A Comprehensive DNA Barcode Database for Central European Beetles with a Focus on Germany: Adding More Than 3500 Identiﬁed Species to BOLD

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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 specimens (92.2%) could be unambiguously assigned to a single known species by sequence diversity at CO1, 1089 specimens (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 widespread 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 distribution 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 (Kohler,€ F Zoology (SNSB-ZSM) with support from the Bavarian unpublished). Only a few specimens were pinned, and State Government (Haszprunar 2009). With an area of few were more than 5 years old. The number of 70 000 km2, Bavaria is the largest German state and pos- specimens analysed per species ranged from 1–26 in sesses all major habitat types in Germany (except coastal Adalia bipunctata (Linnaeus, 1758) (see Appendices S1 regions) and harbours 90% of the terrestrial and and S2, Supporting information), available on Dryad

© 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd. DNA BARCODING OF CENTRAL EUROPEAN BEETLES 797 doi:10.5061/dryad.gg8fg. Individuals were identified to with a minimum length of 500 bp. Neighbour-joining species level either by Frank Kohler€ and/or by other tax- (NJ) trees for each family were calculated following onomic specialists, using appropriate literature (e.g. Fre- alignment based on K2P distances. The ‘BIN Discor- ude et al. 1964–1983; Lohse & Lucht 1989, 1992; Drost dance’ analysis on BOLD was used to reveal species clus- et al. 1992; Pfeffer 1994; Hebauer & Klausnitzer 1998; ters which shared a BIN, and those which were assigned Lompe 2014: http://www.coleo-net.de/coleo/index. to two or more BINs. BOLD groups sequences into clus- htm). ters of closely similar CO1 barcode sequences which are assigned a globally unique identifier, termed a ‘Barcode Index Number’ or BIN (Ratnasingham & Hebert 2013). Laboratory protocols This system can be used to verify the species identifica- A tissue sample was removed from each specimen and tions when taxonomic information is lacking. The BIN transferred into 96-well plates at the SNSB-ZSM for sub- System involves a 3-step online pipeline, which clusters sequent DNA extraction. For specimens with a body similar barcode sequences algorithmically into operational length >2 mm, a single leg or a leg segment was taxonomic units (OTUs). Members of a BIN often belong removed for DNA extraction. The whole voucher was to a single species as delineated by traditional taxonomy used for some very small specimens (e.g. ≤1 mm, Ptilii- (Hausmann et al. 2013). Every ‘disagreement/conflict’ dae), but replacement vouchers from the same locality case is the starting point for re-evaluation of both molecu- were retained. In other cases (vouchers from Malaise lar and morphological data. We follow the concept of inte- traps), DNA was extracted from the whole voucher at grative taxonomy (Padial et al. 2010; Schlick-Steiner et al. the CCDB (Guelph, Canada) using voucher-recovery 2010, 2014; Fujita et al. 2012; Riedel et al. 2013) to infer protocols and the specimens were repatriated to the whether there are previously overlooked species in the SNSB-ZSM for identification and curation. Voucher sample, or if barcode divergence between species is too information such as locality data, habitat, altitude, collec- low or absent to allow the species to be delineated using tor, identifier, taxonomic classifications, habitus images, only CO1. DNA barcode sequences, primer pairs and trace files are publicly accessible in the ‘German Beetles’ data set in Results BOLD (http://www.boldsystems.org – data set DOI: dx. doi.org/10.5883/DS-COLBYGER). Barcode sequences (≥500 bp) were obtained from 63% of Once tissue samples were added, the plates were sent the 25 294 specimens representing 15 948 specimens to the Canadian Centre for DNA Barcoding (CCDB) from a priori identified species with representatives from where they were processed using standard barcoding 97 of 103 families (94%, Table 1) known from Germany. protocols. All protocols for DNA extraction, PCR ampli- Success rates were higher for shorter sequences with fications and Sanger sequencing procedures are available 71% of specimens generating a ≥300 bp barcode (Fig. 3). online under: http://www.dnabarcoding.ca/pa/ge/ Most of the remaining samples (29%) failed to generate a research/protocols. sequence, but a few sequences were flagged by BOLD as All samples were PCR amplified with modified Fol- contaminations or possible pseudogenes as they con- mer primers CLepFolF (50 – ATT CAA CCA ATC ATA tained stop codons. Barcode recovery was very high AAG ATA TTG G) and CLepFolR (50 – TAA ACT TCT across a variety of families when proper amounts of tis- GGA TGT CCA AAA AAT CA) for the barcoding frag- sue were loaded and when specimens were well pre- ment (50 CO1), and the same primers were employed for served. The success in barcode recovery ranged from subsequent Sanger sequencing reactions (see also Ivano- 25% (Eucnemidae) to 91% (Oedemeridae) in families rep- va et al. 2007; Ward et al. 2008). The sequence data and resented by more than 100 species (Table 1). Chi-square trace files were uploaded to BOLD and subsequently also analysis (Table S5, Supporting information) revealed that to GenBank (accession nos KM439102–KM452702). the differences in the success of sequence recovery among families were highly significant (P < 0.00001), suggesting possible problems in primer binding. How- Data analysis ever, no relationship between specimen size and Sequence divergences for the barcode region (mean and sequencing success was observed. Only medium-sized maximum intraspecific variation and minimum genetic (body length 10–14 mm) species with sequences bp were distance to the nearest neighbour species) were calcu- slightly overrepresented (Fig. S4, Table S4, Supporting lated using the ‘Barcode Gap Analysis’ tool on BOLD, information). The impacts of variation in specimen age employing the Kimura-2-Parameter (K2P) distance met- and preservation method (e.g. ethanol concentration) ric (Puillandre et al. 2012). MUSCLE was applied for were not evaluated. Based on our experience, average sequence alignment restricting analysis to sequences barcode recovery was lower (70%) when specimens were

Table 1 The rate of success in recovery of barcode sequences for species belonging to 97 of 103 families of Coleoptera recorded from Germany and Central Europe

% of Central % Success ≥500 bp Species in Species in Species with % of German European Barcodes (sequenced/ Family Germany Central Europe CO1 Barcodes Species Species specimens analysed)

Aderidae 9 11 4 44 36 67 (28/42) Agyrtidae 4 5 1 25 20 29 (2/7) Alexiidae 3 9 2 67 22 75 (21/28) Anthicidae 25 38 10 40 26 86 (57/66) Anthribidae 22 31 13 59 42 67 (66/98) Attelabidae 27 32 19 70 59 79 (81/102) Biphyllidae 2 3 1 50 33 25 (3/12) Boridae 0 1 1 N/A 100 100 (1/1) Bostrichidae 12 31 7 58 23 43 (9/21) Bothrideridae 7 10 2 29 20 54 (6/11) Brachyceridae 10 10 5 50 50 85 (23/27) Brentidae 134 163 85 63 52 77 (446/579) Buprestidae 99 145 65 66 45 69 (211/303) Byrrhidae 25 41 10 40 24 49 (37/75) Byturidae 2 2 2 100 100 82 (18/22) Cantharidae 87 112 58 67 52 82 (392/479) Carabidae 569 827 401 70 48 79 (2398/3046) Cerambycidae 191 259 122 64 47 77 (631/818) Cerophytidae 1 1 1 100 100 100 (2/2) Cerylonidae 6 8 5 83 63 74 (21/26) Chrysomelidae 543 722 281 52 39 78 (1436/1846) Ciidae 45 53 28 62 53 71 (138/195) Clambidae 14 16 10 71 63 84 (47/56) Cleridae 21 27 14 67 52 67 (68/102) Coccinellidae 81 111 57 70 51 74 (382/516) Corylophidae 19 25 10 53 40 65 (41/63) Cryptophagidae 131 158 48 37 30 56 (208/374) Cucujidae 4 5 4 100 80 49 (22/45) Curculionidae 879 1289 473 54 37 64 (1743/2717) Dascyllidae 1 1 1 100 100 83 (10/12) Dermestidae 44 66 17 39 26 62 (79/128) Derodontidae 2 2 2 100 100 100 (6/6) Drilidae 2 2 2 100 100 50 (9/18) Dryophthoridae 5 5 2 40 40 57 (13/23) Dryopidae 14 15 3 21 20 35 (6/17) Dytiscidae 145 163 91 63 56 61 (254/396) Elateridae 146 194 96 66 49 82 (674/826) Elmidae 25 28 21 84 75 95 (81/90) Endomychidae 11 22 4 36 18 62 (36/62) Erotylidae 16 25 9 56 36 55 (44/80) Eucinetidae 2 3 1 50 33 100 (3/3) Eucnemidae 20 26 8 40 31 25 (25/100) Georissidae 3 5 3 100 60 70 (7/10) Geotrupidae 11 12 8 73 67 41 (20/48) Gyrinidae 13 13 6 46 46 86 (18/21) Haliplidae 20 21 17 85 81 78 (57/73) Heteroceridae 14 15 10 71 67 73 (55/75) Histeridae 83 112 32 39 29 37 (102/277) Hydraenidae 53 77 32 60 42 81 (71/88) Hydrophilidae 122 142 73 60 51 75 (279/374) Hygrobiidae 1 1 1 100 100 67 (2/3) Kateretidae 12 15 9 75 60 80 (58/73) Laemophloeidae 20 22 10 50 45 47 (37/79)

Table 1 (Continued)

% of Central % Success ≥500 bp Species in Species in Species with % of German European Barcodes (sequenced/ Family Germany Central Europe CO1 Barcodes Species Species specimens analysed)

