Incurvariapirinella sp. nov., a new species of the vetulella species-group (Lepidoptera, Incurvariidae) from Bulgaria, with release of DNA barcodes for European species of Incurvaria Author Junnilainen, Jari Author Buchner, Peter Author Kaitila, Jari-Pekka Author Mutanen, Marko text Nota Lepidopterologica 2019 42 1 81 100 http://dx.doi.org/10.3897/nl.42.13026 journal article http://dx.doi.org/10.3897/nl.42.13026 2367-5365-1-81 E74584C61E604FC6B11168AF29583F57 Incurvaria pirinella Junnilainen, Kaitila & Mutanen sp. nov. Type material. Holotype: ♂, Bulgaria, Blagoevgrad district, Struma river valley, Stara Kresna 275m a.s.l., 41.795N , 23.157E ; 03.v.2013. J. Junnilainen leg. & coll. with red label "HOLOTYPE of Incurvaria pirinella Junnilainen, Kaitila & Mutanen". - Paratypes 21♂; 6♀: 4♂; 1♀ same locality and data as holotype, Genitalia prep. ♂ No: GPJJ201702, GPPB3334, 2♂ in glycerol, J. Junnilainen leg. & coll.; 7♂; 1♀ Bulgaria, Blagoevgrad district, southern Pirin 1200m a.s.l., 41.528N , 23.584E ; 30.v.2006, Genitalia prep. ♂ No: GPJJ201701, 1 ♂ genitalia in glycerol, J. Junnilainen leg. & coll., 1♂ in. Tiroler Landesmuseum Ferdinandeum, Innsbruck, Austria.; 1♂ Bulgaria, Blagoevgrad district, southern Pirin 1300m a.s.l., 41.574N , 23.656E ; 21-24.vi.2001 with green label DNA sample 24476 Lepid phyl., J. Junnilainen leg. & coll.; 4♂; 3♀ Bulgaria, Blagoevgrad district, Ilindenci road, meadows below barrier 900m a.s.l., 41.675N , 23.278E ; 16.v.2012. J-P. Kaitila & Bo Wikstroem leg, coll. J-P. Kaitila, 1♂, 1♀ in Finnish Museum of Natural History, University of Helsinki, 1♀ in Tiroler Landesmuseum Ferdinandeum, Innsbruck, Austria. 2♀ Bulgaria, Blagoevgrad district, Slavianka, Goleshovo road, highest point 1000m a.s.l., 41.4034N , 23.389E ; 25.v.2012, Genitalia prep. No: GPJJ201704. In coll. J-P. Kaitila; 5♂ Bulgaria, Blagoevgrad district, Ilindenci 900m a.s.l., 41.67N , 23.27E ; 30.v.2012. Bo Wikstroem leg. & coll. Genitalia prep. No: GPJJ201701, GPJJ201702, GPJJ201704, GPPbf3327, GPPbf3328, GPPbm3329, GPPbm3330, GPPbm3334. All paratypes with red label "PARATYPE of Incurvaria pirinella Junnilainen, Kaitila & Mutanen". Deposition of types. The holotype of Incurvaria pirinella is deposited in the research collection of J. Junnilainen. Paratypes are deposited in the Finnish Museum of Natural History, University of Helsinki, Tiroler Landesmuseum Ferdinandeum (Innsbruck, Austria) and in the research collections of J. Junnilainen, J-P. Kaitila & Bo Wikstroem . The holotype is available on loan by request through the Finnish Museum of Natural History, University of Helsinki or directly from the first author. Etymology. The name of the new species is derived from the Pirin mountain range, where the new species is widely distributed. Diagnosis. Considering similar forewing ground colour and markings, Incurvaria pirinella (Figs 3-5) is externally closest to I. circulella (Fig. 14), but is easy to separate from it by the dark grey-brown fringe and distinct white spot in the forewing fringe present in I. circulella . Incurvaria pirinella is also widely allopatric as I. circulella occurs only in northern latitudes in Europe, e.g. the northern part of Sweden and Finland. Although I. triglavensis is rather variable externally, it is always easy to separate from I. pirinella based on its relatively narrow forewing shape, paler ground colour and differences in forewing markings (Figs 6-11); see details in Suppl. material 2. Incurvaria pirinella is easy to separate externally from Scandinavian I. vetulella (Figs 12-13) by a white narrow dorsal spot situated 2/5 from base and extending more than halfway across the forewing in I. pirinella . North European I. vetulella also typically have a distinctly broader dorsal spot, sparser forewing scaling and usually a pale medial area in the fringe, which is always absent in I. pirinella . Male genitalia of I. pirinella are most similar to those of I. triglavensis , but small constant differences are present. I. pirinella has a stouter valva and vinculum. The valva of I. pirinella is broader in middle