The hitherto unknown males of Zygaenoprocris (Zygaenoprocris) eberti (Alberti, 1968) were attracted by sex pheromones in Afghanistan. Conspecificity with the female holotype was confirmed by using DNA barcoding. A description of the male and data on the habitat of this species are provided. A key to the subgenera of the genus Zygaenoprocris Hampson, 1900, and the species of the subgenus Zygaenoprocris Hampson, 1900, is given.

The subfamily Procridinae (Lepidoptera, Zygaenidae) includes some genera with externally very similar species, e.g. Fuscartona Efetov & Tarmann, 2012 (Efetov 1997a), Chrysartona Swinhoe, 1892 (Efetov 2006), Illiberis Walker, 1854 (Efetov 1997b; Efetov et al. 2004), Hedina Alberti, 1954 (Efetov 1997b; Efetov 2010; Efetov and Tarmann 2012), Goe Hampson, 1893 (Efetov 1998), Adscita Retzius, 1753 (Efetov 2001b; Efetov and Tarmann 2012), Jordanita Verity, 1946 (Efetov 2001b; Efetov and Tarmann 2012) etc. Species of the mentioned genera can often be identified only by examination of genitalic structures (Efetov and Tarmann 1999), chaetotaxy of the first instar larvae (Efetov et al. 2006; Efetov and Hayashi 2008), karyotypes (Efetov 2004; Efetov et al. 2004) or DNA analysis. Some species are known only from the type specimens and sometimes only one sex is known. The identification of material of the other sex needs to be verified by molecular methods. One such genus is Zygaenoprocris Hampson, 1900 (Efetov and Tarmann 1994; Efetov 1996; 2001a), which is currently represented by 13 species (Efetov and Tarmann 2012).

Hitherto Zygaenoprocris eberti (Alberti, 1968) was known only from the holotype, a female labelled ‘Z.-Afghanistan, Koh-i-Baba, S-Seite Shahtu-Pass, 3000 m, 17.–19.7.1966, G. EBERT leg.’ and collected on the south side of the pass known as Kotal-e Shahtu that crosses the Koh-i Baba main chain between Panjao and Yakolang (Figs 4, 10). The discovery of the males of this species became possible by the use of two molecular methods: attraction by sex pheromones followed by confirmation of conspecificity of the collected males with the female holotype by DNA barcoding.

Two esters of fatty acids, 2-butyl (7Z)-dodecenoate and 2-butyl (9Z)-tetradecenoate, were found in the female pheromone glands of Illiberis (Primilliberis) rotundata Jordan, 1907 and both (R)- and (S)-enantiomers of each compound were synthesized (Subchev et al. 2010). These compounds and their mixtures were screened in the field and proved to be sex attractants for different species of the genera Illiberis Walker, 1854 (Subchev еt al. 2012; 2013), Zygaenoprocris Hampson, 1900 (Efetov et al. 2011), Adscita Retzius, 1783, and Jordanita Verity, 1946 (Efetov et al. 2010; Subchev еt al. 2010).

During an expedition to Afghanistan in 2011, Axel Hofmann, using baits containing the above-mentioned female sex attractants and their mixtures, collected a series of males of Zygaenoprocris eberti in the vicinity of the lakes of Band-i Amir (ca. 40 km north-east of the type-locality) in the central Koh-i Baba in Hazarajat in Afghanistan (Efetov et al. 2012). Male specimens were attracted to (2R)-butyl (7Z)-dodecenoate and the mixture of (2R)-butyl (7Z)-dodecenoate and (2R)-butyl (9Z)-tetradecenoate (Fig. 2). The pin-label data are as follows: “Afghanistan, Prov. Bamiyan, Band-i Amir, Jarkushan N, Canyon, 3130–3280 m, 5.VII. 2011, [15/11], leg. A. HOFMANN”. At this locality Z. eberti was syntopic with Z. chalcochlora Hampson, 1900 (Figs 3, 10). Amongst the 38 collected males 37 were Z. eberti and only one male was Z. chalcochlora. In other localities in Afghanistan which were visited during the same collecting tour by A. Hofmann in 2011, only Z. chalcochlora could be found. Males of the latter species were seen resting (Fig. 9) and actively flying but a clear preference to any of the presented sex attractants could not be verified.

Figures 1, 2. 

1. Male of Zygaenoprocris eberti (Alberti, 1968). Afghanistan, Prov. Bamiyan, Band-i Amir, Jarkushan N, 3130–3280 m, 5.vii.2011, leg. A. Hofmann. Photo S. Heim. 2. Mixture of (2R)-butyl (7Z)-dodecenoate and (2R)-butyl (9Z)-tetradecenoate (‘R12+R14’) attracting two males of Zygaenoprocris eberti (Alberti, 1968). The two males were so sexually excited that they copulated with each other. Afghanistan, Prov. Bamiyan, Band-i Amir, Jarkushan N, 3130–3280 m, 5.vii. 2011. Photo A. Hofmann.

