New data on the early stages and behaviour of the endangered species Callophrys mystaphia (Lepidoptera: Lycaenidae) and its first larval parasitoid, Cotesia sp. (Hymenoptera: Braconidae)

ACTA ENTOMOLOGICA MUSEI NATIONALIS PRAGAE www.aemnp.euISSN 1804-6487 (online) – 0374-1036 (print) RESEARCH PAPER New data on the early stages and behaviour of the endangered species Callophrys mystaphia (Lepidoptera: Lycaenidae) and its fi rst larval parasitoid, Cotesia sp. (Hymenoptera: Braconidae) Erdem SEVEN Batman University, Faculty of Tourism, Department of Gastronomy and Culinary Arts, 72060, Batman, Turkey; e-mail: [email protected] https://orcid.org/0000-0002-7587-5341 Abstract. The early stages of a lycaenid butterfl y, Miller’s Green Hairstreak Callophrys mystaphia Miller, 1913, are photographed and presented for the fi rst time. Callophrys mystaphia is known to be endangered and is regarded to be endemic to Turkey based on recent studies. The species is considered to be local and isolated, with the likelihood of being one of the rarest butterfl y species in Turkey. The distribution of this species and its food plant, Rheum ribes L. (Polygonaceae), in Turkey are investigated and the possible range of this butterfl y species is highlighted. New data on the early stages, phenology, vertical distribution, and behaviour of adult and larva of C. mystaphia are presented. The larva was found to have been attacked by a newly discovered hymenopteran parasitoid species of the genus Cotesia Cameron, 1981 (Braconidae); the cocoons and the adult parasitoid are illustrated. Key words. Lepidoptera, Lycaenidae, Hymenoptera, Braconidae, biodiversity, biology, endemic, host plant, larval parasitoid, larval stages, Turkey, Palaearctic Region Zoobank: http://zoobank.org/urn:lsid:zoobank.org:pub:F828F6F0-A1AB-48DE-A260-9D0871E2257C © 2022 The Authors. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Licence. Accepted: 26th June 2022 Published online: 16th December 2022 2022 62(2): 391–399 doi: 10.37520/aemnp.2022.021 type specimens of C. mystaphia were known. W & V O (1998) viewed C. mystaphia and C. paulae as being conspecifi c, and incorrectly classifi ed both species as C. mystaphia. However, investigations conducted since 1995, including a revision by T H & M (2010) analysing the genus Callophrys, have supported that C. mystaphia (based on samples only from SW Iran) should be classifi ed as a separate species based on morphology, ecology (especially plant preference), and mtDNA COI barcodes. The species was originally thought to be distributed in Turkey, Iran, and Afghanistan (H et al. 1995). It was later realized that the populations from south- -western Iran were erroneously identifi ed as C. mystaphia by T H (2006) due to the similarity of the morphological characters. A comparison with the type specimens of C. mystaphia convinced K & K (2013) that specimens from south-west Iran represent a diff erent species described as new, C. mystaphioides Krupitsky & Kolesnichenko, 2013. However, T et al. (2014) refer to the presence of C. mystaphia mystaphia (probably in north-west) and C. mystaphia mystaphioides (south-west) in Iran. Furthermore, the identifi cation of Introduction The genus Callophrys Billberg, 1820 is characterized by green colour and the presence (or absence) and arrangement of white spotting on the ventral surfaces of the wings. Within the Palaearctic Callophrys, seven partially heterogeneous groups of species could be identifi ed according to a phylogeny based on the mitochondrial COI gene, with C. mystaphia Miller, [1913], being placed in the C. rubi-group (T H & M 2010). According to K & K (2018), the genus is represented by six species in Turkey: Callophrys mystaphia, C. chalybeitincta Sovinsky, 1905, C. danchenkoi Zhdanko, 1998, C. herculeana Pfeiff er, 1927, C. paulae Pfeiff er, 1932, and C. rubi (Linnaeus, 1758). Miller’s Green Hairstreak, Callophrys mystaphia, was described from the Aras River Valley, Kars, Kağızman, Eastern Turkey, near the Armenian border. W & V O (1998) stated that the locus typicus, ‘Mystaph’, is in the same place as ‘Mesta’ near the village of ‘Novoja Nicolaewka’, which is identical to the village known today as Akçay (right bank of the Arax, on the border between the Kars and Erivan Governments at an altitude of around 1800 m). For a long period, only the SEVEN: Early stages and behaviour Callophrys mystaphia (Lepidoptera: Lycaenidae) 392 specimens from Afghanistan (Paghman Mts.) mentioned in H (1954) as C. mystaphia, was