Lampyridae 3 3 4 133 133 77 (17/22) Latridiidae 84 105 36 43 34 48 (154/324) Leiodidae 154 201 64 42 32 66 (239/348) Limnichidae 3 5 2 67 40 59 (10/17) Lissomidae 1 1 1 100 100 100 (2/2) Lucanidae 7 7 5 71 71 49 (24/49) Lycidae 7 9 6 86 67 87 (27/31) Lymexylonidae 2 3 2 100 67 50 (13/26) Melandryidae 33 41 19 58 46 69 (97/140) Meloidae 18 37 3 17 8 100 (6/6) Melyridae 59 107 44 75 41 90 (271/300) Monotomidae 23 26 16 70 62 64 (83/129) Mordellidae 81 106 25 31 24 77 (130/182) Mycetophagidae 17 17 14 82 82 63 (56/117) Mycteridae 1 2 1 100 50 100 (2/2) Nemonychidae 3 3 2 67 67 100 (2/2) Nitidulidae 126 163 77 61 47 61 (381/630) Nosodendridae 1 1 1 100 100 100 (1/1) Noteridae 2 2 2 100 100 57 (4/7) Oedemeridae 25 35 23 92 66 91 (155/171) Omalisidae 1 1 1 100 100 86 (12/14) Phalacridae 22 25 11 50 44 62 (65/105) Phloiophilidae 1 1 1 100 100 100 (1/1) Platyopodidae 2 2 1 50 50 33 (2/6) Prostomidae 1 1 1 100 100 100 (7/7) Psephenidae 1 1 1 100 100 100 (6/6) Ptiliidae 82 108 23 28 21 50 (81/162) Ptinidae 93 137 49 53 36 59 (199/337) Pyrochroidae 3 3 3 100 100 88 (35/40) Pythidae 2 2 1 50 50 100 (1/1) Salpingidae 14 14 7 50 50 72 (41/57) Scarabaeidae 159 240 77 48 32 71 (373/529) Scirtidae 24 28 18 75 64 67 (96/144) Scraptiidae 28 41 20 71 49 90 (162/180) Silphidae 22 25 14 64 56 69 (100/145) Silvanidae 13 18 7 54 39 49 (25/51) Sphaeriusidae 1 1 1 100 100 100 (9/9) Sphindidae 3 4 2 67 50 62 (13/21) Staphylinidae 1621 2139 726 45 34 63 (3333/5307) Tenebrionidae 85 125 46 54 37 56 (263/469) Tetratomidae 3 3 3 100 100 32 (6/19) Throscidae 11 12 8 73 67 76 (40/53) Trogidae 8 9 3 38 33 37 (7/19) Trogossitidae 10 12 6 60 50 49 (16/33) Zopheridae 19 27 12 63 44 67 (70/104) collected in traps, whereas freshly collected material in sequence clusters in the neighbour-joining (NJ) tree 95% ethanol provided high success rates (>85%). revealed high congruence with morphology-based Subsequent analyses were restricted to sequence identiﬁcations. In fact, 92.2% of the species could be records ≥500 bp (Fig. 1A and B). These 15 948 sequences unambiguously identiﬁed by their CO1 sequence. How- provided coverage for 3514 German species (53%) that ever, in a total of 176 cases, 1089 specimens (6.8%) were were assigned to 3687 BINs (Fig. 2). In total, 524 new represented by more than one BIN, producing a total of BINs were added to BOLD, most representing new spe- 395 BINs for these taxa. Another 42 species pairs and one cies entries for the BOLD system. Inspection of the species trio shared BINS meaning that 409 specimens

Latridiidae Coccinellidae Cryptophagidae Anthicidae Chrysomelidae (A)

Oedemeridae

Noteridae

Eucnemidae

Cucujidae

Histeridae

Silvanidae

Clambidae

Cerambycidae

Ciidae

Curculionidae

Elateridae

Lampyridae

Cantharidae Tenebrionidae

Silphidae

Scarabaeidae Staphylinidae

Leiodidae

Hydraenidae

Hydrophilidae Carabidae

Gyrinidae

Haliplidae

(B)

Fig. 1 (A) Circular neighbour-ioining phylogram from analysis of the whole data set (performed on the BOLD database) – different colours indicate beetle families. Sequence records (sequences >500 bp) from 15 948 specimens submitted for analysis designated to 3515 species (53% of the German fauna) and 3687 BINs, including representatives for 97 of the103 families (94%) recorded from Germany. (B) NJ tree for the family Attelabidae. All other family trees are provided in Appendix S3 (Supporting information), available on Dryad doi:10.5061/dryad.gg8fg.

Fig. 2 Accumulation curve for the 3514 species and 3687 BINs with DNA barcodes from Central European species. The accumulation curve (from BOLD database; randomized; 100 iterations) is based on data for the 15 948 barcoded individuals (>500 bp).

(2.6%) were involved in BIN sharing. Many of these spe- 18 000 cies pairs (28) could be separated by CO1 because their 16 000 members were assigned to different subclusters, but 155 14 000 specimens (0.97%) including 15 pairs and one trio pos- 12 000 sessed identical or overlapping CO1 barcode sequences 10 000 that prevented their discrimination. 8000 Appendix S3 (Supporting information) provides 6000 neighbour-joining trees for all families with more than 4000 one species. Besides the species name, a voucher 2000 0 number, region and country of origin are given for <150 bp 250 bp 350 bp 450 bp 550 bp 658 bp each specimen (available under doi: 10.5061/dryad. gg8fg). Fig. 3 Success rate in N specimens in recovering CO1 ampli- cons of varied length.

BIN sharing and paraphyletic species separated by the form of the head which has angular Our analyses revealed 33 morphologically distinct margins beneath the eyes in A. dubia, but round ones in species clusters (155 specimens) involving 15 pairs A. reyi (Bense 1995). Sequences do not recover either and one triplet that either have very low divergence species as a monophyletic cluster (Fig. 4A). or that share a haplotype (Table 2). As a consequence, Mycetophagus piceus (Fabricius, 1777) and M. salicis the NJ tree failed to recover these taxa as separate Brisout de Barneville, 1862 are widespread European monophyletic lineages. We consider two examples hairy fungus beetles (Mycetophagidae) which can be here. separated by the colour of the dorsal surface of their Anastrangalia dubia (Scopoli, 1763) and A. reyi (Hey- pronotum and elytra and the form of the male genitalia, den, 1889) are longhorn beetles (Cerambycidae) that median lobe and parameres (Lompe 2014). The species occur throughout Europe with the latter taxon also found are found in different habitats as M. salicis lives in in Turkey, Iran, Central Asia, China and Mongolia. The bracket fungus on softwood trees such as willow while two sexes as well as local populations of the species may M. piceus feeds on bracket fungus overgrown by myce- have different colours (black, brown and red). The two lium on hardwood trees such as oak. Sequences within species are morphologically very similar, but can be the BIN do not recover either species as a monophyletic

Table 2 Cases of BIN sharing involving 42 pairs and one triplet of congeneric species of German Coleoptera (409 specimens). The minimum pairwise distance (PD) for each pair or triad is shown as well as their BIN

Family Species 1 Species 2 Species 3 Min PD % Shared BIN

Carabidae Bembidion atrocaeruleum Bembidion varicolor 0 BOLD:AAO0687 Carabidae Bembidion guttula Bembidion mannerheimii 0 BOLD:AAW3426 Cerambycidae Anastrangalia dubia Anastrangalia reyi 0 BOLD:AAJ2081 Mordellidae Mordella brachyura Mordella holomelaena 0 BOLD:ACG2749 Mycetophagidae Mycetophagus piceus Mycetophagus salicis 0 BOLD:AAK8784 Oedemeridae Anogcodes dispar Anogcodes fulvicollis 0 BOLD:ACI6135 Phalacridae Olibrus affinis Olibrus flavicornis 0 BOLD:AAQ0763 Staphylinidae Aleochara bipustulata Aleochara verna 0 BOLD:AAJ2457 Staphylinidae Atheta malleus Atheta volans 0 BOLD:AAN2267 Staphylinidae Bledius filipes Bledius terebrans 0 BOLD:AAO4729 Staphylinidae Euplectus kirbyi Euplectus nanus 0 BOLD:AAO0162 Staphylinidae Haploglossa nidicola Haploglossa villosula 0 BOLD:AAK5150 Staphylinidae Leptacinus formicetorum Leptacinus intermedius Leptanicus sulcifrons 0 BOLD:AAO1025 Staphylinidae Myllaena infuscata Myllaena minuta 0 BOLD:AAX2901 Staphylinidae Tachyporus chrysomelinus Tachyporus dispar 0 BOLD:AAN9511 Carabidae Dyschirius agnathus Dyschirius politus 0.15 BOLD:AAO3582 Curculionidae Anthonomus humeralis Anthonomus pomorum 0.15 BOLD:AAO1532 Staphylinidae Gyrophaena polita Gyrophaena strictula 0.15 BOLD:AAO0297 Carabidae Agonum micans Agonum striatum 0.16 BOLD:AAN9978 Chrysomelidae Phratora polaris Phratora tibialis 0.3 BOLD:ABA3280 Curculionidae Miarus campanulae Miarus ajugae 0.3 BOLD:ABW4930 Carabidae Harpalus anxius Harpalus subcylindricus 0.31 BOLD:ABZ8076 Carabidae Dyschirius arenosus Dyschirius obscurus 0.46 BOLD:AAP8268 Curculionidae Miarus abnormis Miarus ajugae 0.46 BOLD:ABW4930 Dytiscidae Bidessus delicatulus Bidessus minutissimus 0.46 BOLD:AAB7463 Staphylinidae Lithocharis nigriceps Lithocharis ochraceus 0.49 BOLD:AAN6200 Carabidae Harpalus servus Harpalus subcylindricus 0.61 BOLD:ABZ8076 Carabidae Pterostichus cristatus Pterostichus hagenbachii 0.61 BOLD:ACG2758 Curculionidae Microplontus campestris Microplontus millefolii 0.66 BOLD:AAO1007 Carabidae Agonum afrum Agonum duftschmidi 0.77 BOLD:AAO3378 Cerylonidae Cerylon ferrugineum Cerylon histeroides 0.83 BOLD:AAN9867 Carabidae Amara ovata Amara similata 0.92 BOLD:AAJ5377 Staphylinidae Amischa analis Amischa bifoveolata 0.92 BOLD:ABA5313 Carabidae Harpalus attenuatus Harpalus rubripes 1.07 BOLD:AAI2024 Staphylinidae Lathrobium quadratum Lathrobium terminatum 1.07 BOLD:AAX0188 Carabidae Carabus alpestris Carabus sylvestris 1.08 BOLD:ABU7358 Dytiscidae Rhantus bistriatus Rhantus suturellus 1.13 BOLD:AAQ1027 Chrysomelidae Galerucella aquatica Galerucella nymphaeae 1.23 BOLD:AAG4421 Carabidae Dyschirius thoracicus Dyschirius arenosus 1.29 BOLD:AAP8268 Staphylinidae Heterothops niger Heterothops stiglundbergi 1.7 BOLD:AAX0829 Carabidae Limodromus assimilis Limodromus longiventris 1.85 BOLD:AAN9741 Attelabidae Rhynchites auratus Rhynchites bacchus 1.86 BOLD:ACG5878 Carabidae Panagaeus cruxmajor Panagaeus bipustulatus 2.03 BOLD:AAO4843