and the margin of the sacculus is more bulged (Figs 21-22). The vinculum of I. pirinella is relatively short and broad with clearly concave lateral margins. In I. triglavensis , the apex of the valva is more elongated and the vinculum is clearly longer and more slender with straight or slightly concave lateral margins (Figs 1 a-b , 17e, 19b, 20e). The lateral arms of the transtilla are more robust (Fig. 20a), and submedial anterior projections shorter in I. pirinella (Figs 1c, 20d). The length of the transtilla's medial knob with anterior submedial projection is 0.26-0.27 mm in I. pirinella and 0.29-0.33 mm in I. triglavensis . The juxta is longer (0.95-0.98 mm) in I. pirinella than in I. triglavensis (0.8-0.85 mm) (Fig. 16, Suppl. material 1). Also, the ratio between the length of the juxta versus the transtilla's medial knob with its anterior projections is different between the species, being 3.56-3.77 in I. pirinella and 2.76-2.83 in I. triglavensis (see Suppl. material 1). The shape and angle of the distal thorn/hook-shaped projection of the phallus show variation, that is at least partly due to the position of the phallus in genitalia preparations (Figs 15d1, 18 a-b ); thus, this does not provide a good characteristic. However, the ratio between the maximum length of phallus versus the length of the distal projection seems to be different between the species as well (Figs 18 a-b ). In the examined samples, it was 3.34-3.78 for I. pirinella and 3.82-4.07 for I. triglavensis . Shape, size and ratio between length/width of abdominal segments T8 and S8 are different: See shape in Figs 23, 24, 25 and size and ratio measurement values in Suppl. material 1. Although I. triglavensis is a rather variable species, additional slightly different but sometimes overlapping features are found in other parts: details shown in Suppl. material 1. Genitalic structures of both sexes are overall more strongly sclerotised in I. pirinella . In the female genitalia, differences between I. triglavensis and I. pirinella are found in size of the oviscapt, especially distance from tip to bottom of basal excavation. In the examined material, it was 0.12 mm in I. pirinella and 0.09-0.11 mm in I. triglavensis (Fig. 2). The length of the apophyses posteriores is 2.70 mm in I. pirinella and 2.48-2.55 mm in I. triglavensis . Segment VIII also shows differences, but this feature shows overlap and therefore is not useful for determination of a single specimen. Differences between membranous parts of the bursa are not included as diagnostic characteristics, because they may show variation due to the age of specimens and may be altered after copulation. For more details, see Suppl. material 1 for morphometrics of genitalia of I. triglavensis and I. pirinella and photos of female genitalia (Figs 26-28, 30) of both species. Figure 1. Incurvaria pirinella , green and I. triglavensis magenta. Shape of male genitalia compared: 1a. left valva; 1b. vinculum; 1c. knob of transtilla with anterior projections. Figure 2. Incurvaria pirinella and c-f. I. triglavensis length of female oviscapt compared with coloured horizontal lines. Slide numbers a. GPPbf3327; b. GPPbf3328; c. GPPbf3324; d. GPPbf3325; e. GPPH-Inc17; f. GPPbf3335. Scale bar: 1 line = 0.01 mm. Figures 3-5. Incurvaria pirinella habitus of adult. 3. Male (Holotype). 4. male (Paratype). Ilindenci road. Coll. Kaitila 5. Female (Paratype). Slavianka. Coll. Kaitila. Figures 6-11. Incurvaria triglavensis habitus of adults. 6. Male. Austria Osttirol-Defereggen Gebirge 2200m 2013.vii.22 H. Deutsch leg.; 7. Male. Slovenia Julijske Alpe Kamin, Sudseite 1700m 19.vii.1997 H. Deutsch leg.; 8. Male. Slowenien-Steiner Alpe 1500m 1992.viii.12 Habeler leg.; 9. Female. Slovenia, Steiner A. Krvavec Veli zwoh 20.vii.1992 1950m Habeler leg.; 10. Female. Austria Salzburg Nockgebiet Matchanshoehe 1900m 24.vi.1999 Habeler leg.; 11. Female. Austria Steiermark Woeitzer Tauern Greimberg 1700m 26.vii.2001 Habeler leg. All in coll. TLMF. Figures 12, 13. Incurvaria vetulella habitus of adults. 12. Male. Finland EnL Enontekioe Tuolljehuhput 7676:8265, 13.