Material and methods
The taxa P. (A.) shirkuhensis (Iran, Yazd Province, Shirkuh Mts., Deh-Bala village, 2900-3150 m, 12 July 2005, samples J299-1, J299-2 and J299-3, J302 and J304) and P. (A.) bogra birjandensis(Iran, South Khorasan Province, 26 km N of Birjand, 1900-2000 m, 14 July 2005, samples J305, J306, J307, J307-1, J307-2, J307-3, J307-4, J315, J318 and J319) were collected exactly in their type localities.
Fresh (not worn) adult males were used to investigate the karyotypes. After capturing a butterfly in the field, it was placed in a glassine envelope for 1-2 hours to keep it alive until we processed it. Testes were removed from the abdomen and placed into a small 0.5 ml vial with a freshly prepared fixative (ethanol and glacial acetic acid, 3:1). Then each wing was carefully removed from the body using forceps. The wingless body was placed into a plastic, 2 ml vial with pure 96% ethanol. The samples are kept in the Zoological Institute of the Russian Academy of Sciences.
Testes were stored in the fixative for 1-12 months at +4°C. Then the gonads were stained in 2% acetic orcein for 30-60 days at +18-20°C. Different stages of male meiosis were examined by using a light microscope Amplival, Carl Zeiss. We have used an original two-phase method of chromosome analysis (Lukhtanov and Dantchenko 2002, Lukhtanov et al. 2006).
A 643 bp fragment of mitochondrial gene cytochrome oxidase subunit I (COI) and 592 bp fragment of nuclear internal transcribed spacer 2 (ITS2) were used to analyze clustering of the specimens. Primers and the protocol of DNA amplification were given in our previous publication (Lukhtanov et al. 2008). The sequences were edited and aligned using BioEdit 7.0.3 (Hall 1999). Since P. icarus (Rottemburg, 1775) and P. stempfferi (Brandt, 1938) were earlier inferred as outgroups to the subgenus Agrodiaetus (Talavera et al. 2013), we used them to root the phylograms.
Sequences of the following additional representatives of the subgenus Agrodiaetus were found in GenBank (Wiemers 2003, Wiemers and Fiedler 2007, Wiemers et al. 2009, Kandul et al. 2004, 2007, Lukhtanov et al. 2005) and used for phylogenetic inference: P. (A.) ainsae (Forster, 1961), P. (A.) achaemenes Skala, 2002, P. (A.) actinides (Staudinger, 1886), P. (A.) admetus malievi (Dantchenko et Lukhtanov, 2005), P. (A.) aereus Eckweiler, 1998, P. (A.) alcestis karacetinae (Lukhtanov et Dantchenko, 2002), P. (A.) altivagans (Forster, 1956), P. (A.) antidolus (Rebel, 1901), P. (A.) ardschira (Brandt, 1938), P. (A.) baltazardi (de Lesse, 1963), P. (A.) baytopi (de Lesse, 1959), P. (A.) bilgini (Dantchenko et Lukhtanov, 2002), P. (A.) birunii Eckweiler et ten Hagen, 1998, P. (A.) caeruleus (Staudinger, 1871), P. (A.) carmon carmon (Herrich-Schäffer, 1851), P. (A.) carmon munzuricus (Rose, 1978), P. (A.) ciscaucasicus (Forster, 1956), P. (A.) cyaneus (Staudinger, 1899), P. (A.) dagestanicus (Forster, 1960), P. (A.) dagmara (Grum-Grshimaïlo, 1888), P. (A.) damocles (Herrich-Schäffer, 1844), P. (A.) damon (Dennis et Schiffermüller, 1775), P. (A.) damone altaicus (Elwes, 1899), P. (A.) damone damone (Eversmann, 1841), P. (A.) damone