MORPHO-ANATOMICAL AND MOLECULAR INSIGHTS INTO PTILOCEPHALA BIROI (REBEL, 1909), AND RELATED TAXA (LEPIDOPTERA: PSYCHIDAE) Edgardo Bertaccini 1 , Axel Hausmann 2 , Željko Predovnik 3 , Jurij Rekelj 4 1 Via del Canale 24, I-47122 Roncadello di Forlì (FC), e-mail: edgardobertaccini@gmail.com 2 Staatliche Naturwissenschaftliche Sammlungen Bayerns, Zoologische Staatssammlung München, Münchhausenstr. 21 D-81247 München, Germany e-mail: hausmann.a@snsd.de 3 Ob železnici 82, 3313 Polzela, Slovenia. e-mail: predovnik1@gmail.com 4 Struževo 35, 4000 Kranj, Slovenia, e-mail: jurij.rekelj@gmail.com Abstract – This study presents a comprehensive morphological and molecular analysis of Ptilocephala biroi (Rebel, 1909), comparing it to P. vesubiella (Millière, 1872), historically regarded as its closest congener. We also examine P. biroi in rela- tion to P. pyrenaella ducalis Bertaccini, 2017, which is geographically nearest to the type locality of P. biroi in Croatia (Velebit). Our molecular analyses of representa- tive populations from the southern Dinaric Alps (Biokovo) revealed a 2.35% genetic divergence among populations. Notably, both populations showed a closer genetic affinity to P. pyrenaella (Herrich-Schäffer, 1852) from its type locality in the Pyrenees than to P. pyrenaella populations from other regions. In light of these findings, we reasses the taxonomic status of Oreopsyche monteneg- rina Gozmány, 1960, which was previously synonymized with P. pyrenaella by Bourgogne (1967). We propose reclassifying this taxon as Ptilocephala biroi ssp. montenegrina (Gozmány, 1960) (status revised). Additionally, we present and describe the females of P. biroi for the first time. Key words: DNA barcoding, genetic divergence, taxonomic revision, biogeography, morphology, Croatia, Montenegro, Dinaric Alps 125 ACTA ENTOMOLOGICA SLOVENICA LJUBLJANA, DECEMBER 2024 Vol. 32, øt. 2: 125–138 Izvleček – MORFOLOŠKO-ANATOMSKI IN MOLEKULARNI VPOGLEDI V VRSTO PTILOCEPHALA BIROI (REBEL, 1909) IN SORODNE TAKSONE (LEPIDOPTERA: PSYCHIDAE) Ta študija predstavlja celovito morfološko in molekularno analizo Ptilocephala biroi (Rebel, 1909) ter primerjavo s P. vesubiella (Millière, 1872), ki je zgodovinsko vel- jala za njenega najbližjega sorodnika. Prav tako smo preučili P. biroi v povezavi s P. pyrenaella ducalis Bertaccini, 2017, ki je geografsko najbližja vrsta tipičnemu naha- jališču P. biroi na Hrvaškem (Velebit). Naše molekularne analize reprezentativnih populacij iz južnega Dinarskega gorstva (Biokovo) so razkrile 2,35 % genetske raz- like med populacijami. Zanimivo je, da sta obe populaciji pokazali tesnejšo genetsko sorodnost s P. pyrenaella (Herrich-Schäffer, 1852) iz njenega tipičnega nahajališča v Pirenejih kot s populacijami P. pyrenaella iz drugih regij. Ob upoštevanju teh ugotovitev ponovno ocenjujemo taksonomski status Oreopsyche montenegrina Gozmány, 1960, ki je bil prej sinonimiziran s P. pyrenaella s strani Bourgogne (1967). Predlagamo, da se ta takson ponovno razvrsti v Ptilocephala biroi ssp. montenegrina (Gozmány, 1960) (status revidiran). Poleg tega prvič pred- stavljamo in opisujemo samice P. biroi. Ključne besede: DNA barkodiranje, genska analiza, taksonomska revizija, bio- geografija, morfologija, Hrvaška, Črna Gora, Dinaridi Introduction The genus Ptilocephala Rambur, 1858 includes approximately 24 described species from the Palaearctic region (Sobczyk, 2011; Arnscheid & Weidlich, 2017). These species are predominantly distributed on the Iberian Peninsula, in the Pyrenees, the Alps, and