Accepted Article reference libraries will togrow, libraries continue andgaps reference biodivers for method effective an constitutes that Malaise we traps, using demonstrate BINsfurther andOTUs delineation studies. taxonomic Usingintegrative bulk from derived collections DNA made byextracts detailed, subsequent usefulfor foundation a provide will which fauna, Dipteran German a years this within few study of systemtaxonomic anintermediate the for provided half have taxonomicallybeen ones, inaccessible becaus species of Diptera . Until now, most of these families, especially the most diverse known 9,544 theof 50% than more covered, were Germany from recorded (75%) families species without assignment, level clusters Overall, so called88 taxa”. 117 out of “dark haplotype CO1 2,700 including BINs, of 5,200 total a species comprising named 2,453 for records libraryresultant The includes DNAbarcode specimens. 45,040 involving of analysis Abstract: Article typeArticle : Resource STEFAN SCHMIDTDR. ID : (Orcid 0000-0001-5751-8706) :0000-0001-9167-6409)ID MORINIÈRE(Orcid JÉRÔME MR. This article by article rightsprotected This reserved. is All copyright. 10.1111/1755-0998.13022 doi: lead to differences between this version the Versionand of Record. Please cite this article as pa typesetting, copyediting, through the been publication for accepted hasbeen article This goals: main library study aDNA three BINs barcode provide has Diptera; (1) for (2) 5,200 of present The resolution. taxonomic incremental gain will by metabarcoding assembled lists A DNA barcode library for5,2 A DNAbarcodelibrary (Insecta: Diptera)itsimplications and metabarcoding- for Jérôme Morinière Jérôme Hausmann 2 –Zoological Research Museum Alexander Koenig - Leibniz Institute for Biodiversity, Adenauerallee 160, Bonn This study results summarizes of a DNA 1 ,

Lars Hendrich Lars 1 , Michael Balke 1 – SNSB-Zoologische Staatssammlung München, Münchhausenstraße 21, 81247 Germany Germany 81247 21, Münchhausenstraße München, Staatssammlung – SNSB-Zoologische 3 – Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada 1 , Ledis Regalado Ledis , 1 , Dieter Doczkal based biomonitoring biomonitoring based 1 , Björn Rulik Björn , 1 , Matthias F. Geiger Hebert ity studies using ity studies DNAAs metabarcoding. the gination and proofreading process, which which may andproofreading process, gination and undergone full and review buthas peer full undergone not

2 , Stefan Schmidt Stefan , barcoding on campaign German Diptera, 3 00 German andmidges flies 00 German inthespecies catalogueBIN will be filled, e of the lack of specialists. By contrast, specialists. By contrast, of lack the of e 2 , Laura A. Hardulak A. , Laura 1 , Johann-Wolfgang Wägele Johann-Wolfgang , 1 , Gerhard Haszprunar , Gerhard 2 , Paul D. N. D. , Paul 1 , Axel Accepted Article 89 8107 300 advances ranges. The failure totrack the status of indi hasdeclines general for if spanfailed toascertain impacts groups allinsect size all and evidence the However, 2018). al., et (Lister years few a within or 2017) even et al., Hallmann biomassin by the reductions of flying by 75%overinsects decades (Sorg afew al.,2013; et evidenced general as declines be may suggest the methods collecting employing studies mass Other 2013). Initiative, Pollinators the and Vanbergen 2010; al., et (Potts degradation and fragmentation habitat change, climate contamination, pesticide to bees wild in declines Detailedresearch oneconomicallyconcern. im Introduction: DNA Metabarcoding, Germany, diversity, mitochondrial cryptic Keywords Corresponding authors . unresolved their because of projects groupsthat ar species-rich studieson for method OTUs obt and onBINs names based interim that and solution impediment’; totheaccurate (3) demonstrate ‘taxonomic a pragmatic, provide labelled withglobally unique clusters, barcode extractions bulk of bythe example demonstrate This article by article rightsprotected This reserved. is All copyright. seriously underestimated, a conclusion reinforced by the extraordinarily high numbers of of numbers high extraordinarily bythe reinforced conclusion a underestimated, seriously 2004). etal. Moreover, th is (Völkl it likely twogroupscomprise these respectively, Germany & et 2011)and9,600 Blank, (Schumann (Dathe al., species2004) in 9,500 recorded 1999, (Klausnitzer Hymenoptera and Diptera the orders, insect hyperdiverse two the for is particularly serious research, baseline ecological hampers groups activity, many faunistic and taxonomic 2017). Cruaudet al., Even2015; inGermany, than250yearsa country of withmore (Wheeler is limited et species Riedel 2004; 2013;Pante al., et 2014; et et al., al., Brix al., Recent evidence for major declines in insect populations ininsect has populations majordeclines Recent provoked intensepublic evidence for

in taxonomic practices (e.g. integrative taxonomy), our knowledge of of taxonomy), insect integrative practices in taxonomic most our knowledge (e.g. : Barcode library, DNA barcoding, CO1, Diptera, biodiversitymonitoring, biodiversitymonitoring, : Barcode library, DNA Diptera, barcoding, CO1, : Jérôme Morinière, [email protected] & at the true diversity of these two groupsis these true diversity of at the vidual lineages reflects the fact despitethat remain poorly known. This gap, which poorly Thisgap, known. which remain identifiers (such as OTUs and/or BINs), portant groups, like pollinators, have linked linked have pollinators, like groups, portant from a Malaise Trap experiment, that DNA Trapexperiment, Malaise a from e inbiodiversity usually neglected research ained through metabarcoding is an effective effective is an metabarcoding through ained over half of its insect alpha diversity diversity alpha insect its of half over , 2006; Geiger et al., 2016a). With at least at With 2016a). al., et Geiger , 2006; Dieter Doczkal, [email protected] Fax: +49 Accepted Article al., 2018). The analysis of specimens from two Malaise traps deployed for a single summer single a for deployed twoMalaisetraps specimens from of analysis The 2018). al., 2016;Doczkal, 2005; Hebert et 2017;al., Karlsson Hallmann etal., et al., Ssymank 2017; et 1971; (Matthews & Matthews, caught taxa commonly most beingamongthe Diptera with especiallyarthropodsand flyinginsects for biodiversity assessments in terrestrial ecosystems, receivedlittle have Malaise attention. traps widelyare used method ofas choice to collect Sphaeroceridae, Chloropidae, Anthomyiidae) Ceratopogonidae, Phoridae, Cecidomyiidae, se but explored, well are fairly 2011) al., et (Ssymank Syrphidae the and 2018), al., et (Wolff Asilidae the (https://mueckenatlas.com/), Culicidae asthe such families few A 2012). al., et byFontaine defined sense the (in taxonomists unpaid) voluntary(i.e. often experts, two or one just had families of 1/3 for lacking were experts that shows 2018) 18th December accessed workinggroup(http://www.ak-diptera.de/index.htm, Dipterologist’s (de asthetaxonomic known Carvalho German impediment et the 2007). al., For example, material.unidentified Rapid progress byin is inventorying alackhampered experts,of also RulikDoczkal with expected many amongmore 2016; unpublished), unpublished, Rulik, the & Heller 2015; & Rulik, (Reimann material identified among the Germany to new families species various 100 from than more resulted in barcoding campaigns different for efforts collecting the and 24.3%, of increase an of Lestremiinae, 34species adds (2009) Jaschhof 123species (Meyer &Stark, Germany,Germany to anincreasenew 2015) added of 12.5%, of very checklist certainly theinventoryA recent for the Empidoidea incomplete. is country, European any from recorded species Diptera of number highest the is this Although 2009). (Pape, https://fauna-eu.org/) Europaea, Fauna in defined (as for recorded species 2012). et al., 2014;Fujita etal., 2010, 2010;Schlick-Steiner al., et (Padial taxonomy integrative and methods genetic molecular modern suchas approaches, new of adoption the without resolved be not groupwill this in impediment taxonomic described each orare Diptera the year the species million from awaiting more description, of species new 2017) al., et (Santos 1,000 Asonly about 2016). et al., Morinière (e.g. sites from (HTS throughput sequencing - metabarcoding) byclusters retrieved DNA simultan barcode This article by article rightsprotected This reserved. is All copyright. the of all half than slightly more species, by 2,500 represented being order diverse most was the Diptera here trends, similar revealed http://biodiversitygenomics.net/projects/gmp/) - (GMTP Program Trap Malaise Global the within Germany in The known dipteran fauna of Germany includes roughly half of the almost 20,000 20,000 almost the of roughly half Germany includes of fauna dipteran known The veral of the species-richest families (e.g. (e.g. families species-richest the of veral eous analysis of using arthropods high of analysis eous the and the other dipteran that most families, insect collections at single monitoring monitoring atsingle insect collections

Accepted Article biodiversity monitoring that relies upon meta upon relies that monitoring biodiversity a such alsosupportslibrary Furthermore, large-scale limited. are expertise taxonomic and where personnel time, many those identificationsreliable cases species in and fast enables a 2016a).(Geiger al., Acurated science et to known species, species overlooked thatare or they newto referenced represent even may be either will or BINs) (OTUs units character-based molecular unnamed to belong that (ideally specimens specimens type specimens) knowledge. Duringand expert process, this usingspecialists areverse based approach, taxonomy on museum accurately identified taxa Sequenced subsequentlycan beassoci (Page, taxa” 2016). “dark species socalled challenging – taxonomically and unknown it show Herein we also that is registering additionally 2015). for an efficient method al., et 2003; Gwiazdowski et al., Hebert known species(e.g. of specimens identify to required studies DNA-barcoding provide abasis estafor Usually costs. its reduce dramaticallytask and this speed both DNAfragment, standardized a of sequence characterization the barcoding, DNAapproaches alone. morphological through be completed cannot biodiversity of inventory the However, initiatives. specimens representsidentified the foundat species2008), acomprehensive & Bernstein, (Chivian inventory basedonaccurately than the species are order. for more described currently highit too be may estimate Although this alone. midges) (gall Cecidomyiidae the family, one in just species 1.8 million of presence possible suggestinghigher, the much be species could of number that the actual propose , et DNA (2016), toCanadian al. et Hebert applying 2011;Borkent barcoding al., 2018). al., (Papeet species named 160,000 versusapproximatelyspecies 400,000–800,000 from range species Diptera undescribed global (Pape of 2009)with of al., estimates et species diversity al., 2016a). et (Geiger analyzed were that (26,189) individuals all of 70.3% and were collected that species This article by article rightsprotected This reserved. is All copyright. 2012). al., et Yu 2016; al., et Morinière 2015; Knowlton, & Leray Shokralla et thoseal., 2012), 2012; obtained Malaise like (Gibsonfrom traps etal., 2014; At a when hundredstime or thousa possibly working have long aware Diptera on been of Taxonomists the immense well number