determined to conform perfectly to C. afghana Krupitsky, Pljushtch & Pak, 2012 (K et al. 2012). Thus, the distribution area of C. mystaphia became limited only to a small area, the east and southeast of Turkey. Callophrys mystaphia and C. mystaphioides are thought to be closely related to the C. suaveola species group by K & K (2013). In contrast, T H & M (2010, misidentifi ed as C. mystaphia) discovered that C. mystaphioides and C. rubi have quite similar DNA barcodes despite signifi cant morphological and genitalic diff erences between these two butterfl y species. However, phylogenetic reconstructions of Callophrys were based solely on the COI genes, which are not always suffi cient (S & V 2011). Little is known about the biology of C. mystaphia; the only such study was undertaken by K (2009), employing video to investigate the developmental stages. Furthermore, there is no previously known record of any parasitoid species attacking the larval stages of C. mystaphia; here, details are provided of a braconid species of the genus Cotesia Cameron, 1891 newly discovered as a parasitoid of Miller’s Green Hairstreak. Material and methods Material examined. Three fully grown larvae of C. mystaphia were collected from Rheum ribes growing in a fi eld alongside the Maden road, Şirvan district, Siirt Prov., southeast Turkey on 1 June 2020, at 1400 m, 38°04′30″N 42°07′11″E. Larvae were also observed in the fi eld on 18 May 2021. Alongside these, some adult specimens collected by S (2010) were examined: 3 ♂♂ 2 ♀♀, Maden road (the same location as above), 1380–1480 m a.s.l., 26.iv.2009; 14 ♂♂ 5 ♀♀, 1.v.2009, 1 ♂, Hesko (38°08′07″N 42°01′15″E), 975 m a.s.l., 3.v.2009; and 1 ♂, Maden junction (38°03′51″N 42°04′02″E), 950 m a.s.l., 25.iv.2009, all leg. E. Seven. The larvae, together with the food plant, were transported to the laboratory in a transparent box. Larval growth was monitored daily and throughout that process, a Fujifi lm Finepix HS30EXR camera was used to photograph the various stages of development; the images were later arranged in Adobe Photoshop CS6. In addition, some adult specimens collected by S (2010) from the same location were also investigated. The material of adults and larvae is deposited in the Entomology Laboratory of Batman University (BTU), Batman, Turkey. Terminology follows M (1913), K (1977), H et al. (1995), W & O (1998), T H (2006), T H & M (2010), T-  (2011, 2014), K & K (2013), and K & K (2018). Results and discussion Diagnosis. Adult (Fig. 1). Callophrys mystaphia is the smallest member of the genus Callophrys in Turkey. Special features distinguish it from other Callophrys species, namely those of exceedingly small size, sharply rounded outer margins of all wings. Callophrys mystaphia is recognized from all the congeners by the complete absence of the lobe of the hind wings, the black upper surface of the wings, the perpendicular curved white submarginal line and the orthogonally curved white submarginal line (T H 2006, K 2009, T H & M 2010). The wingspan is 17–18 mm. Antennae are black with white rings at the base of the segments. Greenish-white scales are surrounded by brown eyes with small, pale brown hairs. The brown androconial spot is small and rounded. The ground colour of the underside of the forewing is emerald green, and the basal area has a few greenish hairs. White postdiscal spots are mostly distinct and rounded. Adult variation. The white row of dots on the underside of the wings of C. mystaphia shows signifi cant variation in Turkey (H et al. 1995, K 2009, S 2010, T 2011, T 2022). It is quite prominent in certain individuals while being less so in others. Due to these intraspecifi c variation, the identifi cation of some species of this genus is diffi cult and confusing. External morphological and genital characters have been used to classify the populations as separate taxa in Iran (K & K 2013, T et al. 2014). Larva (Figs 2, 3). Callophrys mystaphia larvae resemble woodlouse, like the majority of other members of their family. Adult larvae have a large, plump body that becomes fl attened at the ends and sharply indented between the segments. The larvae have completely smooth green colour; however, they start to turn brown as they approach pupation. It has a head and 10 fl attened segments. The head is round in dorsal view and slightly angular in lateral view. The segments have a prominent dorsal hump, concave sides, and a dilated lateral ridge. The head partially retracts Fig. 1. Habitus of Callophrys