DNA barcoding as a screening tool for cryptic diversity: an example from Caryocolum, with description of a new species (Lepidoptera, Gelechiidae) Author Huemer, Peter Author Karsholt, Ole Author Mutanen, Marko text ZooKeys 2014 404 91 111 http://dx.doi.org/10.3897/zookeys.404.7234 journal article http://dx.doi.org/10.3897/zookeys.404.7234 1313-2970-404-91 E1B904C8A4AE42E990B060E0B1D83042 Caryocolum amaurella (Hering, 1924) Figs 4-5, 8-9, 12-13, 16-17 Lita amaurella Hering 1924 : 82, Figs 11-12. Lita viscariae Schuetze 1926 : 171. Material examined. Lectotype ♂[with nine labels]: 'Fennia Ab Bromarf' 'R. F: tius' '21.7.21' [piece of celluloid where genitalia was mounted] 'Type ♂' [red] 'Lita amaurella m. det. Mart. Hering ♂' 'Mus. Zool. H:fors spec. typ. No 7016 Lita amaurella Hering' 'Lita amaurella m. ♂ Sch.-Armatur Bromarf 21.7.21 Fabritius' 'LECTOTYPE O. Karsholt design.'. Finland: 1 ♂, Ab, Naantali, 25.8.1965, leg. Karvonen, slide Karsholt 2719; 2 ♂, N, Ekenaes , 17.-20.7.1980, leg. Fibiger; 1 ♂, N, Helsinki, 25.7.1982, leg. Schnack; 1 ♂, N, Borga lk., Tirmo, 19-20.7.1980, leg. Fibiger; 5 ♂, same data, but 1.-2.8.1982, leg. Schnack; 2 ♂, U, Sluntle, 18.-31.7.1982, leg. Karsholt (all ZMUC); 6 ♂, 5 ♀, U, Porvoo, 6698:3426 Anaes , e.l. 2012 ( Lychnis viscaria ), leg. Hirvonen (ZMUO); 4 ♂, V, Dragsfjaerd , 664:3249, 2008, leg. Mutanen & Vaelimaeki (ZMUO); 1 ♂, U, Hanko, 6642:3289, 2007, leg. Mutanen & Vaelimaeki (ZMUO). Sweden: 2 ♂, Sk, Maglehen, 10.7.1965, leg. Svensson (TLMF, ZMUC); 1 ♂, Sm, Hoegsby , 13.7.1968, leg. Johansson; 1 ♂, Oel , Oedeshoeg , 17.7.1972, leg. Karsholt, slide Karsholt 1806; 1 ♂, St. Alvar, Tornroer , 25.7.1997, leg. Hendriksen, slide Hendriksen 1953; 2 ♂, Oel , Gardby , 2.8.1999, leg. Hendriksen, slide Hendriksen 2411, 2415; 1 ♂, same data, but 22.7.2000; 1 ♂, Gtl., Hejnum Haeller , 30.7.1977, leg. Hendriksen, slide Hendriksen 1944; 1 ♂, Oeg , Oedeshoeg , 17.7.1972, leg. Karsholt; Upl., Film, 12.7.1995, leg. Hendriksen (all ZMUC). Norway: 2 ♂, On, Vinstra, 19.-29.7.1983, leg. Karsholt & Michelsen, slide Karsholt 4294, 4295; 2 ♀, same data, but 4.-5.7.1987, leg. Karsholt, slide Hendriksen 2099; 2 ♂, same data, but 9.8.1996, leg. Hendriksen (all ZMUC). Denmark: 1 ♂, Bornholm, Ro , 7.1892, leg. Gudmann, slide Wolff 2593; 5 ♂, 1 ♀, same data, but 28.7.1978, leg. Schnack, slide Schnack 1118; 1 ♂, Bornholm, Gudhjem, 1 ♂, 2 ♀, 29.6-3.7.1920, leg. Gudmann, slide Wolff 2625, 3682; 1 ♂, 1 ♀, same data, but e.l. 5.1921 ( Lychnis viscaria ), bred 21. & 28.6.1921, leg. Gudmann, slide Wolff 3681(all ZMUC); 6 ♂, 8 ♀, Bornholm, Hammeren, 18.7.1977, leg. Karsholt & Schnack, slide Hendriksen 1767, Karsholt 2948 TLMF, ZMUC); 2 ♂, same data, but 25.7.1977, leg. Schnack; 4 ♂, same data, but 16. -25.7.1978, leg. Schnack; 4 ♂, same data, but 19.-22.7.1979, leg. Hendriksen; 6 ♂, same data, but 29 -30.7.1981, leg. Hendriksen, slide Hendriksen 385, 561, 722; Bornholm, Randklove , 1 ♂, 22.7.1977, leg. Schnack; Bornholm, 1 ♀, Ringe Bakker, 16.7.1978, leg. Schnack (all ZMUC). Germany: 1 ♂, 1 ♀, Lausitz, Umg. Bautzen, e.l. 1935 ( Lychnis viscaria ), leg. Starke (BMNH); 2 ♂, Thueringen , Bad Blankenburg, 14.7.1964, leg. Steuer (TLMF); 1 ♂, Thueringen , Bad Blankenburg, 8.7.1972, leg. Steuer (TLMF). France: 2 ♂, Alpes Maritimes, Col de la Cayolle, 2200-2300 m, 29.