Molecular Ecology Resources (2015) 15, 795–818 doi: 10.1111/1755-0998.12354 A comprehensive DNA barcode database for Central European beetles with a focus on Germany: adding more than 3500 identified species to BOLD 1 ^ 1 LARS HENDRICH,* JEROME MORINIERE,* GERHARD HASZPRUNAR,*† PAUL D. N. HEBERT,‡ € AXEL HAUSMANN,*† FRANK KOHLER,§ andMICHAEL BALKE,*† *Bavarian State Collection of Zoology (SNSB – ZSM), Munchhausenstrasse€ 21, 81247 Munchen,€ Germany, †Department of Biology II and GeoBioCenter, Ludwig-Maximilians-University, Richard-Wagner-Strabe 10, 80333 Munchen,€ Germany, ‡Biodiversity Institute of Ontario (BIO), University of Guelph, Guelph, ON N1G 2W1, Canada, §Coleopterological Science Office – Frank K€ohler, Strombergstrasse 22a, 53332 Bornheim, Germany Abstract Beetles are the most diverse group of animals and are crucial for ecosystem functioning. In many countries, they are well established for environmental impact assessment, but even in the well-studied Central European fauna, species identification can be very difficult. A comprehensive and taxonomically well-curated DNA barcode library could remedy this deficit and could also link hundreds of years of traditional knowledge with next generation sequencing technology. However, such a beetle library is missing to date. This study provides the globally largest DNA barcode reference library for Coleoptera for 15 948 individuals belonging to 3514 well-identified species (53% of the German fauna) with representatives from 97 of 103 families (94%). This study is the first comprehensive regional test of the efficiency of DNA barcoding for beetles with a focus on Germany. Sequences ≥500 bp were recovered from 63% of the specimens analysed (15 948 of 25 294) with short sequences from another 997 specimens. Whereas most speci- mens (92.2%) could be unambiguously assigned to a single known species by sequence diversity at CO1, 1089 speci- mens (6.8%) were assigned to more than one Barcode Index Number (BIN), creating 395 BINs which need further study to ascertain if they represent cryptic species, mitochondrial introgression, or simply regional variation in wide- spread species. We found 409 specimens (2.6%) that shared a BIN assignment with another species, most involving a pair of closely allied species as 43 BINs were involved. Most of these taxa were separated by barcodes although sequence divergences were low. Only 155 specimens (0.97%) show identical or overlapping clusters. Keywords: barcode library, CO1, Coleoptera, cryptic diversity, DNA barcoding, Germany, mitochondrial DNA Received 10 September 2014; revision received 23 November 2014; accepted 26 November 2014 there is a very active community of professional and Introduction amateur entomologists studying their taxonomy and Beetles are the most diverse order of insects with nearly ecology. This is especially true for the Central European 400 000 described species (Zhang 2011). The fauna of fauna which has a taxonomic history that predates Germany includes 6631 species in 103 families while Linnaeus. Because their extreme species richness is cou- 8985 species occur in Central Europe (Kohler€ & Klausnit- pled with morphological, ecological and behavioural zer 1998). These species possess enormous morphologi- diversity, beetles are widely used for environmental cal and ecological diversity and provide numerous impact assessments (EIAs), ecological studies and moni- ecosystem services – with many species wood boring, toring activities (Foster et al. 1989; Lindhe et al. 2005; Jurc herbivorous or carnivorous and adapted to both terres- 2012; Bicknell et al. 2014). While their value for EIAs is trial and aquatic habitats (Kohler€ & Klausnitzer 1998), undisputed, there remains a need to better understand whereas many larvae are inhabitants of subterranean habitat requirements, especially of their larvae, as well as habitats. The beetle fauna of Europe is well known as interactions between species (Rainio & Niemela€ 2003). This often requires improved taxonomic tools. Correspondence: Lars Hendrich, Fax: +49 89 8107 300; Certain beetle species are serious pests in agriculture E-mail: [email protected] and forestry, with some causing economic losses of 1Joint first authors. billions of euros (Germain et al. 2013; Jordal & © 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 796 L. HENDRICH ET AL. Kambestad 2014). Invasive species, such as the Asian freshwater species known from Germany. Due to its ladybird, Harmonia axyridis Pallas, 1773, are now a threat diverse habitats, Bavaria also hosts approximately 70% to the native European Coccinellidae fauna (Fischer 