mystaphia Miller, 1913 (a – 29.iv.2020 during a fi eld study, b–c – stretched specimen). Acta Entomologica Musei Nationalis Pragae, volume 62, number 2, 2022 393 into the fi rst segment when in rest and feeding, and is the same shade of green as the body. There is no longitudinal line medially on dorsum. The legs are pale green. Larvae are bearing setae, the entire dorsal surface of the head, thorax, and abdomen is heavily covered with whitish, fi nely serrated spines of various length. There are no spines on the limbs. Raised reticulations cover the whole surface of body, and numerous tiny lenticles can be seen along the spiracular region. Spiracles are lighter in colour, they are whitish and distinct. Except for Callophrys rubi, information on the larval development of the majority of species in this genus is still unknown or very limited. According to E (2022), the ground colour of C. rubi is amber-brown, checkered and speckled with black which is most conspicuous as transverse markings on the abdomen. The colour of segments is a brilliant pure green, with a series of sub-dorsal oblique citrine-yellow markings commencing on the third segment, and a lateral line of the same colour. Callophrys rubi is rather densely covered with brown spinous hairs of various lengths (E 2022). The larva of C. mystaphia is similar to C. rubi, but C. mystaphia is completely smooth green and has no transverse markings on the abdomen. Also, it has whitish spinous hairs. Pupa (Fig. 4). In the pupal stage of C. mystaphia, the abdomen is rounded and smooth brown and rather fat compared with the head and thorax. The ventral part of abdomen is lighter brown. Pupae have a characteristic brown, sparse setation, except for the ventral part of the pupa. The general pupal morphology of C. mystaphia is similar to that of the Callophrys genus in general: some species have distinctive darker spots (such as C. rubi), especially on abdomen, but C. mystaphia is almost unicolorous brown. Biology. Larva. Three larvae of Callophrys mystaphia were found near Maden road at 1400 m a.s.l., on 1 June 2020 on the host plant Rheum ribes. Two of them pupated on 13 and 15 June 2020, respectively. One adult emerged on 23 April 2021. The second one did not emerge from the pupa which probably dried out due to the high temperature in summer months. The third larva was found parasitised (see below). Further seven larvae were found during investigations in the area of the host plant on 18 May 2021. It seems that larvae can be found from late May to mid-June in the study area. The associations of lycaenid larvae and pupae with ants, termed myrmecophily, are mediated by epidermal glands whose secretions modify the ants’ behaviour. The most widespread type of lycaenid-ant interactions involves Fig. 2. Feeding larvae of Callophrys mystaphia Miller, 1913 on Rheum ribes host plant. (a–b – fully grown larvae, 20.vi.2020; c–d – fi nal instar larvae approaching pupation, 4.vi.2020). SEVEN: Early stages and behaviour Callophrys mystaphia (Lepidoptera: Lycaenidae) 394 Fig. 3. Larvae of Callophrys mystaphia Miller, 1913 on host plant from various perspectives. Fig. 4. Pupae of Callophrys mystaphia Miller, 1913 on 13.vi.2020 (a – dorsal, b – ventral, c – lateral view). Fig. 5. Larvae of Callophrys mystaphia Miller, 1913 forming ‘feeding windows’ on the host plant. Acta Entomologica Musei Nationalis Pragae, volume 62, number 2, 2022 395 phytophagous caterpillars while they are feeding on their host plants. These caterpillars are visited by ants to harvest their secretions. Life history data are available for only a fraction of Turkish Lycaenidae species. In many cases, these data have not been collected in Turkey but in Central Europe or the Western Mediterranean. Only a few records of myrmecophily have been documented in Turkey, and the type of ant interaction involving C. mystaphia remains unknown (F 1995). No such interaction has been observed on the larvae in the study area, either. However, myrmecoxenous caterpillars are not associated with ants, and the caterpillars of Lycaenini are generally not tended by ants as in C. rubi (F 1990, 1995). Perhaps C. mystaphia larva is also myrmecoxenous. The larvae of C. mystaphia showed no tendency to cannibalism in their natural environment or laboratory conditions as in C. rubi (E 2022). Pupa. The pupa is not fi xed, it has a thick, rounded, and dumpy shape. The pupal stage lasted 10 months and 17 days in laboratory conditions, which is from about mid- -June to the middle