first evidence for presence of P. (A.) poseidon (Herrich-Schäffer, [1851]) in Georgia.

The samples used for molecular and chromosomal analysis were collected in Georgia (Akhaltsikhe, 41.60N, 43.06E, 1000 m alt., 18 July 2014, V. Lukhtanov et V. Tikhonov leg., samples 2014VL56, 2014VL57, 2014VL58, 2014VL62, 2014VL63, 2014VL64, 2014VL65, 2014VL68, 2014VL69, 2014VL70). The methods of DNA sequencing, chromosomal analysis and phylogenetic inference were described previously (Lukhtanov and Dantchenko 2002a, Lukhtanov et al. 2008, 2014, Vershinina and Lukhtanov 2010, Przybyłowicz et al. 2014). Additional samples of Polyommatus belonging to P. (A.) poseidon species complex (Kandul et al. 2007) were used for comparison.

The species P. (A.) poseidon (= Lycaena poseidon var. mesopotamica Staudinger, 1892, synonymized with P. poseidon by Schurian et. 1992) is known to be an endemic of the Middle East sporadically distributed from Kütahya in West Turkey to Artvin in North-East Turkey (Hesselbarth et al. 1995). Phenotypically similar, but chromosomally distinct species P. (A.) putnami (Lukhtanov & Dantchenko, 2002) was described from East Turkey (provinces Erzurum and Ağri) (Lukhtanov and Dantchenko 2002b). The last taxon is allopatric in distribution with P. (A.) poseidon and differs from P. poseidon by chromosome number and karyotype structure (Lukhtanov and Dantchenko 2002b). P. (A.) poseidon has relatively low haploid chromosome number (from n=19 on the south and east of the distributional range to n=21 in the north), all the chromosomes form a gradient size row with no especially large or small chromosomes (de Lesse 1963, Kandul and Lukhtanov 1997). Chromosome numbers n=22 and n=23 were also found in the northern population as intraindividual occasional deviations from the basic n=21 (de Lesse 1963). P. (A.) putnami has higher chromosome numbers (from n=24 to n=27, with n=26 as a distinct mode). Its karyotype is asymmetrical and includes chromosomes of two distinct classes: class of large chromosomes and class of small chromosomes (Lukhtanov and Dantchenko 2002b). Currently P. (A.) putnami is treated as a distinct species (Lukhtanov and Dantchenko 2002b, Wiemers 2003, Wiemers and Fiedler 2007) or a subspecies of P. (A.) poseidon (Tshikolovets 2011).

The taxon P. (A.) deebi (Larsen, 1974) discovered in Lebanon and Syria is often considered as a subspecies of P. (A.) poseidon (e.g. Tshikolovets 2011), however, it differs in chromosome number (n=17, Larsen 1975) and may represent a different species (Eckweiler and Häuser 1997). The taxon P. (A.) damocles krymaeus (Sheljuzhko, 1928) was also considered as subspecies of P. (A.) poseidon (Hesselbarth et al. 1995), however, with respect to mitochondrial genes COI and COII it is very distant from P. (A.) poseidon and was shown to be a subspecies of P. (A.) damocles (Herrich-Schäffer, [1844]) (Lukhtanov et al. 2005, Kandul et al. 2007).

Males of P. (A.) poseidon have plesiomorphic (Kandul et al. 2004, Lukhtanov et al. 2005) blue colouration of the upper side of the wings with no specific morphological characters. Therefore their morphological discrimination from phenotypically similar P. (A.) caeruleus (Staudinger, 1871), P. (A.) damocles and P. (A.) damonides (Staudinger, 1899) is difficult. With respect to COI barcodes, P. (A.) poseidon is indistinguishable from P. (A.) hopfferi (Herrich-Schäffer, [1851]) and P. (A.) putnami (Wiemers & Fiedler, 2007). As it was stated above, the chromosome number varies within P. (A.) poseidon (de Lesse 1963, Kandul and Lukhtanov 1997, Lukhtanov and Dantchenko 2002b) and thus overlap with chromosome numbers found in P. (A.) elbursicus (Forster, 1956), P. (A.) cyaneus (Staudinger, 1899), P. (A.) ectabanensis (de Lessse, 1963), P. (A.) hamadanensis (de Lesse, 1959), P. (A.) alcestis (Zerny, 1932), P. (A.) altivagans (Forster, 1956), P. (A.) mithridates (Staudinger, 1878), P. (A.) shirkuhensis ten Hagen et Eckweiler, 2001 and P. (A.) pierceae (Lukhtanov & Dantchenko, 2002) (Kandul et al. 2007, Lukhtanov et al. 2014).

A population of blue butterflies which were morphologically similar to P. (A.) poseidon (Fig. 1) was discovered near Akhaltsikhe in Georgia in 2013 by V.Tikhonov and I. Kostyuk. In 2014 the locality was visited again in order to collect material available for molecular and chromosomal study. Molecular analysis of this material revealed that COI barcodes were completely identical or nearly identical (barcode gap from 0 to 0.6%) in population from Akhaltsikhe and other populations of P. (A.) poseidon and P. (A.) putnami (Fig. 2).

Figure 1.

Polyommatus (Agrodiaetus) poseidon from Akhaltsikhe, Georgia. a male, upperside b male, underside.

Figure 2.

Bayesian tree of the species close to Polyommatus (Agrodiaetus) poseidon inferred from COI sequences. Posterior probability values >50% are shown.

The haploid chromosome number n=19 was found in MI and MII cells of three studied individuals (2014VL57, 2014VL58, 2014VL62) (Fig. 3). All chromosome elements formed a gradient size row. The karyotype contained no exceptionally large or small chromosomes. In this respect, the population from Akhaltstikhe is indistinguishable from populations of P. (A.) poseidon from Amasya (de Lesse 1963) and Artvin (Kandul and Lukhtanov 1997), but differs from P. (A.) putnami (n=26) (Lukhtanov and Dantchenko 2002b).

Figure 3.

Male karyotype of Polyommatus (Agrodiaetus) poseidon from Georgia. a sample 2014VL57, metaphase I, n = 19 b sample 2014VL62, metaphase II, n = 19. Bar = 10 μm.

Thus, although in the studied case neither the DNA barcodes nor chromosomal numbers are species-specific characters, their combination clearly indicates that the population from Akhaltsikhe should be identified as P. (A.) poseidon. This is the first evidence of P. (A.) poseidon for Georgia and for Caucasus region at whole.

We thank Igor Kostyuk (Kiev State University) for participation in the trip to Georgia The financial support for this study was provided by the grant from the Russian Science Foundation N 14-14-00541 to Zoological Institute of the Russian Academy of Sciences.