clade, highlighting the need for an in-depth analysis of nearly all of these 254 specimens formed monophyletic the status of these two species (Fig. 4B). clusters in the neighbour-joining (NJ) tree and within the BINs (Fig. 5A–D). In the following paragraph, we discuss four cases of well-known species showing low BIN sharing with monophyletic species barcode divergences in three families. Table 2 summa- Another 56 species clusters (254 specimens in 28 pairs) rizes all cases of BIN sharing found in this study. share a BIN, but all of these could be discriminated by The peach weevil Rhynchites bacchus (Linnaeus, 1758) barcodes as they showed minimum K2P distances rang- and the cherry weevil R. auratus (Scopoli, 1763) (Attelabi- ing from 0.16% to 2.03% between the specimens belong- dae) are serious pests in fruit orchards (Hassler & Rhein- ing to the two different species in each BIN. Moreover, heimer 2010). R. bacchus has a black snout while

(A)Anastrangalia dubia (BFB_Col_FK_4770) (B) Mycetophagus salicis (BC ZSM COL 02542) Germany (Bavaria) Germany (Rhineland Palatinate)

Anastrangalia dubia (GBOL_Col_FK_6701) Italy (South Tyrol) Mycetophagus salicis (BC ZSM COL 01944) Germany (North Rhine Westphalia) Anastrangalia dubia (GBOL_Col_FK_6702) Italy (South Tyrol) Mycetophagus piceus (GBOL_Col_FK_4670) Germany (North Rhine Westphalia) Anastrangalia dubia (GBOL_Col_FK_6482) Italy (South Tyrol) 2% Mycetophagus piceus (BC ZSM COL 00576) Anastrangalia dubia (BFB_Col_FK_7292) Belgium (Flemish Brabant) Germany (Bavaria)

Anastrangalia dubia (BFB_Col_FK_4608) Mycetophagus piceus (BC ZSM COL 02832) Germany (Bavaria) Germany (North Rhine Westphalia)

Anastrangalia dubia (GBOL01866) Mycetophagus salicis (BC ZSM COL 02543) Germany (Bavaria) Germany (Rhineland Palatinate)

Anastrangalia dubia (GBOL_Col_FK_7090) 2% Austria (Tyrol) Mycetophagus piceus (GBOL_Col_FK_0971) Germany (Saxonia) Anastrangalia reyi (GBOL_Col_FK_6796)

Austria (Salzburg) 4 Mycetophagus piceus (BFB_Col_FK_2933)

8 Germany (North Rhine Westphalia)

Anastrangalia reyi (GBOL_Col_FK_6982) 7

Austria (Tyrol) 8 K Mycetophagus piceus (BFB_Col_FK_7404)

A Belgium (West Flandern)

A :

Anastrangalia dubia (GBOL_Col_FK_7291) D Germany (Bavaria) L Mycetophagus piceus (BFB_Col_FK_5284)

O Germany (Saarland) Anastrangalia reyi (GBOL_Col_FK_7246) B Austria (Tyrol)

Anastrangalia reyi (GBOL_Col_FK_7135) Austria (Tyrol)

Anastrangalia reyi (GBOL_Col_FK_7245) Austria (Tyrol)

Anastrangalia reyi (GBOL_Col_FK_7009) Austria (Tyrol)

Anastrangalia reyi (GBOL_Col_FK_7028) Austria (Salzburg)

Anastrangalia (GBOL_Col_FK_6797) Austria (Salzburg)

Anastrangalia dubia (GBOL_Col_FK_7255) Austria (Tyrol)

1 Anastrangalia dubia (BFB_Col_FK_4607) 8

0 Germany (Bavaria) 2 J Anastrangalia dubia (GBOL_Col_FK_6962)

A Italy (South Tyrol)

: D

L Anastrangalia dubia (GBOL_Col_FK_7611)

O Slovenia B

Fig. 4 NJ subtrees for species pairs that share a barcode sequence. NJ trees were obtained from BOLD.

R. auratus has a metallic snout. The punctation on the southwards to Switzerland. The three specimens of pronotum of R. bacchus is coarser than in R. auratus, A. scitulum examined in this study shared a distinctive while the eyes in R. bacchus are strongly prominent, but- haplotype, but the 14 individuals of A. micans form a pa- more inserted in R. auratus. Although they are morpho- raphyletic assemblage (Fig. 5B). logically and ecologically distinct species, they share a Harpalus anxius (Duftschmidt, 1812) and H. subcylin- BIN but show a minimum K2P distance of 1.86% dricus Dejean, 1829 are ground beetles (Carabidae) which (Fig. 5A). share a BIN but each species forms a distinct cluster Agonum scitulum Dejean, 1828 and A. micans (Nicolai, (Fig. 5C). H. subcylindricus is restricted to dry, warm, 1822) are two morphologically very similar ground bee- sparsely vegetated habitats such as arid and semi-arid tles (Carabidae) which can be only separated by the form grasslands although it occasionally co-occurs with of male genitalia (Schmidt 2006). A. micans is a Eurosibe- H. anxius. However, H. anxius is only found on sandy rian boreotemperate species, strongly associated with soils, while H. subcylindricus occurs elsewhere. Males can wet alder forests on water-logged river and lakeshore be distinguished by the differing shape of their median soils. It occurs across Europe except in the south, and lobes, but females cannot be discriminated morphologi- east into western Siberia. A. scitulum is hygrophilic cally (Butterweck et al. 2000). ground beetle, favoring wet woodlands and well-vege- The sibling species Galerucella aquatica (Geoffroy in tated marshes, particularly adjacent to tidal rivers. It Fourcroy, 1785) and G. nymphaeae (Linnaeus, 1758) occurs throughout Europe from western France (Chrysomelidae) employ differing host plants. G. nym- and Great Britain, to Russia and Romania in the east, phaeae feeds on Nuphar and Nymphaea (water lilies) while

(A) (B) Agonum micans (BFB_Col_FK_7685) Germany (North Rhine Westphalia)

8 Rhynchites auratus (BFB_Col_FK_11745)

7 Germany (Rhineland Palatinate) 8 Agonum micans (ZSM_COLA_2011_296)

5 Germany (North Rhine Westphalia)

G C

A Agonum micans (ZSM_COLA_2011_344) : Rhynchites auratus (GBOL_Col_FK_8301) Germany (North Rhine Westphalia)

D Germany (North Rhine Westphalia) L

O Agonum micans (GBOL_Col_FK_6075)

B Germany (Rhineland Palatinate) 2% Rhynchites auratus (BFB_Col_FK_11746) Agonum micans (BC ZSM COL 00353) Germany (Rhineland Palatinate) Germany (Rhineland Palatinate) Agonum micans (ZSM_COLA_2011_345) Germany (North Rhine Westphalia) Rhynchites bacchus (GBOL_Col_FK_6611) Agonum micans (BCZSM_COL_WL_0025) Czech Republic (Norhern Bohemia) Germany (Bavaria)

1 % Agonum micans (ZSM_COLA_2011_346) Germany (North Rhine Westphalia) Agonum micans (BFB_Col_FK_3817) Germany (Rhineland Palatinate) Harpalus subcylindricus (BFB_Col_FK_9650) Germany (Baden-Wuerttemberg) Agonum micans (GBOL_Col_FK_2679) Germany (North Rhine Westphalia) Harpalus subcylindricus (BFB_Col_FK_11237) Germany (Saxony-Anhalt) Agonum micans (GBOL_Col_FK_3997) Germany (North Rhine Westphalia) Harpalus subcylindricus (BCZSM_COLA_01638) Germany (Brandenburg) Agonum scitulum (ZSM_COLA_2011_301) Germany (North Rhine Westphalia) Harpalus subcylindricus (BFB_Col_FK_11239) Agonum scitulum (BCZSM_COLA_01391) Germany (Saxony-Anhalt) Germany (North Rhine Westphalia) Agonum scitulum (ZSM_COLA_2011_300) Germany (North Rhine Westphalia) Harpalus subcylindricus (BFB_Col_FK_11237)