of the following March. They pupated on the ground in captivity and were concealed on leaves of the broom they consumed as food. They surely pupate on the ground among moss or other suitable cover in nature. In addition, it is thought that pupae maintain their moisture balance deep in the soil to protect themselves from the dry and hot summer months in the study region. Phenology. Callophrys mystaphia is univoltine, fl ying from late April to mid-June (M 1913, H et al. 1995, K 2009, S 2010, T 2011). In the study area, the population was high in numbers in local sites where R. ribes is abundant at the beginning of May. Towards the beginning of June, the number of fl ying adults decreases as the weather gets warmer and the plants start to wither. Callophrys mystaphia was not observed in the study area later than mid-June. The species fl ies for 15–20 days on average. The temperature is about 19℃ at the end of April when the species begins to fl y, increasing to around 32℃ in mid-June (TSMS 2021). At the Maden road habitat, C. mystaphia was co-occurring with the following identifi ed species of butterfl ies: Anthocharis cardamines (Linnaeus, 1758), A. gruneri Herrich-Schäff er, [1851], Callophrys suaveola (Staudinger, 1881), Colias crocea (Geoff roy, 1785), Issoria lathonia (Linnaeus, 1758) (in high numbers), Pieris persis Verity, 1922, Polyommatus agestis ([Denis & Schiff ermüller], 1775) (in high numbers), P. icarus (Rottemburg, 1775) (in high numbers), Pontia chloridice (Hübner, [1813]), Vanessa cardui (Linnaeus, 1758), and Zerynthia deyrollei (Oberthür, 1869). In addition, the following species were fl ying together with C. mystaphia in the habitat of Ahlat district of Bitlis (O. Başbay, pers. comm.): Coenonympha pamphilus (Linnaeus, 1758), Polyommatus thersites (Canterer, [1835]), and Vanessa cardui. Behaviour. During the daytime investigation in the natural environment of C. mystaphia, it was discovered that larvae always feed on the underside of the leaves of R. ribes. Larvae move very slowly on the leaves and feed by grazing on the leaf surface, creating ‘windows’ on the host plant leaves, except for the thick veins (Fig. 5) that are presumably more diffi cult to consume and digest, and lower in nutrients compared with newly-fl ushed apical leaves. Thus, they form many ‘windows’ of diff erent sizes on plant leaves. In rainy weather, the adult specimen shelters under the broad leaves of its host plant (O. Başbay, pers. comm. 2021) with wings being mostly closed. It was observed that adults seek nectar from the host plant’s fl owers in preference to other sources. Adults were fl ying only short distances from R. ribes host plants. The butterfl y is small, and has much the same colour as the leaves, making observation diffi cult; its habit of remaining in close proximity to the host plant most probably serves as a camoufl age against predators. Thus, its potential to disperse is assumed to be limited, as it rarely strays more than a few metres away from the host plant. Parasitoids (Fig. 6). During fi eld studies at the Şirvan habitat on 1 June 2020, it was found that a fully-grown larva of Callophrys mystaphia had been attacked by a parasitoid. The parasitised larva continued its normal feeding on the fi rst day of observations and turned yellowish colour. Then, its movements slowed and it became motionless, prior to the emergence of the parasitoid larvae and the subsequent formation of cocoons leading to larval death two days after formation of cocoons. The presence of cocoons alongside its body was found on 3 June 2020 (Figs 6a–c) and adult parasitoid wasps emerged on 9 June 2020. Male wasps emerged from two out of the fi ve cocoons (Figs 6d–g). The other parasitoids could not emerge from the pupa due to fl uid fl owing from the dead larva. From photographs, the parasitoid was confi rmed by Donald Quicke and Mark Shaw (pers. comm. 2021) as a species of Cotesia, Cameron, 1981 (Hymenoptera, Braconidae, Microgastrinae). The Microgastrinae subfamily is the most signifi cant group of parasitoid wasps that attack caterpillars, with many species being utilized or regarded as biocontrol agents against lepidopteran pests in agriculture and forestry (W 1997). The genus Cotesia is known to parasitize many species of butterfl ies. In addition, Cotesia inducta (Papp, 1973) is reported to parasitize Callophrys rubi (S et al. 2009). Distribution (Fig. 7). Formerly, C. mystaphia was claimed to be widespread in Turkey, Iran and Afghanistan (H 1954; H et