Deep intraspecific DNA barcode splits and hybridisation in the Udea alpinalis group (Insecta, Lepidoptera, Crambidae) – an integrative revision

Abstract The analysis of mitochondrial COI data for the European-Centroasian montane Udea alpinalis species group finds deep intraspecific splits. Specimens of U. austriacalis and U. rhododendronalis separate into several biogeographical groups. These allopatric groups are not recovered in the analyses of the two nuclear markers wingless and Elongation factor 1-alpha, except for U. austriacalis from the Pyrenees and the French Massif Central. The latter populations are also morphologically distinct and conspecific with Scopula donzelalis Guenée, 1854, which is removed from synonymy and reinstated as Udea donzelalis (Guenée, 1854) stat. rev. Furthermore, Udea altaica (Zerny, 1914), stat. n. from the Mongolian central Altai mountains, U. juldusalis (Zerny, 1914), stat. n. from the Tian Shan mountains of Kazakhstan, Kyrgyzstan and NW China, and U. plumbalis (Zerny, 1914), stat. n. from the Sayan Mountains of Northern Mongolia are raised to species level, and lectotypes are designated. Evidence of introgression of U. alpinalis into U. uliginosalis at three localities in the Central Alps is presented. A screening for Wolbachia using the markers wsp, gatB and ftsZ was negative for the U. alpinalis species group, but Wolbachia was found in single specimens of U. fulvalis and U. olivalis (both in the U. numeralis species group). We do not find evidence for the conjecture of several authors of additional subspecies in U. rhododendronalis, and synonymise U. rhododendronalis luquetalis Leraut, 1996, syn. n. and U. r. ventosalis Leraut, 1996, syn. n. with the nominal U. rhododendronalis (Duponchel, 1834).

The U. alpinalis species group is characterised by a homogenous wing colouration with an inconspicuous maculation. Species of this group exhibit sexual dimorphism, with females having shorter, more acute forewings and the dorsal side of the hindwings being usually darker than in males. Furthermore, the U. alpinalis group is distinguished from other Udea species groups by the presence of a sclerotised protrusion of variable shape on the posterior phallus apodeme. The species inhabit montane regions, and the larvae exhibit a range of feeding habits from monophagy to polyphagy on a variety of herbaceous plants (Lhomme 1935;Huemer and Tarmann 1989;Slamka 2013).
Several authors suspect that the actual species diversity in the U. alpinalis group in Europe is higher than formal descriptions in the literature indicate, specifically in relation to U. austriacalis and U. rhododendronalis . This suspicion is based on small differences in genitalia structure and wing maculation (Zerny 1914, Filipjev 1925, Leraut 1996, Slamka 2013. For U. alpinalis, Galvagni (1933), Weber (1945), and Panigaj and Kulfan (2012) found considerable variability in the forewing maculation. In preliminary COI Barcode cluster analyses, several specimens that have been identified as U. uliginosalis based on wing maculation clustered with U. alpinalis, raising questions about the correct species identification.
In this study, these taxonomic suspicions are addressed through the analysis of morphological as well as mitochondrial and nuclear genetic data. We present, investigate, and, where possible, explain this unsettled question of intraspecific diversity of U. rhododendronalis, U. austriacalis and the U. uliginosalis-U. alpinalis species pair.

Materials and methods
The study is based on adult specimens of Udea alpinalis, U. austriacalis, U. cretacea, U. rhododendronalis, and U. uliginosalis, collected at different localities in Europe and Central Asia. The genetic dataset was complemented with sequences of U. bourgognealis, U. carniolica, U. murinalis, and U. nebulalis. Udea ruckdescheli Mally, Segerer & Nuss, 2016 of the U. numeralis species group (sensu Mally and Nuss 2011) served as outgroup. The morphologically investigated material is summarised in the 'material examined' sections of the respective species in the taxonomic results, the genetic data are summarised in Table 2. Molecular data from three different genes were used for the dataset: the 5' half of the mitochondrial Cytochrome c oxidase subunit 1 (COI) gene (the "DNA Barcode"), 657 base pairs (bp) in length, the 5' part of the nuclear Elongation factor 1-alpha (EF1a) gene (780 bp), and the nuclear Wingless gene (372 bp). In addition, a screening for molecular traces of Wolbachia infections was done by amplifying the bacterial markers Wolbachia surface protein (wsp), aspartyl/glutamyl-tRNA(Gln) amidotransferase subunit B (gatB) and Filamenting temperature-sensitive mutant Z (ftsZ).
COI Barcode sequences and specimen data for the Udea species of interest were obtained from ongoing Barcoding projects of PH and MN on the Barcoding of Life Database (BOLD, www.boldsystems.com), Version 4. The DNA lab protocols at the Canadian Centre for DNA Barcoding (CCDB) are available at http://www.ibolproject. org/resources.php. Barcodes with less than 500 bp were excluded, and public records retrieved from NCBI GenBank were included. In addition, DNA Barcode sequences were obtained for several specimens through PCR and sequencing in the DNA labs of the Senckenberg Natural History Collections Dresden, Germany (SNSD) and the Institute of Biology at the University of Bergen, Norway (UiB).
For DNA lab protocols at SNSD see Mally and Nuss (2011). The DNA lab protocols at UiB are as follows: The abdomen was detached from the dried specimen and DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's protocol. Gene sequences were amplified in 25 µl reactions from 2 µl DNA extract using 400 nM of each primer, 800 µM dNTP mix, 2.5 µl Taq buffer (incl. MgCl 2 ), 0.75u TaKaRa Ex Taq DNA Polymerase and distilled water added up to 25 µl in total per reaction. COI primers were HybLCO (forward) and HybNancy (reverse) (Folmer et al. 1994, Wahlberg andWheat 2008), EF1a primers were HybOscar-6143 (forward) and Bosie-6144 (reverse) (Wahlberg andWheat 2008, Haines andRubinoff 2012), and Wingless primers were HybLepWg1 (forward) and HybLepWg2 (reverse) (Wahlberg and Wheat 2008). Those primers contained the universal primer tail pair T7/T3 ('Hyb' in the primer names; Wahlberg and Wheat 2008), which were used for sequencing. The wsp gene was amplified using the primers WspecF and WspecR (Werren and Windsor 2000). In cases of lacking amplification success the internal primers INTF1 and INTR2 (Sakamoto et al. 2006) were used. The genes gatB and ftsZ were amplified using the primers of Baldo et al. (2006) in combination with the universal forward (T7 promoter) and reverse (T3) tails (Wahlberg and Wheat 2008 Sakamoto et al. (2006) was used for For wsp, and the protocol of Baldo et al. (2006) for gatB and ftsZ.