Germany (Saxony-Anhalt) Agonum micans (BFB_Col_FK_3483) 8

Harpalus subcylindricus (BFB_Col_FK_11238) 9 Agonum micans (BFB_Col_FK_8514)

Germany (Saxony-Anhalt) N

Germany (Saxony Anhalt)

A A Agonum micans (GBOL_Col_FK_1722) : Harpalus anxius (GBOL_Col_FK_4415) D

Germany (Rhineland Palatinate) L

Germany (Rhineland Palatinate)

O B Harpalus anxius (BFB_Col_FK_9874) Germany (Rhineland Palatinate) 0,2% Harpalus anxius (GBOL_Col_FK_3323) Germany (Rhineland Palatinate) (D) Galerucella nymphaeae (BFB_Col_FK_3976) Harpalus anxius (BFB_Col_FK_10685) Germany (Rhineland Palatinate) Germany (Rhineland Palatinate)

Galerucella nymphaeae (BFB_Col_FK_8267) Harpalus anxius (GBOL_Col_FK_3359) Germany (Rhineland Palatinate) Germany (Rhineland Palatinate)

Harpalus anxius (BC ZSM COL 01414) Galerucella nymphaeae (GBOL_Col_FK_9152) Germany (Rhineland Palatinate) Germany (North Rhine Westphalia)

Galerucella aquatica (BC ZSM COL 02210) Harpalus anxius (BC ZSM COL 01413) Germany (Rhineland Palatinate) Germany (Rhineland Palatinate) 1%

Harpalus anxius (GBOL_Col_FK_3324) Galerucella aquatica (BC ZSM COL 02080) Germany (Rhineland Palatinate) Germany (Rhineland Palatinate)

Galerucella aquatica (GBOL_Col_FK_9160)

Germany (North Rhine Westphalia)

2 4

Galerucella aquatica (BC ZSM COLA 00027) 4

Germany (Bavaria) G

: D

Galerucella aquatica (GBOL_Col_FK_8722) L

Germany (Baden-Wuerttemberg) O B

Fig. 5 NJ subtrees for species pairs with low interspeciﬁc variation. NJ trees were obtained from BOLD.

G. aquatica consumes Polygonaceae (Lopatin & High genetic divergences within species and cases of Nesterova 2005). While males can be identiﬁed by the cryptic diversity different structure of their genitalia, females cannot be discriminated without knowledge of the host plant One thousand and eighty-nine specimens in 176 species (Fig. 5D). However, they can now be recognized using clusters were assigned to two or more BINs because of DNA barcoding (minimum K2P distance = 1.23%). their relatively high intraspeciﬁc genetic divergences

© 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd. DNA BARCODING OF CENTRAL EUROPEAN BEETLES 805 with maximum pairwise K2P distance ranging from a Procraerus tibialis (Lacordaire, 1835), another elaterid, low of 1.60% [Harpalus dimidiatus (Rossi, 1790) repre- belongs to a monotypic genus in Europe and occurs in the sented by two BINs] to 15.75% [Athous vittatus Fabricius, cavities of deciduous trees infested by Rhamnusium bicolor 1793 represented by three BINs]). In total, the 1089 speci- (Schrank, 1781) (Cerambycidae) and cossonine weevils mens were assigned to 395 BINs (851 specimens in 146 (Husler & Husler 1940). The adults are only active at species clusters with two BINs, 121 specimens in 17 spe- night, remaining hidden in crevices or under bark during cies clusters with three BINs, 81 specimens in nine spe- the day. Adults can be reared from dry wood of old oaks cies clusters with four BINs, 26 specimens in two species (Nemeth & Merkl 2009). A single specimen from southern clusters with five BINs and 10 specimens in one spe- Bavaria possessed a Max ISD of 13.64% from a cluster of cies cluster with six BINs) (Table 3). The balance of nine specimens (Table 3) collected in different regions of this section considers 12 of these cases from various Germany including northern Bavaria (Fig. 6E). As one families, breaking them into two categories. junior synonym (Ampedus subcarintus Germar, 1844) exists for this species, further specimens from Europe should be Species with high intraspecific variation without correlated dif- barcoded including the type specimens of both taxa to ferences in morphology. Four species of Haliplidae (Hali- clarify the taxonomic status of the barcode lineages. plus furcatus Seidlitz, 1887, H. immaculatus Gerhardt, 1877, Hydrobius fuscipes (Linnaeus, 1758) is one of the most H. ruficollis (De Geer, 1774) and H. variegatus Sturm, 1834) widespread water scavenger beetles (Hydrophilidae) in show high intraspecific divergences from 1.23% in H. var- lentic habitats across the entire Holarctic region includ- iegatus to 2.66% in H. immaculatus (Table 3) (Fig. 6A). ing North America, China and Mongolia (Hansen 1987). However, in contrast to H. lineatocollis (Marsham, 1802) Three morphological variants of this species from Ger- (see below), morphological analysis of the voucher speci- many were named in the past, but were later synony- mens (performed by the first author) did not reveal evi- mized and have not been ranked as species or subspecies dence of morphological divergence between the in recent faunal treatments (Drost et al. 1992; Hebauer & specimens in each cluster. Klausnitzer 1998). Taking into account the geographical The false ladybird Endomychus coccineus (Linnaeus, scale and number of specimens studied (n = 15), the bar- 1758) (Endomychidae) belongs to a monotypic genus code results suggest that at least four (probably) allopat- that is widespread in Central European forests. The lar- ric species occur in Germany (Table 3), with an vae are often prevalent on fungi growing on birch and additional taxon in Greece (Fig. 6F). The occurrence of beech stumps. The maximum intraspecific divergence these lineages needs to be investigated across the whole (ISD) between the two BINs is 3.12% (Table 3) (Fig. 6B). distribution of this probable species complex. Morpho- Trichodes apiarius Herbst, 1792 a checkered beetle, is metric data and additional molecular markers were the most common species of the genus in Central recently assembled for a comprehensive study examin- Europe. Its larval stage parasitizes bees, predating on the ing the boundaries of this species complex in Fennoscan- larvae and nymphs of both solitary bees (e.g. Osmia and dia. Their study includes examination of available type Megachile) and honey bees. The adults feed on the pollen material and support the presence of four species in of flowers, mainly Apiaceae, in May through June (Nie- addition to the barcode clusters found in our study, huis 2013). The maximum intraspecific divergence (ISD) including H. arcticus and the formally established mor- between the two BINs is 3.44% (Table 3) (Fig. 6C). phological variants of H. fuscipes (Fossen 2014; E. I. Fossen, T. Ekrem, J. Bergsten & A. Nilsson, unpublished). Possible cases of cryptic diversity. The following paragraph Corymbia rubra (Linnaeus, 1758), the red-brown long- provides details for nine possible cases of cryptic diver- horn beetle (Cerambycidae), is a well-known xylopha- sity involving species clusters, most with more than 5% gous inhabitant of coniferous forests throughout Central divergence. For Corymbia rubra (Linnaeus, 1758) and Lep- Europe whose larvae feed in the dead wood of spruce tinus testaceus Muller,€ 1817 minimum pairwise distances and pine trees. The distinctive colour of this species has are 3.45% and 2.49%, respectively (Table 3), but no clo- traditionally allowed a straightforward identification sely related sister species are known from Central (Bense 1995). As a result, it is remarkable that this taxon Europe. likely includes two species as evidenced by the presence Athous vittatus Fabricius, 1793 is a common click bee- of 3.45% divergence (Table 3) between members of the tle (Elateridae) with variable dorsal coloration (Laibner two clusters (Fig. 6G). However, there remains a need 2000). Several colour forms have been described and for more comprehensive, integrative investigations to named. The fact that specimens of this species were validate this conclusion. divided into three clusters with a maximum divergence The well-known thistle tortoise beetle (Chrysomeli- of 15.75% suggests that this taxon is actually composed dae) Cassida rubiginosa Muller,€ 1776 feeds on Canada of two if not three species in Germany (Fig. 6D). thistle (Cirsium arvense) and was intentionally introduced

Table 3 176 cases in which high intraspeciﬁc divergence (ISD) led to the assignment of conspeciﬁc individuals to two or more BINs (1089 specimens)

Family Species Country BIN Max ISD

Anthicidae Cyclodinus humilis DE BOLD:ACD0890 2.65 DE BOLD:ACG8457 Anthribidae Araecerus fasciculatus NL BOLD:AAH0210 3.75 NL BOLD:ACC2424 Attelabidae Attelabus nitens DE BOLD:AAO4568 4.68 DE BOLD:ACG7055 Bothrideridae Teredus cylindricus DE BOLD:AAO4189 2.47 DE BOLD:AAO0452 Brentidae Eutrichapion punctigerum DE BOLD:ABX3008 1.48 DE BOLD:ACE5095 Protapion fulvipes DE BOLD:ACF5056 1.85 DE BOLD:AAX8468 Pseudapion moschatae DE BOLD:ACD0419 3.28 DE BOLD:AAO1152 Stenopterapion tenue DE BOLD:ACG4488 6.8 DE BOLD:AAO3660 DE BOLD:ACA7465 DE BOLD:ACG9508 Buprestidae Agrilus viridis DE BOLD:ACE6725 1.85 DE BOLD:ACE5550 Byrrhidae Cytilus sericeus BE/DE BOLD:ACG4339 4.09 DE BOLD:ACG3560 AU/IT BOLD:AAJ5113 DE BOLD:ACG3559 Cantharidae Cantharis sudetica FR BOLD:ACB8167 3.13 FR BOLD:ACB9515 Cantharis tristis IT BOLD:ABU8563 3.25 DE/FR BOLD:AAW8261 Malthodes mysticus DE BOLD:ACC5434 3.46 DE BOLD:ABU9145 FR BOLD:ACB8851 Carabidae Bembidion cruciatum AU BOLD:ACJ4302 1.68 AU BOLD:AAZ8054 Calathus fuscipes CZ BOLD:ACG8624 3.43 DE BOLD:AAO4635 SL BOLD:ACG9156 Carabus problematicus DE BOLD:ACF2388 3.61 DE BOLD:ACF0018 DE/BE BOLD:ACD8256 FR BOLD:ABX1098 DE BOLD:AAK2572 DE BOLD:ABY6094 Cychrus caraboides AU/DE BOLD:AAI9527 3.61 DE BOLD:ACG8586 Dromius quadrimaculatus DE BOLD:AAF6663 2.14 BE/ DE BOLD:ABY8411 Harpalus dimidiatus DE BOLD:ACF2823 1.6 DE BOLD:ABY1286 Oodes gracilis SL BOLD:ACD0995 2.49 DE BOLD:AAY8254 Ophonus ardosiacus DE BOLD:ACB9418 2.79 DE BOLD:AAP8381 Cerambycidae Anaesthetis testacea DE/IT BOLD:ACJ7189 2.32 DE BOLD:ACD4240