irinae (Dantchenko, 1997), P. (A.) dantchenkoi Lukhtanov et Wiemers, 2003, P. (A.) demavendi (Pfeiffer, 1938), P. (A.) dizinensis (Schurian, 1982), P. (A.) dolus vittata (Oberthür, 1892), P. (A.) ectabanensis (de Lesse, 1964), P. (A.) elbursicus (Forster, 1956), P. (A.) eriwanensis (Forster, 1960), P. (A.) erschoffii (Lederer, 1869), P. (A.) faramarzii Skala, 2001, P. (A.) femininoides (Eckweiler, 1987), P. (A.) firdussii (Forster, 1956), P. (A.) fulgens (Sagarra, 1925), P. (A.) glaucias (Lederer, 1870), P. (A.) gorbunovi (Dantchenko et Lukhtanov, 1994), P. (A.) haigi (Dantchenko et Lukhtanov, 2002), P. (A.) hamadanensis (Lesse, 1959), P. (A.) hopfferi (Gerhard, 1851), P. (A.) huberti (Carbonell, 1993), P. (A.) iphidamon (Staudinger, 1899), P. (A.) iphigenia (Herrich-Schäffer, 1847), P. (A.) iphigenides (Staudinger, 1886), P. (A.) karatavicus Lukhtanov, 1990, P. (A.) karindus (Riley, 1921), P. (A.) kendevani (Forster, 1956), P. (A.) kermansis (de Lesse, 1963), P. (A.) khorasanensis (Carbonell, 2001), P. (A.) klausschuriani ten Hagen, 1999, P. (A.) kurdistanicus (Forster, 1961), P. (A.) lorestanus Eckweiler, 1997, P. (A.) lukhtanovi (Dantchenko, 2005), P. (A.) luna Eckweiler, 2002, P. (A.) magnificus (Grum-Grshimaïlo, 1885), P. (A.) masulensis ten Hagen et Schurian, 2000, P. (A.) mediator (Dantchenko et Churkin, 2003), P. (A.) menalcas (Freyer, 1837), P. (A.) merhaba De Prins, van der Poorten, Borie, van Oorschot, Riemis et Coenen, 1991, P. (A.) mithridates (Staudinger, 1878), P. (A.) mofidii (de Lesse, 1963), P. (A.) ninae (Forster, 1956), P. (A.) peilei (Bethune-Baker, 1921), P. (A.) pfeifferi (Brandt, 1938), P. (A.) phyllides (Staudinger, 1886), P. (A.) phyllis (Christoph, 1877), P. (A.) pierceae (Lukhtanov et Dantchenko, 2002), P. (A.) poseidon (Herrich-Schäffer, 1851), P. (A.) poseidonides (Staudinger, 1886), P. (A.) pulcher (Sheljuzhko, 1935), P. (A.) putnami (Dantchenko et Lukhtanov, 2002), P. (A.) ripartii (Freyer, 1830), P. (A.) ripartii paralcestis (Forster, 1960), P. (A.) rjabovi (Forster, 1960), P. (A.) rovshani (Dantchenko et Lukhtanov, 1994), P. (A.) sennanensis (de Lesse, 1959), P. (A.) shahkuhensis (Lukhtanov, Shapoval et Dantchenko, 2008), P. (A.) shahrami Skala, 2001, P. (A.) shamil (Dantchenko, 2000), P. (A.) sorkhensis Eckweiler, 2003, P. (A.) surakovi (Dantchenko et Lukhtanov, 1994), P. (A.) tankeri (de Lesse, 1960), P. (A.) tenhageni Schurian et Eckweiler, 1999, P. (A.) transcaspica (Heyne, 1895), P. (A.) turcicolus (Koçak, 1977), P. (A.) turcicus (Koçak, 1977), P. (A.) urmiaensis Schurian et ten Hagen, 2003, P. (A.) vanensis sheljuzhkoi (Forster, 1960), P. (A.) vaspurakani (Lukhtanov et Dantchenko, 2003) and P. (A.) zarathustra Eckweiler, 1997.
Bayesian analysis was performed using the program MrBayes 3.2.2 (Ronquist et al. 2012). A GTR substitution model with gamma distributed rate variation across sites and a proportion of invariable sites was specified before running the program for 5,000,000 generations with default settings. The first 1250 trees (out of 5000) were discarded as a burn-in prior to computing a consensus phylogeny and posterior probabilities.