al. 1995; T H 2006, 2010). However, it became clear that the populations in Iran and Afghanistan were not conspecifi c (for details see Introduction; K et al. 2012, K & K 2013). The species has been reported in 9 provinces from the east and south-east of Turkey according to the literature records: Kars (Kağızman) (M 1913, H et al. 1995), ?Aksaray (Ihlara), Iğdır (Aktaş) (H et al. 1995; see below), Van (Erek Mt.) (K 2009), Siirt (Şirvan) (S 2010), Adıyaman, Kahramanmaraş (T-  2011), Hakkari, and Tunceli (Ovacık) (K & K 2018). It was also photographed by butterfl y watchers from Bitlis (Ahlat), Elazığ, Gaziantep, Giresun, Kahramanmaraş, Malatya, Muş, Osmaniye, Siirt, Tunceli SEVEN: Early stages and behaviour Callophrys mystaphia (Lepidoptera: Lycaenidae) 396 and Van Provinces (T 2022). Callophrys mystaphia has not been observed at the type locality and other previously published localities for many years (M 1913, H et al. 1995). After its fi rst description in 1913, the species was fi gured from Kars (Kağızman) and Iğdır (Aktaş) by H et al. (1995). But they listed these samples as uncertain (‘status incertus’) because no adults could be found matching with the description and the lectotype (W & V O 1998). However, since Rheum ribes is known in these areas, it is believed that the species may still be present (K & W 2011). There were also unconfi rmed specimens recorded from the valley of Ihlara (Aksaray) in H et al. (1995). They had some doubts about whether the two males found by Wagener in 1985 at 1200 metres on a yellow-fl owered Apiaceae in Aksaray (Ihlara Valley) were C. mystaphia or a diff erent taxon. Moreover, there was some controversy concerning the diagnoses of the Callophrys species in Turkey when H et al. (1995) released the study. Callophrys paulae was presented as a junior subjective synonym of C. mystaphia while W & V O (1998) were comparing the lectotype and the original description with material collected in the surrounding area of the type locality in June 1996 (W & V O 1998); therefore, they associated the habitat of C. mystaphia with Onobrychis cornuta, which is actually the habitat of C. paulae, and this result is based on a misleading taxonomical interpretation as they caught only C. paulae specimens near the type locality of C. mystaphia. Furthermore, the records of the sites in H et al. (1995), the current observations (T 2022) and the research reports (M 1913, H et al. 1995, K 2009, S 2010, T 2011, K & K 2018) do not match exactly. Hence, the locations of the recorded species and food plant must be carefully established to determine the exact distribution of C. mystaphia. This should be supported by extensive molecular studies of mitochondrial, nuclear, and ribosomal genes, including the populations in Turkey in case diff erent taxa for this species may be present. As indicated by K & K (2013), the status of isolated populations of butterfl ies similar to C. mystaphia inhabiting Adıyaman and Kahramanmaraş and cited in T H (2006) and T (2011) still remains controversial because of the likelihood that they belong to undescribed taxa. Additionally, a new plant species of rhubarb, Rheum telianum, was recently discovered in Adıyaman (I & K 2020). Therefore, Adyaman’s populations must be thoroughly examined, together with the other populations of the species in the south-east of Turkey, especially by means of detailed molecular analysis. Habitat (Fig. 8). All recognized habitat types have much in common, largely consisting of eroded steppe with rhubarb plants. The habitat is given by T (2011), as ‘mountain slopes and gorges with sparse vegetation and dominance of Onobrychis cornuta and Rheum spp. from 2000 up to 3000 m’. In the study area, Callophrys mystaphia inhabits mountainous and eroded stony steppe formations. Its vertical distribution ranges from 1400 m up to approx. 