Nota Lepi. 45 2022: 191-205 | DOT 10.3897/n1.45.80106 Research Article Biology and DNA barcode analysis of Coleophora lessinica Baldizzone, 1980 and Coleophora impalella Toll, 1961 (Lepidoptera, Coleophoridae) with description of their larval cases ATTILA TAKACS!, CSABA SZABOKY’, GUSZTAV BOLDOG?, SANDOR JORDAN’, MIKLos Bozso°, DAvip FULOP®, BALAZS TOTH’ 1 Government Office of Fejér county, Major Department of Agriculture Plant Protection and Soil Conservation Department, Orszdg ut 23, H2481 Velence Hungary; molyirto@gmail.com Bécsi ut 88, H1034 Budapest, Hungary; szabokycs.50@gmail.com Haladas ut 4, H5600 Békéscsaba, Hungary; bolgus@gmail.com Egyetem tér 1, H4032 Debrecen, Hungary; jordansanyi@gmail.com Plant Health and Molecular Biology Laboratory, National Food Chain Safety Office, Directorate of Plant Protection, nA BW NHN Soil Conservation and Agri-environment, Budaorsi ut 141-145, H1118 Budapest, Hungary; mikitv.bozs@gmail.com 6 Centre for Agricultural Research, Plant Protection Institute, Nagykovacsi tit 26-30, H1029, Budapest, Hungary; ocypus(@gmail.com 7 Hungarian Natural History Museum, Baross utca 13, H1088, Budapest, Hungary; toth.balazs@nhmus.hu http://zoobank. org/6A BA523B-08 1D-44D8-9466-A 909DBB243D6 Received 4 January 2022; accepted 31 March 2022; published: 21 April 2022 Subject Editor: Lauri Kaila. Abstract. Host plants and cases of several species in the genus Coleophora Hubner, 1822 have remained unknown until now, while the latter structures provide important characters for identification. Host plants and cases of Coleophora lessinica Baldizzone, 1980 and Coleophora impalella Toll, 1961 were discovered by the authors and are presented here for the first time. New data on the distribution and biology of the two species in Hungary are given. Coleophora lessinica is recorded as new for the fauna of Romania. DNA barcode sequencing was performed for both Coleophora species and loaded into the BOLD System and to GenBank. Introduction Members of the family Coleophoridae are distributed worldwide but the number of described species is highest in the Palaearctic region. The family currently includes more than 1500 described species in total (Baldizzone et al. 2006; Baldizzone and van der Wolf 2011, 2015, 2020a, b; Baldizzone 2019; Baldizzone 2021). Based on the latest phylogenetic research (Bauer et al. 2012), only three genera are generally accepted. In Europe, three species are known in the genus Augasma Herrich-Schaffer, 1853, four in Ischnophanes Meyrick, 1891 and 621 in Coleophora Hubner, 1822 (Rennwald and Rodeland 2021). In Hungary the family comprises 211 species: one in Augasma and 210 in Coleophora (Pastoralis and Buschmann 2018; Tabell and Kosorin 2020; Szaboky and Takacs 2021). Knowledge of the biology of Coleophora species 1s well-known in northern Europe but rather limited in southern Europe. The spectrum of host plants is wide and includes Asteraceae, Betulaceae, Copyright Zakdcs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1:92 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella Caryophyllaceae, Cistaceae, Chenopodiaceae, Ericaceae, Juncacae, Fabaceae Lamiaceae, Poaceae, Rhamnaceae, Rosaceae, but the host plants of many species are still unknown (Baldizzone 2019). Coleophora lessinica Baldizzone, 1980 is a southern European species described on the basis of specimens collected in the Lessini mountain near Verona (Baldizzone 1980). Itis known from Bulgaria (Richter 2017), Croatia, France, Italy, and North Macedonia (Baldizzone 2019). The first Hungarian specimen was collected in Fejér county, Bucka-hegy near Csakberény (26.vii1.2000) (Szaboky et al. 2009). Biology of this species is unknown, according to Baldizzone (2019) and Buschmann et al. (2014). The species is univoltine, flying from August to September tn xerothermic habitats. Coleophora impalella Toll, 1961 was described from