nd comprehensive DNA barcode reference library barcode DNA reference nd comprehensive ated withated established binomens by taxonomic ion for all conservation and biodiversity and conservation all ion for barcoding bulk samples (Hajibabaei et al., 2011, 2011, al., (Hajibabaei et barcoding samples bulk blishing the reference blishing reference libraries the sequence is very likelythat this single family includes nds of species become extinct year become extinct each of species nds Accepted Article (Pohjoismäki et al., al., 2016). et (Pohjoismäki Tachinidae and 2015) etal., (Jordaens Syrphidae 2009), Currie, & (Rivera Simuliidae 2016), al., et 2016;Heller 2016;Heller &Rulik,et et Eiseman al., 2015; al., al., (Latibari 2016; et Sevcik Sciaridae 2015), et al., Nzelu 2014; al., Gutierrez et 2012; et al., Kumar 2011; al., et (Krüger Psychodidae 2012), al., et (Renaud Muscidae 2013), al., (Nagy et Hybotidae 2015), al., et 2006;Kumar al., (Cyvinska 2007; al., et Wang et 2012;Ashfaq al., et al., Versteirt et 2014; Cranston 2013; 2016), 2010;Stur&Ekrem, et 2011; et Montagna al., al., Culicidae al., et et & Gresens, 2008;Ekrem et 2007, 2007; Pfenninger 2007;Ekrem Sinclair al., al., 2005, al., et (Carew Chironomidae 2014), Borkent, & (Stur Ceratopogonidae 2013), al., et Jordaens Calliphori for e.g. thereof: units smaller revise this encompassing highlyentire orderha diverse no study Diptera, comprehensive for performed DNAConcerning studies barcoding 2015). al., 2011; Wesener et al., et Myriapoda (Spelda 2016), and al., et (Astrin Opiliones and 2017), Araneae et al., (Hawlitschek Orthoptera 2014), etal., (Morinière Neuroptera (Hausmann Lepidoptera 2011b), 2019), al., al., et 2011a, 2015, et 2017; Schmid-Egger al., (Raupach 2014;Havemann (Schmidt et et 2018),Hymenoptera Heteroptera al., et al., 2017), et (Morinière al. Trichoptera and Plecoptera 2018;Rulik et al., Ephemeroptera, 2016, 2017), et onbarcoding Raupach2015; reported Coleoptera et al., (Hendrich have results for al., researchers represents the second-most compre ZSM- bythe created library DNAbarcode the Currently, 2007). Hebert, & Ratnasingham (BOLD, Life www.boldsystems.org,Data System of Barcode the in maintained records These represent to thea contribution is library global DNAmajor that barcode (ZFMK). Bonn KoenigAlexander Forschungsmuseum the Zoologisches at curated specimens 180,000 approximately and www.barcoding-zsm.de) München (ZSM, see Staatssammlung theanalysis at of specimens that are SNSB-Zoologische the reflecting 250,000 curated nearly DNA over ofspecies 2009, barcodes 23,000 from MetazoaGerman have been assembled, Since 2016b). al., et Geiger (GBOL, www.bolgermany.de, Life’project of Barcode ‘German 2009)andthe ,Haszprunar, (BFB,www.faunabavarica.de Bavarica’ Fauna ‘Barcoding This article by article rightsprotected This reserved. is All copyright. (Schumann far so published additions three the 1999) and et al., (Schumann Diptera German of tothe the According fauna checklist known Diptera Germany. 55% of from approximately covers It 2013). Hebert, & - Ratnasingham Numbers Index BINs (Barcode 5,200 for coverage The results reported in reported this study results derive The This publication presents of publication results This the Diptera first the campaign anditprovides dae (Nelson et al., 2007; Reibe et al., 2009; 2009; et al., Reibe 2007; et al., dae (Nelson hensive library of any nation. Prior studies Prior studies any nation. of hensive library from two major DNA twomajor barcoding campaigns: from s been published, but data have been used to to used been have butdata published, been s Accepted Article filled, BIN lists assembled by metabarcoding will gain incremental taxonomic resolution. resolution. taxonomic incremental gain will BIN lists assembled by metabarcoding filled, will togrow, libraries continue gaps reference biodivers for method effective an constitutes that species demonstrate delineation using names based onBINsinterim and OTUs traps, further bywe made Malaise collections derived bulk from Using extracts DNA 2015; Schmidt et Mutanen et approaches etal., 2009; 2016). al., al., (Packer conventional biases, taxonomic so operational it can provide more objective identifications than by less is affected approach of delineating automated species this Moreover, activities. barcoding DNA byprevious prioritized processedless and treated been have which taxa”, “dark less-explored of inventorying genetic and classification processing, the in role special play a will latter The morphology). (i.e. methods usingtraditional specialists by identified on namedspeci studies to based equivalent are Furtherm studies. taxonomic integrative detailed, subsequent for foundation valuable become a thus wayand sustainable and transparent, structured, a system in taxonomic interim of an creation the BINs enable sequences. barcode using DNA unambiguouslytheir be BOLD identified can to database, the matches BIN on or morphology based identified species, studied the of 88% than more that find, however We 2006). al., (Meier et gene CO1 the on variability genetic interspecific and intra- between overlap wide since barcoding, DNA by identified reliably could be Diptera of families speciescovering 12 450 about of acomposition of than 70% thatless shown, was it Diptera Pohjoismäki et 2012; the 2016). al., In oneof few studies dealing with DNA in barcoding 2007; et Tachinidae (e.g. et are the Calliphoridae Whitworth Nelson and al., al., the genera, some least for at gaps, barcode to due species delineation in problems of examples Further 2006; et al., 2006). mustal., et Rojo be (Mengual inmanyconsulted genera assignment genes aproblem, here since thesyrphidsgroups additional represent a as for such clear type well-studied In particular, Diptera. within barcodes DNA throughspecies determining in insect Hausmann 2013), orders most al., other et there reportingstudies (e.g. are difficulties Although ithasbeen taxa”). shown, that BINs closely species tobiological ofcorrespond namesspecies interim possess or either orfamily) (genus higher-level taxonomy (“dark just and2,700 BINs, which identifications, species a totalofreliable 2,453 includes library now of species 2004, Dipterahave2010) 9,544 2002, recorded Germany.from been Diptera The This article by article rightsprotected This reserved. is All copyright.

ity studies using ity studies DNAAs metabarcoding. the in the species catalogue will cataloguein thespecies will subsequently be es, i.e. where the underlying are specimens where the i.e. es, ore, the BIN system enables analyses that the ore, Accepted Article interest, most of which derived from the huge malaise trap experiments in the framework of framework in the experiments trap malaise huge the from derived which of most interest, the ofFrom specimens collections. potential entire collection, 3,000,000 approximately SNSB-ZSM inthe nowstored specimens are and samples All countries (18). European Central andother (70), (106), (147), Republic (222),Czech in collected were Germany others (44,511), from derived vouchersfor species sequence diversity Most submittedthe of characterization. maximize Samples werescreened to was morphologically in placed Rhineland-Palatinate. trap one mostly across seven (2011-2017) years of each deployed in100Malaise traps were samples.Malaise to been extracted trap 20 from have by andpitfall-trapping.and However, specimens mostvoucher Malaise-, netting, window- by sweep collecting, were hand ofDiptera collected million specimens than five more dates available in is and sites on information collection Detailed “Landskrone”, directive site as well as alpine the Nationalparksas “Bayerischer Wald” and “Berchtesgadener Land”, the EU habitats such protected public lands in and state areas moreforests, than 683localities included sites study The 2016. 2009 – from deployed were which traps, by Malaise collected were >500bp) sequences with COI of 45,040 out (94.5% - 42,587 specimens ). Most France, Republic, Czech Belgium, (Austria, countries European surrounding from also but states, German D the to specimens contributed and collected &Taxonomy: Specimens Fieldwork, Methods: & Material in biodiversity neglected usually pr research method effective is an metabarcoding through inte that (3) demonstrate and impediment’; OTUs BINs),as provide a and/or pragma la clusters, DNA barcode that experiment, Trap based (2) bulkon the of BINs Diptera; of demonstrate, example extractions a Malaise from This article by article rightsprotected This reserved. is All copyright. & 2000a, b; Darvas, Papp 2011). 1998, FromSchumann material, et al., therein; this 1997, litera specialists using appropriate experienced by BRand extent aminor DDandto mostly by level tofamily identified were GMTP, the The studyThe present hasthree main goals: (1)provide a DNA library5,200 barcode for A network of 130 (professional and voluntary) of and 130citizen(professional A network taxonomists and scientists tic, accurate solution to the ‘taxonomic ‘taxonomic the to solution accurate tic, rim names based on BINs and OTUs obtained andOTUsobtained on BINs based names rim ojects because of their unresolved their of ojects taxonomy. because regions in heights of up to 2,926 m (Zugspitze). (Zugspitze). m 2,926 to up of heights regions in NA barcoding projects, primarily from various various primarily from projects, NA barcoding for studies on species-rich groups that that are groups species-rich on studies for belled with globally unique identifiers (such belled withglobally identifiers unique ture (e.g. Oosterbroek, 2006 Oosterbroek, ture and references(e.g. habitats in Bavaria and Baden-Wurttemberg, inBavaria and habitats or ZFMK except for a few held in privateZFMK for few in or a held except Appendix S1 Appendix . Since 2009, 2009, Since . Accepted Article stored in dataset private the stored „ (http://www.boldsystems.org – dataset Life of &German Barcode - GBOL Bavarica Fauna (BFB- Barcoding Diptera of German library reference Barcode DNA - A DIPBFGBL pairs, specimens and trace files 40,753 for primer are publiclyin the accessible “ classifica taxonomic identifier, collector, altitude, habitat, data, as locality such information 2018). Voucher 2008, etal., deWaard 2006; Ivanova al., (seealso et reactions sequencing Sanger bidirectional subsequent for wereemployed and (5’ same etal., primers 2014), the CO1) (seeHernández-Triana fragment barcode the CA)for AAT AAA GGA TGTCCA TCT ACT – TAA (5’ CLepFolR and primers –ATT CLepFolFCAA CCAATC Folmer ATA AAG (5’ modified ATA TTG G) (ccdb.ca/resources/). under: PCR withAll were samples cocktail a amplified standard of and online available are Sanger sequencing procedures and PCRamplifications, DNA extraction, (CCDB)Barcoding where they were processed plates the DNA extraction with samplestissue to were sent Canadian the for Center DNA and curation. ZFMKand SNSB-ZSM identification were to the for repatriated specimens using Canada) “ (Guelph, CCDB atthe voucher the whole from was extracted DNA traps), Malaise from (vouchers butreplacement vouchers the from Sciaridae), for somevery small specimens (e.g. used was The wholevoucher removed DNA leg or for extraction. segmentwas leg, a single a mm >2 body with length a For specimens DNAextraction. subsequent theSNSB-ZSM for at protocols: Laboratory aspossible. identifications as many species-level were wasavailable, toobtain specialists specimens sent to expertise i.e. 1,356, 1 to from than five specimens more old. years number of The 4% rangedwere per species analyzed Specimen ranged ages generally extraction. (>99% Mostsamples projects. DNA barcoding international and national in involvement its support ZSM to at the established specimen) each high for acquisition imaging andmetadata quality sample preparation, (including specimens were 59,000 submitted for sequence analysis through theDNA barcoding pipeline This article by article rightsprotected This reserved. is All copyright.