2800 m (H et al. 1995, K & W 2011). The type material of C. mystaphia was caught at an altitude of about 1800 m (M 1913). S (2010) reported this species between 950–1480 m in the Şirvan district of Siirt, and only two specimens were caught around 950 metres in Hesko and Maden junction; however, most of the adult samples were collected above 1380 m (see examined materials). Rheum ribes was not found in the Hesko and Maden junction locations. However, the presence of food plants in those habitats is undetermined because of the diffi culty in accessing the steep slopes. Almost the entire research area in Şirvan (Siirt Province, SE Turkey) comprises mountainous terrain and has no Fig. 6. a – Parasitised larva of Callophrys mystaphia Miller, 1913; b–c – cocoons of the parasitoid; c – intentionally removed larva to reveal the cocoons, d–g – reared parasitoids of the genus Cotesia (Braconidae: Microgastrinae) from various perspectives. Acta Entomologica Musei Nationalis Pragae, volume 62, number 2, 2022 397 signifi cant wide valleys or plains. The district is divided by the Kezer stream in the west and the Botan River in the east. In the south, the vegetation is largely steppe, whereas in the north, stands of oak are concentrated in mountainous regions. After a rather short spring, precipitation reduces, and the hot, dry air fl ow from Basra (Iraq) continues until October. Herbaceous plants outside the stream border begin to dry out in early June, leaving practically solely woody and bushy plants (S 2014). The deep gorges of Botan, Harbur, and Zab in Southeast Anatolia, south of Van Lake, along with their rich vertical structures, provide a favourable refuge for various tertiary relicts and elements of the Turk-Iranian Zagros fauna and fl ora, which are not found in other Turkish locations (W 2005). Maden road’s location contains herbaceous plant and small shrub species such as Cirsium, Gundelia (both Asteraceae), Astragalus, Medicago, Onobrychis, Trifolium, and Vicia (all Fabaceae), which are common in spring. Sparsely found woody plants in the area are Quercus (Fagaceae), Salix, Populus (both Salicaceae), Crataegus, Rubus (both Rosaceae), and Paliurus spina-christi and Rhamnus (both Rhamnaceae). The habitats in Kahramanmaraş and Bitlis (Ahlat) Provinces are defi ned as barren, steppe, mostly dominated by R. ribes (O. Başbay, pers. comm. 2021). Host plant. Polygonaceae: Rheum ribes L. (K 2009, this paper). Prangos lophoptera Boiss. (Apiaceae) was mentioned as a possible host plant in the original description by M (1913). W & V O (1998) specifi ed that C. mystaphia was linked with the subalpine thorn-cushion plant formation associated with Onobrychis cornuta (L.) (Fabaceae). However, larvae were found on Rheum ribes L. (Polygonaceae) in Erek Mt., Van Prov. of Turkey (K 2009). The species is also determined as feeding on R. ribes in Siirt Prov., in the research area, and under laboratory conditions. Additionally, Iranian C. mystaphioides was stated to be associated with Rheum sp. in known localities – both in the type locality in Esfahan Province as well as in Fars Province (Iran). Rheum persicum is considered a potential host plant of C. mystaphioides Fig. 7. Distribution map of Callophrys mystaphia Miller, 1913 (black circle: records based on scientifi c papers, empty circle: records of butterfl y watchers) and Rheum ribes (square). Fig. 8. Habitat of Callophrys mystaphia Miller, 1913 in Şirvan district, Siirt Prov., south-eastern Turkey, 1400 m a.s.l. (a – 1.vi.2020, b – 29.iv.2021). SEVEN: Early stages and behaviour Callophrys mystaphia (Lepidoptera: Lycaenidae) 398 (T H 2006). Furthermore, C. titanus Zhdanko, 1998 uses Rheum sp. as host plant in Tajikistan (Z 1998). As in the species mentioned above, Rheum spp. is the only known food plant for some closely related species of this genus. On the other hand, C. rubi is extremely polyphagous and feeds on a large variety of plants from about 10–15 diff erent plant families (F 1990). Rheum ribes L. (in Turkish: “Işgın”) is an edible wild species of rhubarb and is an element of the Iran-Turan Phytogeographic Region ranging from Israel, Palestine, Lebanon, Armenia, and Northern Iraq to Iran and the Eastern Anatolian Region of Turkey (C 1996). In Turkey it is reported in the following areas: Ağrı (Tahir- -Eleşkirt, Kavurman Mt.), Bingöl (Çirişli Pass, Kop Mt., Bingöl-Erzurum provinces border), Bitlis (Ahlat, Kambos Mt.), Eskişehir (Anadolu University Campus), Elazığ (Harput, Kup and Mastar Mts., Pekinik), Erzincan (Keşiş Mt., Guleman), Erzurum (Ilıca), Hakkari (Otluca, Sat Mt.), Hatay (İskenderun, Arsuz Amanos Mt.), Kahramanmaraş (Göksun-Geben Kayranlı Mt.), Kars, Mardin (Hessena), Sivas, Van (Erek Mt., Değirmenköy, Gölardı, Bahçesaray, Gürpınar), Tunceli (Munzur Mt., Ovacık) (C 1966, M et al. 2000, K et al. 2016, I & K 2020, A & K 2021). The provinces of Ağrı, Bingöl, Eskişehir, Erzurum, Erzincan, Hatay, Mardin, and Sivas are areas where the food plant is known but C. mystaphia is yet to be found there, as it is most likely present. The population status, distribution, and current distribution limits of the butterfl y species need to be confi rmed by investigating