Southern Russia (Toll 1961) based on a single old male specimen. Later the female was described from the mid-section of the river Vol- ga by Anikin (2004). In addition, there is a population in the Karadag Nature Reserve in Crimea (Budashkin and Puzanov 2017) Outside the Volga region and Crimean Peninsula it has been found only in Bélmegyer, Hungary, from where both sexes were re-described (Baldizzone and Tokar 2008). The adults are diurnal and not attracted to artificial light (Szaboky et al. 2009). Other aspects of the species’ biology are unknown. DNA based identification is often useful, not just for species identification by non-specialists, but also to reveal association between different life stages and sexes of the same species which otherwise might be a very time demanding task. Here we give some details about the biology of two Coleophora species and describe their larval cases. Materials and methods Field work, record and rearing All field work was undertaken in Hungary and in Romania near the Hungarian border (Map 1). Larvae of the studied species, including overwintering stages, were reared on their host plants planted in the gardens of the authors. Escape of larvae was prevented with a tulle bag attached around the host plant. Shortly before the expected time of emergence each case was put in a separate plastic vial to maintain appropriate humidity. Specimens are deposited in the private collections of G. Baldizzone, Cs. Szaboky and A. Takacs. Three specimens of Coleophora impalella Toll, 1961 are to be deposited in the Hungarian Natural History Museum after the publication of this paper. Acronyms Bldz: genitalia preparation of Giorgio Baldizzone (Asti, Italy) HNHM: Hungarian Natural History Museum, Budapest IgR: genitalia preparation of Ignac Richter (Mala Causa, Slovakia) LB: genitalia preparation of Balazs Toth (HNHM) Morphological examinations Photography and genitalia preparation Images of intact larvae, adults and larval damage were prepared with a Canon 450 D camera, applied to a Carl Zeiss Stemi-2000 binocular stereomicroscope; they were edited with software Adobe Photosop CS6. The habitat image was taken with a Sony A7R2 camera. Nota Lepi. 45: 191-205 193 Bratislava is Budapest udapest | Map 1. Collection localities of Coleophora specimens used in this study. Black dots: C. lessinica, black triangles: C. impalella. Map by Péter Gabor Sulyan (Budapest). Methods for preparation of the genitalia follow van Nieukerken et al. (2012, 2018). Images were adjusted with the software Adobe Photoshop CS6. Molecular methods analysis Four larvae of the two species were selected for molecular analyses (Table 1). Specimens were killed and stored in 70% alcohol. The genomic DNA was isolated from a piece of the larval body with Quick-DNA Tissue/Insect Miniprep Kit (Zymo Research) according to the recommended protocol of the manufacturer. Amplification of the standard COI barcode region was performed with the primers LCO-1490 and HCO-2198 (Folmer et al. 1994). The PCR mixture was prepared and optimised according to the recommended protocol of the manufacturer (HotStarTaq Plus PCR Handbook, Qiagen). The PCR mixture contained 2ul of template DNA, 7.5ul of 2x HotStart Taq Plus Master Mix (HotStart Taq Plus Master Mix Kit, Qiagen), 0.5u1 of each primer (1O0UM) and double distilled water to a final volume of 15u1. The amplification profile was determined based on the European and Mediterranean Plant Protection Organization PM 7/129 (2) protocol (Anonymous 2021) and modified for the use of the HotStart Taq Plus Master Mix Kit as follows: the profile consisted of a preheating step of 5 min at 95 °C followed by 5 cycles of 95 °C for 30 sec, 45 °C for 30 sec and 72 °C for 1 min, and additional 35 cycles at 95 °C for 30 sec, 51 °C for 1 min and 72 °C for 1 min 194 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella Table 1. Coleophora