Discussion
P. (A.) shirkuhensis is the only species of the subgenus Agrodiaetus known from Shirkuh Mts massif in Central Iran (province Yazd) (ten Hagen and Eckweiler 2001). Immediately after its description, it attracted attention of lepidopterists (Skala 2002) because of its unusual combination of morphological characters such as loss of the white streak on the underside of the hind wings (most important apomorphy of the subgenus Agrodiaetus as a whole) and exaggerated elements of the wing underside pattern. A similar wing pattern is known in three other Agrodiaetus species from Central and Eastern Iran: P. (A.) eckweileri, P. (A.) baltazardi and P. (A.) bogra Evans, 1932. From these three species, P. (A.) bogra has the white streak on the hind wing underside, whereas P. (A.) eckweileri and P. (A.) baltazardi do not (Eckweiler and Häuser 1997, ten Hagen and Eckweiler 2001, Skala 2002). All four species are allopatric in their distribution ranges (ten Hagen and Eckweiler 2001).
Ten Hagen and Eckweiler (2001) hypothesized that P. (A.) shirkuhensis was a taxon closely related either to P. (A.) eckweileri (distributed in province Esfahan) or to P. (A.) baltazardi (distributed in province Kerman). P. (A.) baltazardi, in its turn, was treated by them as a taxon close to East Iranian – Pakistani species P. (A.) bogra.
However, analysis of COI clusters in the Bayesian tree (Fig. 1) showed that none of these hypotheses was true. Among the major species groups recognized within the subgenus Agrodiaetus by Kandul et al. (2004, 2007) (Table 1), P. (A.) eckweileri is recovered by us as a member of P. pfeifferi (Brandt, 1938) – P. ardschira (Brandt, 1938) – P. luna Eckweiler, 2002 species complex belonging to P. erschoffii (Lederer, 1869) group.
P. (A.) baltazardi is found to be a member of P. (A.) poseidon (Herrich-Schäffer, [1851]) group and, thus, is not related to P. (A.) bogra The latter species has very isolated position within the P. erschoffii group. The karyotypes of P. (A.) baltazardi and P. (A.) bogra are also different (Table 1).
Finally, our target species, P. (A.) shirkuhensis, is found to be a member of P. (A.) cyaneus (Staudinger, 1899) group and is especially close to P. (A.) kermansis (de Lesse, 1962), P. (A.) sennanensis (de Lesse, 1959) and P. (A.) cyaneus (Fig. 1). The position of P. (A.) shirkuhensis on the ITS2 tree (Fig. 2) also does not contradict the conclusion that P. (A.) shirkuhensis belongs to P. (A.) cyaneus species group.
From P. (A.) kermansis, P. (A.) cyaneus and P. (A.) sennanensis, which possess closest COI haplotypes, P. (A.) shirkuhensis differs by blue upper side of wings in males (it is deep violet in P. (A.) kermansis, violet in P. (A.) cyaneus and whitish in P. (A.) sennanensis) (see figures in Eckweiler and Häuser 1997). The wing color in P. (A.) shirkuhensis is similar to those found in P. (A.) mofidii (de Lesse, 1963) and P. (A.) sorkhensis Eckweiler, 2003 (see figs 18–25 in Eckweiler 2003), two other members of the P. (A.) cyaneus group. P. (A.) mofidii, P. (A.) sorkhensis and P. (A.) shirkuhensis are allopatric in their distribution ranges (ten Hagen and Eckweiler 2001, Eckweiler 2003) and significantly different in their karyotypes (Table 1).
To conclude, our study demonstrates that four allopatric taxa known from Central and East Iran, P. (A.) shirkuhensis, P. (A.) eckweileri, P. (A.) baltazardi and P. (A.) bogra birjandensis, which possess significant elements of morphological similarity, are not only specifically distinct from each other, but even belong to different distantly related groups of species within the subgenus Agrodiaetus.