Table 3 (Continued)

Family Species Country BIN Max ISD

Calamobius ﬁlum DE BOLD:ABW9572 5.38 DE BOLD:ACG4102 GR BOLD:ACI8020 Corymbia fulva DE BOLD:ACC1170 2.49 FR BOLD:ABX1256 Corymbia rubra DE BOLD:AAI8975 3.45 AU/DE BOLD:AAJ8299 Dinoptera collaris AU/D BOLD:ACF0404 2.33 AU/CZ/DE/FR/IT BOLD:AAI9899 Evodinus clathratus AU BOLD:ACJ5695 3.07 DE BOLD:AAP7231 Exocentrus lusitanus DE BOLD:ABW5082 3.27 SL BOLD:ACJ6112 Leiopus linnei AU/DE BOLD:AAJ1830 2.81 DE BOLD:ACI6275 Leptura quadrifasciata AU/DE BOLD:ABW4710 4.41 DE BOLD:AAX5760 Phytoecia nigricornis DE BOLD:AAO1346 5.98 DE BOLD:ABA1320 Pogonocherus hispidulus DE BOLD:ABX1236 2.48 DE BOLD:ACC7276 Rhagium bifasciatum DE/IT BOLD:ACJ7014 1.71 DE BOLD:AAE3490 Stenurella melanura AU/DE/BE BOLD:AAP7171 3.45 DE BOLD:ACC3140 Stenurella nigra DE BOLD:ABA3171 4.28 DE BOLD:AAO4415 Chrysomelidae Aphthona cyparissiae DE BOLD:AAO3078 1.27 DE BOLD:ACF4286 Bruchus ruﬁmanus DE BOLD:AAI8460 1.83 DE BOLD:ACF8090 Cassida rubiginosa DE BOLD:AAO0522 5.08 DE BOLD:ABV8043 DE BOLD:ACE9461 DE BOLD:ACA7777 Cassida sanguinosa DE BOLD:ABX8850 2.81 DE BOLD:ACC1852 DE BOLD:ACC2802 Cassida vibex AU/DE/FR BOLD:AAO0525 11.93 DE BOLD:ACJ6497 Clytra quadripunctata AU/DE/IT BOLD:AAM0292 3.61 DE BOLD:AAP6595 DE BOLD:AAO2910 Crepidodera aurata DE BOLD:AAN9961 6.94 DE BOLD:ACI5824 DE BOLD:ACC4115 Hypocassida subferruginea DE BOLD:ACF3354 4.1 DE BOLD:ACC1658 Luperus viridipennis FR BOLD:ACK8176 1.84 AU/IT BOLD:ABA3534 Plateumaris sericea DE BOLD:ABX1859 6.09 DE BOLD:ACC7173 DE BOLD:AAO1505 DE BOLD:ACJ6609 IT BOLD:ABV8921 Ciidae Cis fagi BE/DE BOLD:AAO1205 2.83 DE BOLD:ACE2859

Table 3 (Continued)

Family Species Country BIN Max ISD

Cleridae Trichodes apiarius AU/DE/SL BOLD:ACC1596 3.44 IT/SL BOLD:ACJ6483 Coccinellidae Adalia bipunctata IR/DE BOLD:AAH3316 4.43 DE BOLD:ACE3479 Calvia quatuordecimguttata DE BOLD:AAN3361 2.33 DE BOLD:ABY9024 Halyzia sedecimguttata DE BOLD:AAN9603 7.99 DE/FR BOLD:ABW2151 DE BOLD:ABW2152 DE BOLD:ABA0551 Hippodamia alpina IT BOLD:ACC4209 3.13 BOLD:ABA4696 Hippodamia notata AU/CZ/DE/FR/IT BOLD:AAX1230 3.62 DE/FR BOLD:ABW2193 Myrrha octodecimguttata DE BOLD:AAX2942 2.33 DE BOLD:ACF5104 Nephus quadrimaculatus DE BOLD:ACC4840 5.61 SL BOLD:ACD1125 Psyllobora vigintiduopunctata DE/FR BOLD:AAO1034 5.24 BOLD:ACI8392 Scymnus pallipediformis DE BOLD:ACJ7446 3.91 DE BOLD:ACG3470 Vibidia duodecimguttata DE BOLD:ACD6271 13.33 DE BOLD:ACC4348 Corylophidae Sericoderus lateralis DE BOLD:ABA2914 4.38 DE BOLD:ABX2891 Curculionidae Acalles hypocrita DE BOLD:AAI1000 7.26 DE BOLD:AAI1001 Amalus scortillum DE BOLD:ACI3319 4.85 BOLD:AAR3684 Anthonomus rubi DE BOLD:ABW7258 2.48 DE BOLD:AAO1528 Bagous collignensis DE BOLD:AAO0882 3.77 BOLD:ABA7599 Barypeithes araneiformis DE BOLD:ACG9395 2.33 BE BOLD:AAO4505 Caenopsis waltoni BE BOLD:ACG8575 2.16 BE BOLD:AAO4639 Gnathotrichus materiarius DE BOLD:AAY1105 1.86 DE BOLD:ACC4547 Hypera nigrirostris IT BOLD:ACJ6974 2.97 DE BOLD:ABY6916 DE BOLD:AAP7014 Hypera suspiciosa DE BOLD:AAO4907 6.08 AU/DE BOLD:ACD0692 DE BOLD:ABX1943 Larinus brevis SL BOLD:ACJ5753 2.81 DE BOLD:ABX1248 Larinus planus DE BOLD:AAO1182 3.12 DE BOLD:ACC2725 Lixus ﬁliformis DE BOLD:ACD0808 2.17 SL BOLD:ACJ6008 IT BOLD:ACI1756 Lixus iridis DE BOLD:ABX7915 7.15 DE BOLD:ACC4221 Peritelus sphaeroides DE BOLD:AAO3260 4.42 DE BOLD:ACC1643

Table 3 (Continued)

Family Species Country BIN Max ISD

Rhinoncus pericarpius DE BOLD:AAJ4509 3.13 DE BOLD:ABA0636 Sciaphilus asperatus DE/IT BOLD:AAD5565 2.81 DE BOLD:ACD1894 Sitona sulcifrons DE BOLD:AAO0185 5.92 IT BOLD:ACJ8004 DE BOLD:ACC6979 DE BOLD:ACC4292 Strophosoma capitatum DE BOLD:ABZ5882 2.82 BE BOLD:AAN9983 Tachyerges salicis IT BOLD:ACJ7931 3.62 DE BOLD:ACA9356 Trachyphloeus angustisetulus DE BOLD:ACG9952 1.58 BOLD:ACG9953 Tychius breviusculus DE BOLD:ACD0652 2.65 DE BOLD:ACC1422 Zacladus geranii AU/DE/IT BOLD:AAH8451 2.33 DE BOLD:ACC3456 Dytiscidae Graptodytes granularis DE/SK BOLD:AAL3240 3.29 DE BOLD:AAP9843 DE BOLD:AAY8784 DE BOLD:AAI6621 Hydroporus memnonius DE BOLD:AAY8850 3.48 DE BOLD:AAX2774 Elateridae Adrastus montanus DE BOLD:ACG8745 2.67 BOLD:ACD0291 Adrastus rachifer DE BOLD:AAO3117 3.78 DE BOLD:AAM0496 Ampedus pomorum DE BOLD:ABW5182 3.92 DE/SL BOLD:ACC3229 Athous vittatus BE/DE BOLD:AAN4289 15.75 DE BOLD:ACC1206 DE BOLD:AAN4288 Dalopius marginatus DE BOLD:AAO1078 2.85 DE BOLD:ACC4634 Denticollis linearis DE BOLD:ACI8269 3.12 DE/BE BOLD:AAN4145 DE BOLD:ABY9036 Oedostethus quadripustulatus DE BOLD:ACC3266 2.17 DE BOLD:ACC3267 Procraerus tibialis DE BOLD:ABA3476 13.64 DE BOLD:ACC4293 Quasimus minutissimus DE BOLD:ACD0326 4.9 DE BOLD:ACD0327 Selatosomus aeneus FR/SL BOLD:ACI5545 3.41 DE BOLD:ACJ5854 Selatosomus latus DE BOLD:AAO0051 3.77 DE BOLD:ACG4631 Stenagostus rhombeus DE/SL BOLD:AAV6940 2.59 DE BOLD:ABY6965 Synaptus ﬁliformis DE BOLD:AAO4364 13.41 AU/DE BOLD:ACC2821 Elmidae Elmis rioloides DE BOLD:ABY6700 1.73 DE BOLD:AAF0004 Endomychidae Endomychus coccineus AU/DE BOLD:AAO3281 3.12 BOLD:AAO3282