specimens from Hungary used for molecular analyses. Species Host plant Locality GPS Date of Collector Stage GenBank coordinates collection Accession number C. lessinica Artemisia alba Csakberény 47°20'49"N, = 12.x.2018 Attila Takacs Larva MZ662875 182.1319" EB C. lessinica Artemisia alba Gant-Granas 47°21'11"N, 09.x.2018 Attila Takacs Larva MZ664319 18°22 E, C. lessinica Artemisia alba Nagyharsany 45°51'24"N, 04.x1.2018 Attila Takacs Larva MZ662874 18°24'52"E C. impalella_ Galatella sedifolia Szabadkigyés, 46°34'54"N, 17.viii.2021 Attila Takacs Larva OK329945 Roka kaszin6. =. 21°05'00"E and a final extension at 72 °C for 10 min. Reaction was performed in a PTC-100 DNA thermal cycler (MJ Research). Products of amplification were analysed in 2% agarose gel with addition of ethidium bromide and visualised under UV light. The PCR products were purified using the USB ExoSAP-IT PCR Product Clean-Up reagent (Affymetrix) and amplicons were sequenced bidirec- tionally (BaseClear B.V., The Netherlands). The sequences were assembled with Staden Package 2.0.0b9 (Staden et al. 1998). Sequences were inspected and translated in ExPASy Bioinformatics Resource Portal (Artimo et al. 2012) to verify that they are free of stop codons and that they are not pseudogenes. To identify the larvae, two independent approaches were used. First nblast search was carried out in GenBank (www.ncbi.nlm.nih.gov/Genbank) optimized for highly similar sequences (megablast) (Accessed on 11/12/2021). Also, the identification engine of the BOLD homepage (www.barcodinglife.org) was used to search in the species level barcode library (Accessed on 11/12/2021). The graphical results obtained were visualised and edited with FigTree 1.4.4 (Rambaut 2018) and the final versions were created by Inkscape version 0.92 (Inkscape Project 2021). Results and discussion Rearing young larvae is difficult and cumbersome but has the advantage of limiting parasitism compared with starting with more advanced larvae. However, young larvae are very sensitive to the quality and quantity of food as well as to disturbance. Larvae in closed containers without ventilation are easily killed by mould, whereas in an open container the food plant easily becomes too dry to be palatable. Perhaps the most sensitive period is when the larva changes its case. Natural conditions should be simulated as much as possible to enhance rearing success. The best approximation is to grow the host plants in a garden setting, place cases on them, and cover them with tulle bags to prevent larvae from escaping and to protect them from parasitoids. Success is not assured even when larvae reach the final L5 stage at which point they need to make a U-turn inside the case in preparation for pupation; many larvae fail to perform this turn and die. After overwintering, the larval stage lasts for an additional 4—5 months, with pupation taking place only 3-4 weeks before emergence of the adult. Despite of these difficulties, rearing is still the most efficient way to study the biology of these species. Nota Lepi. 45: 191-205 195 Material examined Coleophora lessinica Baldizzone, 1980 Figs 1-5, 7, 10-14 Adults. 1 9°, Hungary: Fejér county, Készarhegy, 16.viii.2020, light trap. 1 2, same locality, 17.viu1.2020, light trap; det. Giorgio Baldizzone (Asti), slide No. Bldz 17234 (Figs 10, 11). 1 9, Veszprém county, Litér, Mogyoroés-hegy, 21.viti.2017, leg. Cs. Szabdky. Cases. Hungary: 11 cases, Fejér county, Gant-Granas 09.x.2018, on Artemisia alba Turra, leg. A. Takacs. 23 cases, same locality but 15.x.2019, on Artemisia alba, leg. A. Takacs. 15 cases, same locality but 22.x.2020, on A. alba, leg. A. Takacs. 25 cases, Fejér county, Csakberény, Bucka-hegy 10.x.2018, on A. alba, leg. A. Takacs. 45 cases, same locality but 16.x.2019, on A. alba. leg. A. Takacs. 10 cases, same locality but 22.x.2020, on A. alba, leg. A. Takacs. 