-15.vii.2009 Marko Mutanen leg. & det.; 13. Female. Finland EnL Enontekioe Saana 767:825, 16.vii.2012, Marko, Nestori & Anttoni Mutanen leg., coll. Marko & Tomi Mutanen. Description. Male (Figs 3-4). Wingspan 16.5-17.0 mm. Labial palp covered with brown and ochre-yellow scales, most pronounced on inner surface, 2nd segment equal in length to diameter of eye, pointed forward, 3rd segment half as long, turned upward. Antenna faintly ringed with dark brown and paler greyish-brown scales, slightly serrate, reaching three-quarters length of forewing. Head with yellow tufted scales. Thorax and tegula brown. Forewing relatively broad and roundish, ground colour grey-brown; two subtriangular yellowish-white dorsal spots, first narrower, 2/5 from base, extending over half width of forewing, second smaller, close to termen; a small yellowish-white triangular costal spot rather close to apex, sometimes a second usually smaller quadrangular yellowish-white spot close to the first one; fringe as ground colour. Hindwing and fringe fuscous. Abdomen and legs pale greyish-brown. Ventral side of forewing and fringe fuscous throughout except for distinct yellowish-white costal and dorsal markings. Female (Fig. 5). Wingspan 16.0-16.5 mm. Differs from male in darker grey-brown ground colour of forewings; two subapical costal spots always present; female dorsal spots larger and extending even more than 2/3 across forewing towards costa, the second white dorsal spot at tornus extending across the wing and sometimes reaching inner costal spot forming a complete fascia. Antennae not serrate. Male genitalia (Figs 1, 15, 23, 25). Seven specimens examined. Uncus (Fig. 15b2) and tegumen (Fig. 15b1) short, forming together a relatively broad subrectangular plate, posterior margin of uncus somewhat roundish, medially and sublaterally slightly concave. Socii (Fig. 15b3) distinctly sclerotised, round-shaped and hirsute. Transtilla (Figs 15a3-a5) a conspicuous structure; medial knob (Fig. 15a3) and its lateral arms (Fig. 15a5) comparatively robust and strongly sclerotised; arms ending laterally to relatively low and broad subrectangular plate-like structure (Fig. 17c); submedial anterior projections (Fig. 15a4) digitate and relatively short. Valva relatively short and robust; costal margin comparatively slightly concave; dorsal margin of sacculus (Fig. 15a2) hirsute and somewhat obtuse-angled at 2/3 from base (Figs 21b, 22b); cucullus (Fig. 15a1) hirsute, moderately roundish with group of strong setae subdorsally and second smaller group of strong setae subdorsally near sacculus; apex hirsute and not significantly elongated (Fig. 21a). Vinculum V-shaped and relatively stout; lateral margins strongly concave (Fig. 15a6); posterior half strongly tapering towards slender distal half; apex moderately broad and roundish. Juxta (Figs 15c, 16) bifurcate, moderately long (length 0.95-0.98 mm), tip of lateral projections relatively pointed (Fig. 16a), medial part relatively narrow (Fig. 16b), basal part arrowhead-shaped and moderately strongly sclerotised (Fig. 16c). Phallus (Fig. 15d) relatively long (length without distal hook 1.21-1.27 mm); distally hook-shaped, also a relatively long (0.32-0.38 mm) strongly sclerotised projection (Figs 15d1, 18a-b), basal part broad plate-like, forming about 90° angle with hooked distal part; vesica with moderately short and broad irregular sclerotised plate at base (Fig. 15d2). Abdominal segments S8 and T8 (Figs 23, 25) as subrectangular plates. S8: narrow posterior margin slightly concave; wide anterior margin roundish; lateral margins slightly convex. T8: short and broad; posterior margin concave; anterior and lateral margins convex. Figure 14. Incurvaria circulella male, Finland KiL. Muonio 7540:8362, 4.vii.2014, Marko, Anttoni & Nestori Mutanen leg., coll. Marko & Tomi Mutanen. Figure 15. Incurvaria pirinella male genitalia unrolled: a. vinculum, transtilla and valvae; a1. apex of valva; a2. margin of sacculus; a3. medial knob of transtilla; a4. anterior submedial projection of transtilla; a5. strongly