the Apennine Peninsula. Fewer species are known from Central Europe, the Balkans, and Anatolia (Arnscheid & Weidlich, 2021). Ptilocephala biroi (Rebel, 1909) was first recorded as a single male specimen col- lected in the Velebit Mountains (Croatia), near Raduč, approximately 23 km south- east of Gospić, on July 28, 1893, by the Hungarian entomologist Lajos Biró. This specimen, now preserved in the Hungarian Natural History Museum (HNHM), was formally described by Rebel in 1909 under the name Oreopsyche biroi, in honor of its collector. Additional unverified findings of this species have been reported across the Balkans (Kozhanchikov, 1956; Arnscheid & Weidlich, 2017). Nevertheless, the status of this species has remained uncertain for over a century. Until now, conducting internal morphological and genetic studies on P. biroi has been nearly impossible. The available material has been extremely limited, and the age of the specimens has caused significant DNA degradation. Moreover, DNA extraction and internal morphological analysis often involve partially destructive techniques, such as removing body parts, piercing the chitin, or immersing speci- mens in chemical solutions; methods, that are unsuitable or unfeasible for rare or type specimens. For the first time, we now have the opportunity to carry out comprehensive mor- phological and, crucially, genetic comparisons using a sufficient amount of fresh Acta entomologica slovenica, 32 (2), 2024 126 material reliably attributed to P. biroi. This study does not attempt to resolve the complex taxonomic issues surrounding the entire P. pyrenaella (Herrich-Schäffer, 1852) complex; rather, it focuses on detailed comparisons between P. biroi, P. b. montenegrina (Gozmány, 1960), and their close relatives, from both morpho- anatomical and molecular perspectives. Materials and methods Collecting and rearing. This work is based on material from the genus Ptilocephala, primarily collected by the authors, along with specimens from other private collections and the Hungarian Natural History Museum (Table 1). Most of the material was obtained by collecting young larvae, which were then reared in cap- tivity until they reached the adult stage. The caterpillars are polyphagous and were fed a variety of herbs during their development. All procedures were conducted under external conditions to ensure that the environment closely resembled their nat- ural habitat. Additionally, searching for larval cases proved to be an effective method for sample collection. These cases were kept in breeding boxes that were lightly moistened with water daily until the adults emerged. Morphology and illustrations. For genitalia preparation, each specimen was boiled in 10% KOH for approximately 5–7 minutes. Photographs of the specimens were taken using a Nikon D3300 digital camera, with high-resolution images cap- tured through a 20 mm f/2.8 D lens and an Auto Extension Tube Set DG. The images were then edited using Adobe Photoshop. All photographs are the work of the first author, except for those depicting the paratype of O. montenegrina, for which copy- right belongs to the Hungarian Natural History Museum (Lepidoptera collection). Nomenclature follows Arnscheid & Weidlich (2017). Molecular methods. For the DNA barcoding analyses, one or two legs were removed from each dried specimen and transferred to lysis plates. Molecular studies using mitochondrial DNA (mtDNA) and the COI 5’ DNA barcode were conducted at the Canadian Centre for DNA Barcoding in Guelph, Ontario, Canada (CCDB). Sequence divergences within and between species were calculated using the Kimura 2-parameter model, as implemented in the Barcode of Life Data System (BOLD v4, www.barcodinglife.org) (Ratnasingham & Hebert, 2007). Table 1: List of specimens used in this study, along with corresponding BOLD sequence Ids. Edgardo Bertaccini, Axel Hausmann, Æeljko Predovnik, Jurij Rekelj: Morpho-Anatomical and Molecular Insights into Ptilocephala 127 Species Stadium/Sex Gen bank Accession Locality Legit and collection Ptilocephala biroi male BC_ZSM_Lep_117091 Croatia, Velebit, Visočica, 1470–1500 m, 6.VII.2021 leg. J. Rekelj in coll. Bertaccini Ptilocephala biroi male BC_ZSM_Lep_117092 Croatia, Velebit, Visočica, 1500–1550 m, 12.VII.2021 leg. Ž. Predovnik in coll. Bertaccini Ptilocephala biroi montenegrina male BC_ZSM_Lep_117093 Croatia, Biokovo, Sveti Jure, 1570–1600 m, 3– 7.VII.2021 leg. J. Rekelj in coll. Bertaccini Abbreviations Acta entomologica slovenica, 32 (2), 2024 128 Ptilocephala biroi montenegrina male BC_ZSM_Lep_117094 Croatia, Biokovo, Sveti Jure, 1550–1600 m,1.VII.2021 leg. J. Rekelj in coll. Bertaccini Oreopsyche montenegrina Holotype male Montenegro, Durmitor,Jakšiča Katuni, 1800m,ex l. 21.VII.1958 leg. Dr. Gozmány in coll. HNHM Ptilocephala vesubiella male BC_ZSM_Lep_81548 Italy: Liguria, Colla Melosa, (IM), 1800 m, 01.VIII.2013 leg. E. Bertaccini in coll. Bertaccini Ptilocephala pyrenaella falsevocata male BC_ZSM_Lep_84810 Italy: Piemonte, Laghi Clot Foiron (TO), 2140 m, ex l. 25.VII.2014 leg. E. Bertaccini in coll. Bertaccini Ptilocephala pyrenaella ducalis male BC_ZSM_Lep_59255 Italy: Appennino Tosco- Emiliano, Campolino (PT), 1828 m, 29.VII.2016 leg. E. Bertaccini in coll. Bertaccini Ptilocephala pyrenaella ducalis male BC_ZSM_Lep_add_ 00208 Italy: Appennino Tosco- Emiliano, dint. Passo della Vecchia (PT-MO), 1800 m, ex o. 11.V.2016 leg. E. Bertaccini in coll. Bertaccini Ptilocephala pyrenaella ducalis larva BC_ZSM_Lep_91965 Italy: Emilia, dint. Passo della Vecchia (MO), 1800 m, 21.VII.2015 leg. E. Bertaccini in coll. Bertaccini Ptilocephala pyrenaella ducalis male BC_ZSM_Lep_59256 Italy: Appennino Tosco- Emiliano: dint. Passo della Vecchia (PT-MO), 1800 m, ex o. 26.V.2016 leg. E. Bertaccini in coll. Bertaccini Ptilocephala pyrenaella male CWA BC0025 Spain: Lerida, Porto del Canto, 1730 m, 24.VI.2012 leg. W.R. Arnscheid in coll. Arnscheid Ptilocephala pyrenaella male CWA BC0024 Spain: Girona, Nuria, 1900m, 28.VI.2012 leg. W. R. Arnscheid in coll. Arnscheid BIN barcode index number BOLD barcode of life data system COI Cytochrome C Oxidase subunit I coll. collection DNA deoxyribonucleic acid ex l. ex larva (from the larva) ex o. ex ovo (from the egg) leg. legit (si.) or legerunt (pl.) from legere in Latin (to collect) HNHM Hungarian Natural History Museum GP genital preparation Results Field observations. In 2019, Croatian lepidopterist Damir Šešok discovered a freshly attached larval case, believed to belong to an unidentified species of Psychidae, near the peak of Visočica in the Velebit mountain range. This discovery renewed interest in Ptilocephala biroi, and three years later, new insights into the species’ biology and morphology were published based on field observations in Croatia (Predovnik & Rekelj, 2022). Around the same time, additional investigations were conducted in other areas of the Balkan mountains. In 2019, the third author, Ž. Predovnik, found an old male larval case and two old female cases on the wall of a mountain lodge just below the