A tissue sample was removed from each specimen and transferred into 96 plates into well transferred wasand each specimen removed sample from A tissue Megaselia rufa Megaselia voucher-recovery DS-DIPBFGBP - A DNA Barcode reference German DNABarcode of library -A DS-DIPBFGBP (Wood, 1908) (see 1908)(see (Wood, DOI; XXX ≤ 1 mm, e.g. small Cecidomyiidae, Chironomidae, Chironomidae, Cecidomyiidae, small 1mm,e.g. tions, habitus images, DNA barcode sequences,DNA barcode images, tions, habitus from 1-5 years (43,112 specimens – 96%), only only –96%), specimens (43,112 years 1-5 from ” protocols (deWaard et al., 2018) and theand 2018) (deWaard al., et protocols ” same locality were retained. In other cases cases other In retained. were locality same ) were stored in 96% EtOH before DNA 96%EtOH before in stored ) were using standard protocols. All protocols for protocols All using protocols. standard ), whereas 4,420 specimen records willbe records specimen 4,420 whereas ), Appendix S1 ” onBOLDdataset ” ). When taxonomic). When DS- Accepted Article (Padial et al., 2010; Schlick-Steiner et al., 2010, 2014; Fujita et al., 2012) to infer, whether 2010, et 2012) et toinfer, Schlick-Steiner 2014; Fujita et al.,al., al., 2010; (Padial Taxonomy Integrative of concept the Wefollow data. morphological and molecular both as“dark of taxa” well. families in occur also may but 2016), etal., Pohjoismäki 2012; al., et Nelson 2007; al., et Whitworth 2006; al., et Rojo 2006; al., et (Mengual species tachinid syrphid and calliphorid, as beenwell-documented et (Meier has phenomenon sharing or barcode gapthe - & Hubert see 2015). Hanner, Within Diptera, the this due to barcodes, high intra- or low inter-specific (e.g. distances genetic cryptic diversity, BIN onDNAthe species based delineation with problems throughout insects exhibit families Hausma Lepidoptera, for (e.g. taxonomy traditional specimenssharingorders, repr BINvery a often (OTUs) units byinsect being studied majority “named” For the taxonomic number. of a operational into algorithmically sequences barcode similar clusters which pipeline, online identifications when taxonomic information is lacking. The BIN System involves a 3-step orNumber” BIN (Ratnasingham & Hebert, 2013). This system enables tentative species sequences, which are assigned aglobally unique identifier, termed a “Barcode Index into Sequences BINs. grouped ormore are two differentto shared species a BIN, and cases those particular where a species was toassigned The “ distances. onK2P based alignment were following calculated (NJ) trees joining Neighbor 500bp. aminimum of length with sequences restricting to analysis sequence alignment for was applied MUSCLE2012). al., et (Puillandre metric distance (K2P) Parameter calculated using the “ dist genetic minimum and variation intraspecific Data analysis: taxonomists. publication future for records & Bavarica (BFBFauna ofLife) -Barcoding GBOL - Diptera Barcode German private – This article by article rightsprotected This reserved. is All copyright. only COI-characteristics. valid species speciesabsent between using toallow delineated to is too lowor divergence be sample, species (“cryptic inthe or are previously there taxa”) overlooked whether barcode BIN Discordance Sequence divergences for the COI-5P barcode region (mean and maximum maximum and (mean region barcode COI-5P the for divergences Sequence Every other “disagreement/conflict” case is the starting point for re-evaluation of ” reveal onBOLDanalysis to used caseswherewas species assigned Barcode GapAnalysis Barcode ” for subsequent publication by the authors and associated authors and bythe subsequent publication for ” ” tool on BOLD, employing the Kimura-2- the employing on BOLD, ” tool esent esent a species-proxyclose by delineated as ance to the nearest-neighbor species) were nearest-neighbor species) tothe ance clusters of closely similar COIbarcode of closely clusters similar nn et al., 2013). However, some genera or genera some et 2013). nn al., However, al., 2006), at least in some families, such least some at in 2006), al., Accepted Article level (family, subfamily or genus), we we genus), or subfamily (family, level This article by article rightsprotected This reserved. is All copyright. protocol) metabarcoding throughput sequencing was performed on an Illumina MiSeq using v2 (2*250bp, 500 cycles, cycles, 500 (2*250bp, using v2 MiSeq an Illumina on performed was sequencing throughput COI.High- mitochondrial region of coding long 313-bp a targeting 2013) al. et (Leray NGS primers reverse by and using PCR, amplified forward was region A mini-barcode Germany). Hilden, (Qiagen, kit &Tissue DNEasy the Blood with was performed extraction K. DNA Proteinase 10% and Buffer Lysis Insect 90% of solution a overnight, using was performed sample size-sorting tissuestep), and powderpertrap ofinsect lysis (no pestle Allspecimens macroinvertebrates. samples of of 90bulk total a monthly, producing twice werechanged bottles Trap in 2016. September) period (May- vegetated Bayerischer Park its during the the and surroundings NationalWald in traps deployed Malaise were thisstudy, nine In prep). in al. et (Hardulak metabarcoding on samplesinvasive field warning pest an -focusing systemspecies with and basedearly on for Analysis: Data & Bioinformatic Metabarcoding performed. were analyses all after tables metadata BOLD the in field status” “Voucher the into subsequently were names moved specialists. Interim by identification taxonomic for BINs Mala from afterlegs retrieval and/or antennae genus vouchers the or level, and where species sp. BOLD:ACP6151 Melanagromyza on BOLDwith public data e.g. -using (suchas the aBINmatch same identification when BIN taxonomythe specimens genus example other free” had the within that - for name, only thoseuploaded (e.g. as“D taxonomy lower lackwhich records, specimen For necessary. where theinterimtaxonomy updating after same timethe allspecimens at on wasconducted analysis This assignments . BIN with those genetic of divergences among Linnaean species species andto distribution both asasigned in the“ sp.BOLD:AAG2112 Maximilians UniversityMaximilians (LMU, , Germany). (see Ludwig- Unit of the the of chemistry 20M Service Sequencing at reads) maximum When sequenced specimens could onlyWhen beassigned toa speciescategory the above The potential utility of the DNA barcode library for biomonitoring Diptera wastested Diptera forbiomonitoring DNA library ofthe barcode utility potential The Reverse-taxonomy approach: Reverse-taxonomy approach: Barcode Gap Gap Barcode Analysis ) based on the corresponding BIN, so these specimens could be included be could specimens these so BIN, corresponding on the based ) ” in order to provide more comprehensive estimates of the of estimates comprehensivemore provide to order in ” ). All specimens, which not). Allspecimens, be could identified to a employed interim species names (such as as (such names species interim employed ise trap), were selected using the corresponding usingthe selected were trap), ise were dried and ground with a stainless a steel and ground with were dried were in acceptable condition (e.g. unbroken unbroken (e.g. condition acceptable in were iptera”), we applied the highest “conflict- highest the we applied iptera”), Appendix S5 Appendix for a more detailed detailed more a for TachIntGen1 Accepted Article sequences (86.23%) were recovered. Length recoveredthe sequence of varied with the /developing DNA a reference library: barcoding Results: “ the using cutadapt, with removed primers were reverse Forwardand 313bpsequences. yielding approximately theVSEARCH with merged program “f in reads were sample and reverse 2011). each Forward v1.14 (Martin cutadapt 2016) and This article by article rightsprotected This reserved. is All copyright. sequences <658bp> with (41,339) 69.95% yielding sequenced unidirectionally (43,533) rest were the while bp) barcode (658 full-length 12.54% specimens) (7,410 we protocol; sequencing not detected at >= 90% identity. Quality filtering was done detected at with the not Quality done >= 90%identity. “ was filtering VSEARCH,expectedwith sequences zero keeping errors (“f with 0% “dark taxa”. (R version 3.4.4 (2018-03-15), R Core Team, R 2018). (R taxa”. Team, 0% Core (2018-03-15), with “dark 3.4.4 version of families exclusion and inclusion the with both Germany, in reported species of number bodythe with versus mid-range “dark taxa” size percentageof the to estimate calculated created; were study metabarcoding the within ( BIN respective the to assigned then OTUs were the analysis. from removed per sample were reads summed below readofthe 97% below 0.01% numbers total and with entriesidentifications All results table was combined then by filtered Hi The pipeline. bioinformatic bythe table generated OTUthe with and combined Geneious and species genus, family, value, %-ID information for each OTUwasexported from Hit- BIN, included which csvresulting file The (2016). al. et Morinière in described methods following the -NewZealand) Auckland in -Biomatters, (v9.1.7 Geneious BIN information BOLD and downloadedfrom database taxonomy including Diptera the against BLASTed “ employing identity using “ file the fasta from were removedsequences Chimeric de-replicated. then was which file, fasta with“ dereplicated Sequence processing was performed with the VSEARCH v2.4.3 suite (Rognes et al. et al. (Rognes suite with the VSEARCHv2.4.3 processing wasperformed Sequence