Table 3 (Continued)

Family Species Country BIN Max ISD

Georissidae Georissus crenulatus FR/DE BOLD:AAI7429 3.77 FR BOLD:ABX8614 Haliplidae Haliplus furcatus DE BOLD:ABX6809 2.17 DE BOLD:AAY9093 Haliplus immaculatus DE BOLD:AAQ4054 2.66 DE BOLD:ACB2425 Haliplus lineatocollis DE BOLD:AAI6900 6.87 DE BOLD:AAI6901 Haliplus ruficollis DE BOLD:AAI6902 1.23 DE BOLD:ACE4212 Haliplus variegatus DE BOLD:AAQ3485 1.23 DE BOLD:ACL2388 Heteroceridae Heterocerus fenestratus DE BOLD:AAK5782 2.32 DE BOLD:ACF5413 Heterocerus obsoletus DE BOLD:ABY1569 2.33 DE BOLD:ACG9430 Histeridae Paromalus flavicornis DE BOLD:ACC2501 6.95 DE BOLD:AAP7321 Hydrophilidae Cercyon unipunctatus SL/DE BOLD:AAO0405 1.69 DE BOLD:ACE6297 Hydrobius fuscipes DE BOLD:AAP9350 11.54 DE BOLD:ACB2991 GR BOLD:ACI9011 DE BOLD:AAC5899 DE BOLD:AAC5901 Kateretidae Heterhelus solani AU/DE BOLD:ABX9939 7.76 DE BOLD:ACC7098 Laemophloeidae Placonotus testaceus DE BOLD:ACH1309 5.25 DE BOLD:AAP6258 DE BOLD:AAP6257 Lampyridae Lampyris noctiluca DE BOLD:AAM9184 6.41 SL BOLD:ACG9491 Luciola italica DE BOLD:ACJ6941 14.7 SL BOLD:ACJ7149 Latridiidae Corticaria obscura DE BOLD:ACG1289 4.41 DE BOLD:ACC2954 Leiodidae Anisotoma castanea DE BOLD:AAR3436 3.08 DE BOLD:ABX3025 Catops subfuscus DE BOLD:ACG5141 2.33 DE BOLD:AAI7295 Catops tristis DE BOLD:ACE5421 1.7 BE/DE BOLD:AAB5796 Leptinus testaceus BE BOLD:AAP6306 2.49 DE BOLD:ACG2597 Melandryidae Abdera quadrifasciata DE BOLD:ACG3028 4.56 DE BOLD:AAV9939 Anisoxya fuscula DE BOLD:ABY0112 12.19 DE BOLD:AAP8275 Phloiotrya vaudoueri DE BOLD:ACG1400 5.76 DE BOLD:ACG1401 Meloidae Mylabris quadripunctata FR BOLD:ABX1228 4.24 FR BOLD:AAF9407 Melyridae Axinotarsus ruficollis DE BOLD:ABY3101 2.01 IT/SL BOLD:ACJ7109 Clanoptilus geniculatus DE BOLD:ACG1503 2.33 SL BOLD:ACJ5605

Table 3 (Continued)

Family Species Country BIN Max ISD

Hypebaeus ﬂavipes D/IT BOLD:AAN9928 2.97 DE BOLD:ACG1492 Mycetophagidae Litargus connexus BE/DE BOLD:AAK8818 2.66 DE BOLD:ACC4244 Mycetophagus populi DE BOLD:AAP8304 1.17 DE BOLD:ACI7788 Nitidulidae Meligethes aeneus DE BOLD:ACF5199 2.14 DE BOLD:ACE3713 DE BOLD:ACF3253 Omosita depressa DE BOLD:ABY0372 6.02 DE BOLD:ACC8857 Oedemeridae Ischnomera cyanea DE BOLD:ACC0981 2.17 DE BOLD:ACB7830 Phalacridae Olibrus liquidus DE BOLD:AAX0584 2.83 DE BOLD:ABY2922 Ptilidae Pteryx suturalis DE BOLD:ACI9764 4.09 DE BOLD:AAX7272 DE BOLD:ABA3638 DE BOLD:ACC2916 Ptiniidae Ptilinus pectinicornis DE BOLD:AAX7240 5.54 DE BOLD:ACJ6649 Scarabaeidae Anomala dubia DE BOLD:ACB8563 3.46 DE BOLD:AAM0455 Aphodius granarius DE BOLD:ACF5375 1.09 DE BOLD:AAM7733 Cetonia aurata FR/IT BOLD:ABX1134 5.3 DE BOLD:AAP7239 DE BOLD:ACD0702 DE BOLD:ACC2933 Onthophagus fracticornis CZ/DE/SL BOLD:ACC1236 4.11 DE BOLD:ACC7883 Psammodius asper AU/DE BOLD:AAO3228 6.58 DE BOLD:AAO3229 Rhyssemus germanus DE BOLD:AAO3170 2.49 DE BOLD:AAO3169 Tropinota hirta FR/SL BOLD:ACJ5694 1.7 DE/SL BOLD:AAO4477 Scirtidae Cyphon padi DE BOLD:ACB2997 3.77 DE BOLD:AAJ3273 Scraptiidae Scraptia fuscula DE BOLD:ACA8229 7.1 DE BOLD:AAP9091 Silvanidae Silvanus unidentatus DE BOLD:ACC4444 6.62 DE BOLD:ACG2269 DE BOLD:AAO0158 Staphylinidae Acrotona parvula DE BOLD:ACG2276 7.4 DE BOLD:ACG2451 DE BOLD:ACA8283 DE BOLD:ACC8301 Aloconota cambrica FR BOLD:ACD0287 3.61 DE/IT BOLD:ABW9256 Anomognathus cuspidatus BE/DE BOLD:AAO0339 11.08 DE BOLD:ACA9191 Anotylus insecatus DE BOLD:AAR3352 2.87 CZ/DE BOLD:ACE2796 Atheta laticollis DE BOLD:AAO3818 5.39 BE/DE BOLD:ACG4481

Table 3 (Continued)

Family Species Country BIN Max ISD

Atheta nigripes DE BOLD:ACE3701 1.54 DE BOLD:AAP7355 Atheta ravilla BE/DE BOLD:ACG1718 1.86 DE BOLD:ACG1719 DE BOLD:AAP7354 Batrisus formicarius DE BOLD:ACC2922 6.51 DE BOLD:AAW2913 Bibloplectus pusillus DE BOLD:AAO3528 3.27 DE BOLD:AAO3527 Carpelimus fuliginosus DE BOLD:AAW4282 13.57 DE BOLD:AAO0558 Carpelimus obesus DE BOLD:AAP8806 1.15 DE BOLD:ACF5911 Coryphium angusticolle DE BOLD:AAR9114 5.23 DE BOLD:ACC6197 Creophilus maxillosus DE BOLD:ABW4723 1.7 DE BOLD:ABZ0392 Cryptobium fracticorne BE/DE/IT BOLD:ABA3745 8.65 DE BOLD:ABY3593 Euplectus karsteni DE BOLD:ABA0629 4.2 DE BOLD:ACF0201 DE BOLD:AAO0164 Eusphalerum luteum AU/DE/FR/IT BOLD:AAP8206 3.3 DE BOLD:ACD0364 Gabrius osseticus DE BOLD:ACG2959 1.99 DE BOLD:ACC6884 Gyrophaena afﬁnis BE/DE BOLD:ABW9049 3.1 DE BOLD:AAO0291 Myllaena intermedia DE BOLD:ACC2212 4.42 DE BOLD:ABX7762 Ocypus ophthalmicus AU BOLD:ABU7317 7.04 DE BOLD:ACG7002 DE BOLD:ACG9247 Phacophallus parumpunctatus DE BOLD:ACG9238 3.12 DE BOLD:ACG3941 Philonthus subuliformis DE BOLD:AAY1007 4.25 BOLD:ABY2531 Planeustomus palpalis DE BOLD:AAO1482 4.48 DE BOLD:ABA3531 Quedius mesomelinus BE/DE BOLD:AAO1235 4.44 AU/DE BOLD:ACC3013 Quedius umbrinus DE BOLD:ACG8748 2.84 DE BOLD:ABW8822 Rugilus orbiculatus BE/DE BOLD:AAO1222 3.42 DE BOLD:AAX9476 Stenus eumerus AU BOLD:ACC6651 2.56 AU BOLD:ACD0860 Stenus guttula DE BOLD:ABU6199 2.81 FR BOLD:ACD0375 Tachinus corticinus DE BOLD:ACF2045 2.17 DE BOLD:AAH0107 Tenebrionidae Lagria hirta DE BOLD:AAP8224 4.27 DE BOLD:ABA0559 Melanimon tibiale DE BOLD:ACG2696 3.12 BOLD:ACG8851 Scaphidema metallicum DE BOLD:AAR4116 3.45 CZ BOLD:ACJ8093

Table 3 (Continued)