peak of Sveti Jure in Biokovo Nature Park. Subsequent entomological expeditions, carried out in collaboration with the fourth author, J. Rekelj, resulted in the collection of new material in early larval stages, which were successfully reared to adults. The breeding of both populations (from Visočica and Sveti Jure) in the same sea- son did not reveal significant differences in the behavior of the larvae, despite iden- tical rearing conditions. The larvae are polyphagous; in captivity, both populations were fed with the same food, and they showed a preference for Trifolium pratense L., Taraxacum officinale Web., and Plantago lanceolata L. without noticeable dif- ferences. The only significant difference observed was in the emergence of the adults. The population from Sveti Jure began emerging a week later than the population from Visočica, despite identical conditions. This suggests that these are genotypes adapted to their specific environments and represent genetically fixed ecotypes. This adapta- tion is justified and further supported by our field observations, which noted that the Biokovo locality is cooler and approximately 100 meters higher in elevation than Visočica. Such adaptation allows the adults to be on the wing later in the summer, when temperatures are more stable. Genetics. A Neighbor Joining tree is provided for barcodes of P. biroi and P. biroi montenegrina, together with barcodes of the other related European species P. pyrenaella, P. pyrenaella falsevocata (Bourgogne, 1979), P. pyrenaella ducalis Bertaccini, 2017 and P. vesubiella (Millière, 1872), (Fig. 1). Sequences of all 12 specimens used in this study (Table 1) formed six different BINs. Each species or subspecies has its own BIN and is recognizable by its DNA barcode. Prior to this study, P. biroi was considered a closely related species to P. vesubiel- la, as argued by the most authoritative specialists in this field (Rebel, 1909; Kozhanchikov I.V., 1956; Bourgogne, 1967; Arnscheid & Weidlich, 2017). This analysis reveals highly divergent evolutionary lines between P. biroi and its two con- geners, P. vesubiella (with a pairwise distance of 9.77%) and P. pyrenaella ducalis (with a pairwise distance of 8.77%). Comparison P. biroi with other species revealed the closest evolutionary lines to P. pyrenaella, showing pairwise distances ranging from 4.53% to 4.69%. Additionally, the phenotypes of ssp. montenegrina exhibited even more modest dis- tances, falling between 3.46% and 3.84%. While we did not observe major differ- Edgardo Bertaccini, Axel Hausmann, Æeljko Predovnik, Jurij Rekelj: Morpho-Anatomical and Molecular Insights into Ptilocephala 129 ences in the morpho-anatomical aspect between the two entities (biroi - montenegri- na), the two populations still exhibit distinct molecular characteristics (minimum pairwise distance = 2.35%), prompting a change in the original taxonomic status. Therefore, Oreopsyche montenegrina Gozmány, 1960 which was downgraded to synonymy of Ptilocephala pyrenaella by Bourgogne (1967) is now revived at sub- species rank: Ptilocephala biroi montenegrina (Gozmány, 1960), stat. rev. Table 2: Matrix of pairwise distances in the P. biroi - montenegrina complex and its closely related congeners, based on the CO1 gene barcode region and computed using the Kimura 2-parameter method. No Sample 1 2 1 P. biroi (BC_ZSM_Lep_117091)     2 P. biroi montenegrina (BC_ZSM_Lep_117093) 2,35   3 P. pyrenaella (CWA BC0025) 4,69 3,46 4 P. pyrenaella (CWA BC0024) 4,53 3,84 5 P. pyrenaella ducalis (BC_ZSM_Lep_59255) 8,77 8,19 6 P. pyrenaella falsevocata (BC_ZSM_Lep_84810) 8,21 7,29 7 P. vesubiella (BC_ZSM_Lep_81548) 9,77 9,17 Acta entomologica slovenica, 32 (2), 2024 130 Fig. 1: A neighbor Joining tree of Ptilocephala biroi and selected closely related species. Samples are submitted with sequence ID (BOLD), scientific name and bar- code index number (BIN). Numbers at the branches represents bootstrap percent- ages, scale bar represents 1% K2P divergence between sequences. Morphology. Males. In the table below the actual values of morphological char- acteristics are presented, observed in various taxa under examination. In Gozmány’s (1960) original description, it was noted that the wingspan of males falls within the range of 19–22 mm, while the holotype, which is currently preserved at the HNHM, measures approximately 18 mm. Table 3: A comparison of morphological characteristics of biroi-montenegrina- vesubiella complex. Females. Females of the nominate species, as well as subspecies, have not yet been described. Only one source mentions females, stating that despite extensive efforts, no females were found among the larger numbers of male larval cases (Vasić et al. 1990). All female members of the genus Ptilocephala are very similar in appearance, they are apterous, antennae and legs are fully reduced. Here we provide the first description of the females of P. biroi and its subspecies P. biroi monteneg- rina stat. rev.: Ptilocephala biroi (Rebel, 1909) Material: Croatia, Srednji Velebit, Visočica, 1500–1550 m: 9♀♀ with larval cases, leg. Ž. Predovnik, 13.VI.2020 (ex l. 14.VI.–12.VII.2021), coll. Ž. Predovnik; 16♀♀ with larval cases, leg. J. Rekelj, 13.VI.2020 (ex l. 14.VI.–12.VII.2021), 13♀♀ coll. J. Rekelj, 3♀♀ coll. Bertaccini. 4♀♀ with larval cases, leg. J. Rekelj, 6.VII.2021, 2♀♀ coll. J. Rekelj, 2♀♀ coll. Bertaccini. Description of female: Specimens measure 11–12 mm in length and have a diam- eter of 5–6 mm, with these measurements taken after they were freed from their rigid pupal envelopes (Fig. 18). Head small, shiny, turned below the thoracic segments, directed posteriorly. Rudiments of antennae absent, the eye rudiments appear as two small but clearly visible black dots. Wingless, rudiments of legs absent. The abdomen has a very light yellowish color, both thoracic and abdominal segments covered with dense, short hairs. The thoracic segments strongly sclerotized dorsally and laterally, giving them a shining appearance. Boundaries between segments indis- Complex: biroi-montenegrina-vesubiella Male biroi Croatia, Velebit montenegrina Montenegro, Durmitor montenegrina Croatia, Biokovo vesubiella Italy, Liguria ducalis Italy, Emilia Wingspan (objective data) 17-20 mm 19-22 mm 18 (holotype) 17-20 mm 19-27 mm 16-19 mm Antennalsegments 25-27 26 25-27 26-30 25-27 Larval case ♂ ♀ 12-21 16-21mm 12-19 mm 24-28 mm 21mm 17-21 mm 15-22 16-22mm 15-17 mm 12-18 14-18mm 12-16 mm Edgardo Bertaccini, Axel Hausmann, Æeljko Predovnik, Jurij Rekelj: Morpho-Anatomical and Molecular Insights into Ptilocephala 131 tinct. The ovipositor everted. The pupa exhibits a semicircle of rigid spines in the anal area on the dorsal side (Figs. 14, 16). Ptilocephala biroi montenegrina (Gozmány, 1960) Material: Croatia, Biokovo, Sv. Jure, 1550m, 9♀♀ with larval cases, leg. J. Rekelj, 14.VI.2020 (ex l. 3–7.VII.2021, 20–22.VIII.2021), 5♀♀ coll. J. Rekelj, 4♀♀ coll. Bertaccini. Description of female: Specimens measure 10–11 mm in length with a diameter of 4–5 mm (Fig. 19). Other characters are