Figures 3, 4. 

3. Habitat of Zygaenoprocris eberti (Alberti, 1968), and Zygaenoprocris chalcochlora Hampson, 1900. Afghanistan, Prov. Bamiyan, Band-i Amir, Jarkushan N, 3130–3280 m, 5.vii. 2011. Photo A. Hofmann. 4. Near the type locality of Zygaenoprocris eberti (Alberti, 1968). Afghanistan, Prov. Bamiyan, Koh-i Baba, Shahtu pass, 3000 m. The exact type locality is not known. Photo A. Hofmann.

DNA barcodes were obtained by sampling legs from dry specimens. Legs were prepared in the Department of Biological Chemistry of the Crimean State Medical University (Simferopol). All specimens were identified by К. А. Еfetov & G. M. Tarmann. PCR amplification and DNA sequencing were performed at the Canadian Centre for DNA Barcoding following standard high-throughput protocols (which can be accessed at http://www.dnabarcoding.ca/pa/ge/research/protocols), where all obtained DNA extracts are stored now. All sequences were deposited in GenBank according to the iBOL data release policy. Complete specimen data (images, voucher deposition, geographic coordinates, sequence and trace files) can easily be accessed in the BOLD in public project ZYGMO [http://www.boldsystems.org/index.php/MAS_Management_OpenProject?code=ZYGMO]. Sequence divergences for the barcode region were calculated using the Kimura 2 Parameter model by the analytical tools on BOLD.

The conspecificity of the collected males with the holotype of Zygaenorpocris eberti was confirmed by the analysis of the DNA barcode, 658-bp region of the cytochrome c oxidase I mitochondrial gene (Fig. 5). The treeless habitat at Band-i Amir, Jarkushan N, Hazarajat, 3130−3280 m (Fig. 3), was dominated by Acantholimon (Plumbaginaceae), Cousinia, Artemisia (Asteraceae) and Astragalus (Fabaceae) species. As we know from other Zygaenoprocris (Zygaenoprocris) species, Acantholimon and Cousinia may be the larval host-plants for the studied species. We provide below a description of the hitherto unknown male of Z. eberti.

Figure 5. 

Neighbour-joining tree (K2P) of the DNA barcodes for the different Zygaenoprocris (Zygaenoprocris) species.

Zygaenoprocris eberti (Alberti, 1968)

Description of male

(Fig. 1). Length of body: 5.8–6.5 mm; length of forewing: 8.6–8.9 mm, width: 3.3–3.4 mm; length of antenna: 4.6–5.0 mm. Frons and occiput green with submetallic sheen. Antenna strongly clubbed, thickly covered with shining scales, bipectinate, length of pectination in middle part of antenna 0.7 mm, last segments of antenna with pectination reduced, antennal shaft strongly thickened distally, ratio of width of 4th segment from apex to width of 15th segment is 4. Proboscis well developed, yellow. Tegulae and patagia green with submetallic sheen. Thorax thickly covered with green shiny scales. Forewing upperside bright green with submetallic sheen, thickly covered with shiny scales; underside of forewing grey; fringe grey. Hindwing upper- and underside grey, fringe concolorous. Legs green, thickly covered with shiny scales, foreleg with long tibial epiphysis, hind tibia with one pair of spurs (apical). Abdomen greenish black, thickly covered with shiny scales. The long black hair covers the head, labial palpi, thorax, legs and abdomen.

Male genitalia

(Figs 6, 8). Uncus heavily sclerotized, nearly equal in length to tegumen. Valva without any process. Juxta long, 1.5 times longer than uncus. Phallus slightly curved, long, approximately 3 times longer than uncus, with one long straight cornutus, its length 0.5 times length of phallus, distal part of cornutus very slender, with pointed apex.

Figure 6. 

Male genitalia of Zygaenoprocris eberti. Afghanistan, Prov. Bamiyan, Band-i Amir, Jarkushan N, 3130-3280 m, 5.vii. 2011, leg. A. Hofmann.

Differential diagnosis

Zygaenoprocris eberti is syntopic and synchronous with Zygaenoprocris (Zygaenoprocris) chalcochlora Hampson, 1900, the type species of the subgenus Zygaenoprocris Hampson, 1900. Externally these two species cannot be distinguished but both differ significantly in their genitalia morphology (Figs 6–8; Efetov and Tarmann 1999, figs 108, 166). In the male the phallus is longer in Z. eberti and the cornutus is large with a characteristic shape (with broad basal part and ending somewhat abruptly in a pointed tip distally). The female of Z. eberti has long, broad and strongly curved (twisted) ductus bursae, whereas Z. chalcochlora has short, narrow and straight ductus bursae.

Figure 7. 

Female genitalia of the holotype of Procris eberti Alberti, 1968 (= Zygaenoprocris eberti), v – ventral view, d – dorsal view. ‘Z.-Afghanistan, Koh-i-Baba, S-Seite Shahtu-Pass, 3000 m, 17.–19.7.1966, G. EBERT leg.’