Family Species Country BIN Max ISD

Throscidae Aulonothroscus brevicollis BE/DE/FR BOLD:AAZ4368 1.25 DE BOLD:ACE2852 Zoopheridae Coxelus pictus DE BOLD:ACI7867 1.55 DE BOLD:ABA3749 to the United States and New Zealand to control thistles suggest (Table 3) that the Central European Leptinus (Asadi et al. 2013). Given its importance as a biological fauna is more diverse than expected although more sam- control agent, it is interesting that it is represented by at ples need to be examined to confirm this hypothesis least three distinct clades in Germany (Fig. 7A). (Fig. 7E). Lixus iridis (Olivier, 1807), one of the largest (body length = 17 mm) weevils (Curculionidae) in Germany Discussion (Hassler & Rheinheimer 2010), is represented by two barcode clades. All voucher specimens have been carefully This study establishes that DNA barcoding is an effective checked by specialists to exclude misidentification. The tool for fast, reliable species identification of Central Bavarian specimen differs by 7.15% (Table 3) from a European beetles. Other studies, focusing, for example clade with seven specimens from Rhineland Palatinate on Bavarian, German or European species of Lepidop- (Germany) (Fig. 7B). Interestingly, the exceptional speci- tera (Hausmann et al. 2011a,b, 2013), Hymenoptera men shows sequence congruence with unpublished (S. Schmidt, C. Schmidd-Egger, J. Moriniere, A. Haus- records for this species from Finland. mann, G. Haszprunar & P. D. N. Hebert, in prep.), Neur- Ocypus ophthalmicus (Scopoli, 1763) is an easily identi- opterida (Moriniere et al. 2014), Heteroptera (Raupach fied rove beetle (Staphylinidae) because of its shiny blue et al. 2014), Myriapoda (Spelda et al. 2011) and Orthop- coloration and large size with recent records from all tera (O. Hawlitschek, J. Moriniere, S. Schmidt, F. Glaw, states in Germany (Kohler€ & Klausnitzer 1998) from A. Hausmann & G. Haszprunar, in prep.) have obtained deciduous forests and xeric habitats, including urban similar results, strongly suggesting the efficacy of DNA areas (Kleeberg & Uhlig 2011). From most parts of Ger- barcoding for all arthropods. Approximately 42% of the many, only the nominotypical taxon is recorded, but nine Central European fauna of beetles and 53% of the species synonyms have been designated from other regions of known from Germany are now represented in the pub- Europe, and six subspecies are recognized (Drugmand licly available reference library on BOLD. Despite the 1998). Although our data only include six records fact that taxonomic research on German Coleoptera has (Table 3), they suggest that more than one species occurs been underway for more than 200 years, our study in Germany (Fig. 7C). Additional material from all parts of revealed 209 cases where the results from DNA barcod- the country is needed to clarify the status of this species. ing and traditional taxonomy are discordant. These con- Haliplus lineatocollis (Marsham, 1802) is a common, flicts involved 176 cases of deep intraspecific divergence mainly lotic and, compared to most other members of (1089 specimens) and 31 species clusters (155 specimens) the family Haliplidae, easy to identify species which which shared their barcodes, taxa which should be espe- occurs in alkaline, slow-flowing streams, ditches and cially targeted for further sampling in future barcoding gravel pits. The larvae, and perhaps also the adults, feed projects in Europe. These results indicate both to the on filamentous algae (van Vondel 1997). According to presence of very recent species and potentially over- the leading taxonomic specialist for this group (Bernhard looked species diversity, cases are summarized in van Vondel, pers. comm.) subtle morphological differ- Tables 1 and 2. Such incompatibilities between barcodes ences support the presence of three species in Europe and traditional taxonomy are not surprising as evolution with two occurring in Germany (Fig. 7D). It will be a is an ongoing process and lineage idiosyncratic perfor- complex task to resolve their taxonomic status because mance of DNA barcoding or the molecular biodiversity more than 14 junior synonyms (see Nilsson & van Von- assessment approach have been well documented del 2005) will need to be studied, and it is desirable to (Hendrich et al. 2010). The relatively high number of extend the genetic analyses as well. 1089 specimens, representing 176 potentially overlooked Leptinus testaceus is a small, pale, eyeless beetle of the species was surprising, especially because many of these family Leiodidae that lives in the nests of small mammals cases did not involve the specious and taxonomically throughout Central Europe (Kreissl 1988). In the western difficult Staphylinidae, Curculionidae and Elateridae but Palearctic region, six species of the genus have been the well-studied Carabidae, Cerambycidae, Haliplidae described (Besuchet 1980). The current barcode results and Hydrophilidae.

2% Haliplus furcatus (BC ZSM COLA 00727) Germany (Mecklenburg Western Pomerania) Endomychus coccineus (BC ZSM COLA 00296) Germany (Bavaria) Haliplus furcatus (BC ZSM COLA 00729) (B) BOLD:ABX6809 Endomychus coccineus (GBOL_Col_FK_9127) (A) Germany (Mecklenburg Western Pomerania) Austria (Tyrol) 2% BOLD:AAO3281 Haliplus immaculatus (BCZSMAQU00978) Germany (Bavaria) BOLD:ACB2425C Endomychus coccineus (BC ZSM COLA 00378) Germany (Bavaria) Haliplus immaculatus (BCZSMAQU00978) Germany (Brandenburg) BOLD:AAQ4054 Endomychus coccineus (BC ZSM COLA 00462) Germany (Bavaria) BOLD:AAO3282 (BC ZSM AQU 00166) Germany (Bavaria) BOLD:ACEE4212 (BC ZSM AQU 00099) Germany (Bavaria) Trichodes apiarius (GBOL_Col_FK_6784) (BC ZSM AQU 00983) Austria (Salzburg) Germany (Bavaria) BOLD:AAI6902 Trichodes apiarius (GBOL_Col_FK_7249) Austria (Tyrol) Haliplus variegatus (BC ZSM COLA 00666) Trichodes apiarius (GBOL_Col_FK_7003) Germany (Mecklenburg Western Pomerania) Austria (Tyrol) Haliplus variegatus (BC ZSM AQU 00668) 5 (C)

8 Trichodes apiarius (GBOL_Col_FK_7089) 6 4 Germany (Mecklenburg Western Pomerania) 9

3 Austria (Tyrol) 5

1 Haliplus variegatus (BC ZSM AQU 00492) Q Trichodes apiarius (GBOL_Col_FK_7283)

C Germany (Mecklenburg Western Pomerania) A 1% Germany (Bavaria) C

Haliplus variegatus (BC ZSM AQU 00667) D Trichodes apiarius (GBOL_Col_FK_2930)

Germany (Mecklenburg Western Pomerania) Slovenia L

B Trichodes apiarius (BFB_Col_FK_9527) Slovenia B Haliplus variegatus (BC ZSM AQU 00491) Germany (Mecklenburg Western Pomerania) Trichodes apiarius (GBOL_Col_FK_6481) Italy (South Tyrol) Trichodes apiarius (GBOL_Col_FK_6763) BOLD:AAN42888 Slovenia Athous vittatus (BC ZSM COL 03083) BOLD:ACJ6483 (D) Germany (Thuringia) Athous vittatus (BC ZSM COLA 00040)

Germany (Bavaria) Procraerus tibialis (GBOL_Col_FK_5389) 6 2% Germany (Rhineland Palatinate) 7 Athous vittatus (GBOL02073) 4 Germany (Bavaria) (E) Procraerus tibialis (GBOL_Col_FK_5388) 3 Germany (Rhineland Palatinate) A Athous vittatus (GBOL_Col_FK_3293) Procraerus tibialis (BFB_Col_FK_11719) B

A Germany (North Rhine-Westphalia) Germany (Rhineland Palatinate) :

Athous vittatus (GBOL_Col_FK_3261) Procraerus tibialis (BC ZSM COL 02829) L

Germany (North Rhine-Westphalia) Germany (North Rhine Westphalia) O BOLD:ABX68099 B Athous vittatus (BC ZSM COL 02810) Procraerus tibialis (BFB_Col_FK_11740) Germany (North Rhine-Westphalia) Germany (Rhineland Palatinate) Athous vittatus (BC ZSM COL 00498) Procraerus tibialis (BC ZSM COL 02828) Belgium (Vlaams-Brabant) Germany (North Rhine Westphalia) Athous vittatus (BC ZSM COL 02809) Procraerus tibialis (BFB_Col_FK_11763) Germany (North Rhine-Westphalia) Germany (Rhineland Palatinate) Procraerus tibialis (GBOL_Col_FK_5398) BOLD:AAN4289 2% Germany (Rhineland Palatinate) Procraerus tibialis (BFB_ZSM_COLA_1634) Germany (Bavaria) Procraerus tibialis (BFB_ZSM_COLA_1657) Hydrobius fuscipes (BCZSMAQU01007) 9 Germany (Bavaria) Germany (Bavaria) 9 8 BOLD:ACC4293 Hydrobius fuscipes (BCZSMAQU01006) 5 Germany (Bavaria) C (F) A

Hydrobius fuscipes (BC ZSM AQU 00527) :

Germany (Bavaria) D

Hydrobius fuscipes (BCZSMAQU01008) O

Germany (Bavaria) B Corymbia rubra (GBOL_Col_FK_2880) Hydrobius fuscipes (BC ZSM COLA 00632) Germany (Saxonia) Germany (Mecklenburg Western Pomerania) (G) BOLD:AAP9350 Corymbia rubra (BC ZSM COL 02326) Hydrobius fuscipes (BC ZSM COLA 00697) Germany (Rhineland Palatine) Germany (Mecklenburg Western Pomerania) Corymbia rubra (BFB_Col_FK_4765)

1 Hydrobius fuscipes (GBOL00993)

9 Germany (Brandenburg) Germany (Bavaria)

2 Hydrobius fuscipes (GBOL00997) Corymbia rubra (GBOL02151) 9

Germany (Brandenburg) 8

C Germany

A A Hydrobius fuscipes (GBOL00998)