PCR results were examined via gel electrophoresis on a 1 % agarose gel and GelRed as dye agent. Successful PCR samples were cleaned with ExoSAP and subsequently amplified in Sanger-sequencing PCR reactions for both primers using the BigDye kit and this setup: 0.5-3.0 µl of PCR sample (depending on the sample's band thickness on the agarose gel), 160 nM primer, 1 µl buffer, 0.5 µl BigDye, and adding up distilled water to 10 µl in total per reaction. Sequencing was conducted at the sequencing facility of UiB, Dept. of Molecular Biology. PCR and sequencing PCR were performed on a Bio-Rad 1000 thermal cycler; ExoSAP clean-up was done on a MJ Research PTC-200 thermal cycler.
Dissection of genitalia was performed according to Robinson (1976), with modifications. In order to preserve the tympanal organ, the abdomen was cut open longitudinally along one pleural membrane, detached from the genitalia, cleaned, and embedded under a separate cover slip next to the cover slip with the genitalia. Morphological structures were investigated using a Leica M125 stereomicroscope. Photographic documentation of imagines was done with a Canon EOS 60D in combination with a Canon EF 100mm 1:2,8 Macrolens and Canon EOS Utility Version 2.10.2.0 on a Windows PC. A Leica CTR6000 microscope in combination with a Leica DFC420 camera and Leica Application Suite programme (Version 3.8.0) on a Windows PC was used for documentation of the genitalia. Images were edited in GIMP 2.8.6. The distribution maps were generated with DIVA-GIS version 7.5.0.0 (Hijmans et al. 2001) and SRTM 90 m digital elevation data (Jarvis et al. 2008).

Abbreviations
The abbreviations of the insect collections follow Evenhuis (2017).

GTR
General time reversible substitution model (see Rodriguez et al. 1990

Molecular results
In total, genetic data were analysed for 80 specimens (see Table 2) of the U. alpinalis group, specifically for U. alpinalis, U. austriacalis, U. rhododendronalis and U. uliginosalis. COI data were available for 77 specimens, EF1a for 31 specimens, and wingless for 29 specimens. The low coverage of nuclear genetic data is due to the age of most specimens, with the nuclear genome being too fragmented to be sequenced with the classical Sanger approach. Analysis of COI resulted in a gene tree ( Fig. 1) with several deep intraspecific, geographically coherent clades for U. austriacalis and U. rhododendronalis. Udea austriacalis splits into three groups: (aus1) Pyrenees and the French Massif Central (green clade in Fig. 1), (aus2) the French and Italian Maritime Alps (red clade), and (aus3) the Central and Eastern Alps as well as the Balkan Mountains (black clade). A fourth clade within U. austriacalis is represented by a single specimen of U. cretacea, indicating that U. austriacalis is non-monophyletic. Udea rhododendronalis splits into three COI clades: (rho1) Pyrenees (orange clade in Fig. 1); (rho2) Alps (black clade); (rho3) Southern Balkan Mountains (blue clade). Seven specimens of U. uliginosalis (marked in pink in Fig. 1) group with the U. alpinalis clade instead of with the other U. uliginosalis specimens. These seven specimens originate from three different localities in the Central Alps (see Tab. 2); the specimens collected at Hahntennjoch (Tyrol, Austria) group together, while the specimen from Styria (Austria) groups with the specimen from Belluno (Italy). All seven mismatched U. uliginosalis specimens are males. The two specimens identified as U. juldusalis are sister to the clade U nebulalis + U. murinalis.
In the ML analysis of EF1a under the GTRGAMMA model, values for alpha were often above 10, and the EF1a dataset was analysed with the GTR model in-stead. In the resulting EF1a gene tree (Fig. 2), two U. austriacalis clades are found: one clade containing all three successfully sequenced specimens from group aus1, originating from the Pyrenees and the Massif Central (green clade), and one clade containing specimens from groups aus2 and aus3 (U. austriacalis samples marked in red and black). In comparison to the COI results, U. rhododendronalis does not group into distinct clades, and specimens from groups rho1 and rho2 form a single clade instead; no specimen from the group rho3 could be sequenced successfully. All specimens of U. uliginosalis, including those that group with U. alpinalis in the COI gene tree, form a monophyletic clade that is sister to the monophyletic U. alpinalis clade.
In the wingless gene tree (Fig. 3), specimens of the U. austriacalis clade aus1 form a monophylum on a long branch that is nested in the clade containing specimens from group aus3 and the single successfully amplified specimen from group aus2. Udea rhododendronalis specimens of all three COI Barcode groups (rho1-3) form a single monophyletic clade. Udea alpinalis and U. uliginosalis are not distinguished in two clades but form a common clade instead, including the U. uliginosalis specimens with the COI Barcode mismatch.
The three bacterial markers wsp, gatB, and ftsZ were used in order to screen for Wolbachia in a number of Udea specimens. Sequencing was successful in only two of the twelve tested specimens: a specimen of U. fulvalis from Crete collected 5.44 years before DNA extraction, and a specimen of U. olivalis from Armenia collected 4.26 years before DNA extraction. For the other ten tested specimens, PCR amplification produced a band in some cases, but sequencing failed.