5 cases, Csakvar, Haraszt- hegy, 15.x.2021, on A. alba, leg. A Takacs. 35 cases, Baranya county, Nagyharsany, Szarsomly6, 11.x1.2018, on A. alba subsp. canescens, leg. Cs. Szaboky, B. Toth. 5 cases, same locality but 05.x.2019, on A. alba subsp. canescens, leg. Cs. Szaboky, B. Toth. 3 cases, same locality but 22.x.2020, on A. alba subsp. canescens, leg. Cs. Szaboky, B. Toth. 3 cases, Veszprém county, Balatonftired, Nagymezo, 10.x.2020, on A. alba, leg. Cs. Szabdky. 12 cases, Varpalota, Barbély- volgy, 18.x.2018, on A. alba, leg. Cs. Szaboky. Coleophora impalella Toll, 1961 Figs 6, 8, 9, 18-26 Adults. 1 ¢, Hungary: Békés county, Bélmegyer, Fas puszta 29.iii.2021, ex larva, on Galatella sedifolia (L.), leg. A. Takacs. 1 9, same data but 31.iii.2021. 1 9, same data but 13.iv.2021. 1 ¢, same data but 03.v.2021. 3 ¢ and 5 9, same locality but 12.v.2021, leg. Cs. Szaboky and A. Takacs. 2 3 and 8 9, Békés county, Szabadkigyos, Kigyosi puszta, 12.v.2021, leg. Cs. Szaboky and A. Takacs, slide Nos IgR31860 (Fig. 6), TB2148f, TB2149f (Figs 8, 9). Cases. Hungary: 10 cases, Bélmegyer, Faspuszta 24.1x.2020, on G. sedifolia. 3 cases, Békés county, Szabadkigyos, Kigyosi puszta, 17.viii.2021, on G. sedifolia, leg. A. Takacs. 1 case, Békés county, Elek, 06.x.2021, on G. sedifolia, leg. G. Farkas. Romania: 1 case, Arad county, Graniceri, 06.x.2021, on G. sedifolia, leg. G. Farkas. Coleophora lessinica Baldizzone, 1980 Larval morphology and recorded host plants. Case and larval behaviour. Initial case is made of a dry inflorescence of A. alba. The larva consumes the centre of the inflorescence, remaining parts are strengthened with silk (Fig. 12) and thus the remains of the inflorescence retain their original shape. Depending on its size, up to three or four larvae can feed on the same individual plant. Several inflorescences are consumed in the protection of this case, then the larva in L4 stage prepares a tubular silk case beneath the older one. Later on, the achaenia fall off from the tubular case (Figs 13, 14), and the larva seeks a still intact inflorescence, attaches the case to it, chews itself 196 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella Figures 1-5. Larval morphology of Coleophora lessinica. 1. Head and thorax, dorsal view; 2. Head and thorax, lateral view; 3. Head and thorax, ventral view; 4. Prolegs, ventral view; 5. Anal plate, dorso-lateral view. Scale bar: 1 mm, cranial direction top, caudal direction bottom, photos by A. Takacs. inside the inflorescence, and performs its last moult in this shelter. Finally, it descends to the base of the host plant and attaches its case to it. The mouth of case is at a right angle to the longitudinal axis of the case. Anal end of case is trivalved. Colour of case is light brown before overwintering, becoming dark brown after winter. Nota Lepi. 45: 191-205 197 Figures 6—11. Genitalia of C. impalella and C. lessinica. 6. Male genitalia of C. impalella, slide No. IgR 31860 (phallotheca right); 7. Male genitalia of C. /essinica, slide No. IgR 23392 (phallotheca right); 8. Female genitalia of C. impalella, signum broken, flattened, slide No. TB2148f; 8a. Intact signum of C. impalella (enlarged), slide No. TB2149f; 9. Abdominal segments of C. impalella, slide No. TB2149f; 10. Female genitalia of C. /essinica, slide No. Bldz 17234; 11. Abdominal segments of C. /essinica. Scale bars: 0.5 mm. Figs 6, 7: photo by Ignac Richter, figs 10, 11: photo by Janos Babics, figs 8, 9: photo by Balazs Toth. Overwintering happens at the base of the host plant. The larva climbs on the stem up to the ba- sal-most leaves during August and September. There it attaches its case permanently to the stem, and this is the place where the larva turns around inside the case. The early case, as well as the overwintering one, is very similar to that of Coleophora absinthii Wocke, 1877 (Figs 15-17), but the early case of C. /essinica is constructed from only 198 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella Figures 12-17. Cases of C. lessinica and C. absinthii. 