DNA Barcoding

For comparison of Cytochrome c Oxidase, subunit-I (DNA Barcode), the barcode data of Problepsis species (Stadie & Stadie 2016) were obtained from Barcode Of Life Database (BOLD). Subsequently, DNA of freshly collected specimens from Iran was extracted, their DNA barcode region amplified and sequenced according to standard protocols (Ivanova et al. 2006, Hebert et al. 2003) in the molecular laboratory of State Museum of Natural History, Stuttgart. In total 658 bp of DNA Barcode were aligned using BioEdit (ver. 7.2.5.) (Hall 1999). A list of all specimens used for DNA analysis along with their GenBank accession numbers are presented in Table 1 (appendix). Neighbor-joining tree and genetic distances were calculated by MEGA6 (Tamura et al. 2013).

3.1 Morphology

Wing pattern. (Figs. 1-3) Wingspan 39-41 mm (length of forewing: 20 mm). Background of forewing creamy light-brown mixed with ochreous, weakly glossy scales, costal area slightly darker. A large rounded eyespot in median area is characteristic, bordered with a thin black outer line, encircled an olive-brown thicker inner band, toward center a second thick black line shadowed with silvery scales, central area separated into two part, the upper two third light brown and the lower third black. Hind wing lighter than forewing. Eye-spot elliptical, without triangle black shapes internally; a small black patch near the dorsum, sometimes splitted. Underside creamy light, with a dull print of all patterns of upper-side (for more details see Stadie & Stadie 2016).

Male genitalia (Fig. 9). (based on specimens from Pakistan). Socii thin, weakly sclerotized connected from both sides except apical part. Tegumen ventrally more sclerotized with nine prominent spines, usually angled near socii. Aedeagus slender, tubular, more sclerotized, forming a long two-head cleft, one more bent, shorter with two teeth. Vesica as two diverticuli; without any cornuti.

Figs. 9-13.

Genitalia structure. 9-10. ♂, 9. Problepsis cinerea, 10. P. ocellata; 9. P. cinerea ♂; 10. P. ocellata ♂ (10a-2 lateral view); 11-13. ♀; 11-12. P. cinerea (Iran, Pakistan respectively); 13, P. ocellata (Turkey), a, armature, b, aedeagus and vesica, c, sternite 8, de. sclerotized dentation, ma. Mappa, so. Socii; Scale-bar: 1 mm.

Figs. 14-19.

Structure of hind leg and hair pencil. 14. Problepsis cinerea, 15-19. Problepsis ocellata, 15-16. general view of spoonshaped hind tibia with long hair pencil; 17. close-up view of tubular hollow scent scale; 18. Flattened scales in comparison with normal body scales, 19. Mixture of both tubular and flattened scales of hair pencil.

Female genitalia. (Fig. 11). Papillae anales in moderate size, broad, with sparse hairs. Anterior apophyses slightly shorter (80%) than posterior apophyses. Antrum strongly sclerotized, tubular, medially narrower. Ductus bursae very short, heavily sclerotized. Corpus bursae membranous, apple-shaped; signum as a sclerotized spotted patch covering more than half of the length of corpus bursae.

Fig. 20.

Un-rooted neighbor-joining tree of four species Problepsis ocellata, P. cinerea, P. sihvoneni, P. asira (with Kimura 2-Parameter model). Iranian specimen clustered with P. cinerea.

Diagnosis. The most similar species to P. cinerea is P. ocellata (Frivaldszky, 1845) (Figs. 4-5). The distribution pattern and the habitats of these two species are well separated and different (P. ocellata is distributed in Israel, Palestine, Lebanon, eastern Syria and south eastern Turkey towards Greece and Cyprus, whereas P. cinerea is known from northern Oman and southern Iran in the west towards eastern Afghanistan and Pakistan (see below). Vertical distribution of these two species is also different: P. ocellata is distributed in altitudes from sea level up to 1000 m, P. cinerea occurs much higher between 1000-2200 m a. s. 1. (mostly above 1800 m) (Stadie & Stadie 2016). Additionally, P. ocellata has paler, greyish wings, with a light-yellow spot on the upper half of the eye pots, and has more yellowish inner band in both wings. In male of P. ocellata, 8^th sternite anteriorly wider (narrower in P. cinerea) (Fig. 9c, 10c). Tegumen more sclerotized, round-shaped ventrally (thinner in P. cinerea). Apical part of aedeagus and subapical dentation less sclerotized and not clearly visible (strongly sclerotized dentation in P. cinerea) (Figs. 9-10). In female genitalia of P. ocellata, anterior apophyses much shorter than posterior apophyses (50%), (whilst being 80% in P. cinerea). Ductus bursae wider, corpus bursae smaller than those in P. cinerea (Figs. 11-13).