sclerotised lateral arm of transtilla; a6. strongly concave lateral margin of vinculum. b. Unrolled tegumen and uncus; b1. tegumen; b2. uncus; b3. socii. c. Juxta. d. Phallus from semilateral and lateral view; d1. hook-shaped distal projection of phallus; d2. sclerotised plate in vesica. Scale bar: 1 line = 0.05 mm. Figure 16. Incurvaria pirinella (left) and I. triglavensis (right) juxta: a. tip of lateral arm; b. medial part of lateral arm; c. basal part. Scale bar: 1 line = 0.05 mm. Figure 17. Incurvaria pirinella left and I. triglavensis right male genitalia compared: a. apex of valva; b. medial knob of transtilla; c. subrectangular plate-like structure in lateral end of transtilla arm; d. anterior submedial projection of transtilla; e. lateral margin of vinculum; f. apex of vinculum. Scale bar: 1 line = 0.05 mm. Figure 18. a. Incurvaria pirinella and b. I. triglavensis phallus from semilateral and lateral view. mp3a-c. indicate measurement points parts of phallus. Scale bar: 1 line = 0.05 mm. Female genitalia (Figs 2, 26-28, 30). Three specimens examined. Tip of oviscapt triangular, lateral margins concave, 9-11 small distinct teeth on both sides; basal notch somewhat U or V-shaped; lateral projection pointed and relatively short (Fig. 29), shape and size of lateral margin teeth and basal notch variable. Apophyses relatively long (Figs 26-27, 30). Segment VIII a strongly sclerotised subrectangular plate (Figs 26a, 27); posterior 2/5 strongly tapering towards posterior end, anterior margin rounded. Vestibulum a subrectangular membranous sack (Fig. 28a), about twice as long as wide, posterior 1/3 angular, medially somewhat narrower, anterior half with rounded expansions on both lateral margins (expansions not clearly visible in photo because they are turned behind vestibulum), anteriorly membranous (Fig. 28b) funnel-shaped structure probably belonging to ductus bursa, 1/6 the length of remaining part of ductus bursae (Fig. 28c). Structure of ductus bursae complex, posterior 2/5 narrow tube-like, slightly widening anteriorly, anterior 3/5 broad sack-shaped, anterior end strongly tapered towards corpus bursae. Spermatheca spiral-shaped. Corpus bursae (Fig. 28d) a roundish sack, signa absent. Abdominal segments S8 and T8 relatively large subrectangular plates (Fig. 31). S8: posterior margin roundish and hirsute; anterior margin slightly concave; lateral margins almost straight. T8: posterior margin concave; broader anterior margin straight; lateral margins somewhat roundish. Figures 19, 20. Male genitalia from ventral view. 19. Incurvaria pirinella left and I. triglavensis right: a. margin of uncus; b. lateral margin of vinculum. 20. Male genitalia free-floating ventral view. I. pirinella left and I. triglavensis right: a. robust and strongly sclerotised lateral arms of transtilla; b. apex of valva; c. medial part of valva; d. medial knob of transtilla together with it anterior submedial projections; e. lateral margins of vinculum; f. apex of vinculum; mp1a-b. are measurement points length of vinculum. Scale bar: 1 line = 0.05 mm. Second smaller scale bar parallel to transtilla knob: 1 line = 0.01 mm. Figures 21, 22. Male genitalia from lateral view. 21. Incurvaria pirinella left and I. triglavensis right: a. apex of valva; b. sacculus; c. medial knob of transtilla; d. uncus; e. tegumen). 22. Male genitalia free-floating lateral view. I. pirinella left and I. triglavensis right: a. apex of valva; b. sacculus; c. vinculum; d. medial part of valva; mp2a-b. are measurement points length of valva. Scale bar: 1 line = 0.05 mm. Figures 23, 24. Male abdominal tergites T8 and S8. 