similar to the nominate species. The pupa bears a sinuous line of stiff spines in the anal area on the dorsal side (Figs. 15, 17). Discussion This study illustrates the evolutionary relationships and taxonomic status of Ptilocephala biroi and its subspecies montenegrina. Through DNA barcoding and morphological analyses, we could conclude that biroi, along with its subspecies, is more closely related to pyrenaella than previously recognized. The observed genetic distances 4.53% between biroi and pyrenaella, and 3.46% between montenegrina and pyrenaella suggest that these species share a more recent common ancestor than biroi does with vesubiella, which exhibits a greater pairwise distance of 9.77%. These findings raise important questions about the historical biogeography of these species. The proximity of the Dinaric Mountains to the Pyrenees suggests potential historical connectivity, possibly during postglacial periods, which could explain the close evolutionary ties between biroi and pyrenaella. Historically, the classification of these taxa has relied heavily on morphological characteristics, a common practice in taxonomy. However, the results of this study emphasize that speciation is not always accompanied by significant morphological changes. The lack of major morphological differences between P. biroi and mon- tenegrina suggests that traditional methods of species delineation may overlook cryptic diversity. This finding aligns with recent discussions in taxonomy, which argue that the true number of biological species is likely greater than currently rec- ognized, primarily due to the reliance on morphological traits that may not accurately reflect genetic divergence (Weidlich and Arnscheid, 2021). In all definitions of sub- species, there is unanimous agreement that subspecies are geographically defined and diagnosable by at least one presumably heritable character. This is clearly evi- dent in our case. The genetic divergence observed between the Velebit and Biokovo populations, with a minimum pairwise distance of 2.35%, indicates that even geo- graphically proximate populations can exhibit distinct molecular characteristics. Differences in the timing of adult emergence further support the hypothesis that a genetically fixed ecotype can become a subspecies if sufficiently pronounced genetic and phenotypic differences develop between it and other populations of the same species. A subspecies is typically defined by stable characteristics inherited by indi- viduals within that group, which may be adaptations to specific environmental con- ditions. If this ecotype reproduces separately from others and maintains its unique Acta entomologica slovenica, 32 (2), 2024 132 traits over multiple generations, it can be formally classified as a subspecies. In the future, it would be intriguing to identify and rear other populations for behavioral comparison. This molecular differentiation, reproductive isolation, and specific envi- ronmental adaptations, despite minimal morpho-anatomical differences support the recognition of montenegrina as a distinct subspecies, thereby reinstating its taxo- nomic status after previous synonymizing. Furthermore, our study highlights the unexpected genetic diversity present within the Ptilocephala complex, raising concerns about potential regional and taxonomic biases in current diversity estimates. The pronounced genetic divergence observed in geographically distant populations such as those from the Dinaric Alps and the Pyrenees invites further