all the locations where the host plant is documented (Fig. 7). Studies on the current subject are still in progress. Rheum ribes is reported to be a food source for a variety of insects. Noctuid moth Xylena exsoleta (Linnaeus, 1758) feeds on R. ribes leaves (K et al. 2008), and Apochima diaphanaria rjabovi (Wehrli, 1936) (Geometridae) on the blooms (A & K 2021). Also, weevil Petrocladus sp. (Curculionidae), jewel beetle Capnodis marquardti Reitter, 1913 (Buprestidae), and leaf beetle Labidostomis brevipennis Faldermann, 1837 (Chrysomelidae) are insects associated with R. ribes in eastern Turkey (K et al. 2016). Moreover, hymenopter species Kokujewia ectrapela Konow, 1902 (Argidae) feeds alongside C. mystaphia on R. ribes leaves in Şirvan district (S 2022). These are all plant-specifi c herbivores, and the majority appear to be native to Turkey. Additionally, it was observed that the larvae of some other insect taxa from diff erent groups (such as Hymenoptera, Coleoptera and Noctuidae) also feed with C. mystaphia on R. ribes in the fi eld. Further research is pending, and is hoped to be reported upon later. Conservation status. Callophrys mystaphia is a small, local and monophagous species and probably one of the scarcest butterfl ies in Turkey (K 2009). It generally prefers to live around the host plant, making only short fl ights, and its distribution is thus limited by the presence of food plants in Turkey. The species is endangered due to the restricted distribution and pressure on the food plant (K & W 2011). The larval food plant, Rheum ribes L. (Polygonaceae) is a perennial herbaceous wild plant species and has medicinal importance (B 1999). In traditional Anatolian folk medicine, extracts from various parts of the plant are commonly used to treat a variety of diseases (Y et al. 2020). Since R. ribes roots are considered to be benefi cial for the treatment of diabetes, they are dug up by the local people (S 2010). Collection and sale of food plants as vegetables in local markets are uncontrolled, and young shoots are consumed fresh by the public. However, excessive picking of food crops, habitat loss, and plant destruction due to consumption and overgrazing are some of the most crucial threats to the species, and these may lead to the extinction of R. ribes, not a very common plant with a highly specifi c assemblage of insects, most of which are endemic to Turkey (K et al. 2016). Therefore, it is essential to preserve the long-term sustainability of R. ribes harvest, while at the same time maintain control over collection and marketing activities. The species’ records are restricted, as the duration of its fl ight period is short, the number of the populations is low, and investigating the host plant, which grows on steep stony slopes at high altitudes, is challenging. Primarily, it may be easier to identify the food plant to determine the possible locations of the butterfl y species. However, this may not be satisfactory because the low butterfl y population is commonly concentrated on small numbers of plants and sites. The study region, Maden road location, is estimated to have one of the largest populations of the species. Population reports were extremely scarce in almost all other habitats in Turkey (T 2022). Also, the populations in Kahramanmaraş and Bitlis provinces are stated to be rare (O. Başbay, pers. comm. 2021). K & W (2011) described C. mystaphia as ‘near endemic’, taking into account the populations in eastern Turkey and southwestern Iran. However, the population in southwestern Iran was afterwards described as a diff erent species (K & K 2013). As C. mystaphia occurs only in a restricted region with small local populations and has a short fl ying time, the species is thought to be endangered. Callophrys mystaphia was included in “Lycaenidae protected by legislation in Europe” (C 1987). Taking into account all of these factors, it is recommended that the species and its only host plant, R. ribes, should be protected. Acknowledgments It is a pleasure to thank Dr. Mark Shaw (National Museums Scotland) and Dr. Donald Quicke (Thailand) for identifying the Microgastrinae parasitoid. I am also grateful to Onat Başbay (Turkey) for information relating to the population status, phenology, behaviour and habitats of C. mystaphia in Turkey. I thank Eddie John (UK) for reviewing the manuscript and for suggestions. The map used in Fig. 7 is a modifi ed version of http://d-maps.com. 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