Systematic Entomology (2012), 37, 686–705

DNA barcoding and morphology reveal three cryptic of (: : ) in North America, all distinct from their European counterpart

ZHAOFU YANG1,9, JEAN-FRANC¸ OIS LANDRY2,LOUIS HANDFIELD3, YALIN ZHANG1,M.ALMASOLIS4, DANIEL HANDFIELD5, BRIAN G. SCHOLTENS6, MARKO MUTANEN7, MATTHIAS NUSS8 and PAUL D. N. HEBERT9

1Key laboratory of Plant Protection Resources and Pest Management, Ministry of Education; Entomological Museum, Northwest A&F University, Yangling, China, 2Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, C.E.F., Ottawa, Ontario K1A 0C6, Canada, 3133 rue Messier, #301, Mont-Saint-Hilaire, Quebec´ J3H 2W8, Canada, 4Systematic Entomology Laboratory, USDA, c/o Smithsonian Institution, National Museum Natural History, Washington, DC 20013-7012, U.S.A., 5Chemin des Grands Coteaux, Saint-Mathieu-de-Beloeil, Quebec,´ Canada, 6Department of Biology, College of Charleston, SC, U.S.A., 7Department of Biology, University of Oulu, Zoological Museum, Oulu, Finland, 8Museum of Zoology, Senckenberg Natural History Collections Dresden, Konigsbr¨ ucker¨ Landstrasse 159, 01109 Dresden, Germany and 9Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Abstract. (Hufnagel), a Holarctic species of pyraustine crambid , has long been treated as having two geographically separated subspecies – the nominotypical Anania coronata in the Palaearctic Region and Anania coronata tertialis (Guenee)´ in the Nearctic Region. Maximum likelihood and Bayesian inference analysis of mitochondrial DNA barcodes both recover four well-supported, reciprocally monophyletic groups within Anania coronata. Qualitative and quantitative analyses of genital structures reveal diagnostic differences that correspond to the four barcode lineages. On the basis of both molecular and morphological evidence, we conclude that Anania coronata is actually a complex of four species. Anania coronata (Hufnagel) is restricted to Europe, whereas three species occur in North America: Anania tertialis (Guenee),´ Anania plectilis (Grote & Robinson) and Anania tennesseensis sp.n. Yang.

Introduction taxa have long been regarded as synonyms of Anania coronata tertialis: Botys plectilis Grote & Robinson and Anania coronata (Hufnagel) is a Holarctic species that ranges Botys syringicola Packard (Dyar, 1903; McDunnough, 1938; from Europe through Asia to Japan, and to North America Munroe, 1976; Munroe et al., 1995; Hodges et al., 1983). (Munroe, 1976; Inoue, 1982; Speidel, 1996; Sinev, 2008). Munroe (1976) indicated that more than one species might Munroe (1954, 1976) and Munroe et al. (1995) treated be included within Anania coronata because of its Holarctic North American populations as a distinct subspecies, Anania distribution and morphological variation, but he did not analyse coronata tertialis (Guenee).´ Two other North American it in detail. Following an examination of some North American specimens in the Museum´ d’histoire naturelle de Paris, Leraut Correspondence: Yalin Zhang, Key laboratory of Plant Protection (2005) raised both tertialis and plectilis to full species rank Resources and Pest Management, Ministry of Education, Entomolog- separate from coronata and illustrated male genital characters ical Museum, Northwest A&F University, Yangling 712100, China. distinguishing them from the nominal coronata and from each E-mail: [email protected] other. However, Leraut did not examine any type specimens

© 2012 The Authors 686 Systematic Entomology © 2012 The Royal Entomological Society DNA barcoding reveals three cryptic species of Anania 687 and appeared to rely entirely on the identifications of specimens European samples, and to clarify the nomenclature of this in the Paris Museum to revise the status of these species. species complex. He retained Botys syringicola as a synonym of Anania tertialis. Despite the great value of genital characters for species Material and methods recognition, the preparation of genitalia is so time consuming that it limits the use of this approach for large-scale specimen Taxon sampling sorting and examination. As a consequence, cryptic species are commonly overlooked, particularly in taxa with broad Eighty-nine specimens of Anania coronata from Europe distributions (Smith et al., 2006; Burns et al., 2007; Kristensen and North America (Fig. 1) were analysed as well as 20 et al., 2007). The recent integration of morphological and specimens from three congeneric taxa – Anania quebecensis DNA-based approaches has revealed an effective way to (Munroe), (Hubner)¨ and Anania stach- accelerate species discovery and description (Dayrat, 2005; ydalis (Germar). Table 1 summarizes information on these Lumley & Sperling, 2010; Padial & De La Riva, 2010; specimens whereas more complete details including images, Padial et al., 2010; Schlick-Steiner et al., 2010), as well as GPS coordinates and information on the institution hold- assist in detecting previously unsuspected cryptic species ing each specimen are available in the project ‘Ana- (Hebert et al., 2004; Wilson et al., 2010; Mutanen et al., nia coronata ANAN’ on BOLD (www.boldsystems.org). 2012). Comprehensive studies of lepidopteran faunas have A paralectotype of Ebulea tertialis in the USNM and revealed 90–98% discrimination of species in varied settings the holotype of Botys plectilis in the AMNH were also (Hajibabaei et al., 2006a; Hebert et al., 2010; Dinca et al., analysed. 2011; deWaard et al., 2011). The present investigation was prompted by results obtained Morphological characters and morphometric analysis during an effort to barcode all Lepidoptera species in the Holarctic region (see http://www.lepbarcoding.org/) which Eight female genitalia characters and 14 male genitalia revealed that specimens of Anania coronata from North characters were compared among individuals in each of America and Europe separated into four sequence clusters. the four different lineages of the Anania coronata com- This paper examines the geographic distribution of these plex (7 traits for females and 11 characters for males were four lineages and the morphological divergence among them. analysed morphometrically) (Fig. 2). In total, 11 females and We compare phenotypic characters and COI sequences to 23 males (Table 2) were dissected, including the female illuminate species differences among North American and holotype of Botys plectilis and a female paralectotype of

Fig. 1. Distribution of collection localities of analysed specimens of Anania coronata species complex in this study. Each lineage is represented by a symbol. The symbol refers to the geographical range to which the genetic lineage belongs: European lineage (22); North American lineage (47); Eastern North American lineage (19); Tennessee, USA (1).

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 688 Z. Yang et al. GenBank acession Sequence length specimens included in the sequence. Anania Bold project codeLGSMGCGUKBCGUKC Process ID LGSMG172-07FBLMS CGUKB608-09FBLMU BGS03493 CGUKC074-09LEFIR UKLB18A09LEFIR FBLMS201-09 Specimen ID UKLB23A04 FBLMU401-09LEFIRPYRG BC ZSM LEFIA713-10 Lep BC 23012PYRG ZSM Lep LEFIA714-10 Hap1 27051 Hap2RDNMH Hap2 LEFIE721-10 Haplotype Hap2 MM01868RDNMH Hap2 PYRG083-09 MM01869CGUKA Country PYRG084-09 RDNMH541-09 United MM09813 UnitedCGUKC States Kingdom Germany RDNMH543-09 UnitedCGUKA BC Kingdom MTD CNCLEP00057860 Germany 00171 England CGUKA485-09CGUKB BC MTD CNCLEP00057862 00172 Tennessee Hap2 England CGUKC224-09CGUKB Hap2 UKLB6B04 State/province Hap2 CGUKA820-09CGUKB Hap2 Hap2 UKLB24F02 Bavaria Hap2 CGUKB328-09CGUKB Bavaria Hap2 UKLB9F10 CGUKB606-09 FinlandCGUKD Germany UKLB15A11 CGUKB607-09 FinlandFBLMS Germany UKLB18A07 Poland CGUKB787-09FBLMS Hap3 Finland UKLB18A08 Germany Hap4 CGUKD126-09RDNMH UKLB19H12 Depository FBLMS057-09 Hap5 BMNHZYPAN USNM UKLB34B05 Savonia Hap5 Saxony australis FBLMS200-09 BMNH UnitedBBLPE Kingdom Savonia Hap5 RDNMH542-09 Saxony australis BC UnitedBBLPE ZSM Kingdom Lep Podlachien Hap5 Aland 22980 Baden-Wuerttemberg 658 BC EnglandBBLPE ZSM 658 CNCLEP00057861 ZYPAN066-10 RCTG United Lep Kingdom Hap5 23011 United England 658 ZSC KingdomBLTIB Hap6 BBLPE524-09 Hap5 United Kingdom CNCLEP00074266LGSMG Hap8 Hap7 BBLPE498-09 England United KingdomLGSMB 658 UO BBLPE507-09 09BBELE-2524 JQ348024 England United MTD JQ348047 KingdomLGSMB Hap9 UO 09BBELE-2498 JQ348023 Germany BLTIB483-08 658 England United Kingdom LGSMG267-07LPOKA 09BBELE-2507 Germany Germany England LGSMB024-04LPOKC MTD England Hap9 BL732 BGS03588 HM901988 MTD LGSMB025-04LPSOD 658 MTD 658 United Hap10 States DNA-ATBI-0873RDLQD LPOKA445-09 658 GU707126 BMNH Hap11 Bavaria UO DNA-ATBI-0874MECD BMNH LPOKC816-09 Saxony Bavaria 658 Canada MDOK-0445MEC LPSOD766-09 658 Minnesota Canada Hap14 658 GU700962 HM386857 BMNH RDLQD767-06ZYPAN 596 MDOK-2893 Canada Hap15 Hap13 640 HM386858 BMNHZYPAN 08BBLEP-00548 MECD251-06 658 MDH002015 Hap12 BMNHZYPAN BMNH GU679045 United States 658 BMNH GU700961 MEC583-04ZYPAN Hap16 ZYPAN061-10 GU679039 United United Newfoundland States States 658 jflandry2823 and JQ348031 BMNH LabradorZYPAN Newfoundland ZYPAN062-10 Hap18 Hap17 and JQ348030 Labrador 658 Canada 658 CNCLEP00074261 Newfoundland ZYPAN070-10 North HM874441 and Carolina Labrador BIO 658 jflandry0583 United Hap19 CNCLEP00074262 ZYPAN063-10 RCAH States BIO North JQ348028 Tennessee Carolina 658 Canada CNC United CNCLEP00074270 ZYPAN069-10 Hap21 RCTG MTD BIO States JQ348027 CNCLEP00074263 Hap19 Hap22 JQ348026 JQ348022 Canada Oklahoma CNCLEP00074269 636 Hap22 JQ348025 658 Ontario Canada Oklahoma 658 Hap23 624 Hap20 JQ348029 658 658 Canada Canada 658 Hap24 Ontario RCBS Canada RCBS GU706308 Canada Canada HM416114 Quebec HM416092 GU706543 United CC HQ987640 States GU679038 Newfoundland HM416100 658 and Labrador Quebec Newfoundland 591 and Labrador BIO Quebec CNC Florida Newfoundland BIO Quebec CNC and UG Labrador 658 GU089510 GU089511 CNC BIO 658[1n] 658 658 658 JQ348040 658 RCDH HQ987635 658 658 JQ348043 CNC HQ987636 656 GU801599 CNC CNC JQ348041 CNC HQ987637 656 JQ348044 JQ348039 658 658 597 JQ348042 HQ987644 HQ987643 GU096009 Sample information for the Table 1. Taxon tennesseensis coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata coronata plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis plectilis