Map.

Distribution map of Problepsis cinerea and P. ocellata in the Middle East. The European part of distribution of P. ocellata is not shown.

Remark. Stadie & Stadie (2016) regarded two subspecies for Problepsis ocellata (ssp. ocellata distributed in east Mediterranean Sea, i.e. Greek islands, Crete and coasts of west Turkey and ssp. cypria in Cyprus). These two subspecies were based on geographic separation of slightly different in wing pattern (e.g. darker ground colour with wider median shade and presence of a small blackish horizontal line in basal part of forewing near dorsum in ssp. cypria (lighter ground colour and missing the horizontal line in nominate ssp.). These two subspecies show no genetic distance in DNA-barcodes, which confirms that they belong to the same species. For evaluation of these two subspecies further larval studies, biological and ecological investigations are recommended.

Morphological note. A close-up view of the structure of the genus Problepsis is shown (Fig. 14-19). At least two different modified scales in this hair pencil are recognizable: most of them are apically flattened, while some tubular hollow scales are scattered in between (Figs. 17-19). Spoon-shaped hind tibia created a chamber, which conceal the tubular scent scales with the help of flattened ones (Figs. 14, 15). Hypothetically, during courtship those flattened scales uncover the tubular ones.

3.2 DNA-Barcoding

The unrooted neighbor-joining tree (Fig. 20) shows the clustering of the Iranian specimen with P. cinerea among the specimens from Pakistan and Oman. Genetic mean distances between Problepsis species in the Middle East are shown in table 1.

Table 1.

Genetic mean distances (COI) between Problepsis species in the Middle East resulted from neighbor-joining analyses with Kimura 2-Parameter model (see NJ tree in Fig. 20).

3.3 Distribution and habitat

Problepsis cinerea is confined to evergreen and sclerophyllous woodlands mostly with Olea species in North Oman, South Iran, eastern Afghanistan and Pakistan. Vertically it is distributed between 1000-2200 m (in N Oman and S Iran in altitudes over 1800 m).

The newly recorded species in Iran was found in the Khalijo-Omanian ecological zone, consisting of both dry southern coastal plains and mountainous parts with higher humidity. The community harbors both plants of tropical and palaearctic-eremic origin. Habitats of this zone are mostly covered with Artemisia, Acacia, Prosopis, Ziziphus, Avicennia, Rhizophora, Populus, Astragalus, Amygdalus, Dodonaea, Pistacia and Olea (Zohary 1973, Heshmati 2007).

Its habitat in southern Iran is located in the Geno protected area (30 km northwest of Bandar Abbas) (Figs. 6–8), with a relatively warm climate (the annual mean temperature 26.8 degree Celsius) ( http://taziyaniha.blogfa.com/post-76.aspx). Variable climate conditions, geological characters and altitudes in the Geno area generate ideal habitat for a vast range of plants. Generally, three different vegetation communities may be recognized in this area: 1) Acacia community in slopes (e.g. Acacia ehrenbergiana, A. nilotica, Andrachne aspera, A. maxima); 2) Amygdalus community in the middle elevations (e.g. Amygdalus scopario, A. wendelboi, A. eburnea): 3) Juniperus community in higher altitudes and peaks (e.g. Juniperus excelsia, J. polycarpos, Olea europaea cuspidata) ( http://www.ettelaat.com/etiran/?p=163643; Mojdeh Raam, personal comm.).

Most Problepsis species feed on Oleaceae species (Stadie & Stadie 2016). Asadpoor (2005) claimed that there is no Oleaceae species in southern Iran, therefore the presence of Problepsis species in Iran questioned by Stadie & Stadie (2016). However there are several cultivate but also wild Oleaceae species in southern Iran. As Olea europaea cuspidata is the only wild olive species in Geno protected area, therefore we suggest this tree as potential natural host plant of P. cinerea in the south of Iran (Figs. 6, 7, 8).