In this paper, by using combination of molecular and chromosomal markers, populations of Polyommatus (Agrodiaetus) karindus (Riley, 1921) from north-west and central Iran are analyzed. It has been found that taxon usually identified as P. (A.) karindus is represented in Iran by two geographically separated groups of individuals, strongly differentiated by their karyotypes and mitochondrial haplotypes. It is demonstrated that populations from NW Iran have the haploid chromosome number n = 68, while the haploid chromosome number of P. (A.) karindus from central Iran is found to be n = 73. Phylogenetic analysis revealed that these groups also differ by at least eight nucleotide substitutions in a 690 bp fragment of the mitochondrial COI gene and form separated groups of clusters in Bayesian inference tree. Thus, population entities from central Iran are described here as a new subspecies Polyommatus (Agrodiaetus) karindus saravandissp. n. Strong chromosomal and molecular differentiation are confirmed between P. (A.) karindus and its sister species, P. (A.) dama (Staudinger, 1892).

COI , Iran, karyotype, molecular marker, chromosome number

Agrodiaetus Hübner, 1822 is the most species-rich subgenus within the genus Polyommatus Latreille, 1804 (Talavera et al. 2013a, Lukhtanov et al. 2015a). It consists of approximately 130 species distributed in the western Palearctic (Vila et al. 2010, Lukhtanov et al. 2008, 2014, Vershinina and Lukhtanov 2010, Przybyłowicz et al. 2014, Lukhtanov and Tikhonov 2015). Today Agrodiaetus has become a model group in studies of speciation (Lukhtanov et al. 2005, 2015b), intraspecific differentiation (Dincă et al. 2013, Przybyłowicz et al. 2014, Lukhtanov et al. 2015a), and rapid karyotype evolution (Lukhtanov et al., 2005, Kandul et al. 2007). From the point of view of taxonomy, Agrodiaetus is a very complicated group. Many Agrodiaetus taxa display extremely similar phenotype (Hesselbarth et al. 1995) and, in contrast to other Lepidoptera taxa, genitalia offer only few distinctive features. Furthermore, many taxa represent allopatric populations which differ only slightly in morphology, and a conclusion on their status as distinct species or subspecies is controversial and can be misleading (Wiemers 2003, Lukhtanov et al. 2015a). This resulted in description of numerous polytypic species based on geographic distribution and classic morphological characters (Forster 1956, 1960a, b, 1961).

In particular, Polyommatus (Agrodiaetus) dama (Staudinger, 1892) was traditionally regarded as a polytypic species that included two subspecies: Polyommatus (Agrodiaetus) dama dama (Staudinger, 1892) (orig. comb. Lycaena Dama) and Polyommatus (Agrodiaetus) dama karindus (Riley, 1921) (orig. comb. Lycaena dama subsp. karinda). P. (A.) dama dama has only been found in South Anatolia (a few localities in Malatya, Maraş, and Mardin provinces (Turkey), while P. (A.) dama karindus distribution range is restricted to Zagros Mountains in Iran.

The karyotype studies of de Lesse (1957, 1959a, b, c, d, 1960a, b, 1961, 1962a, b, 1963a, b, 1964, 1966, 1968) revealed that Agrodiaetus species exhibit a wide diversity of karyotypes. Karyotyping may provide necessary diagnostic character for many Agrodiaetus species, and therefore become an important requirement for describing new taxa (de Lesse 1960a, b, Lukhtanov and Dantchenko 2002, 2003, Lukhtanov et al. 2008). Karyological investigations showed strong chromosomal differentiation between Turkish and Iranian populations of P. (A.) dama s. l.. De Lesse (1959a) described karyotype of P. (A.) dama dama from Kahramanmaraş and Olivier et al. (1999) confirmed his results from the type locality Malatya. It has an asymmetric karyotype with n = 41 chromosomes, about eleven of them are large, gradually decreasing in size, the others medium–sized; whereas the karyotype of Iranian taxon was determined as n = 68 (Wiemers 2003). Thus, on the basis of karyotype studies, P. (A.) dama s. l. was split into two species, P. (A.) dama and P. (A.) karindus, that can be characterized by species-specific haploid chromosome numbers.

However, the chromosome number of P. (A.) karindus was determined only for one population from NW Iran (Saqqez, Kordestan Province) (Wiemers 2003). Further investigations showed that Iranian species P. (A.) karindus has complicated genetic and phylogeographic structure (Lukhtanov et al. 2015b). Here a combination of molecular mitochondrial (COI) and nuclear chromosomal (karyotype) markers are used to analyze different Iranian populations of P. (A.) karindus. Our study demonstrates that butterflies from central Iran strongly differentiated by their karyotypes and mitochondrial haplotypes from NW Iranian populations. Thus, population entities from central Iran are described here as a separate subspecies Polyommatus (Agrodiaetus) karindus saravandi ssp. n.