Figures 8a–d. 

a, b. Male genitalia of Zygaenoprocris eberti. Afghanistan, Prov. Bamiyan, Band-i Amir, Jarkushan N, 3130–3280 m, 5.vii. 2011, leg. A. Hofmann (Prep. GMT Z 3562). a, Uncus, tegumen, vinculum, saccus, valvae; b, Phallus (Photo S. Heim). c, d. Male genitalia of Zygaenoprocris chalcochlora. Afghanistan, Prov. Kabul, Kabul N, Darreh Estalif, 1880–2200 m, 21. vi. 2011, leg. A. Hofmann (Prep. GMT Z 3564). c, Uncus, tegumen, vinculum, saccus, valvae; d, Phallus (Photo S. Heim).

Remarks

As shown earlier, males of Zygaenoprocris (Molletia) taftana (Alberti, 1939), were attracted by (2R)-butyl (7Z)-dodecenoate (Efetov et al. 2011). Males of Zygaenoprocris (Zygaenoprocris) eberti were attracted by (2R)-butyl (7Z)-dodecenoate (‘R12’) as well as by the mixture of (2R)-butyl (7Z)-dodecenoate and (2R)-butyl (9Z)-tetradecenoate (‘R12+R14’) (Fig. 2). Of the 38 males that were collected at both attractants 28 were taken around or on the R12 pheromone baits and 10 around or on the baits with the mixture of R12+R14. Of the 13 dissected specimens taken at R12, 12 were Z. eberti and only one Z. chalcochlora. All 10 specimens attracted to R12+R14 were Z. eberti. The collecting time was between 13.15–14.15 hours.

Key to the species of the subgenus Zygaenoprocris Hampson, 1900

1	Forewing upperside green, bluish green or coppery, with submetallic sheen Figs 1, 9)	2
–	Forewing upperside light brown, without submetallic sheen	5
2	Cornutus short, slightly sclerotized (Fig. 8d, Efetov and Tarmann 1994: figs 56–60; 1999: fig. 108; Efetov 2001a: fig. 10), ductus bursae not twisted (Efetov and Tarmann 1999: fig. 166; Efetov 2001a: fig. 13)	3
–	Cornutus long, strongly sclerotized (Figs 6, 8b; Efetov and Tarmann 1999: fig. 109), ductus bursae twisted (Fig. 7; Efetov and Tarmann 1999: figs 167, 168)	4
3	Papillae anales narrow, apophyses posteriores long (Mollet and Tarmann 2007: fig. 10)	Z. (Z.) chalcochlora Hampson, 1900
–	Papillae anales broad, apophyses posteriores short	Z. (Z.) khorassana (Alberti, 1939)
4	Cornutus very long (2.5 times longer than uncus), juxta with sclerotized spines ventrally (Efetov 1996: figs 5, 6; Efetov and Tarmann 1999: figs 109, 109a), ductus bursae narrow, distal part of ductus bursae with smooth walls (Efetov 1996: fig.7; Efetov and Tarmann 1999: fig. 167)	Z. (Z.) rjabovi (Alberti, 1938)
–	Cornutus shorter (only 1.8 times longer than uncus), juxta without sclerotized spines (Fig. 6), ductus bursae broad, distal part of ductus bursae with folded walls (Fig. 7; Efetov 1996: figs 8, 9; Efetov and Tarmann 1999: fig 168)	Z. (Z.) eberti (Alberti, 1968)
5	Cornutus 6 times shorter than phallus, apex of cornutus obtuse (Mollet and Tarmann 2007: fig. 6)	Z. (Z.) hofmanni Mollet & Tarmann, 2007
–	Cornutus only 3 times shorter than phallus, apex of cornutus pointed (Mollet and Tarmann 2007: fig. 5)	Z. (Z.) efetovi Mollet & Tarmann, 2007

Figure 9. 

Male of Zygaenoprocris chalcochlora Hampson, 1900, resting on Artemisia. Afghanistan, Prov. Baghlan, Salang pass, north side, Do Shagh N, Chahar Maghzak vic., 2180–2250 m, 22.vi. 2011. Photo A. Hofmann.

Figure 10. 

Distribution map of Zygaenoprocris eberti and Z. chalcochlora in Afghanistan. Blue dots: Z. chalcochlora, yellow dots: Z. eberti (at the northern locality of Z. eberti this species is syntopic with Z. chalcochlora).

We are indebted to Professor Dr Mitko A. Subchev (Bulgaria) for supplying us with the sex attractans, to Professor Dr P. D. N. Hebert (Canada) and Dr R. Rougerie (France) for help in DNA investigation, to Dr Robert Trusch (Germany) for the loan of the holotype of Procris eberti, to Mr P. V. Ruchko and the late Mr V. V. Kislovsky (both Crimea) for their help in the preparation of genitalia drawings, Mr S. Heim and Mr H. Kühtreiber (both Austria) for photographic work and help in compiling the barcoding tree.