From the 59,102 specimens submitted for Sanger sequencing, 50,963 CO1-5P 50,963 Sanger sequencing, submitted specimens for 59,102 the From uchime_denovo Appendix S2 derep_fulllength discard_untrimmed cluster_size ”. The remaining sequences were then clustered into OTUs at 97% 97% OTUs at into clustered then were sequences remaining The ”. ). Presence-absence). overviews of ”, a greedy, centroid-based clustering program. OTUs were OTUs were clustering program. greedy, centroid-based a ”, ”, ”, first atthe level, andthen into sample concatenated a ” option to discard sequences for which primers wereprimers which sequences for todiscard option ” astq_mergepairs one-sided Pearson correlation were coefficients t-%-ID value and total read numbers per OTU. per read and total numbers value t-%-ID 500bp and 3.75% (2,214specimens) with 3.75% 500bp and re bidirectionally sequenced and yieldedsequenced bidirectionally and a re ” with a minimum overlap of 40 bp, of overlap minimum witha ” astq_maxee selected Diptera taxa (BINs) taxa Diptera selected ” ” 1). Sequences were fastq_filter ” in Accepted Article DNA barcode sequences (> DNA barcode For10 old. years subsequentthe analyses we selected specimens 45,040 high with quality Barcodespecimens. recovery successful most forwas EtOH less preserved than specimens theof 13.77% (8,139) recovered from was information Nosequence sequences <500bp. This article by article rightsprotected This reserved. is All copyright. species that shared that species a BIN. ( (1 specimens all of 2.9% representing (6.56%), species 156 Another BINs. to thosefour of oneother BINs and to three assigned were whose members were assigned totwoBINs ( strongare candidates for cryptic overlooked diversity. Mostofthese cases involved species that cases 40 species, another 6-12% in from and species in48 3-6% from ISPranging max 88 species Another lineages. showedphylogeographic higher with considerably divergences secondary betweenvariationcontact from arising or high intraspecific species sibling young either may reflect which cases 1.1-3.0%), = (range 3% than less was (maxISP) species. thes For current 34of to a assigned S3 255BINs ( of inatotal BIN,resulting thanone assigned tomore were specimens studied individuals), (3,951 8.7% of all representing (4.97%), 122 species Another by CO1sequences. their (2,138) discriminated unambiguously 88.67% were BIN matches, free” “conflict identifications and on morphological based Linneanassigned a taxa binomen 2,453 the Among identifications. morphology-based with high congruence revealed 2017), al. (Rulik et incongruences taxonomic detecting for BOLD) theTaxCl-approach on andusing BINs newto the (1,829) were of BOLD.third from Germany ( known 88 families 117(75%)dipteran from of the one representatives or more included BINs These of dataset). the 52.4% specieswhile aspecies designation, lacked 2,700 Linneaen totalof2,453 were a 5,200 assigned BINs (2,500 included approximately dataset This BIN. (Syrphidae), a family that has seen intensive taxonomic study. hasseen afamily intensive that taxonomic (Syrphidae), species were the BINsharing (168) (105/156)thirds of exhibiting two almost majoranae 1758; Linnaeus, identification (e.g. divergences BIN the meaningconsistent within CO1sequencesenabledspecies that ). For purposes of re-identification, the spec the re-identification, of For purposes ). Inspection of the CO1 sequence CO1sequence ofthe usingNJclusters Inspection (created with analytical tools trees Figure 1 Sericomyia lappona Sericomyia 1857 and Rondani, Chrysotoxum bicinctum , Table 1, Appendix S3, S3, 1,Appendix Table 500bp), which fulfilled the requirements for being assigned toa being assigned for requirements the fulfilled which 500bp), Table 2) Linnaeus, 1758and . Ten of of speciesthese pairspossesed. Ten but shallow Paragus pecchiolii Linnaeus, 1758 Linnaeus, ,316 specimens), involved,316 specimens), two or namedmore e taxa, the maximum intraspecific thevariation maximum e taxa, ies in this subset can unambiguously subset also can inthis be ies 112 species), but specimens of nine species nine of but specimens species), 112 Krona graph in Supplement). More a Supplement). than graph in Krona Sericomyia silentis Sericomyia , Rondani, 1857). Interestingly, Interestingly, 1857). , Rondani,

and and Chrysotoxum festivum Table 1, Appendix 1,Appendix Table Harris 1776; Harris