We have found that a taxon usually identified as P. (A.) karindus is represented in Iran by two geographically separated groups of individuals. The first group unites specimens collected in NW Iran, while the second group comprises specimens from central Iran. The representatives of these groups have different chromosome numbers, n = 68 and n = 73 respectively. They also have at least eight fixed nucleotide differences in 690 bp fragment of mitochondrial COI gene. The first group is monophyletic with respect to both COI gene and karyotype (n = 68). The second group has complicated genetic structure, comprises several differentiated populations and is paraphyletic with respect to the COI gene. Despite this gene paraphyly, it appears as a clearly monophyletic group with respect to its karyotype (n = 73). Thus, the NW and central Iranian groups are differentiated by at least five fixed chromosome fusions/fissions. Fixed chromosome differences are often considered as characters associated with reproductive isolation (King 1993). From this point of view, the NW and central Iranian groups could be theoretically treated as a different species. However, our recent studies on Agrodiaetus demonstrated that multiple chromosome fusions and fissions did not block fertility in chromosomal hybrids (Lukhtanov et al. 2015b). In other words, differentiation by five fixed chromosome rearrangements would not guarantee impossibility of blending populations together when they occur in sympatry. Thus, NW and central Iranian groups of populations should be considered as a subspecies rather than separate species.

Since Polyommatus (Agrodiaetus) karindus (Riley, 1921) (orig. comb. Lycaena dama subsp. karinda) was described from NW Iran (type locality is “Harir, Karind, and Karind Gorge, N.W. Persia” according to original description, and “N.W. Persia, Karind Gorge, 6000 ft” according to lectotype designation made by Bálint (1999) (not from central Iran), the name P. karindus karindus should be attributed to the NW Iranian group of populations. The formal description and naming of the central Iranian group is provided below.

Description of the novel taxon

Polyommatus (Agrodiaetus) karindus saravandissp. n.

http://zoobank.org/ADC4F3C8-B804-4869-955A-9F7C7164B0C5

Fig. 1 – map, Fig. 2B karyotype, Figs 3, 4 phylogeny, Fig. 5 – Holotype of P. (A.) karindus saravandi, Fig. 6A, B – Underside and upperside of the male and female wings

Holotype

♂. Forewing length 34.0 mm. Iran, Lorestan province, Zagros Mt., vicinity of Saravand village, 33°22.39'N; 49°10.25'E, 2070 m, 22.07 2009. N. Shapoval and V. Lukhtanov leg. In the Zoological Institute of the Russian Academy of Sciences (St. Petersburg). Specimen field code W064, GenBank code for mitochondrial cytochrome c oxidase subunit I (COI) gene (partial cds) is KT582713.

Figure 4. 

COI Haplotype analysis. A geographical distribution of haplogroups. Number of studied individuals sharing the same haplogroup is given in parentheses B most parsimonious COI haplotype network; h01–h12 are COI haplotypes; GH1–GH5 are COI haplogroups. Number of studied individuals sharing the same haplotype is given in parentheses.

Figure 5. 

Holotype of P. (A.) karindus saravandi, sample W064. Upperside (left) and underside (right) of the male wings.