23. Incurvaria pirinella ; 24. I. triglavensis . Scale bar: 1 line = 0.05 mm. Molecular diagnostics (Fig. 33). Barcode Index Number (BIN, see Ratnasingham and Hebert 2013 ): BOLD:ACW2589. The nearest neighbour of I. pirinella in our dataset is I. triglavensis (BOLD:AAI8097) with a K2P minimum divergence of 4.74%. The next closest species are I. vetulella (BOLD:AAD4279) with 7.06%, I. ploessli (BOLD:AAP5641) with 7.07% and I. oehlmanniella (BOLD:AAD1334) with 8.45% minimum divergences, respectively. Variation. The external habitus of both sexes is rather constant. Females are darker overall than males. One male has a second yellowish white costal spot near forewing apex. Sometimes the tornal spot and inner subapical costal spot form a complete fascia in the female forewing. Genitalic structures of both sexes are only slightly variable, whereas those of I. triglavensis show significant variation both externally and in genitalic structures of both sexes. Distribution. Known from four different localities in the south-west corner of Bulgaria around the Struma river valley and its adjacent regions, which all belong to the Blagoevgrad district and the Pirin mountain range. The elevational range is wide: 200-1200 m, at least. Figure 25. Male abdominal segments of Incurvaria pirinella (left) and I. triglavensis (right). Male abdominal segments. Figures 26, 27. 26. Incurvaria pirinella (left) and I. triglavensis (right). 27. I. pirinella (left) and I. triglavensis (right). Female genitalia from ventral view. Scale bar: 1 line = 0.05 mm. Figures 28, 29. 28. Incurvaria pirinella (left) and I. triglavensis (right) female bursae: a. vestibulum; b. membranous funnel-shaped structure in posterior end of ductus bursa; c. ductus bursae; d. corpus bursae. Scale bar: 1 line = 0.05 mm. 29. I. pirinella (left) and I. triglavensis (right). Oviscapt of female genitalia. Scale bar: 1 line = 0.01 mm. Biology. Many specimens were captured with insect nets during daytime, and females especially were disturbed in the evening from shrubs such as Rosa L. Males were observed swarming early in the morning, but they were also caught with artificial light late at night, which is an unusual collecting method for the species of the vetulella -group in the Alps (P. Huemer pers. comm.) and northern Europe (own observations, and although summer nights are light in the North, I. circulella comes readily to light). Incurvaria pirinella is an early species, flying at lower elevations in early May and at higher elevations in late May and June. The biology of the early stages remains unknown. The food-plant of I. vetulella is reported to be Vaccinium L. ( Ericaceae ), especially V. myrtillus L. ( Klimesch 1961 , Wojtusiak 1976 ), but the life history details for the other members of the vetulella -group remain largely unknown. It is suspected that the other European members of the group are also associated with Vaccinium ( Huemer 1993 ). However, no species of Vaccinium or Erica L. are present, at least not in most localities where we have found I. pirinella , suggesting that they are not its food-plants. Specimens of I. pirinella have been collected from completely different habitats from the other species of the vetulella -group, which in the Alps are always found in alpine areas usually above timberline and especially from north-facing slopes. In Scandinavia, I. vetulella occurs in alpine and subalpine habitats, but also boreal coniferous forest and boggy habitats. Incurvaria pirinella seems to prefer open xerothermic south-facing slopes with rich deciduous forest edges of Quercus L., and Fagus L. (Fig. 34), and a variety of shrubs, including Crataegus L., Corylus L., and Rosa , sometimes also Cornus L., Malus Mill., Pyrus L., and Chamaecytisus Link. Along with I. pirinella , I. masculella , well known as an early flying species, was collected in numbers. Figures 30-32. 30. a. Incurvaria pirinella ; b. and c. I. triglavensis . Female genitalia free-floating from lateral view. Scale bar: 1 line = 0.05 mm. 31. Incurvaria pirinella ; 32. I. triglavensis . Female abdominal tergites S8 and T8. Scale bar: 1 line = 0.05 mm. Figure 33. Maximum likelihood tree as based on DNA barcodes (658 bp fragment of the mt COI gene) of European Incurvaria species and three members of other genera of Incurvariidae . The node support values are based on 500 bootstrap replicates. The tree was rooted on Crinopteryx familiella ( Crinopteryginae ). Figure 34. Habitat of Incurvaria pirinella Slavianka, Southern Pirin 1000 m a.s.l.