For the two successful samples, wsp sequences produced no match in BIGSdb (Jolley and Maiden 2010). For the specimen of U. fulvalis (voucher ZMBN Lep125), the gatB sequence had the closest match with gatB allele 196, found in MLST profile 306; the ftsZ sequence had the closest match with ftsZ allele 36, present in 12 MLST profiles (41,42,109,145,146,150,151,156,157,235,305,374). Both gatB and ftsZ place the Wolbachia strain from the U. fulvalis specimen into supergroup B. For the specimen of U. olivalis (voucher ZMBN Lep156), gatB had the closest match with gatB allele 7, present in seven MLST profiles (19,112,118,261,266,268,272); ftsZ resulted in an exact match with ftsZ allele 3, present in 31 MLST profiles (10,13,14,17,19,24,25,54,80,83,89,91,92,107,112,118,122,123,127,132,133,165,199,234,330,404,432,434,449,451,454). Both gatB and ftsZ place the Wolbachia strain from the U. olivalis specimen into supergroup A. The gatB and ftsZ sequences obtained from U. fulvalis and U. olivalis can be obtained from RM.

Taxonomy
Based on morphological, genetic, and biogeographical data, the following changes in the taxonomy of the species of the U. alpinalis species group are proposed, and redescriptions are provided.     Zerny", Mally prep. no. 1097 (NHMW).
Diagnosis. Outer side of labial palps' 2 nd and 3 rd palpomeres pronouncedly darker than the rest of the labial palps; in U. donzelalis, labial palps' 2 nd and 3 rd palpomeres on outside barely darker than rest of labial palps. Maculation of forewing usually less pronounced than in U. donzelalis, the apical brown streak often narrower; hindwing in females dorsally evenly dark brown, whereas in U. donzelalis the inner area is greyish cream-white, contrasted by a darker outer band. In the male genitalia, the fibula of U. austriacalis is generally a bit narrower and more evenly broad from the base to the subapex; in U. donzelalis the fibula is somewhat broader and elongate triangular. In the female genitalia, the signum of U. austriacalis is 3.4-4.4 times as long as broad (n = 8), whereas in U. donzelalis the signum is on average narrower, being 4.1-5.1 times as long as broad (n = 6) (Tab. 1). Udea austriacalis can furthermore be distinguished by the COI Barcode from all other sequenced Udea species; the two allopatric DNA barcode clades of U. austriacalis, the first confined to the Maritime Alps and the second from other parts of the Alps, the Balkan Mountains and the Caucasus, do not differ from each other morphologically and in the nuclear genes wingless and EF1-alpha, so that they are considered conspecific. The two COI Barcode clades are the nearest neighbours to each other, and they differ by a minimum of 2.38 % p-distance of nucleotide divergence from each other; the Maritime Alps clade also has U. cretacea as nearest neighbour with 2.38% p-distance of minimum nucleotide divergence (Tab. 3).
Redescription. Head. Frons and vertex covered with beige scales; frons evenly convex, covered with beige to light brown scales; labial palps projecting forward, third segment pointed, palps covered with beige scales, outer sides of labial palps' second and third segment light to dark brown; maxillary palps approximately one quarter as long as labial palps, with beige apical scale tuft; compound eyes hemispherical; proboscis well developed, its base covered in pale beige and dark brown scales; antennae filiform, Figure 6. Distribution of investigated specimens of Udea juldusalis (blue), U. altaica (red) and U. plumbalis (yellow) in Central Asia; altitudes ³ 1,000 m are marked in increasingly darker grey shades every 500 m, altitudes ³ 4,000 m are in black. Note that the eastern locality of U. juldusalis and the localities of U. altaica and U. plumbalis are only approximations of the type localities. posterior side covered in pale beige scales, anterior side in males densely covered with cilia shorter than the basal antennal radius, shorter still in females, antennal length approx. 50-60 % of forewing length in males, approx. 70 % in females; ocellus posterior to antenna base.
Thorax. Cream-white, with collar and anterior part of tegulae scales more caramelcoloured; legs cream-white except for dark brown inner side of fore-and midlegs as well as distal half of hindlegs; tibial spurs on fore-/mid-/hindleg 0/2/4, as in other species of the genus; midleg outer spur ca. 2/3 length of inner spur; hindleg outer spurs ca. 2/3 length of inner spurs.
Wings. Forewing length 11-13 mm in males, 8-10 mm in females. Males and females with one frenulum bristle. Females with more acute apex due to the straight costa (distally curved in males). Forewing ground colour glossy cream-white to beige, with maculation more or less prominent: basal two thirds of costa with light brown streak, distal discoidal stigma a diffuse brownish dot, postmedial line brownish, arching around distal discoidal stigma, then turning basad until below distal discoidal stigma, sharply arching back outwards (the typical "Udea loop"), then following course of arch in postmedial line's anterior part and meeting with dorsum at about two thirds of dorsum length; apical streak more or less pronounced, narrow; ends of veins on dorsum in some specimens with minute dark brown dots, but often missing; fringe light creamwhite. Hindwing in males cream-white to brownish, postmedial line brown, more or less clear, terminal band brown, often only at apex; hindwing in females evenly dark brown, some specimens with a slightly darker terminal band. Underside of forewing uniformly brown with a slightly darker distal discoidal stigma and, in some specimens, with a slightly darker postmedial line; underside of hindwing greyish white to grey, with costal area somewhat darker, a diffuse brownish grey postmedial line might be visible in males, in females a more or less pronounced terminal band is present.
Abdomen. Dorsal side of abdomen covered with cream-white glossy scales, ventral side with dark brown scales, interspersed by cream-white scales; male 8 th segment with long beige posterior scales. Tympanum with broad short lobulus; 2 nd sternite with broad U-shaped sclerotisation, more so in females, inner part less sclerotised; 3 rd sternite anterior edge with broad sclerotised lobe on each side of the centre, tapering anterolaterad into thin apex; 4 th sternite anterior edge with oval hole in sternite sclerotisation on each side of the centre; centre of anterior edge of sterites V-VII with broad, short rectangular protrusion; male 8th sternite with U-shaped sclerotisation along borders, posterior ends broadened; male 8th tergite with broad central longitudinal sclerotisation, somewhat broadening anteriorly, leading laterally into pointed triangular process.