12. Flower case of C lJessinica. Csakberény, Bucka- hegy, 20.x.2020, on Artemisia alba; 13. Sheath case before overwintering of C. /essinica. Csakberény, Bucka- hegy, 27.x.2020, on A. alba; 14. Sheath case before overwintering of C. /essinica. Csakberény, Bucka-hegy, 20.x.2020, on A. alba; 15. Flower case of C. absinthii. Pakozd, 25.viii.2021, on A. absinthium. The larva is feeding in the third inflorescence, which will be incorporated to its case; 16. Flower case of C. absinthii. Pettend, 30.viii.2021, on A. absinthium; 17. Sheath case before overwintering of C. absinthii. Pettend, 04.1x.2021, on A. absinthium. Scale bars: 0.5 mm, photos by Attila Takacs. one inflorescence while that of C. absinthii is made of three flower heads that are unified before preparing the final, overwintering case. L5 larva (Figs 1-5). Length 3 mm. Head brown. Body light brown. Thoracic shields (Fig. 1) and spiracular sclerites (Fig. 2) not sclerotised. Thoracic legs uniform light brown as body (Fig. 3). Prolegs Nota Lepi. 45: 191-205 199 on segments A3—A6 with 12—18 crochets in two uniordinal rows; distribution of crochets asymmet- rical (Fig. 5). Anal plate shiny brown (Fig. 4). Anal proleg half-moon-shaped, each with 11 crochets. Habitat. The habitats of C. /essinica differ from each other in terms of bedrock. The bed- rock in the habitats of Eastern Bakony Mountains (K6oszarhegy, Varpalota), the Balaton Uplands (Litér, Balatonfiired) and the Villany Mountains (Nagyharsany) is limestone, that of the Vértes Mountains (Csakvar, Csakberény, Gant-Granas) is dolomite. The bedrock affects the species of- plant associations, but the association-forming species everywhere in these habitats is 4. alba (Borhidi 2003). Coleophora impalella Toll, 1961 Case and behaviour. Construction of the case begins in late August or early September from the leaves of the host plant, Galatella sedifolia. This finding does not support Richter (2021), who has found cases on G. /inosyris (L.). The case 1s made of 2-6 mm long and 2-4 mm wide strips spun to each other with overlap, resulting in a sheath case (Figs 24, 25). Feeding lasts until drying of host plant in autumn. The overwintering case is 12 mm long and is attached to a broader section of the dried plant. The larva continues feeding on fresh leaves in spring. Early case is greyish black, changing to dark brown after overwintering. Mouth directed in 40 ° angle to longitudinal axis of case, case slightly tapering towards anal end, caudal 2/3 part slightly curved, anal end flattened, bivalved. Adults emerge 10—12 days after the permanent attachment of the case. They fly in daytime and remain close to the host plant. Mating was observed between 11 h and 13 h. LS larva (Figs 18—23). Length 5.5 mm. Head brown, adfrontal suture black. Body light brown, with inconspicuous pinacula around prolegs on segments A3—A5. Thoracic shields shiny black, rounded; prothoracic shield ovoid, more than 1.5 times as broad as long, very finely divided along median axis except anterior margin; mesothoracic shield D-shaped, divided to two halves by gap medially, gap being narrowest at middle, shield gradually broadening towards anterior and poste- rior margins, anterior margin straight, lighter than rest of the shield; metathoracic shield made of two separated ovoid plates with dentate inner edges, plates separated by gap slightly broader than the plates (Fig. 18). Spiracular sclerites oval, of equal length, sclerite on prothorax half as broad as those on meso- and metathorax, latter two sclerites with uniform shape and size (Fig. 19). Thoracic legs brown; proximal parts of segments darker than distal parts (Fig. 20). Prolegs on segments A3—A5 with 6—6 crochets in two uniordinal rows; on segment A5 two crochets in anterior, four crochets in posterior row, three crochets in each of remaining row (Fig. 21). Anal plate matt black, heavily irrorated with tiny hollows (Fig. 23). Anal proleg half-moon-shaped, each with 17-19 crochets (Fig. 22). Conditions of collecting. Ten cases of C. impalella were collected in Bélmegyer, Faspuszta (Figs 24, 25), on 24.ix.2020. Dates and numbers of emerged specimens: 1 <, 29.iii.2021, 1 9, S17. 2027, 1°93 tv. 2024, 1 gg2.03-v.2021,, In addition to the Hungarian locality already known, the species was also found in Szabad- kigyos, Kigyosi puszta on 12.v.2021 (Fig. 26). One case was collected in Elek on 06.x.2021 and one case in Graniceri, Romania on 06.x.2021. This latter record is the first one outside Russia and Hungary. New to Romania. Habitat. Coleophora impalella feeds on Asteretum sedifolii Sod 1947 corr. Borhidi 1996 (Pannonic salt steppes and salt marshes), a species that 1s highly influenced by dry continental 200 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella Figures 18—23. Larval morphology of Coleophora impalella. 18. Head and thorax, dorsal view; 19. Head and thorax, lateral view; 20. Head and thorax, ventral view; 21. Prolegs on segments A3—A6, ventral view; 22. Anal prolegs, ventral view; 23. Anal plate, dorsal view. Scale bar: 1 mm, cranial direction top, caudal direction bottom, photos by A. Takacs. Nota Lepi. 45: 191-205 201 climate with extreme temperatures and uneven distribution of precipitation. This habitat type was formed by secondary salinisation (Fig. 27). It is characteristic for the Continental climate zone and can also be found in the Ukrainian and Russian steppe belt (Borhidi 2003). Parasitoids. No cases of C. impalella and C. lessinica specimens have given parasitoid speci- mens in our rearings to date. Molecular analysis and identification The specimens from Artemisia alba all belong to the same haplotype. The alignment of the COI region numbered 536 nucleotide positions. The sequence length of the specimen from Galatella sedifolia was 595 nucleotides in the final data set. she Figures 24-27. Some stages and habitat of C. impalella. 24. L5 case of C. impalella before overwintering, Bélmegyer, 13.x.2020; 25. L4 case of C. impalella before overwintering, Bélmegyer 24.1x.2020; 26. Imago of C. impalella from Szabadkigyos, Kigy6si puszta, 12.v.2021; 27. Habitat of C. impalella in Szabadkigyos, Kigy6osi puszta, 20.v.2021. Scale bar: 3 mm (24); 2 mm (25); 2.1 mm (26). Figs 24, 26: photo by A. Takacs, Figs 25, 27: photo by G. Boldog. 202 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella No exact matches were found in GenBank (Table 2). In the specimen from Galatella sedifolia the closest hit with 99.5% similarity was a C. impalella specimen from Russia (K-X048258). For all other hits, the similarity was below 95%. The specimen was identified by the BOLD System as Coleophora impalella (BOLD:ACB0657) with 99.5% probability (Fig. 28). In case of the specimens from Artemisia alba the closest matches in GenBank were three specimens of Coleophora artemisicolella with 97.57% similarity (Table 3). The BOLD System based on similarity was not able to unambiguously identify our specimens from Artemisia alba because of the high similarity of related species (Table 4). The two possible matches were C. lessinica and C. magyarica. The tree-based identification tool indicated that our specimens belong to C. /essinica(BOLD:ABA2006) (Fig. 29). Molecular identification was confirmed by genitalia dissection (Figs 1—5). The examination of DNA barcodes confirmed that C. /essinica and C. impalella are present with at least one population in Hungary. Based on our analysis, the barcodes of “/essinica” and “impalella” populations form monophyletic clusters. The studied DNA sequences of the Macedonian and Hungarian populations of C. /essinica were identical. The intraspecific distance between studied Russian and Hungarian C. impalella populations was 0.5% (3 nucleotide positions). Identification was confirmed by genitalia dissection (Figs 6—11). We were able to collect C. /essinica specimens from every location where its host plant, Artemisia alba is known to occur in Hungary. This implies that the species is more widespread than the published records suggest. To clarify the distribution of the species the host plant populations Table 2. First five hits in GenBank using MEGABIlast search on the COI sequences of the Hungarian specimen identified as Coleophora impalella. Accessed on 11/12/2021. Scientific Name Max Score Total Query E value Percent Accession Accession Score Cover identity Length number Coleophora impalella 1083 1083 100% 0.00 99:5 658 KX048258 Coleophora striolatella 911 911 100% 0.00 94.29 658 KX049535 Coleophora striolatella 900 900 100% 0.00 93:95 658 KX047871 Coleophora thurneri 889 889 100% 0.00 93.62 652 KX048570 Coleophora sp. JFLOS51 889 889 100% 0.00 93.62 658 HM406336 Hungarian specimen C. impalella Russia C. striolatella C. medelichensis 0.02 8 Figure 28. Part of the Neighbor-joining tree of the barcoded Hungarian specimen later identified as Coleophora impalella. Query sequence is marked with red. Scale bar indicates 2% K2P divergence of nucleotide substitution. Nota Lepi. 45: 191-205 203 Table 3. First five hits in GenBank using MEGABlast search on the COI sequences of the Hungarian specimens later identified as Coleophora lessinica. Accessed on 11/12/2021. Scientific Name Max Total Query Evalue Percent Accession Accession Score Score Cover identity Length number Coleophora artemisicolella 918 918 100% 0.00 957 672 JN248884 Coleophora artemisicolella 918 918 100% 0.00 DES7 658 HM871573 Coleophora artemisicolella 918 918 100% 0.00 97-97. 658 HM875737 Coleophora fasciella 913 913 100% 0.00 97.39 658 MT394458 Coleophora nubivagella 907 907 100% 0.00 97-2 658 KX042273 Table 4. Top 20 matches from the BOLD System identification tool of the specimens later identified as Coleophora lessinica. Genus Species Specimens Similarity Remarks Coleophora lessinica 2 100% Coleophora magyarica 1 99.06% private Coleophora microdon 1 97.94% early-release Coleophora artemisicolella 8 97.57% Coleophora nubivagella 8 97.57% private Hungarian specimens C. lessinica Macedonia C. lessinica Italy C. microdon C. magyarica C. absinthii 0.005 29 Figure 29. Part of the Neighbor-joining tree of the barcoded Hungarian specimen later identified as Coleophora lessinica. Query sequence is marked with red. Scale bar indicates 0.5% K2P divergence of nucleotide substitution. should be investigated during the flowering period. A disjunct distribution of C. impalella is unlikely, due to the widespread occurrence of its host plant, G. sedifolia. With more comprehensive searches, it 1s likely that more populations will be discovered in the future. Our results support that identifying material based on DNA barcodes is often highly reliable for this group and provides an alternative to morphology-based identification. DNA-based identifications are very useful and could be widely used in species identification when different life stages are difficult to connect. Nevertheless, morphological confirmations are still needed, especially when species have highly similar DNA barcodes. 204 Takacs et al.: Discovery of the biology of Coleophora lessinica and Coleophora impalella Acknowledgements We are grateful to those who helped us in searching for cases: Péter Finy, Diana Gy6ori, Istvan Harta and Kristof Antal (Fejér County Government Office, Velence), Gabor Farkas (Békés County Government Of- fice, Békéscsaba), Péter Gabor Sulyan (OMKI, Budapest). We thank Ignac Richter (Mala Causa, Slovakia) for the genital preparations, and Janos Babics for genitalia photos. 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