Accepted Article species reported from Germany (r=0.33, p=0.0037) ( p=0.0037) (r=0.33, Germany from reported species of numbers with positively and withbody p=0.0004) size(r=-0.41, correlated inversely Malaise sites. Syrphidae for trap selected and Chironomidae Cecidomyiidae, 2 ( 71 representatives (1,3 of including families 1,403BINs of atotal weidentified data, Diptera Using the merged. BIN asingle were to DNA barcoding in theidentification Central Diptera, results in DNA barcoding of reinforcingof the European efficiencyof the studydemonstrates The 2016a). al. et Geiger (see research biodiversity other and biology, conservation in work ecology, for handles’ ‘taxonomic useful are lack their aLinnean still Although name, these species BINassignments half fauna. Diptera German the of half for system identification taxonomic an interim study provided this years, few a just within Bycontrast, ofspecialists. the lack of because inaccessible taxonomically some beenof themostdiverse, most especially these have Until now, of families, 2010). 2002, 2004, etal. 1999; Schumann, Germany(Schumann for reported Diptera fauna the 50%of covering approximately to88families, unnamed belonging plus2,700 BINs) BINs 2,500 comprising species named (2,453 BINs 5,200 for records library included reference of 45, characterization onthe work based Discussion: This article by article rightsprotected This reserved. is All copyright. a total of 2,809 OTUs matched to the Diptera database with > with database Diptera the to OTUsmatched 2,809 of total a with sample per 6,928 was count read average BOLD(the from downloaded library barcode in the orhigher toa -5,960 average sample, matching at97% DNAtaxon reads per reads 536,376 comprising OTUs, dipteran revealed 1,735 metabarcoding prep), in et al. (Hardulak samples: trap Malaise of metabarcoding for library reference the of Performance XXXXXXXXXXXXX. DOI: publicly accessible the All under data available is project information. Appendix S2 Appendix illustrates illustrates examples ofpresence/absence overviews for the families Muscidae, Among families containing “dark taxa”, the percentage of unnamed taxa was unnamed of taxa percentage the taxa”, “dark containing Amongfamilies This study summarizes the results of a DNA barcoding campaign on German Diptera, on German barcoding campaign DNAof a results the studysummarizes This Appendix S1 Appendix Among the 90 Malaise trap samples from the Bavarian Forest National Park Forest National the from Bavarian trap samples 90Malaise Amongthe ). Almost one third (498/1403) of these BINs belonged to “dark taxa”. “dark taxa”. to these BINs belonged of (498/1403) third one Almost ). provides species names, sample IDs, BIN assignment, and collection and sample IDs,BIN assignment, names, species provides 040 specimens. The040 DNA resultant barcode specimens. 85 species) within the metabarcoding dataset dataset metabarcoding the within species) 85 Figure 3 - 90%). Multiple OTU matches OTUmatches Multiple 90%). Appendix S4 Appendix ). ). Figure Accepted Article of a higherfamily, the theratio of “dark taxa” ( averag taxa”and of “dark the number between inverse relationship strong was there Overall, waslow. a families these in taxa” “dark (e.g. Opetiidae, Megamerinidae, Phaeomyiidae) known from Germany and the incidence of nearly DNA studies. was coverage families species-poor barcode many earlier for complete This article by article rightsprotected This reserved. is All copyright. certain taxa certain can visualizedeasily be usingpresence-absence maps( overallof biodiversityas phenologiessamplingin well annual as Variation sites between Germany( in Diptera of diversity assessingthe for approach valuable a will be metabarcode bulksamples of that analysis indicates result This record. dipteran OTUs with 1,735 identified Germany,we of region 2016). et 1982;May 2000; Hebert Erwin al. 1988;Ødegaard (e.g. studies several other in conclusion reached a seriously underestimated, hasbeen world the and in Germany, Europe, Diptera diversityof true the conclude study, we more for need Despite butitislargelyDiptera. accelerated, the throughout group unstudied toberate known 2018)another Schmidt, Hansson& al., 2018; et (e.g. Branstetter Hymenoptera from known is haplodiploidy of phenomenon The 2003). during embryogenesis (Normark inactivated - haplodiploidy. Here, paternallygenomes inherited ofreproduction diploid males are of tomode their belinked unusual - might or BINnumber- species extraordinary the (2016), al. et Hebert by As proposed already anincreased evolutionary rate. indicating Figure 2B,it is clear that the Cecidomyiidae show lower a average inter-specific variation, families in theother with studied compared area. When in the Diptera of family largest ( Germany example, as impressive 930BINsencountered we only while 886species are knownfrom most represent the Cecidomyiidae The 1999). al. et Germany (Schumann from known BINcount their as of high levels diversity unexpectedly DNA andTrichoceridae, indicates barcoding Milichiidae &Stark 2015), Meyer - see (Empidoidea group” the “Iteaphila as such Among families, recognized. previously ofbehigher number Germany dipteran thanis likely species the to in indicating much species, overlooked and diversity cryptic suggest incidence of a higher our results body sizes, dataset notbe assigneddataset could Linnean to names,such a species, asinterim BIN assignments, metabarcoding the within OTUs the of third a Although preparation). in al. et (Hardulak large comparison of datasets for feature useful Within the metabarcoded Malaise trap samples themetabarcoded in over Malaise collected Within one season one just Table 1 Table , Schumann et al. 1999; Jaschhof 2009). As such, they represent by far the by far Assuch, 2009). theyJaschhof represent 1999; al. et Schumann , is substantially higher than number of species of number higherthan substantially is e body size - the smaller the average body size theaveragebodysize smaller e bodythe size- Figure 3 and for monitoring beneficial or pest insects orpest beneficial monitoring for and a sequence identity higher than 97% to 97% a than asequence identity higher ). Among families with the smallest smallest the with Among families ). Figure 2 ). This will be a Appendix S2 Appendix ). ). Accepted Article taxonomic experts. Additionally, data generated by ongoing metabarcoding studies, such as such studies, generated metabarcoding by ongoing data Additionally, experts. taxonomic unavailable onoften dependence less and with fast biodiversitydata generate to need the isso too and crucial, is projects biomonitoring upscale ability to The al. 2013). et Ji 2012; al. (Hajibabaeiapproaches 2011; morphological Taberlet classical et Douglas al.2012; et al. et certainly enable species surveys atlarger scal of technologyto biodiversity this will assessments Theapplication time. at specimens a of thousands DNAfragments analyze and of to millions sequencers high-throughput of with anythe life the ability stage, traceenvironment, oreven bodyidentify DNAin fragment DNA namely the the barcoding, to capacity advantages of combines This approach 2018). al. et soilbiology(Oliverio for 2018), and al. et (Chimeno entomology forensic for identification species2018), etal. Hawlitschek et 2014; Barba al. (De dietary for analyses inference feces 2018), et Serrana & Leeseal. 2015; assessingwater (Elbrecht for andstreamhealth sampling macrozoobenthos prep), in al. et Hardulak 2016, Hebert, & (Ashfaq species pest and invasive trap including surv Malaise contexts various in targets as forspecies action. conservation ofdesignation some potential the dipterans status of of and the the proper evaluation permit to first step a study is plant present speciesinEurope. The and animal orendemic threatened rare, of a widerange of conservation the ensures which 1992), flora, and fauna wild of directive (CouncilDirective 92/43/EEC Habitat among the listed ca. 1,000 being accordingspecies protected European tothe Flora-Fauna- is species dipteran a single not but al. 2016), et (Gruttke list Germanred the in included are Thaumaleidae) Syrphidae, Psychodidae, Hybotidae, Empididae, , Dixidae, (Asilidae, 11 families from species 2,000 Approximately inGermany: assessments environmental within under-represented extremely present invery dipteranAt present, species - high are –though numbers sites. overall still are valuable differences betweenfor evaluating or samples environments various from records their genus, familytoassigneda or may be only taxa sequence library. Although such “ usedto track canbe taxa known (<97%) to similarities lowersequence OTUs with sites. to sampling possible compare it make This article by article rightsprotected This reserved. is All copyright. have taxonomic orwhen beenWhile biomonitoring studies annotations available improved. become sequences reference new when lists, species comprehensive more recursively producingreanalyzed, and combined can be projects, biomonitoring national annual from Prior studies have shown the great potential of metabarcoding for biotic assessments biotic metabarcoding for of potential great shownthe have studies Prior dark taxa dark eys (Morinière et al. 2016), biosurveillance of biosurveillance 2016), al. et (Morinière eys ”, species those missing thereference from es, time, shorter lowerand costsrather than Atelestidae, Ceratopogonidae, Chaoboridae, Chaoboridae, Atelestidae, Ceratopogonidae, on the Conservation of on theConservation of natural habitats and Accepted Article specimens are still being identified by external specialists, a process that is labour intensive intensive labour that is process a specialists, by external identified being still are specimens Voucher http://biodiversitygenomics.net/projects/gmp/). (see program Malaise Global the of a component deployed Malaise as traps three obtainedjust from study the were for specimens specimen the of onlya fraction Diptera, groups of ofover German 80%of coverage families. Due to the general of lack taxonomy inmany with Diptera, a of barcoding DNA studies on of the mostcomprehensive one of results presents Diptera. This thepaper groups of research subsequent onthese unexplored for cornerstone a studyrepresents present The taxa". "dark of proportion large a include families diverse of itsmost several that but also order isextremely that species-rich, this confirmed onlyhas not Diptera work on Our expertise. taxonomic more and richness species lower with (e orders than other attention less received represents the largestDiptera andtaxonomically of present study ofa decade The represents animportant work component directed Because species. animal German for library DNA barcode a comprehensive creating toward vouchers these study identifying by for many the " to taxonomists improve identifications dipteran DNA reco barcode voucher-based of thousands bepursued–vigoro they should biodiversity, - thus conserve and - to identify our ability enhance greatly technologies these Because world'sfauna. conserve the to efforts toaid and metabarcoding DNAbarcoding of potential the highlighted also has Ourwork library. reference the of extension future for resource valuable a samples remaining the making material, sorted this within remain library reference the from absent genera currently and species thatmany iscertain It be analyzed. could taxonomy orbetter to a sortedonlyhalf the specimens family that meant constraints resource identify regions that are of mo to patterns biodiversity assess to rapidly ability the enhance can metabarcoding Furthermore, assessmentscomprehensive of biodiversity since even " metabarcoding employed indicator subsets will enable traditionally of small have species, This article by article rightsprotected This reserved. is All copyright. taxa. taxonomically challenging consuming, for especially time and Although this project aimed to to develop aimed library,DNA barcode a comprehensive Although thisproject st significancest for conservation. using reverse taxonomic using approaches. taxonomic reverse .g. Lepidoptera, Coleoptera, freshwater orders) freshwater Coleoptera, Lepidoptera, .g. usly. As our study has provided several provided has study As our usly. s could be identified to species level. Most species level. to identified be s could rds, we invite the global community of of global community the weinvite rds, most challenging insect order, theyhave order, insect challenging most dark taxadark dark taxa ” inour” encountered ” can be tracked. Accepted Article biodiversity studies, and barcoding initiatives at national and international levels. levels. and international national at initiatives and barcoding studies, biodiversity so research, they portal be data taxonomic will for vailable through a community public Our dataspecies. release aims toatbiological accessible results the scientific making these as recognition gain eventually will that entities genetic thediscovery of speed will barcoding many the that finding of specieswe encount the and This conclusion Diptera. for tool species identification asa DNA barcoding of species by defined BINsand traditi between Pohjoismäki et 2014; mostcases, However, Ståhls al., 2016). in there was congruence & al., Haarto Nelson 2012; et 2007; al., et (e.g. Whitworth identification species for needed Tachinidae may where and Calliphoridae Syrphidae, in the markers observed additional be were variation interspecific low of cases Some taxa. mostthe dipteran of identification strongly DNA that will supportthe discoveryresults enableconclusiuon the barcoding and Our order. insect diverse most certainly, and, almost megadiverse, a onDiptera, projects This article by article rightsprotected This reserved. is All copyright. organisms’, but also to solve long-standing taxonomic puzzles. latter students The leads alsototaxonomic long-standing solvework but organisms’, ‘real onlywith not work to of theallowing students advantage additional has approach facilita &Hebert 2007)further (Ratnasingham of the Democratization method, analyticalthe tools, and data through theBOLD database amplicons reliablygenerate sets primer current around the in andbecause been have world implemented many laboratories workflows DNA barcoding Because is not. sequencing genome way that a students in undergraduate to introduction intuitive easy, an provide libraries Itneeds that DNAba phenologies. emphasis wide-rangingseasonal withvaried to generalists taxa micro-endemics of from range Germany. known from diversityDue tothe we families sites, sampling of wideencountered a dipteran most libraryfor areference to we constructed settings, lowland alpine from ranging ecosystems in Malaise traps of year-longbydeployment the collected specimens of pool expertise. the of from By lack excludeddueto selectinga taxonomic morphospecies been have German which would including otherwise of Diptera, families, several highly diverse critical that senior taxonomists thatsenior critical and professors recognizeneed to these possibilities and is it However, the species. bychase potentially describing toend anew and type material, or locations of type search in inpast toregale expeditions literature, probe the historical to Our study presents results from one of one from results Our studypresents In summary, the application of DNAof barc application the In summary, onal morphological methods, supporting the use the supporting methods, morphological onal ered represent “dark taxa” indicates “dark taxa”ered that indicates DNA represent , this method is ideal for educational purposes. purposes. educational ideal for is method , this rcoding and the resultant reference barcode rcodingthe and to molecular genetics, an approach accessibleapproach an genetics, molecular to oding enabled a comprehensive assessment comprehensive a enabled oding tes its world real its The use in tes situations. the most comprehensive DNA barcoding DNA barcoding comprehensive the most Accepted Article Kjaerandsen, J., Kleiner, M., Koehler, F., Koehler, J., Kölbl, N., Koenig, T., Kolbeck, H., Jaschof,M., Jon, T., Kamin,J.,Kappert, Kehlmaier, J., Kilian, C., Kirsch, D., H., Hilbig V., Hierlmeier, K.,R., Hessing, Heller, T., ,Haberberger, S.,J.,Hable, Haeselbarth, Hansen, E., L.O., Hartop, Hawlitschek, E., O., I., Ga S., Gabriel, Fuhrmann, S., Fütterer, J., J.(+), E. M., Fittkau, Fiedler, Fahldieck, J., Diller,A., E., Drozd,P.,Dunz, A., Duschl Miesbach,Dworschak, M., W., Eckert, I.,Esser, Brown, A., E. Burmeister G., Charabidze, D., Chimeno, C., Claussen, C., Dettinger-Klemm, Bo T., Belleuvre, G., Behounek, N., Blick, A., Adaschkiewitz,help of Baranov, R., Bährmann, P. P., Assum,Babiy, the W., V.,Beermann, not would have of species.The thismammoth-task been realization without possible dipteran support who enthusiasts nature and scientists, species Germany. in of thousands unrecorded contains thisfamily single that likely allow for an accuratenot projection for the size speciesof the total seems numbers, it quite et 2016; Borkent et al. (Hebert Cecidomyiidae und enormous to studies pointan Further others. and , Limoniidae, Empidoidea, the extentfor to a lesser and Sphaeroceridae, Cecidomyiidae, Ceratopogonidae, Chironomidae, Anthomyiidae, , the for speciesevident were unrecorded of proportions High thought. formerly higherthan much inGermany iscertainly species dipteran of result of our estimatednumber study important keys.the that Itis particularly a identification up-to-date of lack asbythe well as families these many of for experts of lack bythe slowed collecting efforts for practically every specie Isles, British the byPape (2009) for presented bemust regarded as fragmentary. In accordance with the species accumulation curve fauna Diptera of its knowledge Despite long this effort, half of European fauna. the almost comprising country anyEuropean highest for the species is recorded Diptera of number impediment. taxonomic studentsencourage toembrace their this approach as it offers such a clear solution to the This article by article rightsprotected This reserved. is All copyright. First of all, we want to express our extreme we tothe gratitude taxonomists, our want First of to express all, citizen extreme Acknowledgements: Germany has a tradition of more than 250 Germany research, 250 yearstradition a ofmore than has andthe ofentomological Flügel, H. J.,Forster, W., Forstner, P., Fünfstück, P., Fünfstück, Forstner, W., H.J.,Forster, Flügel, mmelmo, O., Gerecke, R., Glaw, F., Guggemoos, ed this campaign by collecting thousands of of thousands this by campaign collecting ed , D., , D., J.R., Höglund, lz, R., Brandt-Floren, C., C., S.,Brenzinger, Brandt-Floren, lz, R., additional species were revealed from revealed current were from species additional al. 2018; Jaschhof 2018). Whereas our data do2018). data Whereasour Jaschhof 2018; al. s-rich s-rich is “new”Recording species family. er-estimation of the species diversity in the diversity thespecies in of er-estimation Chloropidae, Phoridae, Sciaridae, and Sciaridae, Phoridae, Chloropidae, Höhne, F., Honold, D., D., F., Honold, Höhne, Accepted Article Nord ‘Bezirksregierungen’ Bavarica” Fauna “Barcoding project: [ Bavaria of BIN diversity. BIN diversity. to of the with enable the Munich, KRONAGermany)inspection file GmbH, assisting for (AIM Bozicevic Vedran Dr. to ourthanks to express like Wewould specimens. countless photographing legs and countless picking for (barcoding-zsm.de) projects barcoding thank platform.all the who We in also the ZSM-assisted students BOLD informatics the of development supported and NSERC Innovation and Research of Ministry Ontario the while Institute, Genomics Ontario the through Canada toGenome Canada of Government the byfunding from part, in supported, was sequencing work tools. The management BIN (BOLD; BOLDdatabase the developing for SujeevanRatnasingham to particularly and help and support great their for Canada) (Ontario, team tothe grateful Weare at the Genomics Biodiversity Centre in Guelph 01LI1501). for and 01LI1101 BMBFFKZ Life: 2012-2019, of Barcode (German Research and Education of FederalMinistry German the and BFB) Bavarica, Fauna Barcoding (2009-2018: Arts S.,T., Winqv Windmaisser, Wiedenbrug, Wagner,Warncke,R., K., Weber,D., Weiffenbach,Weigand, H., A.M., Weixler, K., Velte L., Ess, van H., Vallenduuk, , K., Dunk, d. v. M., Tschirnhaus, v. M., Troester, R., Treiber, E., Toussaint, C., Toussaint, A., Telfer, R., K., Tänzler, Tänzler, Stuke, Stur, E., J. F., H., Stoecklein, U., G., Stenhouse, Steigemann, A., Ssymank, M., Spies, J., G., Spelda, A., Sellmayer, Segerer, G., Sedlak, R., Schwemmer, C. S., Schubart, K., Schrott, Schoenitzer, M., A., Schäfer, (+), W. Schacht, C., Salomon, S., Rennwald, K., Richter, B., Riedel, G., Rohrmoser, S., Rozo, P., H.Rudzinksi, G., Ruf, T., Reiso, A., Reimann, N., Reiff, T., Rehm, F., Reckel, V., Pushkar, M., Prozeller, S., Prescher, J., E., Podhorna, Plassmann, M., L., Steven, A., Pötter, Olsen, K. Pavlova, M., Olberg, S., C., Neumann, S., Mueller-Kroehling, R., Mueller, Müller, J., Mortelmans, G., Wallner, W. Lucas, J., O. Loennve, Lindner, S., M., G. Kvifte, K., Kusdas, M., Kuhlmann, F., Kuehlhorn, M., Kuehbandner, W., Kraus, G., Kraus, This article by article rightsprotected This reserved. is All copyright. ’, Axel Schmidt (Koblenz, Germany)]. Germany)]. (Koblenz, Schmidt ’, Axel