Male genitalia. (Figs 23-28) Uncus broadly attached to tegumen, the attachment site laterally constricted; apical part of uncus constricted into slim, strap-like neck leading into flattened oval uncus head, dorsally covered densely with stiff, deeply bifid setae. Tegumen broad, rectangular. Broad, weakly sclerotised gnathos band with a central conical dorsad protrusion. Transtilla arms forming equilateral triangles, dorsal surface sparsely set with thin long simple setae. Vinculum broad, well sclerotised, sides elongate drop-shaped to oval, ventral part mediodorsally forming broad bell-shaped protrusion towards juxta, ventrally forming U-shaped saccus with prominent ventromedial keel. Juxta nearly rhomboidal to almost circular, apex sharply bifid with medial incision about one quarter as long as juxta. Valva long, slender, slightly tapering towards apex; costa slightly concave, broader at base, surface sparsely set with thin, long, simple setae; sacculus broad, roughly oval, dorsodistal edge close to fibula base, ventrodistal part concavely curving towards ventral valva edge; ventral valva edge straight apart from a slightly concave recess in the area of the fibula tip; valva apex rounded towards distal end of costa; slender, evenly broad, strongly sclerotised fibula directed towards sacculus apex, apical half narrowed to pointed, ventrad curved claw, ventral side of claw flat; fibula emerging from oval sclerotised lobe near base of costa, sparsely set with thin, long simple setae. Phallus tubular, slightly curved, evenly sclerotised; posterior phallus apodeme bent sinistrad, dorsally and ventrally with elongate unsclerotised strip, right posterior phallus apodeme forming sclerotised spatulate lobe with medially protruding ridge containing three (rarely two) teeth of variable shape apically.
DNA data. See Table 2. In BOLD, U. austriacalis is represented by the BINs AAD2363 and AAD2364. The seven DNA barcoded specimens forming the clade from the Central and East Alps and Macedonia differ between 0% and 0.95% in p-distance,  Additional material (aus1 i Fig. 1 Diagnosis. Labial palps in males approx. 20% longer than in U. austriacalis and U. cretacea; outer side of labial palps' 2 nd and 3 rd palpomeres barely darker than rest of labial palps. Maculation of forewing usually more pronounced than in U. austriacalis, the apical brown streak often broader; hindwing in both sexes with greyish cream-white inner area contrasted by a prominent dark brown postmedial line and dark brown terminal band, especially in specimens where the postmedial line and the outer band are fused into a broad band; in contrast, the hindwings' upper side is evenly dark brown in females of U. austriacalis. In the male genitalia, the fibula of U. donzelalis is generally broader and elongate triangular, tapering from the broad base towards the apex; in U. austriacalis the fibula is narrower and evenly broad from the base to the subapex. In the female genitalia, the signum of U. donzelalis is 4.1-5.1 times as long as broad (n = 6), whereas in U. austriacalis the signum is 3.4-4.4 times as long as broad (n = 8) (Tab. 1). Udea donzelalis can furthermore be distinguished by the DNA Barcode from all other sequenced Udea species; the nearest neighbour is U. cretacea with 4.29% minimum p-distance.
Redescription. Head. As for U. austriacalis, apart from: frons and vertex covered with cream-white to beige scales; labial palps covered with light brown scales, outer sides of labial palps' second and third segment sometimes slightly darker with dirty light brown scales; maxillary palps approximately one third as long as labial palps, with beige to light brown apical scale tuft; antennal length approx. 60 % of forewing length in males, approx. 70 % in females.
Thorax. As for U. austriacalis, apart from: legs cream-white except for dark brown inner side of fore-and midlegs; hindleg proximal outer spur ca. 2/3 length of inner spur, distal spurs almost equal in length, outer slightly shorter.
Wings. Forewing length 12-13 mm in males, 8-10 mm in females. Males and females with one frenulum bristle. Female forewing with more acute apex due to the straight costa (distally curved in males). Forewing ground colour glossy cream-white, with maculation more or less prominent: a light brown streak parallel to the basal two thirds of the costa, distal discoidal stigma a diffuse brownish area, postmedial line brownish, arching around distal discoidal stigma, then turning basad until below distal discoidal stigma, sharply arching back outwards (the "Udea loop" typical for most species in the genus), then following the course of the arch in the postmedial line's anterior part and meeting with the dorsum at about two thirds of the dorsum length; apical streak more or less pronounced, usually relatively broad; ends of veins on dorsum with minute dark dots; fringe light cream-white. Hindwing in both sexes creamwhite to grey-brown, postmedial line and terminal band dark brown, can be fused into one broad band. Underside of forewing uniformly brown, sometimes with grey-grown strip along cell; underside of hindwing greyish white with a brown tinge, postmedial line brownish grey, rather diffuse; colour of external area as internal area, or brownish grey as postmedial line, with which it can form a broad band.
Abdomen. As for U. austriacalis. Male genitalia. (Figs 29-33) As for U. austriacalis, apart from: juxta nearly rhombical to broad drop-shaped, apex sharply bifid with medial incision about one fifth of juxta length; ventral valva edge straight to slightly convex; elongate triangular, apically tapering, strongly sclerotised fibula directed towards distal sacculus apex, apical half narrowed to pointed, ventrad curved claw, ventral side of claw flat; right posterior phallus apodeme forming sclerotised spatulate lobe with medially protruding ridge containing three teeth of varying shape at posterior end.
Immature stages. Unknown. Distribution. Massif Central (France), Pyrenees (France, Andorra, Spain) (Fig. 4). Food plants. Unknown. DNA data. See Table 2. On BOLD, U. donzelalis is represented by BIN ADB6837. The five DNA barcoded specimens are identical in their DNA barcodes. The nearest neighbour is U. cretacea with 4.29 % minimum p-distance in the COI Barcode (see Tab. 3). The next-closest neighbour is U. rhododendronalis with a minimum interspecific COI p-distance of 4.76 %.
Remarks. Lederer (1863) synonymised donzelalis with U. austriacalis, and following authors (La Harpe 1864, Marion 1973, Leraut 1996 came to the same con-clusion. However, Leraut (1996) mentioned that the specimens from the Pyrenees, conspecific with the revised U. donzelalis, differ from other specimens of U. austriacalis in their clearer maculation on the forewings in both sexes. Based on the investigation of the four syntypes of U. donzelalis, a lectotype and three paralectotypes are designated (see material examined). (Zerny, 1914), stat. n. Figs 6,[15][16][17][18][36][37]47 Pyrausta austriacalis v. altaica Zerny, 1914: 334-335. Diagnosis. Proximal outer spur of hindleg minute (as in U. alpinalis, U. juldusalis and U. plumbalis), whereas in U. austriacalis, U. cretacea, U. donzelalis and U. uliginosalis it is ca. half to two thirds the length of the proximal inner spur. The wing maculation of U. altaica can be confused with that of U. austriacalis, U. cretacea, U. donzelalis, U. juldusalis, U. plumbalis, U. uliginosalis and untypically maculated specimens of U. alpinalis (see Fig. 4 in Panigaj and Kulfan 2012), but it can be distinguished from all those species by the more or less distinct proximal brown section of the postmedial line on the ventral side of the forewing in both sexes (Figs 16,18); in males, the proximal subterminal area of the ventral forewing side is as light brown as the central area (Fig. 16), whereas in the other species it is darker than the central area; on the hindwing ventral side, the subterminal area is only faintly darker than the central wing area in both sexes (Figs 16, 18), whereas the other species have a darker subterminal area, at least in the apex. In male genitalia only distinguishable from U. cretacea and U. uliginosalis by the small dentate ridge-like process on the posterior phallus apodeme, whereas in U. cretacea the sclerotisation at posterior phallus apodeme is a slim, elongate, apically dentate process emerging from the posteriormost end (Fig. 35), in U. uliginosalis a large hooked spine. In the female genitalia, the main signum is 3.1-to 3.4-times as long as broad, whereas in U. austriacalis the main signum is 3.4-to 4.4-times longer and in U. donzelalis 4.1-to 5.1-times longer than its maximum width (Tab. 1). The antrum is conical, widening posteriorly and is about twice as long as broad (Fig. 47), whereas in U. austriacalis and U. donzelalis the sclerotised antrum is predominantly tubular and 1-to 1.5-times as long as broad (Figs 45-46).

Redescription.
Head. As for U. austriacalis, part from: frons and vertex with cream-white scales; distal half of labial palps brown on the outside, basal half and inner sides cream white; maxillary palps cream-white with some brown scales mixed in; antennal length approx. 60 % of forewing length in males, approx. 70 % in females.
Thorax. As for U. austriacalis, apart from: legs cream-white on inner and outer sides; proximal pair of metatarsal spurs with outer spur minute and inner spur long, distal pair ca. half the length of the proximal inner spur, distal inner spur a bit longer than distal outer spur.
Wings. Forewing length 14 mm in males, 11 mm in females. Males and females with one frenulum bristle. Female forewing with more acute apex due to the straight costa (distally curved in males), and with outer wing margin (termen) of hindwing cut straight. Forewing upper side cream white with brown scales interspersed, giving it a dirty appearance; brownish subcostal line along the basal two third of the forewing; cell margin facing the forewing centre demarcated by a thin brown line, less prominent in females; outer medial area with a transverse cream white band lacking the interspersed brown scales; outer margin of cream white band delimited by diffuse grey postmedial line which leaves the costa in a right angle, bends inward at vein M1 and parallels the termen until the line reaches the dorsum; postmedial area homogenous greyish white, apex with more or less darker streak; termen with a slim brown margin and long, cream-white fringes. Hindwing upper side in males pale yellowish brown with diffuse light brown apex, in females light brown with a faint, slightly brighter medial band. Forewing underside in females vivid brown, with a darker, diffuse outer cellular spot and a darker postmedial area, demarcated by the postmedial line, maculation paler in males; slim whitish subcostal line along the basal two third of the forewing; fringes cream-white. Hindwing underside cream white with the subcostal and terminal areas tinted slightly brownish, the postmedial line more or less prominent.
Abdomen. Pale grey dorsally, slightly darker grey ventrally; distal segment margins greyish white, scales on terminal segment pale yellowish. Tympanum without broad short lobulus.
Male genitalia. (Figs 36-37) As for U. austriacalis, apart from: juxta nearly rhombical to broad drop-shaped, with small indention on each side dorsal of its greatest width, apex sharply bifid with narrow V-shaped medial incision ca. 1/4 of juxta length; ventral valva edge convex, with a slight bulge in the area to which the fibula is pointing; valva apex evenly rounded. An elongate triangular, apically tapering, strongly sclerotised fibula directed towards the distal sacculus, apical half narrowed to pointed, ventrad curved claw, ventral side of claw with flat 'blade'; fibula emerging from an oval sclerotised lobe near base of costa which is very sparsely studded with thin long simple setae; posterior phallus apodeme dorsally and ventrally with elongate unsclerotised strip, right posterior phallus apodeme forming a sclerotised spatulate lobe with a medially protruding ridge containing at the posterior end two equal-sized teeth and a third tiny posterior-most tooth.
Immature stages. Unknown.   Kyrgyzstan, Ysyk-Kol, Chong Oruktu, 42.796 77.86, 1900m, 22-Jun-1998 Diagnosis. Udea juldusalis has a wing maculation similar to that of U. altaica, U. plumbalis, U. uliginosalis and untypically maculated specimens of U. alpinalis (see Fig. 4 in Panigaj and Kulfan 2012). It can be distinguished from U. uliginosalis by the minute proximal outer spur of the hindleg, which is well developed in U. uliginosalis and about half to two thirds the length of its proximal inner spur; furthermore, U. uliginosalis has a large hooked spine on the posterior phallus apodeme, whereas the posterior phallus apodeme carries a small dentate ridge-like process in U. juldusalis. Udea altaica specimens have lighter forewings dorsally and ventrally, and the proximal section of the postmedial line is a diffuse, though well visible brown arch in both sexes. Udea plumbalis has darker, broader and more rounded fore-and hindwings (at least in the male). Udea alpinalis is distinguished by the dark subterminal band on the hindwings' dorsal and ventral side that contrasts with the white inner hindwing area. The COI sequences (DNA Barcode) of U. juldusalis are unique and not shared with any other DNA-barcoded organism, and the nearest neighbour is U. uliginosalis with a minimum of 2.86 % p-distance.
Redescription. Head. Frons and vertex covered with cream-white scales; frons evenly convex, covered with beige to light brown scales; labial palps projecting forward, third segment pointed, palps covered with beige scales, outer sides of labial palps' sec- ond and third segment brown; maxillary palps brown on outside, cream-white on the inside apart from subapical area with brown scaling; compound eyes hemispherical; proboscis well developed, its base covered in brown and greyish scales; antennae filiform, dorsal side covered in beige scales, anterior side in males densely covered with cilia shorter than the basal antennal radius, shorter still in females, antennal length approx. 50% of forewing length in males, females unknown; ocellus posterior to antenna base.
Thorax. As for U. austriacalis, apart from: greyish brown ground colour; hindlegs and outer side of fore-and midlegs cream-white, inner side of fore-and midleg brown; proximal pair of metatarsal spurs with outer spur minute and inner spur long, distal pair about half the length of the proximal inner spur, distal inner spur a bit longer than distal outer spur.
Wings. Forewing length 13-14 mm in males, females unknown. Males with single frenulum bristle. Forewing dorsal side pale brownish to brownish yellow white, somewhat darker between cell and costa; veins delimiting cell pale brown; outer medial area behind cell cream white and clear, intersected by the brownish coloured veins R5, M1-3 and Cu1, outer margin of cream white area sharply defined by postmedial line; postmedial line slightly darker than brown ground colour, indistinct at anterior and posterior wing margins, smoothly curving around whitish area of medial wing; outer wing margin with two thin brownish lines separated by a thin yellowish cream-white line; distal part of fringe pale whitish. Hindwing dorsal side with dirty white to brownish ground colour, intersected by the brown-tinted veins; a broad brown subterminal band along the outer margin, blurrily demarcated from the somewhat lighter inner area; hindwing outer margin with a thin yellowish line between subterminal band and brownish basal half of fringe; distal half of fringe whitish. Forewing ventral side homogenous brown, subcostal area and veins delimiting the cell somewhat darker; more or less prominent white line along costal side of cell; outer wing margin and fringe as on dorsal side. Hindwing ventral side with dirty white basal and central area, intersected by the brown-tinted veins; brown subterminal band more prominent and clearly marked-off from the inner area; outer wing margin and fringe as on dorsal side.
Abdomen. As for U. austriacalis, apart from: abdomen pale grey dorsally, dark grey ventrally; distal segment margins on dorsal side cream white, scales on terminal segment pale yellow.
Male genitalia. (Figs 38-41) As for U. austriacalis, apart from: vinculum ventrally forming a roundly V-shaped saccus with a prominent ventromedial keel; juxta nearly rhombical to broad drop-shaped, apex sharply two-pointed with a narrow V-shaped medial incision ca. 1/4 of the juxta length; valva long, relatively broad, slightly tapering towards apex; costa concave, evenly tapering towards apex, surface sparsely studded with thin long simple chaetae; sacculus broad, roughly rectangular, dorsodistal edge close to fibula base, ventrodistal part concavely curving towards ventral valva edge; ventral valva edge straight from mid-sacculus to valva subapex, with a very slight bulge in the area where the fibula is pointing to; valva apex evenly rounded; elongate, apically tapering, strongly sclerotised fibula directed towards the distal sacculus, fibula base  (Mally prep. 1047) 46 U. donzelalis (Mally prep. 1023) 47 U. altaica (Mally prep. 1084). somewhat constricted, apical half narrowed to a pointed, ventrad curved claw; right posterior phallus apodeme forming a sclerotised spatulate lobe with a central raised ridge bearing two small, laterally protruding teeth at its posterior end, sometimes with one or two additional, much smaller teeth posterior to the larger ones.

Distribution.
The species is known from the Küngöy Ala-Too (Kungey Alatau) Range in Kazakhstan and Kyrgyzstan, and from the Yulduz mountains in NW China; both mountain ranges are part of the Tian Shan mountain system (Fig. 6).

Udea plumbalis
Diagnosis. Udea plumbalis can be confused with U. juldusalis, U. uliginosalis, U. uralica and untypically maculated specimens of U. alpinalis (see Fig. 4 in Panigaj and Kulfan 2012). It differs from U. uliginosalis in the minute proximal outer spur of the hindleg, which is well developed in U. uliginosalis; furthermore, U. uliginosalis has a large hooked spine on the posterior phallus apodeme, while U. plumbalis has a small dentate ridge-like process. In U. alpinalis, the forewing apex is more acute and the hindwings have a white inner area (with a more or less broad brown area along the dorsum, sometimes occupying the majority of the inner wing area) contrasted with a clear dark-brown terminal band along the termen, whereas in U. plumbalis the hingwings' dorsal side is evenly brown (Fig. 19), and on the ventral side the inner wing area is only faintly lighter than the subterminal band (Fig. 20). Udea juldusalis has lighter, narrower, and more acute fore-and hindwings in the male, and the hindwings' inner area is lighter. Udea plumbalis is distinguished from U. uralica by the rounded apex and dark fringe in the forewing of males, and by the absence of a prominent bulge in the centre of the ventral valva edge (compare Slamka 2013, pl. 27 fig. 129); it is not clear whether this bulge on the ventral valva edge or its absence is a reliable character to distinguish the two species; the length of the hindlegs' proximal outer spur in comparison to the proximal inner spur is not known for U. uralica.
Redescription. Head. As for U. austriacalis, apart from: frons and vertex with greyish brown scales; proboscis base covered in brown and greyish scales; labial palps directed forward, dirty-to cream white, distal half of the outside greyish brown; maxil-lary palps brown on outside, cream white on the inside; dorsal side of antennae with line of metallic brown scaling; antennal length approx. 60 % of forewing length in males, females unknown.