investigation into the evolutionary processes at play. These findings resonate with the insights of Bickford et al. (2007), who noted that our understanding of biodiversity is often clouded by assumptions based on morpholog- ical criteria. Acknowledgements We would like to extend our heartfelt gratitude to all those who supported us by providing valuable data and borrowing essential materials. A special note of thanks goes to Dr. Wilfried R. Arnscheid from Bochum, Germany, for his valuable advice and for granting us access to some molecular data. We sincerely appreciate the invaluable assistance we received from Zsolt Bálint, Balázs Tóth, and Gergely Katona from the HNHM. Furthermore, we are grateful to the Trustees of the Museum for granting us permission to feature the images of the type specimen of Oreopsyche montenegrina from the HNHM. Our deepest gratitude goes to the “Centre for Biodiversity Genomics, University of Guelph” and the individuals Paul Hebert, Evgeny Zakharov, and Sujeevan Ratnasingham for their exceptional work in sequencing the DNA of the samples. Finally, we would like to express our sincere gratitude to Stanislav Gomboc for commenting on the manuscript, Prof. Dr. Radovan Kranjčev from Koprivnica, Croatia, as well as the park administration, for their invaluable assistance in obtaining the research permit for Biokovo Nature Park. References Arnscheid W.R., Weidlich M., 2017: Psychidae. In: Karsholt, O, Mutanen, M. and M. Nuss [edit.]: Microlepidoptera of Europe. Leiden and Boston (Brill), Vol. 8, p. 1–423. Arnscheid W.R., Weidlich M., 2021: First taxonomic revision of the Ptilocephala albida - species group in Europe (Lepidoptera: Psychidae, Oiketicinae). SHI- LAP Revista de lepidopterología, 49 (195): 417–433. Bertaccini E., 2017: Ptilocephala pyrenaella ducalis nuova sottospecie rinvenuta sull’Appennino Tosco-Emiliano. (Insecta Lepidoptera Psychidae). Quaderno di Studi e notizie di Storia Naturale della Romagna, 46: 155–171. Edgardo Bertaccini, Axel Hausmann, Æeljko Predovnik, Jurij Rekelj: Morpho-Anatomical and Molecular Insights into Ptilocephala 133 Bickford D., Lohman D.J., Sodhi N.S., Ng P.K.L., Meier R., Winker K., Ingram K.K., Das I., 2007: Cryptic species as a window on diversity and conservation. TRENDS in Ecology and Evolution 22(3): 149–156. Bourgogne J., 1967: Materiaux pour une révision du genre Oreopsyche (Psychidae) - Alexanor, 5: 1–40. CCDB [Canadian Centre for DNA Barcoding] (2020) Description of DNA barcode protocols. http://www.dnabarcoding.ca/pa/ge/research/protocols. Accessed 1.12.2020. Gozmány L. A., 1960: Oreopsyche montenegrina sp. n. from Yugoslavia (Psychidae, Lepidoptera). Bulletin du Museum d’Histoire Naturelle de Belgrade, 15: 91. Kozhanchikov I.V., 1956: Fauna of the U.S.S.R. Lepidoptera. Psychidae. Zoologicheskii Akademii Nauk SSSR, N.S., 62, 3(2): 1–517. Predovnik Ž., Rekelj J., 2022: New finds of the bagworm Ptilocephala biroi (Rebel, 1909) from Velebit in Croatia (Lepidoptera: Psychidae). Acta entomo- logica slovenica, 30(2): 107–114. Ratnasingham S., Hebert P.D., 2007: The Barcode of Life Data System. http://www.barcodinglife.org. Molecular Ecology Notes, 7(3): 355–364. Rebel H., 1909: Eine neue Psychiden-Art aus Croatien. Annales Musei Naturalis Hungarici, 7: 344–346. Sobczyk T., 2011: Psychidae (lepidoptera). in: nuss, M. (ed.), World Catalogue of insects, Apollo books, Stenstrup, 10: 1–467. Vasić K., Tomić D., Carnelutti J., Zečević M., Kranjčev R., 1990: Heterocera I. Bombyces et Sphinges (Insecta, Lepidoptera). In: Fauna Durmitora, Vol. 3, Posebna izdanja knjiga 23, Crnogorska akademija nauka i umetnosti, Podgorica, p. 99–137. Weidlich M., Arnscheid W.R., 2021: Faunistic and taxonomic notes on Phalacropterix apiformis (Rossi, 1790) and Phalacropterix restonicae (Fiumi & Govi, 2015) from Corsica, France (Lepidoptera: Psychidae, Oiketicinae). Nota Lepidopterologica, 44: 57–67. Acta entomologica slovenica, 32 (2), 2024 134 Edgardo Bertaccini, Axel Hausmann, Æeljko Predovnik, Jurij Rekelj: Morpho-Anatomical and Molecular Insights into Ptilocephala 135 Figs. 2-6: Ptilocephala spp., wing venation in males: 2): Ptilocephala vesubiella, Italy, Liguria, Colla Melosa (IM), 1650m, 10.VI.2008, ex o., leg. E. Bertaccini in coll. Bertaccini; 3): P. biroi, Croatia, Velebit, Visočica, 1470–1500m, 06.VII.2021, leg. J. Rekelj in coll. Bertaccini; 4): P. biroi montenegrina, Croatia, Biokovo, Sv. Jure, 1550m, 10.VII.2021, ex l., leg. J. Rekelj in coll. Bertaccini; 5): P. biroi mon- tenegrina (Oreopsyche montenegrina (Holotypus, HNHM)); 5a): Original labels of P. biroi montenegrina (HNHM); 6): P. pyrenaella ducalis, Appennino Tosco- Emiliano, dint. Passo della Vecchia (PT MO), 1800 m, 11.V.2016, ex o., leg. E. Bertaccini in coll. Bertaccini. Acta entomologica slovenica, 32 (2), 2024 136 Figs. 7-10: Ptilocephala spp., antennae of males: 7): Ptilocephala biroi, Croatia, Velebit, Visočica, 1470–1500m, 06.VII.2021, leg. J. Rekelj in coll. Bertaccini; 8): P. vesubiella, Italy, Liguria, Colla Melosa (IM), 1650m, 10.VI.2008, ex o., leg. E. Bertaccini in coll. Bertaccini; 9): P. biroi montenegrina, Croatia, Biokovo, Sveti Jure, 1570–1600m 3–7.VII.2021, leg. J. Rekelj in coll. Bertaccini; 10): P. biroi mon- tenegrina, Durmitor (Holotypus, HNHM). Edgardo Bertaccini, Axel Hausmann, Æeljko Predovnik, Jurij Rekelj: Morpho-Anatomical and Molecular Insights into Ptilocephala 137 Figs. 11-17: Ptilocephala spp. morfological characters: 11): Ptilocephala biroi, male, abdominal sternites, tergites and genitalia: Croatia, Velebit, Visočica, 1500– 1550m, 16.VI–12.VII.2021, ex l., leg. Ž. Predovnik in coll. Bertaccini, (GP № 1100 E. Bertaccini); 12): P. vesubiella, male, abdominal sternites, tergites and genitalia: Italy, Liguria, Colla Melosa (IM), 1650m, 10.VI.2008, ex o., leg. E. Bertaccini in coll. Bertaccini, (GP № 694 E. Bertaccini); 13): P. biroi montenegrina, male, abdominal sternites, tergites and genitalia: Croatia, Biokovo, Sv. Jure, 1550m, 10.VII.2021, ex l., leg. J. Rekelj in coll. Bertaccini. (GP № 1101 E. Bertaccini); 14): P. biroi, female, pupa, Croatia, Velebit, Visočica, 1470–1500m, 06.VII.2021, leg. J. Rekelj in coll. Bertaccini; 15): P. biroi montenegrina, female, pupa, Croatia, Biokovo, Sveti Jure, 1570–1600m, 3–7.VII.2021, leg. J. Rekelj in coll. Bertaccini; 16): P. biroi, female pupa, dorsal view, detail of the spines on the 7th abdominal seg- ment, Croatia, Velebit, Visočica, 1470–1500m, 06.VII.2021, leg. J. Rekelj in coll. Bertaccini; 17): P. biroi montenegrina, female pupa, dorsal view, detail of the spines on the 7th abdominal segment, Croatia, Biokovo, Sveti Jure, 1570–1600m, 3– 7.VII.2021, leg. J. Rekelj in coll. Bertaccini. Acta entomologica slovenica, 32 (2), 2024 138 Figs. 18, 19: 18): Ptilocephala biroi, female, Croatia, Velebit, Visočica, 1470– 1500m, 06.VII.2021, leg. J. Rekelj in coll. Bertaccini; 19): P. biroi montenegrina, female, Croatia, Biokovo, Sveti Jure, 1570–1600m, 3–7.VII.2021, leg. J. Rekelj in coll. Bertaccini. Fig. 20: Distribution map of Ptilocephala biroi and P. biroi montenegrina.