Paratypes

87 ♂♂, field codes W061, W062, W063, 21.07.2009; W065, W072, W073, W074, W075, W081, W082, W083, W084, W085, W086, W087, the same locality, date and collectors as the holotype. Field codes W093, W094, W095, W096 23.07.2009, the same locality and collectors as the holotype. Field codes W377, W378, W379, W380, W381, W382, W383, W386, W387 03.08.2009, the same locality and collectors as the holotype. Field codes W370, W371, W372, W373, W374, W375, W376, Iran, Lorestan province, Zagros Mt., vicinity of Nahavand village, 34°02.57'N; 048°20.22'E, 2170 m, 02.08.2009, the same collectors as the holotype. Field codes W388, W389, W390, W391, W392, Iran, Lorestan province, Zagros Mt., vicinity of Nahavand village, 34°02.57'N; 048°20.22'E, 2170 m, 02.08.2009, the same collectors as the holotype. Field codes U217, U218, U219, U220, U223, Iran, Lorestan province, Zagros Mt., vicinity of Nahavand village, 34°02.57'N; 048°20.22'E, 2170 m, 19.07.2011, the same collectors as the holotype. Field codes U228, U229, U230, U231, U232, U233, U234, U235, U236, U237, U238, U239, U240, U256, U257, U262, U263, U264, U265, U266, U267, U278, U279, U280, U281, Iran, Lorestan province, Zagros Mt., vicinity of Nahavand village, 34° 02.92'N; 48° 20.40'E, 2160 m, 20.07.2011 the same collectors as the holotype. Field codes Z381, Z382, Z396, Z397, Z398, Z399, Z400, Z408, Iran, Lorestan province, Zagros Mt., W of Borujerd, Kuh-e Garin mount mount., Vennnai, 33°53.89'N; 48°34.03'E, 2150 m, 21.07.2007, the same collectors as the holotype. Field codes Z412, Z413, Z416, Iran, Lorestan province, Zagros Mt., W of Borujerd, Kuh-e Garin mount., Vennnai, 33°53.89'N; 48°34.03'E, 2150 m, 22.07.2007, the same collectors as the holotype. Field codes V331, 335, V336, Iran, Lorestan province, Zagros Mt., W of Borujerd, Kuh-e Garin mount., Vennnai, 33°53.89'N; 48°34.03'E, 2150 m, 02.08.2014, N. Shapoval and A. Barabanov leg. Field codes U169, U178, U179, Iran, Lorestan province, Zagros Mt., Darreh Takht, 33° 21.19'N; 49° 22.34'E, 2000–2100 m, 18.07.2011, the same collectors as the holotype. 1 ♀, field code U169 Iran, Lorestan province, Zagros Mt., Darreh Takht, 33° 21.19'N; 49° 22.34'E, 2000–2100 m, 18.07.2011, same collectors as the holotype. All paratypes are kept in the Zoological Institute of the Russian Academy of Sciences (St. Petersburg). GenBank accession numbers of the paratypes are presented in the Table 1.

Derivatio nominis

The new taxon is named after the village Saravand, one of the places where it was found.

Description

Male upperside. Forewing length 30–36 mm, ground colour bright blue with azure tint. Discoidal, submarginal and antemarginal marking absent on both fore- and hindwings. Black outer marginal line on forewings and hindwings very narrow; forewing hind margin with long white pubescence. Fringes of both wings dark grey; tips of hindwings veins indicated with fine black.

Male underside. Ground colour light grey, white streak on the hindwings absent. Basal black spots present only on hindwings. Discoidal series of spots present on fore- and hindwings, although the black spots composing it are minute. Postdiscal black marking very narrow, longitudinal, present only on forewings. Submarginal and marginal lunules only faintly indicated.

Female upperside. Ground colour brown with vastly darker veins. Discoidal black spots present on forewings. Submarginal markings dark brown with orange submarginal lunules well developed on forewing and hindwing. Fringe greyish-brown.

Female underside. General design as in males, but ground colour slightly darker.

Genitalia. The male genitalia have a structure typical for other species of the subgenus Agrodiaetus (Coutsis 1986). No specific characters in genitalia are found.

Figure 6. 

Underside and upperside of the P. (A.) karindus saravandi ssp. n. wings. A upperside (left) and underside (right) of the male wings B upperside (left) and underside (right) of the female wings.

Diagnosis

Genetically P. (A.) karindus saravandi differs from all other taxa of Agrodiaetus by fixed substitutions in mitochondrial gene COI. Phenotypically the new taxon is extremely similar to P. (A.) karindus karindus from north-west Iran, but they have different chromosome numbers, n=73 and n = 68 respectively.

Distribution

Central part of Zagros Mountains, Iran.

Flight period

From July to August.

Ecology

Dry slopes, gorges and plateaus with xerophyte or steppe vegetation, sometimes wooded areas from 1800 up to 2800 m. Butterflies fly together with P. (A.) alcestis (Zerny, 1932), P. (A.) cyaneus (Staudinger, 1899), P. (A.) hamadanensis (de Lesse, 1959), P. (A.) lorestanus (Eckweiler, 1997) and P. (A.) zarathustra (Eckweiler, 1997).

The complete financial support for this study was provided by the grant N 14–14–00541 from the Russian Science Foundation to the Zoological Institute of the Russian Academy of Sciences. Postdoctoral fellowship (N 1.50.1617.2013) was provided to N. Shapoval from St. Petersburg State University. We thank A. Barabanov for help in obtaining material during the field expedition to Iran in 2014. The work was partially performed using equipment of the ‘Chromas’ Core Facility and Centre for Molecular and Cell Technologies of St. Petersburg State University.