2013; of the Central European fauna. In 2012, DNA barcoding Lombaert et al. 2014). On the other hand, the ladybird was elevated to a national mission through the establish- Coccinella septempunctata Linnaeus, 1758 is useful for pest ment of the ‘German Barcode of Life (GBOL)’ project control (Bianchi et al. 2013) and the defence secretions of (http://www.bolgermany.de), led by the Zoologisches Harmonia axyridis have medical applications (Rohrich€ Forschungsmuseum Alexander Koenig in Bonn (ZFMK), et al. 2012). but involving strong participation by other German insti- DNA barcoding provides an efficient method for bio- tutions including the SNSB-ZSM. In close cooperation diversity assessment as it meets the need for fast, effi- with the Biodiversity Institute of Ontario (BIO, Guelph, cient and reliable species identification at this time of Canada), the German barcoding projects aim to assemble climate change and massive habitat destruction (Hebert a DNA barcode library for all animal species present in & Gregory 2005; Valentini et al. 2009). This approach is Germany in the framework of the International Barcode arguably the best way to handle the vast diversity of of Life (iBOL) Project. invertebrates which are crucial for ecosystem functioning but often poorly known taxonomically. DNA barcoding Materials and methods also has the power to connect different life stages such as eggs, larvae and adults. As such, it can link hundreds of Fieldwork years of taxonomic, ecological, faunistic and ethological studies with ultra-high-throughput sequencing the geno- A network of taxonomists and citizen scientists collected mic era. The latter approach will have great benefits for specimens from Bavaria and from other German states to many application-oriented fields, especially in agricul- obtain species which are rare in Bavaria. Field work per- ture and forestry where the rapid and reliable identifica- mits were issued by the responsible state environmental tion of bulk samples is critical. offices of Bavaria [Bayerisches Staatsministerium fur€ Strongly validated, reliable species libraries are of Umwelt und Gesundheit, Munich, Germany, project: importance as climatic change and global distribution of ‘Barcoding Fauna Bavarica’]. The study sites included goods facilitate the movement of invasive species, which, more than 537 localities in state forests, public lands and after being introduced, might exhibit greater ecological protected areas such as the National Parks ‘Bayerischer damage outside their native habitat. However, identifica- Wald’ and ‘Berchtesgadener Land’. The general distribu- tion success is only as good as the underlying database tion of all included species, based on literature cited (Kvist 2013). To date, most published DNA barcoding below, can be studied in Fig. S3 and Table S3 (Supporting studies dealing with insects have focused on Hymenop- information). tera and Lepidoptera, while few recent studies have examined the hyperdiverse Coleoptera (Woodcock et al. Specimens and taxonomy 2013; Pentinsaari et al. 2014). Bergsten et al. found that a minimum of 70 specimens needed to be analysed to sam- Vouchers from Germany (13 470), Austria (689), Belgium ple 95% of its intraspecific variation in the species they (515), Northern Italy (436), France (392) and other coun- examined (Bergsten et al. 2012a). However, the local tries (448) are now stored in SNSB-ZSM with the excep- DNA barcode library developed in this study provides tion of a few specimens in private collections. In 5 years, an important foundation for future efforts to better 538 800 specimens of beetles were collected using vari- assess intraspecific variation projects. This publication ous methods (i.e. hand collecting, sweep-netting, Mal- begins to address this deficit by releasing the largest aise-, window- and pitfall-traps) which were deployed in Coleoptera barcode reference library to date, focused on varied aquatic and terrestrial habitats (Koch 1989, 1991, the heart of Europe. The records provide coverage for 1992, 1993, 1994, 1995a,b, 1996; Kohler,€ F unpublished, 15 948 beetle specimens (3514 species), 53% of the Ger- see Figs S1 and S2, Tables S1 and S2, Supporting infor- man and 39% of the Central European fauna (Kohler€ & mation). From this total, 462 550 specimens were sorted Klausnitzer 1998). This release is a direct result of the and identified to a species level and more than 25 000 Barcoding Fauna Bavarica project (http://www.faun- specimens were submitted for sequence analysis, accord- abavarica.de) led by the Bavarian State Collection of ing to an internal catalogue of missing species