-30.7.2005, leg. Skou, slide Hendriksen 5364 (ZMUC). Austria: 1 ♂, Niederoesterreich , Jauerling, 24.7.1935 (TLMF); 2 ♂, Oberoesterreich , Windischgarsten, Veichltal, 23.7.1976, leg. Wimmer (TLMF); 1 ♂, Oberoesterreich , Waldhausen, Schwarzenberg, 6.8.1997, leg. Wimmer (TLMF); 9 ♂, Kaernten , St. Jakob im Lesachtal, Mussen E, 1680-1800 m, 4.8.1999, leg. Huemer & Erlebach (TLMF). Slovakia: 1 ♀, Pol'ana , 28.7.1989, leg. Patocka (ZMUC). Macedonia: 4 ♂, NP Mavrovo, Korab, Korabska jezero, Kobilino pole, 2080-2180 m, 28.7.-1.8.2011, leg. Huemer & Tarmann (TLMF). Turkey: 2 ♂, 1 ♀, prov. Sivas, 10 km W Goeruen , 1650 m, 27.7.1989, leg. Esser & Fibiger, slide Huemer GU 90/130, GU 91/215; 4 ♂, prov. Erzerum, Kop Pass, 1750 m, 15.-16.9.1993, leg. Fibiger, slide Hendriksen 2889, 2894; 1 ♂, prov. Erzincan, Kizildaĝ , Gecidi , 2100 m, 19.8.1993, leg. Schepler, slide Hendriksen 2384 (all ZMUC). Diagnosis. See above. Description. Adult (Figs 4-5). Wingspan 10-14 mm. Segment 2 of labial palpus bone-white on inner and upper surface, blackish grey on outer and lower surface; segment 3 almost black with light tip. Antenna black, indistinctly lighter ringed. Head with light yellow frons and black neck; thorax and tegula black mottled with brown. Forewing blackish grey mottled with some light brown; base black; two indistinct black spots in fold; one oblique spot above it and one in cell; some white scales before and after these spots; costal and tornal spot small, white, rarely fused. Hindwing light grey. Variation. The colour of the forewings varies from greyish to blackish. Worn specimens look lighter than fresh ones. Sometimes there are no white scales in the middle of the wing. Male genitalia (Figs 8-9, 12-13). Uncus subovate; tegumen stout; transtilla membranous; valva short, moderately stout, apex rounded; sacculus short, with angular ventral and convex dorsal margin, apically pointed; posterior margin of vinculum with deep medial emargination and slight medial incision, two pairs of short processes developed; saccus long, comparatively broad at base, distal part gradually tapered; phallus long and slender, weakly curved, with some minute cornuti apically. Female genitalia (Figs 16-17). Segment VIII without processes, subgenital plate sub-triangular, with numerous narrow folds, separated from sclerotized lateral plates by membranous zone; apophysis anterior slightly longer than segment VIII; antrum moderately short, about one-third to one-quarter length of apophysis anterior, broadly funnel-shaped; posterior part of ductus bursae with pair of lateral sclerites, extending to anterior third of apophysis anterior, and with two tiny sclerites anteriorly; signum with crescent-shaped base, short and stout, strongly bent hook. Figures 8-9. Male genitalia. 8 Caryocolum amaurella (Hering), Finland, slide GU 14/1373 P.Huemer; 9 Caryocolum amaurella , Finland, slide GU 14/1374 P.Huemer. Figures 10-13. Details of male genitalia (vinculum-valva-complex). 10 Caryocolum crypticum sp. n., paratype, Italy, slide GU 86/041 P.Huemer 11 Caryocolum crypticum sp. n., paratype, Italy, slide GEL 1215 P.Huemer 12 Caryocolum amaurella , Finland, slide GU 14/1373 P.Huemer 13 Caryocolum amaurella , Finland, slide GU 14/1374 P.Huemer. Figures 14-15. Female genitalia. 14 Caryocolum crypticum sp. n., holotype, slide GEL 1234 P.Huemer 15 Caryocolum crypticum sp. n., paratype, Italy, slide GEL 1232 P.Huemer. Figures 16-17. Female genitalia. 