2% D Germany (Brandenburg) Corymbia rubra (GBOL_Col_FK_0547)

L Hydrobius fuscipes (GBOL00978) Germany (Saxonia) D L

O B Germany (Berlin) Corymbia rubra (GBOL01895) Germany (Bavaria B Hydrobius fuscipes (GBOL00996) Germany (Brandenburg) Corymbia rubra (GBOL_Col_FK_7027) Hydrobius fuscipes (GBOL00994) Austria (Tyrol) Germany (Brandenburg) Corymbia rubra (BFB_Col_FK_4764) Hydrobius fuscipes (GBOL01010) Germany (Bavaria) Germany (Brandenburg) 2% Hydrobius fuscipes (BC ZSM AQU 00233) Corymbia rubra (BFB_Col_FK_5456) Germany (Bavaria) Germany (Rhineland Palatine) Hydrobius fuscipes (BCZSMAQU01009) Corymbia rubra (BFB_Col_FK_6361) Germany (Bavaria) BOLD:AAC5901 Germany (North Rhine Westphalia) Hydrobius fuscipes (GBOL02455) Corymbia rubra (GBOL_Col_FK_7235) Greece (Peloponnese) Austria (Tyrol) Hydrobius fuscipes (GBOL02454) Greece (Peloponnese) BOLD:AAJ8299 BOLD:ACI9011

Fig. 6 NJ subtrees for species pairs with high intraspeciﬁc variation that may represent cases of cryptic diversity. NJ trees were obtained from BOLD.

We encourage the coleopteran scientific community nuclear mitochondrial DNA (NUMTs) is a known to join the DNA barcoding projects on BOLD, to aid problem in DNA barcoding (Jordal & Kambestad 2014), clarification of the status of species and to assist in the we double checked the trace files for the presence of dou- description of possibly overlooked species. Discussion ble peaks and stop codons (the latter regularly carried and commenting on specific specimens on BOLD is out by the CCDB, Guelph, in the sequence editing possible for all registered users. As the presence of process). We could not find any evidence of either

Cassida rubiginosa (BFB_Col_FK_4439) (A) Germany (North Rhine Westphalia) BOLD:ABV8043 Cassida rubiginosa (BC ZSM COL 03058) Germany (Saxonia) BOLD:ABX7915 Lixus iridis (GBOL_Col_FK_8098) Cassida rubiginosa (BFB_Col_FK_5251) BOLD:ACA7777 Germany (Rhineland Palatinate) Germany (Saarland) (B) Cassida rubiginosa (GBOL_Col_FK_2792) Lixus iridis (BFB_Col_FK_8847)

Germany (Saxonia) Germany (Rhineland Palatinate) 2

2 Lixus iridis (GBOL_Col_FK_8128) 5 Cassida rubiginosa (BFB_Col_FK_8125) 0 Germany (North Rhine Westphalia) Germany (Rhineland Palatinate)

O Lixus iridis (GBOL_Col_FK_5058)

A Cassida rubiginosa (GBOL_Col_FK_4503) Germany (Rhineland Palatinate) A

: Germany (Rhineland Palatinate) Lixus iridis (BFB_Col_FK_4177) D

L Cassida rubiginosa (BFB_Col_FK_4194) 2% Germany (Rhineland Palatinate)

O Germany (Rhineland Palatinate) Lixus iridis (BFB_Col_FK_8848) B Cassida rubiginosa (GBOL_Col_FK_0552) Germany (Rhineland Palatinate) Germany (Saxonia) Lixus iridis (GBOL_Col_FK_1683) Cassida rubiginosa (BC ZSM COL 00429) Germany (Rhineland Palatinate) Germany (North Rhine Westphalia) Lixus iridis (BFB_ZSM_COLA_1710) Germany (Bavaria) Cassida rubiginosa (BC ZSM COL 03028) Germany (North Rhine Westphalia) BOLD:ACC4221 Cassida rubiginosa (GBOL_Col_FK_0253) Germany (North Rhine Westphalia) Haliplus lineatocollis (BC ZSM COLA 00551) Germany (Bavaria) Cassida rubiginosa (GBOL01867) 2% Germany (Bavaria) Haliplus lineatocollis (BC ZSM AQU 00510) Cassida rubiginosa (BFB_Col_FK_5013) Germany (Bavaria) Germany (Rhineland Palatinate) (D) Haliplus lineatocollis (BCZSMAQU00981) 2% Cassida rubiginosa (GBOL_Col_FK_3540) Germany (Bavaria) Germany (Rhineland Palatinate) Haliplus lineatocollis (BC ZSM COLA 00552) BOLD:ACE9461 Germany (Bavaria) Haliplus lineatocollis (BC ZSM COLA 00553) Germany (Bavaria)

3 Haliplus lineatocollis (BC ZSM COLA 00500)

Ocypus ophthalmicus (BC ZSM COL 02387) 7 Germany (Bavaria)

Ocypus ophthalmicus (GBOL_Col_FK_6944) A Haliplus lineatocollis (BCZSMAQU00980) : Germany (Bavaria) Austria (Tyrol) D

L Ocypus ophthalmicus (GBOL_Col_FK_6955) Haliplus lineatocollis (BC ZSM AQU 00089) O Germany (Bavaria) Austria (Tyrol) B Haliplus lineatocollis (BC ZSM AQU 00678) Ocypus ophthalmicus (BFB_Col_FK_10420) BOLD:ACG9247 Germany (Bavaria) Germany (Rhineland Palatinate)

Ocypus ophthalmicus (BFB_Col_FK_12320) Haliplus lineatocollis (BCZSMAQU00979) Germany (Rhineland Palatinate) Germany (Bavaria) 2% Ocypus ophthalmicus (BFB_Col_FK_12334) Haliplus lineatocollis (GBOL00999) Germany (Rhineland Palatinate) Germany (Brandenburg) BOLD:ACG7002 Haliplus lineatocollis (GBOL01001) Germany (Brandenburg) Haliplus lineatocollis (GBOL01002) Germany (Brandenburg) BOLD:AAI69000

(E) BOLD:ACG2597 Leptinus testaceus (GBOL_Col_FK_4845) Germany (North Rhine Westphalia)

Leptinus testaceus (GBOL_Col_FK_5141) Germany (Rhineland Palatinate)

Leptinus testaceus (GBOL02064) Germany (Bavaria)

Leptinus testaceus (GBOL_Col_FK_5823) Germany (North Rhine Westphalia)

Leptinus testaceus (BFB_Col_FK_10603) Belgium (East Flanders) Leptinus testaceus (GBOL_Col_FK_4287) Belgium (East Flanders)

Leptinus testaceus (BC ZSM COL 02154) Belgium (Flemish Brabant)

2% Leptinus testaceus (BC ZSM COL 02153) Belgium (Flemish Brabant) BOLD:AAP6306

Fig. 7 NJ subtrees for species pairs with high intraspeciﬁc variation that may represent cases of cryptic diversity. NJ trees were obtained from BOLD.

© 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd. 816 L. HENDRICH ET AL. complexity in the observed cases. All suspicious cases of Bergsten J, Brilmyer G, Crampton-Platt A, Nilsson AN (2012b) Sympatry BIN sharing and cryptic diversity candidates were, and colour variation disguised well-differentiated sister species: Sup- hrodytes revised with integrative taxonomy including 5 kbp of house- moreover, compared with DNA barcode reference keeping genes (Coleoptera: Dytiscidae). DNA Barcodes, 2012,1–18. libraries constructed by colleagues involved in other Besuchet C (1980) Revision des Leptinus palearctiques (Coleoptera: Lep- European DNA barcoding initiatives (FINBOL, Mikko tinidae). Revue Suisse Zoologie, 87, 131–142. Pentinsaari; PASSIFOR, Rodolphe Rougerie). 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Data Accessibility Fig. S1. Diagram displaying different habitat preferences and total numbers of all submitted and successfully sequenced species. Compendium of all German beetle species used in this study including information on their geographical distri- Fig. S2. Diagram displaying different biotope preferences and total numbers of all submitted and successfully sequenced species. bution, specimen count, BIN, mean and max ISD, nearest neighbour(NN) species and distance to NN: Appendix Fig. S3. Diagram displaying the general distribution of all sub- S1 (Supporting information) Dryad doi: 10.5061/dryad. mitted and successfully sequenced species. gg8fg. Fig. S4. Diagram displaying the successfully sequenced beetle Accession nos, BOLD sample IDs and species names species according to their total body length. for all specimens used in this study: Appendix S2 (Sup- Table S1. Total numbers of habitat preferences of all German porting information) Dryad doi: 10.5061/dryad.gg8fg. beetles (Kohler€ & Klausnitzer 1998), total numbers of all DNA sequences: GenBank accessions KM439102– successfully sequenced species and missing species. KM452702. Table S2. Total numbers of biotope preferences of all German Final DNA sequence assembly: Dryad doi: 10.5061/ beetles (Kohler€ & Klausnitzer 1998), total numbers of all success- dryad.gg8fg. fully sequenced species and missing species. Phylogenetic data (a compendium of NJ trees for species in the each of the 97 families. NJ trees were obtained Table S3. General distribution of all German species, successfully sequenced and missing species. from BOLD): AppendixS3 (Supporting information) Dryad doi: 10.5061/dryad.gg8fg; available on BOLD Table S4. Total numbers of species according to their body size, under dx.doi.org/10.5883/DS-COLBYGER BOLD project successfully sequenced beetle species and missing species. DTST. Table S5. Results of the Chi-square analysis. Differences among beetle families in the success of sequence recovery. Supporting Information

Additional Supporting Information may be found in the online version of this article: