Systematic Entomology (2012), 37, 686–705 DNA barcoding and morphology reveal three cryptic species of Anania (Lepidoptera: Crambidae: Pyraustinae) 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. Anania coronata (Hufnagel), a Holarctic species of pyraustine crambid moth, 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), Anania perlucidalis (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 Table 1. Sample information for the Anania specimens included in the sequence. Bold project Sequence GenBank Z. Yang Taxon code Process ID Specimen ID Haplotype Country State/province Depository length acession tennesseensis LGSMG LGSMG172-07 BGS03493 Hap1 United States Tennessee USNM 658 JQ348047 et al coronata CGUKB CGUKB608-09 UKLB18A09 Hap2 United Kingdom England BMNH 658 JQ348024 coronata CGUKC CGUKC074-09 UKLB23A04 Hap2 United Kingdom England BMNH 658 JQ348023 . coronata FBLMS FBLMS201-09 BC ZSM Lep 23012 Hap2 Germany Bavaria RCTG 658 HM901988 coronata FBLMU FBLMU401-09 BC ZSM Lep 27051 Hap2 Germany Bavaria ZSC 658 GU707126 coronata LEFIR LEFIA713-10 MM01868 Hap2 Finland Savonia australis UO 658 HM386857 coronata LEFIR LEFIA714-10 MM01869 Hap2 Finland Savonia australis UO 658 HM386858 coronata LEFIR LEFIE721-10 MM09813 Hap2 Finland Aland UO 658 HM874441 coronata PYRG PYRG083-09 BC MTD 00171 Hap2 Poland Podlachien MTD 658 GU700961 Systematic Entomology coronata PYRG PYRG084-09 BC MTD 00172 Hap2 Germany Baden-Wuerttemberg MTD 658 GU700962 coronata RDNMH RDNMH541-09 CNCLEP00057860 Hap2 Germany Saxony MTD 658 GU679045 coronata RDNMH RDNMH543-09 CNCLEP00057862 Hap2 Germany Saxony MTD 658 GU679039 coronata CGUKA CGUKA485-09 UKLB6B04 Hap3 United Kingdom England BMNH 596 JQ348031 coronata CGUKC CGUKC224-09 UKLB24F02 Hap4 United Kingdom England BMNH 640 JQ348030 coronata CGUKA CGUKA820-09 UKLB9F10 Hap5 United Kingdom BMNH 658 JQ348022 coronata CGUKB CGUKB328-09 UKLB15A11 Hap5 United Kingdom England BMNH 658 JQ348028 coronata CGUKB CGUKB606-09 UKLB18A07 Hap5 United Kingdom England BMNH 658 JQ348027 © coronata CGUKB CGUKB607-09 UKLB18A08 Hap5 United Kingdom England BMNH 658 JQ348026 2012 The Royal Entomological Society, coronata CGUKB CGUKB787-09 UKLB19H12 Hap5 United Kingdom England BMNH 658 JQ348025 coronata CGUKD CGUKD126-09 UKLB34B05 Hap5 United Kingdom England BMNH 658 JQ348029 coronata FBLMS FBLMS057-09 BC ZSM Lep 22980 Hap6 Germany Bavaria RCAH 636 GU706308 coronata FBLMS FBLMS200-09 BC ZSM Lep 23011 Hap7 Germany Bavaria RCTG 624 GU706543 coronata RDNMH RDNMH542-09 CNCLEP00057861 Hap8 Germany Saxony MTD 658 GU679038 plectilis ZYPAN ZYPAN066-10 CNCLEP00074266 Hap9 United States Minnesota CNC 658 HQ987640 plectilis BBLPE BBLPE524-09 09BBELE-2524 Hap9 Canada Newfoundland and Labrador BIO 658 HM416114 plectilis BBLPE BBLPE498-09 09BBELE-2498 Hap10 Canada Newfoundland and Labrador BIO 658 HM416092 plectilis BBLPE BBLPE507-09 09BBELE-2507 Hap11 Canada Newfoundland and Labrador BIO 658 HM416100 plectilis BLTIB BLTIB483-08