Thorax. As for U. austriacalis, apart from: greyish brown thorax; front legs and inner side of mid-and hindlegs cream-white, side of mid-and hindlegs pale brownish with many cream-white scales intermixed; hindleg proximal outer spur minute, inner spur the longest one on the hindleg, distal outer spur ca. 80% the length of the inner spur.
Wings. Forewing length 12-13 mm in males, females unknown. Males with one frenulum bristle. Forewing dorsal side dirty pale brown, distal of the postmedial line brown-grey to dirty greyish. Maculation absent apart from a more or less prominent oval whitish area basal of the postmedial line on the M veins. Area between costa and cell and veins encircling the cell somewhat darker brown. Slim band along outer wing margin consisting of three thin brown lines alternated with two thin pale yellowish brown lines; distal fringe pale white. Hindwing pale brown, apical area somewhat darker; outer wing margin with thin bands as in forewing, outermost band somewhat fainter. Ventral side of forewing uniformly brown, costa blended with darker brown and greyish scales and with a thin whitish line running in parallel on the length of the cell; distal end of cell with a faint darker brown arc; subterminal area slightly darker; dirty greyish brown area on wing apex, running along outer wing margin, narrowing towards posterior end of termen; outer wing margin and fringe as on dorsal side. Ventral side of hindwing dirty brownish grey with a more or less prominent broad pale brown subterminal band; outer wing margin and fringe as on dorsal side.
Abdomen. As for U. austriacalis, apart from: abdomen brownish grey, distal segment margins on dorsal side and scales on terminal segment pale yellow.
Male genitalia. (Figs 42-44) As for U. austriacalis, apart from: juxta rhombical to broad drop-shaped, apex sharply bifid with a narrow V-shaped medial incision 20-25% of the juxta length; Valva long, relatively broad to slim, slightly tapering towards apex; costa concave, central costa somewhat narrowed, costa surface sparsely studded with thin long simple setae; sacculus broad, roughly rectangular, dorsodistal edge close to fibula base, ventrodistal part concavely curving towards ventral valva edge; ventral valva edge straight to convex, with or without a slight bulge in the area where the fibula is pointing to; fibula elongate, apically tapering, strongly sclerotised, directed towards the distal sacculus, dorsal fibula edge inflated to a narrow tube, apical half narrowed to a pointed, ventrad curved claw, ventral side of claw with flat surface; right posterior phallus apodeme forming a sclerotised spatulate lobe with a central raised ridge bearing two to three small, laterally protruding teeth at its posterior end.
Female genitalia. Unknown. Immature stages. Unknown. Distribution. So far only known from the eastern Sayan mountains in the Khövsgöl Province in N Mongolia (Fig. 6).
Food plants. Unknown. DNA data. No data available.

Discussion
Deep intraspecific splits are observed in the results of a phylogenetic analysis of COI sequence data of the U. alpinalis species group. However, further investigation of these clades based on nuclear genetic and morphological data does not indicate species-specific differences, with the exception of the U. austriacalis clade from the French Massif Central and the Pyrenees. We conclude that the latter clade represents a species distinct from U. austriacalis, as it differs in morphology and in all three investigated genetic markers from U. austriacalis. This distinct species is identified as U. donzelalis, after comparison with the respective type material, and consequently revoked from synonymy with U. austriacalis.
In the COI phylogram (Fig. 1), the U. rhododendronalis clade rho1 corresponds with the geographic distribution of Leraut's (1996) subspecies U. rhododendronalis luquetalis, but no morphological character could be found to separate it from the nominate U. rhododendronalis from the Alps. Similarly, the Balkan specimens of U. rhododendronalis, forming clade rho3 in the COI phylogram, are morphologically not distinct from the specimens of the Alps and Pyrenees, and thus not supporting Slamka's (2013) assumption of as potential Balkan subspecies. Specimens from the Maritime Alps, from where Leraut's (1996) subspecies U. r. ventosalis is described, do not form a distinct COI clade, like the U. r. luquetalis specimens, but group with the U. rhododendronalis specimens from the Central Alps instead. Furthermore, the COI groups are not reflected in the phylogenetic results of the two nuclear markers (Figs 2-3). In conclusion, no conclusive evidence for either additional species or subspecies in U. rhododendronalis is found.
Morphological investigation of the type material of Zerny's (1914) three U. austriacalis subspecies reveals that they are good species, and they are consequently raised to species level. For U. juldusalis, this decision is further supported by COI Barcode sequences which are not shared with other Udea species.
Similar cases of deep intraspecific, although not necessarily allopatric, divergences in COI data of Lepidoptera have been observed in other groups, e.g. by Charlat et al. (2009), Dasmahapatra et al. (2010, Muñoz et al. (2011), Mutanen et al. (2012), Nieukerken et al. (2012 and Ritter et al. (2013). In U. rhododendronalis and U. austriacalis, geographically well-separated COI groups are found. The observed mitochondrial genetic divergences could therefore be explained with allopatry, and the absence of (congruent) clades in the nuclear data could be due to slower substitution rates and incomplete lineage sorting. However, the specimens of U. donzelalis form distinct, congruent clades among all three investigated genetic markers. No such congruent pattern is found for the COI subclades of U. austriacalis and U. rhododendronalis, respectively, and the species status is therefore rejected for the named subclades of U. rhododendronalis based on the available data.
The uniformity of male genitalia in the U. alpinalis species group makes species discrimination based on this character complex difficult to impossible, as is also the case for example in the U. fimbriatralis complex (Mally et al. 2016). The female genitalia, however, are suitable for species distinction: the sclerotised parts of antrum, colliculum and signa as well as the ratio of length to breadth of the main signum are useful for species identification. Munroe (1966) points out the usefulness of the hindleg's proximal outer spur for distinguishing species of the U. itysalis group, a character that is also useful for distinguishing species of the U. alpinalis group.

Introgression between U. alpinalis and U. uliginosalis
Some specimens morphologically identified as U. uliginosalis have been found grouping with specimens of U. alpinalis in the COI Barcode ML analysis (Fig. 1). All those 'mismatched' specimens are males, and their genitalia present the diagnostic characters of U. uliginosalis. Analyses of two nuclear markers result in different topologies: The ML analysis of wingless data (Fig. 2) does not provide a differentiation between U. alpinalis and U. uliginosalis. In the ML analysis of EF1-alpha data (Fig. 3), all 'mismatched' specimens are monophyletic with all other specimens of U. uliginosalis and are sister to U. alpinalis. The contradicting placements of those 'mismatched' U. uliginosalis specimens in the mitochondrial and nuclear ML analyses leads us to the assumption that introgression must have occurred between U. uliginosalis and U. alpinalis. This could be explained by the following scenario: A female of U. alpinalis successfully mates with a male of the sympatrically occurring U. uliginosalis. The F1 generation has the mitochondrial genotype of U. alpinalis, and the nuclear genotype is heterozygotic. In concordance with Haldane's rule (Haldane 1922), the sex that is heterogametic for sex factors -in Lepidoptera this is the female -is rare or sterile or absent. The specimens of U. uliginosalis collected at the localities of assumed hybridisation are exclusively male, without a single female. (However, females are less frequently collected than males -they may be poor flyers due to their shorter wings.) Despite this, assuming that a few fertile hybrid females exist in the F1 generation and these mate with males of U. uliginosalis, the F2 (backcrossing) generation retains the mitochondrial genotype of U. alpinalis, since the mitochondrial genome is maternally inherited; the nuclear genotype of the offspring consists to three quarters of that of U. uliginosalis. Additional backcrossings further increase the proportion of the U. uliginosalis nuclear genome, while the mitochondrial genotype remains that of U. alpinalis.
The deep intraspecific splits led us to test for Wolbachia infection which might play a role in the U. alpinalis group. The intracellular bacterium Wolbachia is known to have an impact on the reproduction of a wide range of arthropods, including Lepidoptera (e.g. Werren et al. 2008). The observed deep intraspecific COI Barcode splits in species of the U. alpinalis group could be explained by Wolbachia-mediated cytoplasmic incompatibility between different populations, or by geographic isolation, or a mix of both processes. In the present screening, no evidence of a Wolbachia infection was found in specimens of the U. alpinalis species group. However, several instances of false negative results exist that could obscure the presence of Wolbachia in these species: The mean Wolbachia infection prevalence is 21 % among Crambidae (Ahmed et al. 2015), so that Wolbachia can remain undetected if the sampling of a population is not comprehensive enough. The amplification with wsp primers resulted in sequences from U. fulvalis and U. olivalis that could not be matched with Wolbachia or any other sequences in GenBank or BIGSdb, indicating suboptimal primer sequences which can lead to failure of PCR amplification or amplification of fragments other than the target sequence. Although no evidence for the presence of Wolbachia in the U. alpinalis group was found in the present screening, the possibility of Wolbachia infection and its biological implications for the hosts should be kept in mind for future studies.
The analyses of the two nuclear markers (Figs 2-3) show partially contradictory results: in the wingless gene tree (Fig. 3), a relatively long terminal branch leads to the U. donzelalis clade, whereas in the EF1a gene tree (Fig. 2), the branch of the same clade is much shorter. Further, in the EF1a phylogram U. uliginosalis and U. alpinalis form separate clades, whereas in the wingless phylogram the specimens of those two species share a common clade. This indicates that more nuclear markers are required to reliably reconstruct this contradictory and insufficiently supported part of phylogenetic inference.
These results shed further light on the U. alpinalis species group, but more material is needed from the mountain systems of Central and West Asia to study the morphology and genetics of the species found there, to bring them in phylogenetic context with European species of the U. alpinalis group and to investigate the biogeography of the species group. Additional morphological investigations are encouraged regarding the status of the genetic clades. In a similar case, new techniques, like the eversion of the phallus vesica, allowed the morphological differentiation of hitherto exclusively genetic clades (Zlatkov and Huemer 2017).
Other Udea species groups require revisionary work, like the Palaearctic U. numeralis group that needs the most systematic attention in this region. Mally et al. (2016) described a new species from Crete and placed it in the U. numeralis group, based on a phylogenetic analysis, but several problematic taxa remain to be revised in this group, e.g. the U. fimbriatralis complex and the U. numeralis complex as well as U. praepetalis (Lederer, 1869) and U. bipunctalis (Herrich-Schäffer, 1848). Future systematic studies on this group should include material from the Middle East and the East Palaearctic, especially the taxa described by Amsel (1961Amsel ( , 1970. In North America, the majority of the 25 Udea species belong to the U. itysalis group (Munroe 1966) that requires careful revision with the help of molecular data. A large COI Barcode dataset for Nearctic Udea material has been accumulated, but analysis of the data is pending (pers. comm. Jean-François Landry). Apart from these studies, Udea is poorly investigated in systematic terms. With this integrative revision, the number of Udea species is raised from 217 to 221 species, with 40 of them occurring in Europe.
of Gothenburg, Sweden) and Heiko Stuckas (MTD, Dresden) for helpful comments on the data analysis. PH is most grateful to Paul Hebert and the entire team at the Canadian Centre for DNA Barcoding (Guelph, Canada), whose sequencing work was enabled by funding from the Government of Canada to Genome Canada through the Ontario Genomics Institute. We also thank the Ontario Ministry of Research and Innovation and NSERC for their support of the BOLD informatics platform. PH is furthermore indebted to the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano -South Tyrol for helping to fund the project "Genetische Artabgrenzung ausgewählter arktoalpiner und boreomontaner Tiere Südtirols". We thank Stephen Sutton (Universiti Malaysia Sabah, Kota Kinabalu, Malaysia) for the language checking. Bernard Landry (Muséum d'histoire naturelle, Geneva, Switzerland) contributed helpful modifications and comments as a reviewer.