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 689 GenBank acession Sequence length Bold project codeLBCBBLTIBLBCALPMN Process IDRWWB LBCB325-05LGSMC BLTIB381-08LGSMC LBCA537-05LGSMC LPMN599-08 HLC-21265 Specimen ID RWWB779-10RWWA BL611 LGSMC324-05RWWA HLC-20537 LGSMC323-05RWWA 08BBLEP-01400 RWWA-1778 LGSMC325-05RWWA DNA-ATBI-2324 RWWA588-09RWWA DNA-ATBI-2323 Haplotype Hap25 RWWA632-09RWWA DNA-ATBI-2325 Hap26 RWWA176-09RWWA Hap26 Hap26 RWWA-0606 Hap26 Country Hap26 RWWA200-09XAK Hap26 RWWA-0650 RWWA342-09 CanadaLBCD Hap26 RWWA-0176 RWWA357-09LBCD Canada RWWA-0200 United RWWA390-09 CanadaLBCD States Canada United RWWA-0342 States Hap26 United State/provinceLBCA States RWWA-0357 Hap26 XAK207-06 UnitedLBCA States British LBCD557-05 Tennessee RWWA-0390 Columbia Hap26TMNBB Washington LBCD543-05 Manitoba Tennessee Hap26LBCA Depository British United LBCD542-05 Tennessee Columbia Ontario States Hap26 BIORDLQE 2006-ONT-1202 United LBCA011-05 HLC-23377 States Hap26LBCA United LBCA002-05 HLC-23363 States TMNBB689-06 Hap26 BIO WashingtonLBCF BIO BIO United HLC-23362 States WashingtonLPMN BIO United BIO LBCA001-05 HLC-20011 States Hap26 RDLQE558-06 WashingtonBLTIB MNBTT-1629 BIO United HLC-20002 States 658 WashingtonLPMN UG Hap27 United LBCA536-05 States WashingtonZYPAN BIO Hap27 MDH002561 HLC-20001 658 LBCF023-07 658 658 WashingtonZYPAN BIO Canada LPMN864-08 Hap27 658 658 WashingtonLBCE Hap27 BIO BLTIB387-08 Hap27 HLC-20536 658 CanadaMNAC JQ348054 BIO LPMN841-08 Hap27 07-JDWBC-0072 ZYPAN065-10 Canada 658LBCA BIO 08BBLEP-02223 658 Hap27 ZYPAN068-10 Canada JQ348052 LGSMC BIO BL617 GU089162 HQ971973 Hap27 Ontario Canada 658 CanadaLPSOC BIO GU089161 08BBLEP-01644 JQ348053 CNCLEP00074265 LBCE014-05 658 Hap27 CanadaLPSOC MNAC760-07 GU089160 British Hap27 CNCLEP00074268 Hap27 Columbia 658LPSOB British JQ348051 LBCA924-05 Canada Columbia 658 LGSMC633-05 Hap27 CanadaMEC GU803197 British Hap27 Columbia HLC-23774 658 Hap27 BIO CNCLEP00027498 LPSOC438-08MEC New GU802611 British Brunswick Canada Columbia 658 Canada BIO LPSOD178-09MNAC Canada BIO GU802869 DNA-ATBI-2633 British HLC-20924 Columbia Hap27 BIO LPSOB853-08RDLQD GU802845 Hap28 Canada PPBP-2437 Quebec BIO Canada BIO GU802798 British United Columbia States 08BBLEP-04003 MEC656-04 BIO GU802781 Hap30 Hap28 658 British MEC660-04 PPBP-1852 Columbia MNAC761-07 GU802750 Canada British Manitoba 658 Columbia 658 RDLQD766-06 BIO Canada Minnesota Hap29 658 Nova Hap32 Scotia jflandry0656 Manitoba RCJD 658 658 Hap31 United BIO CNCLEP00027499 States Canada MDH001560 jflandry0660 RCDH 658 JQ348062 Ontario Hap33 Canada JQ348063 JQ348049 Quebec BIO Hap36 Canada Tennessee 658 CNC JQ348064 CNC Canada 658 Hap34 JQ348058 JQ348069 658 BIO British 658 Hap37 Columbia Hap35 JQ348059 Canada Canada British Columbia Ontario 658 JQ348060 658 BIO UG BIO 658 Canada Ontario JQ348073 CNC Canada Canada JQ348071 658 BIO JQ348061 Ontario Quebec JQ348066 HQ987642 655 658 HQ987639 Quebec 658 655 BIO Quebec Quebec BIO JQ348067 606 BIO CNC JQ348065 GU089163 JQ348057 JQ348068 658 CNC 658 JQ348070 RCDH CNC 658 655 JQ348056 658 657 JQ348050 658 JQ348072 JQ348055 GU096010 JQ348048 GU096011 Continued. Table 1. Taxon tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis tertialis

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 690 Z. Yang et al. GenBank acession ´ ebec, Canada; Sequence length sden, Germany; UG, University ollection, Munich, Germany. , Ottawa, Canada; RCAH, Research f Daniel Handfield, Qu specimens listed are available in the Barcode Bold project codeXAEXACZYPANZYPAN Process IDZYPAN XAE382-04CGUKB XAC686-04 ZYPAN064-10CGUKB ZYPAN067-10 SpecimenCGUKC ID ZYPAN071-10 CNCLEP00074264 Moth4382.03CGUKC CGUKB585-09 04HBL006686 CNCLEP00074267LEFIR CGUKB584-09 CNCLEP00074271 Hap40LEFIR CGUKC290-09 UKLB17G10 Hap41CGUKB Haplotype CGUKC928-09 UKLB17G09 Hap38 Hap42LEFIR Hap39 UKLB25C10 LEFIA709-10CGUKC Canada Country UKLB32A10 LEFIA710-10CGUKB United CGUKB780-09 States Hap43 Canada CanadaZYPAN Canada Hap43 MM01864 LEFIF771-10ZYPAN CGUKC239-09 UKLB19H05 Hap43 MM01865 CGUKB781-09CNPYD Minnesota Hap43 United Nova State/province KingdomCNPYD Scotia UKLB24G05 ZYPAN072-10 MM12996 United KingdomCNPYD UKLB19H06 ZYPAN074-10 United Ontario New Kingdom EnglandCNPYD Brunswick Ontario Hap43 Hap43 CNPYD1599-10 United CNCLEP00074272 Depository Kingdom EnglandCNPYD Hap43 CNC CNPYD1601-10 CNC CNCLEP00074274 EnglandLEFIR Hap44 CNC CNPYD1602-10 Pyr001599 Hap46 EnglandLEFIR Hap45 Hap43 CNPYD1603-10 United Finland Pyr001601 Kingdom Hap47LEFIR CNPYD1604-10 Finland Pyr001602 BMNH BIO 658 BIO United Pyr001603 658 Kingdom BMNH England LEFIC910-10 Canada 307 United Pyr001604 Finland Kingdom BMNH LEFID662-10 Canada Hap48 BMNH England LEFIG61710 658 Hap48 Savonia England MM05244 australis 658 568 Hap48 HQ987641 658 Savonia HQ987638 MM06695 australis 658 Hap48 JQ348074 China BMNH UO 658 MM14831 New Hap48 Brunswick China Regio UO aboensis JQ348032 New Brunswick China BMNH JQ348037 GU092590 China GU093571 BMNH Hap49 CNC 658 JQ348034 China UO Hap50 CNC JQ348033 658 638 Hap50 Shanxi 658 658 Finland Shanxi 307 Finland JQ348035 Shanxi 307 658 Shanxi Finland HM386854 JQ348038 Shanxi HM386855 JQ348036 NWAFU JQ348046 Regio aboensis NWAFU JQ348045 Aland HM875455 NWAFU 658 Karelia NWAFU australis UO 658 NWAFU 658 UO 658 658 HM908380 UO HM908381 637 HM908382 658 HM908383 HM908384 658 HM872727 HM876283 HM873424 Continued. tertialis tertialis tertialis tertialis perlucidalis perlucidalis perlucidalis perlucidalis perlucidalis perlucidalis perlucidalis perlucidalis perlucidalis perlucidalis quebecensis quebecensis stachydalis stachydalis stachydalis stachydalis stachydalis stachydalis stachydalis stachydalis Table 1. Taxon tertialis SpecimenIDs are specimen identifiers. ProcessIDs are sequence identifiers. Details of collecting data, images, sequences, and trace files for the 109 of Life Database (BOLD)BIO, (www.barcodinglife.org) Biodiversity in Institute the project ofCollection codes Ontario, of indicated. Guelph, Abbreviations Alfred Canada; for Haslberger, BMNH,RCJD, specimen Research The Teisendorf, depositories: Collection Natural Germany; of History RCBS, Jeremyof Museum, deWaard, Research Guelph, Vancouver, London, Guelph, Canada; Collection UK; Canada; RCTG, of CNC, Research UO, Brian Canadian Collection University of National Scholtens, of Theo Collection Oulu, Charleston, Gruenewald, Oulu, of USA; Germany; Finland; MTD, RCDH, USNM, Museum Research National of Museum Collection Zoology, of Dre o Natural History, Washington, USA; ZSC, Zoological State C

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 691

A

C

B

Fig. 2. Morphological traits measured in the present study. (A) Male genitalia; (B) Phallus; (C) Female genitalia. DASA, diameter of accessory sac; DBUR, diameter of corpus bursae; LAAP, length of anterior apophyses; LDUC, length of ductus bursae; LPAP, Length of posterior apophyses; LPHA, length of phallus; LSAC, length of sacculus; LSIG, length of signum; LUNC, length of uncus; LUTV, length from uncus to vinculum; LVAL, length of valva; LVPR, length of ventral process of editum; PEDI, perimeter of editum; WPHA, width of phallus; WSAC, width of sacculus; WSIG, width of signum; WUNC, width of uncus; WVAL, width of valva.

Ebulea tertialis. Genital preparations were stained with DNA extraction and PCR amplification Orange G in lactic acid, and characters were measured before mounting on a microscope slide in Euparal. Each Barcode records were obtained from 109 specimens by of the slide-mounted genitalia was photographed with a extracting DNA from a single leg from each individual. All Nikon AZ100 Multi-Zoom microscope, and deep-focus images samples were processed at the Canadian Centre for DNA Bar- were produced by stacking approximately 14 images using coding (CCDB) using a silica-based 96-well extraction automa- Combine Z (http://www.hadleyweb.pwp.blueyonder.co.uk) tion protocol for DNA extraction (Ivanova et al., 2006). The (Hadley, 2010). Techniques for preparation of the genitalia 658 base pair (bp) barcode region of COI (Hebert et al., 2003) slides and photography followed Landry (2007) whereas gen- was usually amplified with the LepF1/LepR1 primers (Hebert ital terminology follows Munroe (1976), Kristensen (2003) et al., 2004), but two additional primer pairs LepF1/MLepR1 and Nuss & Speidel (2005). Sets of distance and perime- and MLepF1/LepR1, which target shorter amplicons (307 bp, ter measurements were taken from the structures of female 408 bp), were used for some older specimens (Hajibabaei and male genitalia using ImageJ v1.43u Java (http://rsbweb. et al., 2006b). For the two type specimens, we used whole nih.gov/ij/). Statistical comparisons of female and male gen- abdomens for DNA extraction before removing the geni- italia were performed using Statistica v8.0 for Windows talia for dissection (Knolke¨ et al., 2005). COI sequences were (Statsoft Inc., 1999). Whereas principal components analy- assembled from shorter amplicons using primer sets designed sis (PCA) was performed to summarize patterns of variation for work on specimens with degraded DNA (R. Rougerie and in female and male genitalia, discriminant function analysis S. Prosser, personal communication). (DFA) was used to determine the morphological variables that The 12.5-μL PCR reaction mixes contained 2.5 mm MgCl2, best discriminate individuals of the four barcode lineages. Both 1.25 pm of each primer, 50 μm dNTPs, 10 mm TrisHCl PCA and DFA were performed separately for females and (pH 8.3), 50 mm KCl, 10–20 ng (1–2 μL) of genomic DNA males. Canonical analysis was conducted in order to maximize and 0.3 U of Taq DNA polymerase (Platinum Taq DNA the separation between groups and to determine differences that polymerase; Invitrogen, Burlington, Ontario, Canada). The best separate them. thermocycling profile consisted of one initial denaturation step

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 692 Z. Yang et al.

◦ ◦ Table 2. Genitalia preparations measured in this study. 55 C for 15 s and extension at 60 C for 4 min. Sequences were generated on an ABI 3730xl DNA Analyser (Applied SpecimenID Slide no. Sex Biosystems) after clean-up with Sephadex (Sigma-Aldrich, BGS03493 BIOZY00021 m St. Louis, MO) (Hajibabaei et al., 2005). The sequences CNCLEP00074270 ZY00046 m were managed in SeqScape v2.1.1 (Applied Biosystems) 09BBELE-1930 BIOZY00015 m and Sequencher v4.5 (Gene Code Corporation, Ann Arbor, 09BBELE-2507 BIOZY00018 m MI) and aligned using BioEdit v7.0.5.3 (Hall, 1999) and 09BBELE-2524 BIOZY00019 m MEGA v5.0 (Tamura et al., 2011). Sequences generated in this 09BBELE-2498 BIOZY00020 m study together with collateral information on the specimens jflandry0583 PYR519 m MM01869 BIOZY00022 m are deposited in BOLD and in GenBank (see Table 1 for MM01868 BIOZY00023 m accession numbers). The sequence alignment (FASTA format) MM09813 BIOZY00024 m is available as File S1. CNCLEP00057860 PYR565 m Moth4382.03 BIOZY00001 m DNA-ATBI-2324 BIOZY00003 m Molecular and phylogenetic analysis DNA-ATBI-2633 BIOZY00005 m 2006-ONT-1202 BIOZY00006 m Sequences were aligned using CLUSTAL W and genetic HLC-20001 BIOZY00008 m distances within and among lineages were estimated using 08BBLEP-01400 BIOZY00009 m 08BBLEP-04003 BIOZY00010 m the Kimura 2-parameter (K2P) algorithm in MEGA v5.0, 08BBLEP-01644 BIOZY00013 m including all sites with the pairwise deletion option. Boot- RWWA-0357 BIOZY00016 m strap values were calculated with 1000 replicates (Kimura, RWWA-0176 BIOZY00017 m 1980; Tamura et al., 2011), and neighbour-joining (NJ) and CNCLEP00074268 ZY00045 m minimum-evolution (ME) trees based on distance were con- CNCLEP00027499 PYR521 m structed in MEGA software. We selected Anania perlucidalis, CNCLEP00074266 ZY00044 f A. stachydalis and A. quebecensis, which are morphologically MDOK-0445 BIOZY00011 f and genetically the most similar members of the , as the MDOK-2893 BIOZY00012 f 09BBELE-1953 BIOZY00014 f primary outgroup to root the trees. The number of haplotypes jflandry2823 PYR520 f was calculated with DnaSP v5.10 (Rozas et al., 2003). BioEdit 04HBL006686 BIOZY00002 f v7.0.5.3 in Conservation Plot mode, was used to visualize and DNA-ATBI-2323 BIOZY00004 f analyse diagnostic characters. HLC-20002 BIOZY00007 f Phylogenies were inferred using Maximum likelihood (ML) CNCLEP00027498 PYR522 f and Bayesian Inference (BI), using PhyML v.3.0 (Guindon & BC MTD 00172 AT 31 f Gascuel, 2003) and MrBayes v3.1.2 (Ronquist & Huelsenbeck, CNCLEP00077265 JFL1691 f 2003), respectively. For the parameter values (e.g. sensitivity to codon bias and unequal rates of evolution) considered, the statistical inconsistency of Maximum parsimony (MP) method ◦ ◦ of 1 min at 94 C, followed by five cycles of 40 s at 94 C, can occur and was not performed in this study. For ML ◦ ◦ 40 s at 45 C and 1 min at 72 C, followed by 35 cycles of analyses, we employed the approximate likelihood ratio test ◦ ◦ ◦ 40 s at 94 C, 40 s at 51 C and 1 min at 72 C, with a final (aLRT; Anisimova & Gascuel, 2006) to estimate node support. ◦ extension of 5 min at 72 C. For mini-barcode fragments, we Nucleotide substitution model parameters were estimated used the touch-up profile (Meusnier et al., 2008): a hot start using jMODELTEST v0.1.1 (Guindon & Gascuel, 2003; ◦ ◦ for 2 min at 94 C, followed by denaturation (40 s at 94 C), Posada, 2008). In the Bayesian analysis we produced posterior ◦ ◦ annealing for 1 min at 46 C, extension for 30 s at 72 C, probability distributions by allowing four incrementally heated the last three steps cycled five times, then denaturation for Markov chains (using default heating values) to proceed for ◦ ◦ 40 s at 94 C, annealing for 1 min at 53 C, extension for 4 000 000 generations, with sampling occurring every 1000 ◦ 30 s at 72 C, the last three steps cycled 35 times, followed generations. The first 1000 trees were discarded as burn-in, ◦ by a final extension for 30 s at 72 C. PCR products were and those remaining were used to estimate topology and tree visualized on a 2% agarose E-Gel 96-well system (Invitrogen). parameters, producing a 50% majority rule consensus tree with Unpurified samples revealing faint to strong bands were cycle bipartition frequencies equal to posterior probability values sequenced bidirectionally (with the same primers used for (Ronquist & Huelsenbeck, 2003). the PCR reactions) in 10 μL reaction volumes containing: Abbreviations used in the text and figures are as follows: 0.25 μL of BigDye® v3.1 (Applied Biosystems, Foster City, AMNH (American Museum of Natural History, New York, CA), 1.875 μLof5× ABI sequencing buffer, 5 μL of 10% USA); BMNH (Natural History Museum, London, UK); CNC trehalose, 1 μLof10μm primer, 0.875 μL of ultra-pure water (Canadian National Collection of Insects, Ottawa, Canada); and 1 μL of PCR product. The following thermocycling profile ENA (Eastern North America); EU (Europe); NA (North ◦ was used for all products: initial denaturation at 96 Cfor America); TN (Tennessee, USA); USNM (National Museum ◦ 2 min, followed by 30 cycles of 96 C for 30 s, annealing at of Natural History, Washington, USA).

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 693

Table 3. Kimura 2-parameter genetic distances calculated within (in bold) and between each lineages/species of Anania.

tennesseensis (TN) coronata (EU) plectilis (ENA) tertialis (NA) perlucidalis quebecensis stachydalis tennesseensis (TN) — 0.0063 0.0053 0.0070 0.0108 0.0175 0.0123 coronata (EU) 0.0287 0.0011 0.0050 0.0068 0.0108 0.0175 0.0129 plectilis (ENA) 0.0240 0.0217 0.0046 0.0071 0.0103 0.0161 0.0125 tertialis (NA) 0.0322 0.0331 0.0368 0.0019 0.0098 0.0163 0.0127 perlucidalis 0.0784 0.0714 0.0687 0.0676 0.0003 0.0123 0.0122 quebecensis 0.0849 0.0871 0.0783 0.0809 0.0491 0.0033 0.0166 stachydalis 0.1027 0.1061 0.1039 0.1095 0.0971 0.0835 0.0071

The diagonal row of values (in bold) indicates intraspecific distances, the values below the diagonal indicates mean interspecific distances and values above the diagonal indicates SE estimates obtained by bootstrap procedure (1000 replicates) as implemented in MEGA 5.0. The four lineages of the Anania coronata complex were defined using the 2.0% divergence.

Results A

DNA barcoding and genetic distance analysis

The 109 COI sequences ranged in length from 307 to 658 bp (mean length = 644 bp) and variation was detected at 109 sites (16.6%). The pairwise genetic distances within and between these lineages are shown in Table 3. The mean genetic distance between lineages ranged from 2.17 to 10.95%, whereas intralineage variation ranged from 0.03 to 0.71%. On average, members of different species showed approximately 13× higher divergence (4.16%) than within species (0.30%). The highest genetic distance among species was between the NA lineage of Anania coronata and from B Eurasia. Four distinct and well-supported clades were observed within the A. coronata group and these groups (EU, NA, ENA, TN) are hereafter treated as four putative species. The highest genetic distance (3.68%) among the four A. coronata lineages was less than the mean distance among congeneric species, but the mean genetic distance (2.96%) between different lineages was 11× higher than that within lineages (0.25%). Excluding the TN lineage (not determined because there was a single specimen), the EU lineage (0.11%) showed the lowest intraspecific divergence whereas the NA and ENA lineages had slightly deeper divergences (0.19 and 0.46%, respectively). Geographically separated lineages of A. stachydalis from Fig. 3. (A) Neighbour-joining tree (K2P) for 109 barcodes COI sequences including Anania coronata species complex, rooted with Finland and China showed 1.43% divergence, suggesting Anania perlucidalis, Anania quebecensis and Anania stachydalis as the need for more intensive sampling of this species. In outgroup. The depth of each branch shows divergence within lineages; contrast, the divergence between any pair of A. coronata (B) Neighbour-joining tree (K2P) for 110 barcodes COI sequences lineages regardless of geographic area exceeded 2.00%. In shows that the mini-barcodes of holotype Botys plectilis clustered general, divergence levels of COI within each lineage were in ENA lineages, rooted with A. perlucidalis, A. quebecensis and low, whereas high divergences existed between lineages. ME A. stachydalis as outgroup. The depth of each branch shows divergence yielded the similar results to those of NJ based on K2P model, within lineages. Minimum-evolution (ME) based on K2P yielded the and the same NJ tree topology was produced under K2P and same tree topology as NJ and is not presented. Tamura 3-parameter (T3P) substitution models (Fig. 3A). sequences. Figure 3B shows that the sequence from the holotype of B. plectilis clustered with members of the ENA The assignment of mini-barcodes from 144-year-old type lineage, congruent with the phenetic results below. specimens

A 130-bp fragment of COI barcode region was successfully Phylogenetic analysis of COI gene recovered from the >144-year-old type of Botys plectilis.We associated the sequence from this specimen by including it The overall COI alignment was 658 bp long, including in an overall NJ analysis with all 109 ingroup and outgroup 109 variable and 101 parsimony-informative sites. We detected

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Fig. 4. Majority rule consensus trees based on Bayesian (MB) phylogenetic analyses of COI of 50 haplotypes for this study. The node support: bootstrap ML/Bayesian posterior probabilities. Single values on the MB tree correspond to Bayesian posterior probabilities. Question marks representing the bootstrap values less than 50.

50 different haplotypes among the 109 barcode sequences, bootstrap support and posterior probabilities. The EU and ENA with 42 haplotypes from the four lineages of A. coronata and clades were sister groups with moderate support values, but 8 haplotypes from the three outgroup species (Anania per- TN + (ENA + EU)cladesaswellasNA+ (ENA + EU + lucidalis, Anania quebecensis, A. stachydalis). Seven haplo- TN) clades were observed with high support values. The ENA types from Europe (EU lineage, corresponding to A. coronata) clade included two distinct subclades with a high level of sup- and 35 haplotypes from North America were observed for port, one occurring in Oklahoma and Florida, and the other A. coronata s.l. There was 1 unique haplotype for the TN lin- with a broad distribution in eastern North America. The NA eage (Anania tennesseensis sp.nov., see below), 16 haplotypes clade contained four well-supported subclades: the first two for ENA (corresponding to Anania plectilis) and 18 haplotypes included specimens from Ontario and Tennessee, respectively, for NA (corresponding to Anania tertialis). All haplotype whereas the last two were widely distributed across North sequences were included in the phylogenetic analyses. The America (Table 1, Fig. 4). best-fit model of nucleotide substitution selected by jMOD- In , mitochondrial inheritance can be impacted by ELTEST v0.1.1 was TPM2uf + I with a relative AIC weight symbiont Wolbachia infections associated with male killing, of 0.1747. ML and BI analyses recovered the same topology, cytoplasmic imcompatibility and feminization, as well as high and all haplotypes were assigned to the same major clades divergence in host mtDNA (Braig et al., 1998; Hurst & Jiggins, (Fig. 4). 2005; Frezal´ & Leblois, 2008; Smith & Fisher, 2009; Munoz˜ In ML and BI analyses, the major clades were well- et al., 2011). We considered, but ruled out, the possibility that supported, reciprocally monophyletic groups with high the barcode of the single individual of the TN lineage might

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Table 4. Results of principal component analyses of 11 male genital measurements of 23 specimens of the Anania coronata complex.

Component 1 Component 2 Component 3 Variable importance (power)

Eigenvalues 5.520 1.536 1.227 % total variance 50.180 13.963 11.159 Cumulative eigenvalue 5.520 7.056 8.283 Cumulative % 50.180 64.143 75.302 Component loadings LUNC 0.697 0.159 −0.564 0.830 WUNC 0.795 0.333 −0.188 0.778 DUTV 0.789 −0.105 0.301 0.727 LVAL 0.859 −0.134 0.000 0.757 LSAC 0.939 −0.085 −0.226 0.940 WVAL 0.384 0.785 −0.099 0.773 WSAC 0.043 0.776 0.328 0.709 PEDI 0.672 0.056 0.584 0.795 LVPR 0.647 −0.192 0.490 0.695 LPHA 0.724 −0.237 −0.034 0.582 WPHA 0.784 −0.224 −0.182 0.697

DUTV, distance from uncus to vinculum; LPHA, length of phallus; LSAC, length of sacculus; LUNC, length of uncus; LVAL, length of valva; LVPR, length of ventral process in editum; PEDI, perimeter of editum; WPHA, width of phallus; WSAC, width of sacculus; WUNC, width of uncus; WVAL, width of valva. derive from the recovery of a symbiont such as Wolbachia (as Table 5. Results of principal component analyses of 7 female genital has been shown in butterflies; Narita et al., 2006; Russell et al., measurements of 11 specimens of the Anania coronata complex. 2009; Munoz˜ et al., 2011). In particular, our morphological analysis showed four distinct groups that were congruent with Variable importance the four genetic lineages of the Anania coronata complex as Component 1 Component 2 (power) below. In other words, the validity of none of the species is justified by barcodes only, but all species show diagnostic Eigenvalues 2.907 2.547 morphological differences. % total variance 41.535 36.381 Cumulative eigenvalue 2.907 5.454 Cumulative % 41.535 77.916 Component loadings Morphological characters and morphometric analysis LAAP 0.603 −0.701 0.860 LPAP 0.567 −0.614 0.704 Comparisons of qualitative morphological characters showed LDUC 0.737 −0.343 0.664 four distinct groups that were congruent with the four genetic LSIG 0.604 0.453 0.568 lineages of the coronata complex. For example, the male gen- WSIG 0.638 0.701 0.892 italia of the ENA lineage are strikingly different from those DBUR 0.122 0.906 0.834 of the other lineages as the phallus is deeply divided into two DASA 0.945 0.204 0.932 arms without serrations (Fig. 8B), whereas those of the other DASA, diameter of accessory sac; DBUR, diameter of corpus bursae; three species have three arms with serrations (Fig. 8A, C, D). LAAP, length of anterior apophyses; LDUC, length of ductus bursae; Females of the ENA lineage and the holotype of B. plectilis LPAP, length of posterior apophyses; LSIG, length of signum; WSIG, have very similar genital structure as they lack the spinulose, width of signum. ribbon-like sclerite in the distal part of ductus bursae, present in the EU and NA lineages (similar characteristics are present WPHA, width of phallus) genital features for quantitative in the paralectotype of Ebulea tertialis) (Fig. 9A–D). On this morphological multivariate analyses and added one female basis, we call the ENA cluster Anania plectilis. (RLSB, ribbon-like sclerotized band) and three male characters Based on the qualitative morphological analysis, we selected (NSED, number of scales on editum; NSSA, number of spines 7 female (LAAP, length of anterior apophyses; LPAP, length on sacculus; CLEF, phallus cleft) for supplementary variables of posterior apophyses; LDUC, length of ductus bursae; LSIG, (except that no female of the TN lineage was available length of signum; WSIG, width of signum; DBUR, diameter for analysis). The PCA of these variables is summarized in of corpus bursae; DASA, diameter of accessory sac) and Tables 4 and 5. The results show that the first two components 11 male (LUNC, length of uncus; WUNC, width of uncus; of PCA together explain 77.9 and 64.1% of the total variance DUTV, distance from uncus to vinculum; LVAL, length of for the female and male, respectively. Four female genital valva; LSAC, length of sacculus; WVAL, width of valva; variables (DASA, WSIG, LAAP, DBUR) and six male genital WSAC, width of sacculus; PEDI, perimeter of editum; LVPR, variables (LSAC, LUNC, PEDI, WUNC, WVAL, LVAL) have length of ventral process in editum; LPHA, length of phallus; their highest loadings on the first component (P>0.75).

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 696 Z. Yang et al.

AB

CD

Fig. 5. Principal component analysis (PCA) scatter plot comparing variation of the first two principal components for all morphological characters analysed (A) males; (B) females and scatter plot of the canonical measures calculated after the discriminant function analysis (DFA) for the morphometric data along the frist roots (C) males; (D) females. Four female genital variables (DASA, WSIG, LAAP, DBUR) and six male genital variables (LSAC, LUNC, PEDI, WUNC, WVAL, LVAL) have their highest loadings on the first component (P > 0.75).

This analysis nearly distinguishes lineages ENA, TN, NA the ENA lineage was nearly restricted to the eastern part of andEUfromeachother(Fig.5A,B).InDFA,themost North America. By contrast, the NA lineage was found across significant diagnostic variables for adult males were LUNC the continent. (F = 10.9, P<0.004) and LSAC (F = 6.4, P<0.022). For adult females, there were no significant diagnostic variables after DFA, but combining those variables enables specimens of Anania coronata species complex from the four separate groups to be distinguished. Moreover, the first roots of canonical analysis yielded similar results, All four species recognized here are supported by both separating the lineages into four groups (Fig. 5C, D). molecular and morphological evidence. Morphologically, they possess the diagnostic characters of the genitalia characteristic of Anania: a digitiform sclerotization in the female antrum and Biogeographical patterns a strong, elongated, asymmetric sclerite in the phallic apodeme (Leraut, 2005; Trankner¨ et al., 2009). The coronata complex is more diverse (three species) in Measurements of male and female genitalia are presented in the Nearctic than in the Palearctic (one species) (Fig. 1). Table 6. COI sequence variation in the European lineage was less than in both North American lineages represented by multiple Anania coronata (EU lineage) individuals, and there were no sequence haplotypes shared (Figs 6A, 7A, 8A, 9A) between the two continents. Lineage EU is widely distributed in central and northern Europe (e.g. UK, Germany, Poland, Phalaena coronata Hufnagel, 1767: 616. Type locality: Finland), whereas two of the three North American lineages Berlin, Germany. Type lost. have restricted distributions: the TN lineage was found only in Pyralis sambucalis Denis & Schiffermuller,¨ 1775, 122. Type the area of Great Smoky Mountains, Tennessee, which was a locality: Vicinity of Vienna, Austria. Type lost. major North American Pleistocene refuge and harbours a large Phalaena sambuci Retzius, 1783: 49. Type locality: number of endemic species (Scholtens & Wagner, 2007); and Unknown

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Table 6. Measurements (in mm, means ± SE) of male and female genitalia in four species of the Anania coronata complex.

SpecimenID tennesseensis male holotype coronata tertialis plectilis plectilis female holotype

Male measurements N = 1N= 4N= 12 N = 6

LUNC 0.46 0.49 ± 0.02 0.48 ± 0.01 0.58 ± 0.01 WUNC 0.38 0.43 ± 0.02 0.41 ± 0.01 0.46 ± 0.01 DUTV 1.28 1.28 ± 0.02 1.19 ± 0.03 1.28 ± 0.02 LVAL 1.71 1.73 ± 0.02 1.64 ± 0.03 1.79 ± 0.02 WVAL 0.48 0.50 ± 0.01 0.51 ± 0.01 0.53 ± 0.02 LSAC 0.81 0.86 ± 0.01 0.79 ± 0.02 0.95 ± 0.01 WSAC 0.26 0.26 ± 0.01 0.29 ± 0.01 0.27 ± 0.01 PEDI 1.23 1.28 ± 0.01 1.20 ± 0.02 1.25 ± 0.04 LVPR 0.34 0.36 ± 0.01 0.34 ± 0.00 0.36 ± 0.01 LPHA 1.20 1.34 ± 0.01 1.22 ± 0.02 1.34 ± 0.03 WPHA 0.24 0.22 ± 0.01 0.20 ± 0.01 0.28 ± 0.01 NSED 8–12 12–13 8–12 12–19 NSSA 4 3–5 3–5 4–5 CLEF 3 3 3 2

Female measurements N = 1N= 4N= 5N= 1

LAAP 0.38 0.32 ± 0.01 0.39 ± 0.02 0.35 LPAP 0.42 0.42 ± 0.02 0.58 ± 0.01 0.5 LDUC 2.61 2.47 ± 0.17 2.90 ± 0.19 2.13 LSIG 0.63 0.63 ± 0.01 0.62 ± 0.06 0.44 WSIG 0.77 0.64 ± 0.02 0.66 ± 0.05 0.48 DBUR 1.17 1.32 ± 0.11 1.11 ± 0.12 0.98 DASA 0.63 0.49 ± 0.02 0.60 ± 0.05 0.42 RLSB 1100

Male measurements: CLEF, phallus cleft; DUTV, distance from uncus to vinculum; LPHA, length of phallus; LSAC, length of sacculus; LUNC, length of uncus; LVAL, length of valva; LVPR, length of ventral process in editum; NSED, number of scales on editum; NSSA, number of spines on sacculus; PEDI, perimeter of editum; WPHA, width of phallus; WSAC, width of sacculus; WUNC, width of uncus; WVAL, width of valva. Female measurements: DASA, diameter of accessory sac; DBUR, diameter of corpus bursae; LAAP, length of anterior apophyses; LDUC, length of ductus bursae; LPAP, length of posterior apophyses; LSIG, length of signum; RLSB, ribbon-like sclerotized band; WSIG, width of signum.

Phalaena sambucaria Fabricius, 1787: 186. Type locality: Barcode. Barcodes ranged from 596 to 658 bp long. Unknown Underlined bold nucleotides highlight diagnostic substitutions between species, three autapomorphies shown in italics for this species are located at nucleotide positions 212 C (cytosine), Diagnosis. The fuscous areas of the forewings are darker 476 A (adenine), 619 G (guanine). and more contrasting with the pale patches than in the North AACATTATATTTTATTTTTGGAATTTGAAGAGGAATA American members of the complex. In male genitalia, the GTTGGAACATCTTTAAGTTTATTAATTCGAGCTGAATT apex of the phallus is deeply notched with the edge of AGGAAATCCTGGATCATTAATTGGAGATGATCAAATTT the notches finely serrated and the tip of the projections ATAACACAATTGTTACAGCCCATGCATTTATTATAATTT moderately tapered; the only other member of the complex TTTTTATAGTTATACCTATTATAATTGGAGGGTTTGGAA with deep apical notches is Anania plectilis but the edges are ATTGATTAGTGCCTCTTATAC TAGGAGCCCCTGATATA smooth and the tip of the projections is sharply spiniform; GCATTTCCACGAATAAATAACATAAGATTTTGATTACTA the vesica is armed with several short spiniform setae but CCACCTTCTTTAACCCTTTTAATTTCAAGAAGTATTGTA without large cornuti. (However, cornuti may be deciduous GAAAATGGAGCAGGAACTGGATGAACTGTTTACCCCC and lacking in mated males so their number must be used CTCTTTCTTCAAACATTGCTCATGGAGGAAGATCAGTA with caution in diagnosis.) In female genitalia, the ductus GATTTAGCAATCTTTTCTTTACATTTAGCTGGTATTTCT bursae has a strongly spinulose, ribbon-like sclerotized band TCAATTTTAGGAGCAATTAATTTTATTACTACAATTATT in the anterior section which is extended as a dense and broad AATATACGAATTAATAGATTATCTTTUTUGATCAAATAC patch onto the corpus bursae; a similar ribbon-like band is CATTATTTGTATGAGCAGTAGGAATTACAGCCTTATTAT present in the female of Anania tertialis but with coarser TATTATTATCTCTTCCTGTATTAGCTGGTGCTATTACCAT spinulation. ATTATTAACTGACCGAAATTTAAATACATCTTTTTTTGA TCCGGCTGGTGGGGGAGATCCAATCTT ATACCAACAT Length of forewing. 11.67 ± 0.11 mm (N = 21). CTATTT

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 698 Z. Yang et al.

A D

B E

C F

Fig. 6. Adults of Anania coronata species complexes. (A) Anania coronata (Europe: Finland, male specimen MM09813, forewing length = 12.0 mm); (B) holotype female of Botys plectilis (forewing length = 11.5 mm); (C) Anania plectilis (Canada: Quebec, female spec- imen jflandry2823, forewing length = 11.5 mm); (D) Anania tennesseensis, holotype male (USA: Tennessee, specimen BGS03493, forewing length = 9.0 mm); (E) paralectotype female of Ebulea tertialis (forewing length = 10.8 mm); (F) Anania tertialis (Canada: Quebec, female spec- imen CNCLEP00027498, forewing length = 10.0 mm).

Distribution. Widespread in Europe (Speidel, 1996), east- widest and largest of the four species, tongue-shaped. The apex wards to Russian Far East (Kirpichnikova, 1999; Sinev, 2008). of the phallus affords the most distinctive difference, being However, we studied specimens from UK, Germany, Poland, deeply notched with smooth edges (serrated in the other three Finland only. It is not known whether further cryptic diver- species) and the terminal points are large and sharply spini- sity may be encompassed within the traditional boundaries of form (rounded in Anania coronata, sharp but proportionally Anania coronata over its entire Palearctic range. much shorter in Anania tennesseensis and Anania tertialis). The ductus bursae in the female genitalia is also very distinc- tive, without a ribbon-like sclerotized band (present in Anania Anania plectilis (ENA lineage) coronata and Anania tertialis) and the corpus bursae has a dif- (Figs 6B, C, 7B, 8B, 9B, C) fuse, very finely spinulose patch (patch sharply delineated and Botys plectilis Grote & Robinson, 1867: 27. strongly developed in Anania coronata and Anania tertialis). Phlyctaenia tertialis (Guenee):´ Dyar, 1903: 388; McDun- nough 1938: 15; Munroe 1976: 31; Munroe 1978: 70. Holotype Forewing length. 11.55 ± 0.16 mm (N = 11). in AMNH (examined). Type locality: USA, Pennsylvania. Anania plectilis: Leraut, 2005: 126–128. Barcode. Barcodes ranged from 591 to 658 bp long. The fragment (130 bp) in grey highlight was obtained from the Diagnosis. Larger in size (mean FWL = 11.5 mm) than the holotype of Botys plectilis and italic bold nucleotides show other two North American members of the complex (mean variable sites. Underlined bold nucleotides show diagnostic FWL < 10 mm). The male genitalia have the uncus and sac- substitutions between species, three autapomorphies shown in culus slightly longer than in the other three species. The italics are located at nucleotide positions 232 C (cytosine), apical portion of the sacculus is a smoothly rounded cone, 517 G (guanine), 616 C (cytosine). and the spines in the basal portion are not aggregated into a AACATTATATTTTATTTTTGGAATT TGAAGAGGAATA protrusion (as in the other three species) and somewhat sep- GTTGGAACATCTTTAAGTTTATTAATTCGAGCTGAATT arated from each other. The ventral lobe of the editum is the AGGAAATCCTGGATCATTAATTGGAGATGATCAAATTT

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 699

A B

C D

Fig. 7. Male genitalia. (A) Anania coronata (genitalia slide PYR565, CNC); (B) Anania plectilis (genitalia slide PYR519, CNC); (C) Anania tennesseensis (holotype genitalia slide BIOZY00021, USNM); (D) Anania tertialis (genitalia slide PYR521, CNC).

ATAAC ACAATTGTTACAGCCCATGCATTTATTATAATTT handwritten]; ‘in coll. as P. tertialis Gn.’ [white, handwritten]. TTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAA Genitalia dissected and mounted on slide JFL 1691. TTGATTAGTGCCTCTTATATTAGGAGCCCCTGATATAGC C TTCCCACGAATAAATAACATAAGATTTTGATTACTTCC Distribution. Eastern Nearctic region [United States (Min- ACCTTCTTTAACCCTTTTAATTTCAAGAAGTATTGTAG nesota, Oklahoma, Tennessee, North Carolina, Florida), Canada AAAATGGAGCAGGAACTGGATGAACTGTTTACCCCCC (Ontario, Quebec, Newfoundland, Labrador)] TCTTTCTTCAAACATTGCTCATGGAGGAAGATCAGTAG ATTTAGCAATCTTTTCTTTACATTTAGCTGGTATTTCTTC AATTTTAGGAGCAATTAATTTTATTACTACAATTATTAAT Remarks. Because he did not study types, Leraut (2005) ATACGAATTAATGGATTATCTTTCGATCAAATACCATTA misinterpreted the identity of this species and his diagnosis TTTGTATGAGCAGTGGGAATTACAGCCTTATTATTATTA and illustration of the male phallus (fig. 8, p.126) are those of TTATCTCTTCCTGTATTAGCTGGTGCTATTACAATATTAT Anania tertialis. TAACTGACCGAAATTTAAATACATCTTTTTTTGAC CCA GCTGGTGGGGGAGATCCAATCTTATATCAACATCTATTT Anania tennesseensis Yang, sp.nov. (TN lineage) (Figs 6D, 7C, 8C) Type material examined. Holotype, ♀, USA: Pennsylva- nia (AMNH). Labelled, ‘Pa.’ [brown, printed]; ‘No. 23005 Type material examined. Holotype, ♂, USA: Tennessee, Grote & Robin’ [white, printed with numbers handwritten], Cocke Co., Foothills Pkwy., 2nd pkglot nr. Cosby, 13.VIII. ‘TYPE No. A.M.N.H.’ [red, printed,]; ‘B. plectilis’ [white, 2006 (John W Brown) (USNM). Labelled, BOLD sample ID

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 700 Z. Yang et al.

A

B

C

D

Fig. 8. Phallus. (A) Anania coronata (genitalia slide PYR565, CNC); (B) Anania plectilis (genitalia slide PYR519, CNC); (C) Anania tennesseensis (holotype genitalia slide BIOZY00021, USNM); (D) Anania tertialis (genitalia slide PYR521, CNC). and process ID: BGS03493, LGSMG172-07. [white, printed]; tertialis in being shallowly notched with serrated edges but Genitalia slide number: BIOZY00021. ♂|BIOZY00021 [pale with the notches more pronounced; vesica with two long and green, printed]; ‘HOLOTYPE|Anania tennesseensis|Yang 2011 thin cornuti. | by ZF Yang 2011’ [red, partly printed, partly handwritten]. The holotype is provisionally deposited at the USNM, Wash- Description. Forewing length 9.0 mm. Labial palpus por- ington, DC, pending agreement with the U.S. National Park rect, third segment exposed and porrect, distal part with fus- Service regarding specimen deposition. For additional type cous color. Body and wings pale buff, with strongly con- locality information, see Table 1. trasting pale patches on infuscated ground. Forewing with antemedial line fuscous, somewhat indistinct or obsolete. Post- Etymology. Named after the state where the type specimen medial line fuscous, strongly and regularly dentate. Beyond was collected. the postmedial a fuscous, strongly dentate, subterminal shade. Terminal line fuscous, broken between the veins; fringe concolorous with wing. Hindwing antemedial, medial line Diagnosis. This is the smallest species of the complex and postmedial, subterminal, and terminal markings as on (forewing length 9 mm) based on our sampling, however the forewing. size difference is small, especially compared to Anania ter- tialis (mean FWL 9.8 mm) and would have to be assessed on greater sampling. The uncus is slightly shorter (0.46 mm) and Male genitalia. Uncus subtriangular. Transtilla incomplete narrower (0.38 mm) than in the other three species; the saccu- medially. Juxta lunular. Vinculum ventrally rounded. Valva lus length (0.81 mm) is intermediate between that of Anania tapered somewhat in distal half; base of costa weakly tertialis (shorter at 0.79 mm) and Anania coronata, Anania inflated; sacculus inflated and spinulose dorsally, distal plectilis (both longer at 0.86 and 0.95 mm, respectively); the part with several dorsally-projected small spines, spines dorsal edge of the sacculus has more protruded and more aggregated at base; basal pad of editum with a crest of densely aggregated spines basally with spines of uneven sizes 8–12 dorsally directed scales, ventral process narrow. Phal- and some more protruded, and the distal portion of the saccu- lus with apex broad and moderately indented, with three lus has coarse, blunt serrations (serrations ill-defined in Anania roughly serrated notches; vesica with two slender, spiniform tertialis); the ventral process of editum is narrow, similar to cornuti. that of Anania tertialis; phallus length/width ratio c. 5.0 (6.0 in Anania tertialis); apex of phallus resembling that of Anania Female genitalia. Unknown.

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 701

AB CD

Fig. 9. Female genitalia. (A) Anania coronata (genitalia slide AT31, MTD); (B) holotype of Botys plectilis (genitalia slide JFL1691, AMNH); (C) Anania plectilis (genitalia slide PYR520, CNC); (D) Anania tertialis (genitalia slide PYR522, CNC).

Barcode. A 658 bp barcode was obtained from the holo- Distribution. Known only from the Great Smoky Mountains type. Underlined bold nucleotides highlight those diagnostic in Tennessee, U.S.A. substitutions from other members of the complex, four autapo- morphies shown in italics are located at nucleotide positions Anania tertialis (NA lineage) 25 C (cytosine), 151 C (cytosine), 433 G (guanine), 625 C (Figs 6E, F, 7D, 8D, 9D) (cytosine). Ebulea tertialis Guenee,´ 1854: 364. Lectotype in BMNH AACATTATATTTTATTTTTGGAATC TGAAGAGGAATA (examined, see below). Type locality: North America. GTTGGAACATCTTTAAGTTTATTAATTCGAGCTGAATTA Botys syringicola Packard, 1870: 250. Dyar, 1903: 388 [as a GGAAATCCTGGATCATTAATTGGAGATGATCAAATTTA synonym of tertialis]; McDunnough 1938: 15 [as a synonym of TAACACAATTGTTACAGCCCATGCATTTATTATAATC TT tertialis]. Munroe 1976: 31; Munroe 1978: 70 [as a synonym TTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAAT of coronata tertialis]. Location of type material unknown. Type TGATTAGTACCTCTTATATTAGGGGCCCCTGATATAGCA locality: USA, New York [state]. TTTCCACGAATAAATAACATAAGATTTTGATTACTTCCA Phlyctaenia tertialis; Dyar 1903: 388. CCTTCTTTAACCCTTCTAATTTCAAGAAGTATTGTAGA Phlyctaenia coronata tertialis; Munroe 1954: 429. AAATGGAGCAGGAACTGGATGAACTGTTTATCCCCCT Anania tertialis; Leraut, 2005: 126–128. CTTTCTTCAAACATTGCTCATGGGGGAAGATCAGTAG ATTTAGCAATCTTTTCTTTACATTTAGCTGGTATTTCTTC Diagnosis. Intermediate in size between Anania tennesseen- AATTTTAGGGGCAATTAATTTTATTACTACAATTATTAA sis (9.0 mm) and Anania plectilis (11.5 mm). In male genitalia, TATACGAATTAATGGATTATCTTTCGACCAAATACCATT spines of sacculus aggregated both basally and distally; dorsal ATTTGTATGAGCAGTAGGAATTACAGCCTTATTATTATT lobe of editum somewhat rounded with nearly straight ventral ATTATCTCTTCCTGTATTAGCTGGTGCTATTACCATATTA margin (margin concave in Anania plectilis); apex of phallus TTAACTGACCGAAATTTAAATACATCTTTTTTTGATCCA resembling that of Anania tennesseensis with shallow, serrated GCTGGC GGAGGAGATCCAATCTTATATCAACATCTATTT notches and one sharply spiniform but short point (rounded in

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 702 Z. Yang et al.

Anania coronata, smooth and long in Anania plectilis). Female subspecies are known to be leaf-webbers or shoot borers on genitalia similar to Anania coronata. species (Caprifoliaceae), the same host used by the nominate subspecies Anania coronata in Europe (Goater, Forewing length. 9.80 ± 0.11 mm (N = 15) 1986). There are discrepancies between Packard’s description of syringicola that suggest a difference from the superficial appearance of all nominal species treated here. For instance, Barcode. Barcodes range from 307 to 658 bp long. Under- Packard described the hindwing as ‘yellow, with four sharply lined bold nucleotides highlight diagnostic differences with zigzag dark gray lines’, a characterization that is somewhat other species, five autapomorphies shown in italics are located difficult to reconcile with the extensive dark peppering with at nucleotide positions 91 C (cytosine), 181 G (guanine), a contrasting large pale yellowish (or whitish) patch that 190 G (guanine), 202 C (cytosine), 514 G (guanine). strikingly mark tertialis or coronata complex specimens. AACATTATATTTTATTTTTGGAATTTGAAGAGGAATA These discrepancies coupled with the odd host record suggest GTTGGAACATCTTTAAGTTTATTAATTCGAGCTGAATT that syringicola might be another species altogether, or might AGGTAATCCTGGATCC TTAATTGGAGATGATCAAATTT not even belong to Anania. For the time being, it is better to ATAATACAATTGTTACAGCCCATGCATTTATTATAATTT leave it in its current synonymy until the type is discovered or TTTTTATAGTTATACCTATTATAATTGGGGGGTTTGGGA matching specimens are found. ATTGATTAGTC CCTCTTATATTAGGAGCCCCTGATATAG CATTTCCACGAATAAATAACATAAGATTTTGATTACTTC Distribution. Eastern and western Nearctic region [United CACCTTCTTTAACTCTTCTAATTTCAAGAAGTATTGTA States (Washington, Minnesota, Tennessee), Canada (British GAAAATGGAGCAGGAACTGGATGAACTGTTTACCCCC Columbia, Manitoba, Ontario, Quebec, New Brunswick, Nova CTCTTTCATCAAACATTGCTCATGGAGGAAGATCAGTA Scotia)]. GATTTAGCAATCTTTTCTTTACATTTAGCTGGTATTTCT TCAATTTTAGGAGCAATTAATTTTATTACTACAATTATTA ATATACGAATTAATGGATTATCTTTCGACCAAATACCAT Remarks. Because he did not study types, Leraut (2005) TATTTGTATGAGCGGTAGGAATTACAGCTTTATTATTAC misinterpreted the identity of this species and his diagnosis TACTATCTCTTCCTGTATTAGCTGGTGCTATTACCATATT and illustration of the male phallus (fig. 7, p. 126) are those of ATTAACTGACCGAAATTTAAATACATCTTTTTTTGATCC Anania plectilis. TGCTGGTGGAGGAGATCCAATCTTATACCAACATCTA TTT Discussion

Type material examined. There are two syntypes of Ebulea Many recent commentaries (Goldstein & DeSalle, 2010; Lum- tertialis, one in the BMNH and one in the USNM. Solis ley & Sperling, 2010; Padial et al., 2010) and ever increasing selected the BMNH specimen as the lectotype. studies in Lepidoptera (Hebert et al., 2004; Wilson et al., 2010; Lectotype, ♀, here designated (BMNH). Labelled: ‘Ex. Mutanen et al., 2012) have either discussed and/or illustrated Musaeo|Ach. Guen´ ee’´ [white, printed]; ‘Tertialis|Gn. Am. bor’ the power of ‘integrative taxonomy’. This integrated study uti- [white, handwritten]; ‘Oberthur|Collection’ [pale yellow with lizing morphological and molecular characters revealed that red print]; ‘Ebulea|tertialis,Guenee´ |Sp. G. VIII-446’ [white Anania coronata is really four well-supported, reciprocally with black border, handwritten]; ‘plectilis|gr. Fr.’ [white, monophyletic groups. The molecular analysis indicated that handwritten]; ‘LECTOTYPE ♀|Ebulea|tertialis Guenee,´ 1854| the North American Anania plectilis and Anania tennesseen- det. M. A. Solis 2011’ [red, printed]. sis, and the European A. coronata form a monophyletic cluster Paralectotype, ♀, in USNM. Labelled: ‘Ex. Musaeo|Ach. that is rather distant to the third North American species, Ana- Guen´ ee’´ [white, printed]; ‘Oberthur|Collection’ [pale yellow nia tertialis. The same result was obtained regardless of the with red print]’; ‘Barnes|Collection’ [printed in red]; ‘PAR- method and substitution model. The quantitative morphologi- ALECTOTYPE ♀|Ebulea|tertialis Guenee,´ 1854| det. M. A. cal analysis revealed that genital differences are correlated with Solis 2011’ [blue, printed]; genitalia on slide USNM 130208. the genetic differences. The location of the type of Botys syringicola, which was Integration by congruence has a ‘long tradition in system- described from New York state, is unknown. Despite extensive atics’ and promotes taxonomic stability, but it runs the risk searches in the USNM, as well as inquiries with the AMNH, of underestimating species numbers because characters do not New York State Museum (Albany, NY) and Academy of change at all levels and the rates of character change are hetero- Natural Sciences (Philadelphia), we were unable to locate the geneous (Padial et al., 2010). Interestingly, A. tertialis displays type specimens. The synonymy of syringicola goes back at the closest morphological similarity to A. coronata, in contrast least to Dyar (1903) (not Munroe, 1976, as stated incorrectly by to their marked COI divergence. In contrast to the species Leraut, 2005), but hitherto seems to have remained unverified. group analysed in this study, Trankner¨ et al. (2009) illustrated The type material, probably made up of a single specimen, was that many Anania species in Europe are externally strikingly reared from a larva boring into a branch of lilac (Syringa sp., morphologically divergent. Padial et al. (2010) stated that gen- Oleaceae). Lilac is not native to North America and represents ital structures of arthropods would be the most important char- an unusual host record. Larvae of the North American acters to study because they contribute to the reproduction of

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 703 the species. This has not always been the case in the . Acknowledgements Studies of pyraloid adult morphological characters, most par- ticularly the genital characters upon which the nomenclature We thank colleagues at the Biodiversity Institute of Ontario is based, at taxonomic levels higher than species have yielded for assistance with DNA sequencing. We thank Vazrick Nazari very few informative characters either due to homoplasy or for assistance with photography of the genitalia at the CNC, a dearth of morphological divergence (Solis & Maes, 2002; and Mark Metz for measurement and photography of the Regier et al., in press). In some pyraloid groups, the adults type specimen at the USNM. We thank Andreas Segerer, can exhibit obvious gaps in the external morphology, but no Brandon Laforest, Evgeny Zakharov and Jeremy deWaard appreciable divergence in internal morphology of structures for permission to access their data. We are also grateful such as genitalia, or the reverse. Unfortunately, many genera to Suzanne Rab Green and David Grimaldi (AMNH) for (e.g. ; Mutuura & Munroe, 1970) in the Pyraustinae permission to analyse the type specimen, J. Donald Lafontaine are plagued with a lack of obvious divergence in both external who aided work on the type specimen in the British Museum and internal characters. The use of molecular characters will of Natural History, and Tim McCabe, NY State Museum (Albany), for searching the collection under his care for greatly accelerate the discovery of characters, new species and possible types. This study was supported by The Ministry of relationships between species in the Pyraustinae. Science and Technology of the People’s Republic of China The relatively small haplotype variation was probably due (2011FY120200) and by grants from NSERC, and from the to a limited sample size and collecting efforts in the middle government of Canada through Genome Canada and the and southern Appalachian areas to obtain more specimens Ontario Genomics Institute in support of the International of A. tennesseensis, including females, could provide greater Barcode of Life project to PDNH. Z. Yang thanks the China resolution. Further studies in phylogeographic patterns should Scholarship Council for its support. attempt to examine more specimens from the East Palaearctic Region, such as the Russian Far East, China and Japan. Nuclear markers (e.g. RpS5, CAD, EF-1a and D2 loop of 28S rRNA) Authors contributions would be helpful in further understanding species divergence and relationships within the Anania coronata species complex. Conceived and designed molecular analysis: ZFY, YLZ, This study solves a relatively small problem in species PDNH. Conceived and performed molecular and morphome- numbers that had been suspected for many decades; Munroe tric analyses: ZFY. Provided morphological data: ZFY, JFL, (1976) stated ‘further work may show that more than one LH. Analysed morphological data: ZFY. Studied type speci- species have been included under this name’. It is easy mens: JFL, MAS. Contributed specimens and/or observations: find such statements for most genera of the Pyraloidea in DH, BGS, MAS, MM, MN, PDNH. Wrote and/or edited the the literature. For taxonomists who study hyperdiverse taxa manuscript: ZFY, JFL, YLZ, MAS, MM, PDNH. Prepared the ‘taxonomic impediment’ is the magnitude of known and illustrations: ZFY. The authors have declared that no com- potentially undiscovered species. In hyperdiverse taxa even peting interests exist. genera can consist of hundreds of species. Anania is one of the largest genera in Pyraustinae with 114 species (Nuss et al., 2009; Trankner¨ et al., 2009; Trankner¨ & Nuss, 2010). References Only with a project such as the Lepidoptera Barcode of Anisimova, M. & Gascuel, O. (2006) Approximate likelihood-ratio Life (http://www.lepbarcoding.org/), that to date has produced test for branches: a fast, accurate, and powerful alternative. 624 163 sequences for 73 186 species, can taxonomists who Systematic Biology, 55, 539–552. study hyperdiverse groups such as Lepidoptera (157 424 Braig, H.R., Zhou, W., Dobson, S.L. & O’Neill, S.L. (1998) Cloning species; van Nieukerken et al., 2011) take long leaps to and characterization of a gene encoding the major surface protein pinpoint interesting questions, and, as in this study, discover of the bacterial endosymbiont Wolbachia pipientis. Journal of and validate cryptic species. Bacteriology, 180, 2373–2378. Burns, J.M., Janzen, D.H., Hajibabaei, M., Hallwachs, W. & Hebert, P.D.N. (2007) DNA barcodes of closely related (but morpho- logically and ecologically distinct) species of skipper butterflies Supporting Information (Hesperiidae) can differ by only one to three nucleotides. Journal of the Lepidopterists’ Society, 61, 138–153. Additional Supporting Information may be found in the online Dayrat, B. (2005) Toward integrative taxonomy. Biological Journal of version of this article under the DOI reference: the Linnaean Society, 85, 407–415. 10.1111/j.1365-3113.2012.00637.x Denis, J.N.C.M. & Schiffermuller,¨ I. (1775) Ank¨undung eines system- atischen Werkes von den Schmetterlingen der Wienergegend her- File S1. COI allingment as FASTA file. ausgegeben von einigen Lehrern am k.k. Theresianum. Augustin Please note: Neither the Editors nor Wiley-Blackwell Bernardi, Wien. frontispiece, 1–323, pls. 1–3. [1776 edition seen]. are responsible for the content or functionality of any Dinca, V., Zakharov, E.V., Hebert, P.D.N. & Vila, R. (2011) Com- plete DNA barcode reference library for a country’s butterfly supporting materials supplied by the authors. Any queries fauna reveals high performance for temperate Europe. Philosoph- (other than missing material) should be directed to the ical Transactions of the Royal Society B: Biological Sciences, 278, corresponding author for the article. 347–355.

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 704 Z. Yang et al.

Dyar, H.G. (1903) A list of North American Lepidoptera. Bulletin of Inoue, H. (1982) . of Japan 1+2(ed.byH.Inoue, the United States National Museum (Washington, DC), 52,i–xix, S. Sugi, H. Kuroko, et al.), pp. vol. 1: 307–404; vol. 2: 223–254; 1–723. pls. 36–48, 228, 296–314. Kodansha, Tokyo. Fabricius, J.C. (1787) Mantissa Insectorum sistens species nuper detec- Ivanova, N.V., deWaard, J.R. & Hebert, P.D.N. (2006) An inexpen- tas adiectis synonymis, observationibus, descriptionibus, emenda- sive, automation-friendly protocol for recovering high-quality DNA. tionibus. Christian Gottlieb Proft, Hafniae. Molecular Ecology Notes, 6, 998–1002. Frezal,´ L. & Leblois, R. (2008) Four years of DNA barcoding: Kimura, M. (1980) A simple model for estimating evolutionary rates current advances and prospects. Infection, Genetics and Evolution, of base substitutions through comparative studies of nucleotide 8, 727–736. sequences. Journal of Molecular Evolution, 16, 111–120. Goater, B. (1986) British Pyralid Moths. A Guide to their Identification. Kirpichnikova, V. A. (1999) Pyraloidea [sine Phycitinae]. Key to the Harley Books, Colchester. insects of Russian Far East 5(2) (in Russian) (ed. by P. A. Ler), Goldstein, P.Z. & DeSalle, R. (2010) Integrating DNA barcode data pp. 320–443. Dalnauka, Vladivostok. and taxonomic practice: determination, discovery, and description. Knolke,¨ S., Erlacher, S., Hausmann, A., Miller, M.A. & Segerer, A.H. Bioessays, 33, 135–147. (2005) A procedure for combined genitalia dissection and DNA Grote, A.R. & Robinson, C.T. (1867) Descriptions of American extraction in Lepidoptera. Systematics & Evolution, 35, Lepidoptera. Transactions of the American Entomological Society, 401–409. Philadelphia, 1, 1–30, pls 1–2; 171–192, pl. 4. Kristensen, N.P. (2003) Skeleton and muscles: adults. Lepidoptera: Guenee,´ A. (1854) Species G´en´eral des L´epidopt`eres, Tome Huiti`eme, Moths and Butterflies, Handbook of Zoology, Volume 2: Morphology, Deltoïdes et Pyralites. Librairie Encyclopedique´ Roret, Paris. Physiology, and Development (ed. by N.P. Kristensen), pp. 39–131. Guindon, S. & Gascuel, O. (2003) A simple, fast, and accurate Walter de Gruyter, Berlin and New York, NY. algorithm to estimate large phylogenies by maximum likelihood. Kristensen, N.P., Scoble, M.J. & Karsholt, O. (2007) Lepidoptera Systematic Biology, 52, 696–704. phylogeny and systematics: the state of inventorying moth and Hadley, A. (2010) Combine ZP Sofware, New Version [WWW docu- butterfly diversity. Zootaxa, 1668, 699–747. ment]. URL http://www.hadleyweb.pwp.blueyonder.co.uk/CZP/ Landry, J.F. (2007) Taxonomic review of the leek moth genus News.htmH [accessed on 20 January 2011]. Acrolepiopsis (Lepidoptera: Acrolepiidae) in North America. The Hajibabaei, M., deWaard, J.R., Ivanova, N.V. et al. (2005) Critical Canadian Entomologist, 139, 319–353. factors for assembling a high volume of DNA barcodes. Philosoph- Leraut, P.J.A. (2005) Contribution al’` etude´ de quelques genres et ical Transactions of the Royal Society B: Biological Sciences, 360, especes` de Pyraustinae (Lepidoptera: Crambidae). Nouvelle Revue 1959–1967. d’Entomologie, 22, 123–139. Hajibabaei, M., Janzen, D.H., Burns, J.M., Hallwachs, W. & Hebert, Lumley, L.M. & Sperling, F.A.H. (2010) Integrating morphology and P.D.N. (2006a) DNA barcodes distinguish species of tropical mitochondrial DNA for species delimitation within the spruce Lepidoptera. Proceedings of the National Academy of Sciences of budworm (Choristoneura fumiferana) cryptic species complex the United States of America, 103, 968–971. (Lepidoptera: ). Systematic Entomology, 35, 416–428. Hajibabaei, M., Smith, A., Janzen, D.H., Rodriguez, J.J., Whitfield, J. McDunnough, J. (1938). A Checklist of the Lepidoptera of Canada and & Hebert, P.D.N. (2006b) A minimalist barcode can identify a the United States of America. Memoirs of the Southern California specimen whose DNA is degraded. Molecular Ecology Notes, 6, Academy of Sciences, 1, 1–275. 959–964. Meusnier, I., Singer, G.A.C., Landry, J.F., Hickey, D.A., Hebert, Hall, T.A. (1999) BioEdit: a user-friendly biological sequence align- P.D.N. & Hajibabaei, M. (2008) A universal DNA mini-barcode ment editor and analysis program for Windows 95/98/NT. Nucleic for biodiversity analysis. BMC Genomics, 9, 214. Acids Symposium Series, 41, 95–98. Munoz,˜ A.G., Baxter, S.W., Linares, M. & Jiggins, C.D. (2011) Deep Hebert, P.D.N., Cywinska, A., Ball, S.L. & deWaard, J.R. (2003) mitochondrial divergence within a Heliconius butterfly species is Biological identifications through DNA barcodes. Philosophical not explained by cryptic speciation or endosymbiotic bacteria. BMC Transactions of the Royal Society B: Biological Sciences, 270, Evolutionary Biology, 11, 358. 313–321. Munroe, E.G. (1954) A new Phlyctaenia, with notes on two related Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H. & species (Lepidoptera: Pyralidae). The Canadian Entomologist, 86, Hallwachs, W. (2004) Ten species in one: DNA barcoding reveals 428–431. cryptic species in the neotropical skipper butterfly Astraptes fulger- Munroe, E.G. (1976) Pyraloidea Pyralidae comprising the subfamily ator. Proceedings of the National Academy of Sciences of the United Pyraustinae tribe Pyraustini (part). The Moths of America North of States of America, 101, 14812–14817. Mexico 13.2A (ed. by R.B. Dominick, T. Dominick, D.C. Ferguson Hebert, P.D.N., deWaard, J.R. & Landry, J.F. (2010) DNA barcodes et al.), pp. 1–78, pls 1–4, A-H. E.W. Classey Ltd. and The Wedge for 1/1000 of the kingdom. Biology Letters, 6, 359–362. Entomological Research Foundation, London. Hodges, R.W., Dominick, T., Davis, D.R., Ferguson, D.C., Fran- Munroe, E.G. (1978) Hubner¨ and allied neotropical genera: clemont, J.G., Munroe, E.G. & Powell, J.A. (1983) Check List supplementary notes and descriptions (Lepidoptera: Pyralidae: of the Lepidoptera of North America North of Mexico. E.W. Pyraustinae). The Canadian Entomologist, 110, 673–695. Classey and The Wedge Entomological Research Foundation, Munroe, E.G., Becker, V.O., Shaffer, J.C., Shaffer, M. & Solis, M.A. London. (1995) Pyraloidea. Atlas of Neotropical Lepidoptera. Checklist: Part Hufnagel, J.S. (1767) Fortsetzung der Tabelle von den Nachtvogeln,¨ 2. Hyblaeoidea – Pyraloidea – Tortricoidea (ed. by J.B. Heppner), welche die 3te Art derselben, nehmlich die Spannenmesser (Phalae- pp. 34–105. Association for Tropical Lepidoptera, Gainesville, FL. nas Geometras Linnaei) enthalt.¨ Berlinisches Magazin, 4, 504–527; Mutanen, M., Aarvik, L., Huemer, P., Kaila, L., Karsholt, O. & 599–626. Tuck, K. (2012) DNA barcodes reveal that the widespread Euro- Hurst, G.D.D. & Jiggins, F.M. (2005) Problems with mitochondrial pean tortricid moth manniana (Lepidoptera: Tortricidae) DNA as a marker in population, phylogeographic and phylogenetic is a mixture of two species. Zootaxa, 3262, 1–21. studies: the effects of inherited symbionts. Philosophical Transac- Mutuura, A. & Munroe, E.G. (1970) Taxonomy and distribution of the tions of the Royal Society B: Biological Sciences, 272, 1525–1534. European corn borer and allied species: genus Ostrinia [Hubner]¨

© 2012 The Authors Systematic Entomology © 2012 The Royal Entomological Society, Systematic Entomology, 37, 686–705 DNA barcoding reveals three cryptic species of Anania 705

(Lepidoptera: Pyralidae). Memoirs of the Entomological Society of approach to exploring biodiversity. Annual Review of Entomology, Canada, 71, 1–112. 55, 421–438. Narita, S., Nomura, M., Kato, Y. & Fukatsu, T. (2006) Genetic Scholtens, B.G. & Wagner, D.L. (2007) Lepidoptera of Great Smoky structure of sibling butterfly species affected by Wolbachia infection Mountains National Park: methods and results of the inventory. sweep: evolutionary and biogeographical implications. Molecular Southeastern Naturalist, 6, 193–206. Ecology, 15, 1095–1108. Sinev, S.Yu. (2008) Crambidae. Catalogue of the Lepidoptera of van Nieukerken, E.J., Kaila, L., Kitching, I.J. et al. (2011) Order Russia (ed. by S.Y. Sinev), pp. 170–187. KMK Scientific Press, Lepidoptera Linnaeus, 1758. In: Zhang, Z.-Q. (Ed.) Animal St. Petersburg and Moscow. Biodiversity: An Outline Of Higher-Level Classification and Survey Smith, M.A. & Fisher, B.L. (2009) Invasions, DNA barcodes, and Of Taxonomic Richness. Zootaxa, 3148, 212–221. rapid biodiversity assessment using ants of Mauritius. Frontiers in Nuss, M. & Speidel, W. (2005) Introduction. In: B. Goater, M. Nuss Zoology, 6, 31. and W. Speidel, ed. Pyraloidea I, Microlepidoptera of Europe, Smith, M.A., Woodley, N.E., Janzen, D.H., Hallwachs, W. & Hebert, Vol. 4 (ed. by P. Huemer and O. Karsholt), pp. 7–20. Apollo Books, P.D.N. (2006) DNA barcodes reveal cryptic host-specificity within Stenstrup. the presumed polyphagous members of a genus of parasitoid flies Nuss, M., Landry, B., Vegliante, F. et al. (2009) Global Informa- (Diptera: Tachinidae). Proceedings of the National Academy of tion System on Pyraloidea (GlobIZ) [WWW document]. URL Sciences of the U.S.A., 103, 3657–3662. www.pyraloidea.org [accessed on 29 April 2011]. Solis, M.A. & Maes, K.V.N. (2002) Preliminary phylogenetic analysis Packard, A.S. (1870) Injurious insects, new and little known. Seven- of the subfamilies of Crambidae (Pyraloidea: Lepidoptera). Belgian teenth Annual Report of the Secretary of the Massachusetts Board Journal of Entomology, 4, 53–95. of Agriculture for 1869. pp. 235–263, 1 plate. Wright & Potter, Speidel, W. (1996) Family Pyralidae. The Lepidoptera of Europe, Boston. a Distributional Checklist (ed. by O. Karsholt and J. Razowski), Padial, J.M. & De La Riva, I. (2010) A response to recent proposals for pp. 166–196. Apollo Books, Stenstrup. integrative taxonomy. Biological Journal of the Linnaean Society, StatSoft Inc. (1999) STATISTICA for Windows [Computer Program 101, 747–756. Manual]. StatSoft Inc., Tulsa, OK. Padial, J.M., Miralles, A., De la Riva, I. & Vences, M. (2010) The Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & integrative future of taxonomy. Frontiers in Zoology, 7, 16. Kumar, S. (2011) MEGA5: molecular evolutionary genetics anal- Posada, D. (2008) jModelTest: phylogenetic model averaging. Molec- ysis using maximum likelihood, evolutionary distance, and max- ular Biology & Evolution, 25, 1253–1256. imum parsimony methods. Molecular Biology and Evolution, 28, Regier, J.C., Mitter, C., Solis, M.A. et al. (2012). A molecular phy- 2731–2739. logeny for the pyraloid moths (Lepidoptera: Pyraloidea) and its Trankner,¨ A. & Nuss, M. (2010) Anania ochrofascialis (Christoph) implications for higher-level classification. Systematic Entomology, comb. n. and A. murcialis (Ragonot) comb. n. – two vicarious in press. species from the western Palaearctic region (Pyraloidea: Crambidae: Retzius, A.I. (1783) Caroli Lib. Bar. de Geer ... Genera et species Pyraustinae). Nota lepidopterologica, 33, 59–65. insectorum e generosissimi auctoris scriptis extraxit, digessit, latine Trankner,¨ A., Li, H. & Nuss, M. (2009) On the systematics of quoad partem reddidit, et terminologiam insectorum Linneanam Anania Hubner,¨ 1823 (Pyraloidea: Crambidae: Pyraustinae). Nota addidit. Lipsiae, Apud Sigfried Lebrecht Crusium. lepidopterologica, 32, 63–80. Ronquist, F. & Huelsenbeck, J.P. (2003) MRBAYES 3: bayesian de Waard, J.R., Hebert, P.D.N. & Humble, L.M. (2011) A com- phylogenetic inference under mixed models. Bioinformatics, 19, prehensive DNA barcode library for the looper moths (Lepi- 1572–1574. doptera: Geometridae) of British Columbia, Canada. PLoS ONE, 6, Rozas, J., Sanchez-Delbarrio, J.C., Messeguer, X. & Rozas, R. (2003) e18290. DnaSP, DNA polymorphism analyses by the coalescent and other Wilson, J.J., Landry, J.F., Janzen, D.H., Hallwachs, W., Nazari, V., methods. Bioinformatics, 19, 2496–2497. Hajibabaei, M. & Hebert, P.D.N. (2010) Identity of the ailanthus Russell, J.A., Goldman-Huertas, B., Moreau, C.S. et al. (2009) Spe- webworm moth (Lepidoptera, Yponomeutidae), a complex of two cialization and geographic isolation among Wolbachia symbionts species: evidence from DNA barcoding, morphology and ecology. from ants and lycaenid butterflies. Evolution, 63, 624–640. ZooKeys, 46, 41–60. Schlick-Steiner, B.C., Steiner, F.M., Seifert, B., Stauffer, C., Chris- tian, E. & Crozier, R.H. (2010) Integrative taxonomy: a multisource Accepted 30 April 2012

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