16 Caryocolum amaurella , Finland, slide GU 14/1372 P.Huemer 17 Caryocolum amaurella , Finland, slide GU 14/1371 P.Huemer. Molecular data. The intraspecific divergence of the barcode region is high with mean intraspecific divergence of 3.01% and maximum intraspecific divergence of 4.62% (n=9). The distance to the nearest neighbour Caryocolum mucronatella is 5.21%, the divergence to the morphologically closest Caryocolum crypticum is 6.82%. The extraordinary high intraspecific divergence with 4 haplotypes is partially related to geographical pattern. However, we also found two haplotypes within one population in Finland and morphology does not support cryptic diversity. Distribution. With certainty known from scattered records from northern and Central Europe and Turkey. All the specimens from north of the Alps that we have been able to cross-check are correctly attributed to Caryocolum amaurella . However, recent records from Ukraine ( Bidzilya and Budashkin 2009 ) and Russia (southern Ural Mountains) ( Junnilainen et al. 2010 ) have to be re-examined due to a possible mix-up with Caryocolum crypticum . Records from Switzerland are dubious, and at least in one instance refer to the new species, whereas those from France ( Nel 2003 ) are confirmed (see Huemer and Karsholt 2010 , Fig. 154c). Bionomics. The larva has been recorded feeding on Silene viscaria (L.) Jess (= Lychnis viscaria L. ( Caryophyllaceae ) ( Huemer and Karsholt 2010 ), while the other stated host-plants, namely Silene otites (L.) Wibel ( Burmann 1990 ) and Silene nutans L. ( Huemer 1989a ), refer to Caryocolum crypticum . Schuetze (1926 , 1931 ) gives a detailed account of the life-history. The larva feeds in April and May in the young terminal leaves which are - without spinning - attached to a tube where the larva is hidden. Dark frass is frequently extruded at the tip of the larval dwelling. Later it bores into the stem and the shoots often become swollen and stunted. Pupation takes place on the ground in a cocoon among debris. The adult occurs from late June to early September and it is attracted to light. Caryocolum amaurella is restricted to warm and sunny habitats such as dry meadows and pastures. Vertical distribution: from lowland localities to about 2200 m in the Alps. Remarks. Lita amaurella was described from an unspecified number of specimens of both sexes ('♂, ♀') from Finland (Bromarf) ( Hering 1924 ). In order to stabilize nomenclature, a male, labelled as type, in ZMUH is here designated as lectotype (see data above). Lita viscariae was described from 67 specimens reared from Silene viscaria from Eastern Germany (near Rachlau) ( Schuetze 1926 ). No type material was traced during this and earlier studies ( Huemer 1988 ), but the original descriptions and topotypical material leave no doubt about the identity. Turkish specimens of Caryocolum amaurella examined by us differ from European specimens of this species by the thorax with rusty brown posterior part and the rusty brown tegulae with blackish brown base, similar to Caryocolum crypticum , and they are thus hardly separable from the latter on external characters. The genitalia of both sexes of Caryocolum amaurella from Turkey agree in all details with those of European Caryocolum amaurella and, because no contradicting genetic data is currently available, we consider them as belonging to that species. One of the examined specimens of Caryocolum amaurella from Turkey was collected in the same locality (Kizildaĝ Gecidi , prov. Erzincan) as a specimen Caryocolum iranicum in ZMUC. The latter species, which is only known from a few specimens, differs, as stated above, in characters of the male genitalia.