The project was was project The funded by Bavarian the State Ministry grants from Science of and the Bayerisches Staatsministerium für Umwelt und Gesundheit, Munich, Germany, Germany, Munich, Gesundheit, und Umwelt für Staatsministerium Bayerisches

Field work permits issued work were bypermits Field state responsible the offices environmental ] and Rhineland-Palatinate [‘ Rhineland-Palatinate and ] ; confirmed bythe regional governments ist, K., Woodley, N., E., Zahn, A. E., K.,N., Woodley, ist, Scheingraber, M., Scheler, Schmieder, F., Schödl, Schödl, F., Schmieder, Scheler, M., Scheingraber, www.boldsystems.org) infrastructure and the infrastructure www.boldsystems.org) , Schubert, C., Schubert, W., Schwarz, K., Schwarz, W., Schubert, C., Schubert, , , Mair, K., X., K., Merkel- Mengual, Mandery, rop, J., Voith, J., Volf, M., Wachtel, F., F., M., Wachtel, Volf, J., Voith, J., rop, Struktur- und Genehmigungsdirektion Struktur- Accepted Article Contributed (additions/corrections) to the tothe (additions/corrections) MS: PDNH,Contributed AH,LH, MFG, GH. Wrote thepaper: JM, LAH, SS, MB,DD. Analyzed thedata: JM, LAH, MFG, LR, BR. Conceived anddesigned experiments: the JM, LAH, MG, BR. Collected the samples: DD, BR. GH, AH.,funding: Obtained WW, PDNH.

This article by article rightsprotected This reserved. is All copyright. References: References: contributions: Author Astrin, J.J., Höfer,H.,Spelda, J.,Holstein, J.,Bayer, Hendrich, S., L.,- & ... Ashfaq, M., Hebert, D.N.,P. Mirza, J. H., Khan, A. M., Zafar, Y., & Mirza, M. - - Carew, M. E., Pettigrove, V., Cox, R.L., &Hoffmann, A. A. (2007). DNA - F., new Riehl, Brix, Leese, T., S., and Anew genus & Kihara, T. C. (2015). K. Kapheim, K. R., D., Hopper, K.,A. Cox-Foster, Childers, G., M. Branstetter, - Borkent,A., Brown, B., Adler, P. H.,Amorim, D. D. S., Barber, K., Bickel,D., - - bio-surveillance: for barcodes DNA D. N. P. (2016). Hebert, & M., Ashfaq, - Theutility A. M. Carew, V., A. DNA & Hoffmann, (2005). E., Pettigrove, of harvestmen of Germany. barc a DNA Towards (2016). C. J. Monje, DNA barcoding. barcoding. DNA by Pakistan Analyzing (Diptera: in Culicidae) mosquito (2014). S. diversity Journalof theNorth American Benthological Society identification Tanytarsini of urban Chironomidae). (Diptera: chironomids the EntomologicalSociety ofAmerica abyssdescribed byof means integrative taxonomy. speciesof Desmosomatidae Sars, 1897 (Isopoda) from the easternAtlantic South opinion in insect science of theHymenoptera. Toth,& L.,K. C.M.,Genomes A. Worley, (2018). Costa forest.Rican cloud of apatch in diversityRemarkable (Diptera) (2018). S. R. &Capellari, ... 933–945. pests. plant economicallyimportantregulated and differentiate Australian markers inclassical taxonomy: using oxidasecytochrome Imarkers to 7–61. PloS ONE Cladopelma , PloS ONE PloS 25 Zootaxa , , 65–75. 65–75. , 9 (5), e97268. e97268. (5), , 4402 , (Diptera: midges. Chironomidae) 11 , 98 (9), e0162624 (24 pp, supplements). supplements). (24 pp, e0162624 (9), (1), 53–90. 53–90. (1), (4), 587–594. 587–594. (4), ode and spiders for reference database , Marine BiodiversityMarine 26 (4), 587–600. 587–600. (4), Genome Annals of , Current Current , 59 45 (11), (11), (1), (1), Accepted Article This article by article rightsprotected This reserved. is All copyright. - Miquel, Boyer, DeM., Barba, C., - Dathe, H. H., & Blank, S. M. Nachträge(2004). Verzeichniszum der &M. A. (2000). Andersen, Skuhravá, B., Darvas, - - Cywinska, A.,Hunter, F. F., & Hebert, D.P. (2006). Identifying Canadian - Cruaud, P.,Rasplus,J. Y., Rodriguez, L.&Cruaud, A. J., (2017). High- - Woodford, W.H., Wong, Lim,A.,R.B.H., Heyzer, Y.C., Ang, P.S., Cranston, - DeWaard, J. R., Levesque-Beaudin, V., deWaard, S.L., Ivanova,N.V., - De Carvalho, M. R., et al. (2007). "Taxonomic impediment or impediment to to impediment or impediment "Taxonomic (2007). al. et R., M. - Carvalho, De A. (2008). (Eds.). & Bernstein, E., Chivian, - Chimeno,- C., Morinière, J., Podhorna, J., Hardulak, L., Hausmann, A.,Reckel, - DeWaard, J. R., Ivanova, N. V., Hajibabaei, M., & Hebert, P. D. N. (2008). D. N. (2008). P. Hebert, - & M., Hajibabaei, V., N. Ivanova, R., J. DeWaard, Resources dietaccuracy applicationomnivorous for assessment: to diet. metabarcoding DNA (2014). P. Taberlet, undBerichte Nachrichten Entomologische Entomofauna Deutschlands, Hautflügler Science Herald, Budapest Dipterology and Applied General 1, Vol. Diptera. Palaearctic of ato Manual the Palaearcticregion 20 barcodes. mosquito DNA species through taxonomy. throughput sequencing ofmultiple amplicons barcodingfor and integrative 779–793. Bedok reservoirs. and Pandan Singapore's Meier,ThenuisanceJ.M. (2013). & midgesof Chironomidae) (Diptera: R. ondepends biodiversity arthropod diversity by coupling Malaise traps with DNA barcoding. barcoding. DNA arthropod diversity with by Malaise traps coupling of assessment Hebert, (2019). Expedited &Zakharov, E.V. terrestrial P.D.N. C.N., Sones, J.E., Steinke, C., Telfer, A.C.,Young, A.D.,Young, M.R., Pe S., Naik, R., Miskie, J.T.A., McKeown, taxonomy? A commentary on systematics and the cybertaxonomic-automation Aon systematics the and taxonomy? commentary cybertaxonomic-automation 4029.13869 species. Establishing potentiallyDNA libraryof relevant reference arthropodforensic a F.,Haszprunar, ...& G.DNA (2018).barcoding in forensic entomology – https://doi.org/10.1139/gen-2018-0093 https://doi.org/10.1139/gen-2018-0093 DNA barcodes. Assembling paradigm." (4), 413–424. Journal Sciences ofForensic , Scientific Reports 14 Evolutionary BiologyEvolutionary (2), 306–323. 306–323. (2), . p. 565–649 In: Papp, L. & Darvas, B. L. (eds). In: Darvas, & Papp, p. . 565–649 . Oxford University Press. 542 pp Environmental Genomics , 7 , 41948 (12 pp). pp). 41948 (12 , , 34 F., Mercier, C., Rioux, D., Coissac, E., & D., E., C., Rioux, Coissac, Mercier, F., , (3), 140–143. 140–143. (3), , Germanica Band 4 (Hymenoptera). (1). (1). Germanica 4 (Hymenoptera). Band , (9 pp). https://doi.org/10.1111/1556- rez, K.H.J., Ratnasingham, S., Sobel, S., Sobel, K.H.J., rez, Ratnasingham, 48 Medical and veterinaryMedical and entomology multiplexing validation and of data Raffles Bulletin of Zoology Sustaining life: how human how human health Sustaining life: (3-4),179–182. (3-4),179–182. Agricultural dipteran pests of of pests dipteran Agricultural , 410 , 275–294. 275–294. , Molecular Ecology Ecology Molecular Contribution Contribution Genome , 61 (2), (2), . , , Accepted Article This article by article rightsprotected This reserved. is All copyright. - Fontaine, B.,van Achterberg, K., Alonso-Zarazaga, M. A., Araujo, R., Asche, - DER, R. D. E. G. Richtlinie(1992). 92/43/EWG Rates vom des 21. Mai1992 - T. Erwin, L.Tropical fore (1982). - assessments ecosystem DNA-based CanF. Leese, (2015). & Elbrecht,V. Ekrem, T., Stur, E., & Hebert, P. D. N. -(2010). Females do count: Documenting - sequence DNA A Ekrem, Willassen, T., E., &Stur, E. (2007). comprehensive Heller, K., & Eiseman, C.A new leaf-mining dark-winged S., Rulik, B. (2016). - - Geiger, M.F., Moriniere, J., Hausmann, A., Haszprunar, G.,Wägele, W., - Fujita, M. K., Leache, A. D., Burbrink, F. T., McGuire, J. A., & Moritz, C. Douglas, Y.,W. Ji, Y., Emerson,B. C., Wang, X., Ye, C.,Yang, C., &- Ding,Z. - Vollständigkeit der und Proben der Vorsortierung Doczkal, D. (2017). M., … & Bouchet, P. (2012). New species in the Old World: Europe as a frontier afrontier as Europe Old World: New species the in (2012). Bouchet, &M.,P. … L, pp). (43 206, Reihe Gemeinschaften, Amtsblatt Pflanzen. der Europäischen zur der Erhaltung Lebensräumenatürlichen dersowie wildlebenden Tiere und arthropod species. in biodiversity exploration, a test bed for 21st Century taxonomy. taxonomy. Century 21st for bed test a exploration, biodiversity in e0130324 (16 pp). (16 pp). e0130324 protocol. metabarcoding with innovative an relationships quantify Testing species abundance? primer biomass—sequencebias and Diversity & Evolution Chironomidae (Diptera) species diversity usingDNA barcoding. Phylogenetics and Evolution library is essential for identification with DNA barcodes. Entomological Society of Washington marigold (Ranunculaceae: fungus gnat (Diptera: withSciaridae), notes onother insect associates of marsh 623. Germany? Germany? – How well does the currentbarcode referencelibrary identifyinsects flying in (2016a). B. & Rulik, D.N., P. Hebert, &Evolution inEcology Trends (2012). Coalescent-based species delimi 7 assessment and biomonitoring. biomonitoring. and assessment (2012). Biodiversity soup: metabarcodin der Folge des Klimawandels. Einfal im südwestlichen Bestandsaufnahme desRheint und Dinkelbergrandes Erfassung.Ssymank, In: A. & Doczkal,D. Biodiversität (eds). des südwestlichen (5), e36881 (7 pp). pp). (7 e36881 (5), Biodiversity Data Journal The Coleopterists Bulletin , 10 (5), 397–408. 397–408. (5), , Mauritiana (Altenburg) 34 (Altenburg) Mauritiana 43 palustris , 27(9), 480–488. 480–488. 27(9), , (2), 530–542. 530–542. (2), Methods in Ecology and Evolution inEcologyMethods and sts: their richness in Coleoptera and other other and Coleoptera in richness their sts: , 4, e10671 (22 pp plus supplements). supplements). plus pp (22 e10671 4, , , 118 Testing Globalthe Malaise Trap Program g of arthropods for rapid biodiversity biodiversity for rapid gof arthropods alsGrenzach-Whylen, bei eine (4), 519–533. 519–533. (4), tation in an integrative taxonomy. lstor Deutschlands Deutschlands lstor , 36 (1), 74–75. L.). L.). , 1–910. 1–910. , Proceedings of the PloS ONE für neue Arten in , PLoS ONE Organisms Organisms 3 Molecular (4), 613– , 10 (7), , Accepted Article This article by article rightsprotected This reserved. is All copyright. Hallmann, C.A., Sorg,M., Jongejans, E., Siepel,H.,Hofland, N., Schwan,H., ... - Assessing (2012) S Konynenburg &van S. Shokralla L., J. Spall M., Hajibabaei - - M., Hajibabaei Shokralla S., X., Zhou G.Singer A. & D.Baird J. (2011) Geiger, M. F.,Astrin, J. J., Borsch, T., Burkhardt,- U., Grobe, P., Hand, R., ... & - of species of European the Revision (2018). S. & Schmidt, C., Hansson, - congruent misleading: COI is mtDNA When G.& (2014). A. Ståhls, Haarto, D. (2015). H.& Hebert, L., N. P. E. Maw, G., R. Foottit, A., R. Gwiazdowski, - Gutiérrez, M. A. C., Vivero, R. J., Vélez, I. D., Porter, C. H., &Uribe, S. (2014). - Gruttke, H., Binot-Hafke,-M., Balzer, S., Haupt,Hofbauer, H., N.,Ludwig, G., Janzen, S., Konynenburg, van I., King, T.M., Porter, S., Shokralla, J., - Gibson, - die Fauna Bavarica–eine Chance für Barcoding G. (2009). Haszprunar, - Hardaluk, L. prep.) (in Wald - Metabarcoding theNationalparkin Bayerischer - Ecology preservative ethanol. from ofDNA barcoding destructive environmental biodiversity ofa freshwat insect biomass in protected areas. areas. protected in biomass insect yearsMore than75 percent over 27D. in Goulson,decline& flying (2017). total biomonitoring applications using river benthos. benthos. river using applications biomonitoring anext-g Environmentalbarcoding: Life initiative GBOL (2012–2015). industrializedof of theGerman Barcode phase from nation—lessons the first an tackle inventory for to species molecular the How (2016b). Monje, C. species of Euplectrini. a key with European to Eulophidae), (Hymenoptera, Westwood Euplectrus 58 Entomologie. samples. bulk in invertebrates pest and invasive for screening signal ofmolecular ITS2 marker North morphology European for and barcodes. The Hemiptera (Insecta)of Canada: constructing areference libraryof DNA Phlebotominae) inColombia. DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Vielfalt Deutschlands. Band 4: Wirbellose Tiere (Teil 2). &Ries, M. Liste (2016). Rote gefährdeter Pflanzen Tiere, undPilze United States of America metasystematics. DNA macrobiome andmicrobiome in abulkof sample tropical arthropods through D.H.,Hallwachs, W. & Hajibabaei, M. (2014). Simultaneous assesssment of the (1/2), 45. 45. (1/2), , , 70 12 PLoS ONE (4). (28), 10 pp. Schiener, 1860 (Diptera, Syrphidae). Nachrichtenblatt der bayerischen Entomologen bayer Ent Entomologen bayerischen der Nachrichtenblatt , 10(4), e0125635 (20 pp). pp). (20 e0125635 10(4), , Journal of Hymenoptera Research Hymenoptera of Journal Proceedings of the National Academy of Sciences of the ofof Nationalof Sciences Academy the Proceedings , er benthic macroinvertebrate community through non- through benthic macroinvertebrate communityer 111 , 8007–8012. PloS one PloS ONE Genome , 9 eneration sequencing approach for for approach eneration sequencing (1), e85496. e85496. (1), , , 59 12 (9), 661–670. 661–670. (9), PLoS ONEPLoS (10), e0185809 (21 pp). pp). e0185809 (21 (10), ZooKeys Naturschutz und Biologische und Naturschutz Biologische , 67, 1. , 6 , (4), e17497 (7 pp). (7pp). e17497 (4), 431 , 93–134. 93–134. , BMC BMC , Accepted Article This article by article rightsprotected This reserved. is All copyright. - Heller,K., & Rulik, B. (2016). - Hebert, P.D.N., Ratnasingham, S., Zakharov, E.V., Telfer, A.C.,Levesque- D. A., Ball, Hebert, N.,P. Cywinska, - - Hawlitschek, O., Fernández-González, A., Balmori-de la Puente, A.,& M., Kropf, A. W., Lehmann, C., U. G. Lehmann, J., Morinière, O., Hawlitschek, - - N., Gossner, Havemann, M. M., Hendrich, L., Morinière, Niedringhaus,J., R., Hausmann,- A., Godfray,H. C. J., Huemer, P., Mutanen, M., Rougerie, R., van - Hausmann, A., Haszprunar, G., Segerer, A. H., Speidel, W., Behounek, G., & - Hendrich, L., Morinière, J., Haszprunar, G., Hebert, P. D.N., Hausmann, A., Heller, K.,Köhler, A.,Menzel,- F., Olsen, K.M., Gammelo,& Ø. (2016).Two - Hausmann, A., Haszprunar, G., & Hebe & G., A., Haszprunar, Hausmann, - Society B: Biological Sciences Biological B: Society insects. Canadian barcodes: DNA with species Counting(2016). animal &A., Jannetta, M. ... V., P. Milton, Beaudin, barcodes. DNA identifications through (19 pp). e0201763 mammal. semi-aquatic small a for developed samples meta pipeline for A (2018). J. Castresana, and . Germany onAustria, with focus Europe Central from (Orthoptera) grasshoppers Dunz, A., ... & Szucsich,N. U. (2017). DNA barcodingof crickets,katydids and barcodes. onDNA based Germany molecularof diversity aquatic Heteropteraof Nepomorpha) (Gerromorpha, &water Raupach,J. (2018).From P., toSchäfer, water M. the striders bugs: ONE geometrid moths revealed by (BIN)system.the Barcode IndexNumber Nieukerken, E. ...J., Hebert, D.P. N. (2013).Genetic patterns in European Germany. of moths larger Thebutterflies and NowDNA-barcoded: D.N.(2011b). Hebert, P. PLoS ONE geometridfaunaofBavaria (Lepidoptera):Successes, surprises, andquestions. identified species toBOLD. onGermany:beetles3500 Central with afocus more European than adding for barcode database DNA A comprehensive M. (2015). & Balke, F., Köhler, barcoding. formerlyspeciesofunrecognized Sciaridae revealed (Diptera) byDNA pp). (XX Sciaridae), an exotic invader inGermany? , 8 (12), e84518 (11 pp). pp). e84518 (11 (12), . Series B: Biological Sciences, 270(1512), 313-321. 313-321. SeriesB:Biological 270(1512), . Sciences, , 6(2), e17134 (9 pp). (9pp). e17134 , 6(2), Norwegian Journal of Entomology Journal Norwegian Spixiana Molecular Ecology Resources Ecology Molecular , 34 (1), 47–58. 47–58. (1), Molecular Ecology Resources Ecology Molecular , 371 Ctenosciara al Ctenosciara (1702), 20150333 (XX pp). pp). (XX 20150333 (1702), PeerJ S. L., S. (2003). R. J. Biological & Dewaard, Philosophical Transactions of the Royal rt, P. D. N. (2011a). DNA barcoding the the barcoding DNA D. N. (2011a). P. rt, , Proceedings Proceedings of the Royal Society of barcoding and diet analysis from fecal fecal from analysis diet and barcoding 6 , e4577 (30 pp). pp). (30 e4577 , Biodiversity Data Data Biodiversity Journal , 63 , 17 exanderkoenigi (1), 96–115. (5), 1037–1053. 1037–1053. (5), , 15 PloS ONE (4), 795–818. 795–818. (4), sp.n. (Diptera: , 4 , , e6460 13 PLoS PLoS (8), (8), Accepted Article This article by article rightsprotected This reserved. is All copyright. Jordaens, K., Goergen, G.,Virgilio, M., Backeljau, T.,Vokaer, A., &De Meyer, - Jordaens, K., Sonet,- G., Braet, Y., De Meyer, M., Backeljau, T., Goovaerts, F., Ji, Y.,Ashton, L., Pedley, M.,S. Edwards, D. P., Tang,Y., - Nakamura, A., & ... Deutschlands, Holzmücken der Artenliste aktualisierte Eine M. (2009). Jaschhof, - - & He R., J. Dewaard, V., N. Ivanova, - delineation species barcoding,and DNA N., Hanner, Hubert, R.& (2015). Krüger, A., Strüven, L., Post, R. J., & Faulde, M. (2011). The- sandflies (Diptera: Klausnitzer B Stiefkinder der(2006) Entomologie inMitteleuropa. - - F. (2005). Liljeblad, &A.,K. Ronquist, D., T., J., Johansson, Pape, Karlsson, - Kumar,N. P.,Srinivasan, R., &Jambulingam,(2012). P. barcoding DNA for - Kumar,N. P.,Rajavel,A. R., Natarajan, R., & Jambulingam, P.(2007).DNA - of barcodingmetabarcoding DNA and Knowlton N.Leray M., (2015). Latibari, M.H., Moravvej,- G., Heller,K., Rulik, B., Namaghi,& H. S. (2015). hoverflies (Insecta: Diptera: Syrphidae). of Afrotropical the tothe improve taxonomy DNAbarcoding M. (2015). Chrysomyinae). AmericanWest and European North between differentiation the and barcoding DNA (2013). S. Desmyter, &... via metabarcoding. Larsen, H.T. Reliable, (2013). verifiable and efficient monitoring ofbiodiversity Lestremiinae). Fa der unterbesonderer Berücksichtigung Ecology Notes protocol forautomation-friendly recoveringDNA. high-quality taxonomy: a historical DNAperspective. barcodes, Medicine & Parasitology 2009), as identified by morphology and DNA ‘barcoding’. Psychodidae, Phlebotominae) inmilitary in northern Afghanistancamps (2007– Entomologie Diptera are there in ?. The Swedish Malaise Trap Project, or how many species of Hymenoptera and Resources identification fliesofsand (Diptera: Psychodidae) inIndia. Journalof Medical Entomology barcodes can distinguish species of(Diptera: Indianmosquitoes Culicidae). the National Academy ofSciences pattern reveal samples standardized reinstatementof BradysiaFrey. cellarum New recordsof BlackGnats Fungus (Diptera: Sciaridae)Iran, from the including , 12 , 56 (3), 414–420. 414–420. (3), , Spixiana 6 , 360–368. 360–368. , ZooKeys (4), 998–1002. Ecology Letters Ecology , 32 , , 105 365 (1), 139–151. 139–151. (1), Entomologisk Tidsskrift Entomologisk , 149–174. 149–174. , (2), 163–176. 163–176. (2), , 44

USA , (1), 01–07. 01–07. (1), s of marine benthic diversity. diversity. benthic marine of s Phormia regina Phormia 16 , (10), 1245–1257. 1245–1257. (10), 112 bert, P. D. N. (2006). An inexpensive, An (2006). D. inexpensive, N. bert, P. PloS ONE Studia dipterologica Studia una Bayerns (Diptera, Cecidomyiidae, (Diptera, Bayerns una (7), 2076–2081. 2076–2081. (7), , 3 10 (1), 44-58. 44-58. (1), (Diptera, Calliphoridae, , (10), e0140264. e0140264. (10), 126 , 43–53. 43–53. , Molecular Ecology Ecology Molecular Annals of Tropical Annals ofTropical , 22 (1), 39–44. 39–44. (1), Proceedings of of Proceedings Beiträge zur Molecular Molecular Accepted Article This article by article rightsprotected This reserved. is All copyright. - May, R. M. (1988). Howspecies many May, M. R. onare (1988). there earth? - - Matthews, Matthews,R.The Malaise trap: W.,& its J.R. (1971). utilityand Mills, Ranwez, N., … & V., C.P., Agudelo, Y.J., Leray M., S.C., Meyer, Yang, - - & G., Haszprunar, A., Hebert, P., Hausmann, L., Hendrich, J., Morinière, - Montagna, M., Mereghetti, V., Lencioni, V., & Rossaro, B. (2016). Integrated - (20 A. Stark,H. & Meyer, Vuji G., Ståhls, X., Mengual, - - Meier, R., Shiyang,K., Vaidya,G., &Ng, P.K. (2006). DNAbarcoding and - high-throughput sequences from removes Cutadapt M adapter Martin, (2011). Lister, B. C., Garcia,A. (2018). Climate-driven declines arthropod in abundance - Morinière, J., Hendrich, L., Balke,M., Beermann,A. König,J., T.,- Hess,M., ... Morinière, J., de Araujo,B. C., Lam, A. Hausmann,W., A., Balke, M., Schmidt, - Gruppe”, Oreogetonidae). Oreogetonidae). Gruppe”, Brachystomatidae, Dolichopodidae l.,s. Empididae, Hybotidae, “Iteaphila- Atelestidae, Empidoidea: (Diptera: Deutschlands Tanzfliegenverwandten 4. potentialThe samplingpopulations. for Lakes insect Great Entomologist,4(4), https://doi.org/10.14806/ej.17.1.200 for characterizing coral reef fish gut contents. the mitochondrial for COI metabarcoding metazoanregion diversity: application of fragment short a targeting set primer versatile new A (2013). R.J. Machida, Molecular EcologyResources Molecular stoneflies caddisfliesand (Ephemeroptera, Plecoptera and Trichoptera). Germany DNA for library barcode A A. (2017). Hausmann, & 11 matrix. ascoring and technologies NGS on based DNA barcoding S., ... & Haszprunar,G. (2016). Species id barcoded! Bava Fauna Barcoding A. (2014). Gruppe, ONE of Chironomidae). (Diptera: barcoding DNA midges alpine taxonomy and 26. taxonomy of Iberian identification success. Diptera:taxonomyin atale ofhigh intraspecific variability and low 1441-–1449. sequencing reads. Sciences restructure a rainforestweb. food pp. (5), e0155497 (14 pp). (14 pp). e0155497 (5), , 11 , (3), e0149673. e0149673. (3), 115 PLoS ONEPLoS (44), 201722477 (10 pp). , Merodon Systematic biology Systematic 9 (10), e109719 (8 pp plus supplements). (8ppplussupplements). e109719 (10), Studia Dipterologica Studia EMBnet.journal ć , species (Diptera, Syrphidae). , A., & Marcos-Garcia, M. A. (2006). Integrative Integrative (2006). A. M. Marcos-Garcia, & A., , 17 (6), 1293–1307. 15). Verzeichnis und Bibliografie der der Bibliografie und Verzeichnis 15). Proceedings of Proceedings National the of Academy , entification in Malaise trap samplesMalaiseby entification trap in 55 , rica: 78% of the Neuropterida fauna fauna of Neuropterida the 78%rica: (5), 715–728. 715–728. (5),

Frontiers inZoology Frontiers Supplement 19 Supplement 17 (1), 10 pp. DOI: Science Zootaxa , 376 , pp. , , ′ s mayflies, 241 10( , PloS one PloS 1377 34), 14 (4872), (4872), PLoS PLoS , 1– , Accepted Article This article by article rightsprotected This reserved. is All copyright. - Mutanen, M., Kivelä, S. M., Vos, R. A., Doorenweerd,C., Ratnasingham, S., - Nelson, L. A., Lambkin, C. L., Batterham, P.,Wallman, J. F., Dowton,M., - to barcodes Using COI (2007). M. F., Wallman, L.J. & Dowton, A., Nelson, - Nagy,Z. T., Sonet, G.,Mortelmans, Vandewynkel,J., C., & Grootaert, P. Ødegaard,F. (2000). How many speciesof arthropods? - Erwin'sestimate revised. F., M. Lañas-Rosas, J., M. Arrunátegui-Jiménez, G., A. Cáceres, O., C. Nzelu, - Thegenetic in systems B. ofalternative Normark, evolution B. insects. (2003). - Pante, E., Schoelinck, C., & Puillandre,N. (2014). From integrative to taxonomy - taxadark dark and barcoding and texts. DNAtaxonomy: M. D. Page,(2016). R. - Padial, J. M., Miralles,A., De la Riva, I.,& Vences, M. (2010).The integrative- - Packer,L., Gibbs, J.,Sheffield, C.,&Hanner, R. (2009). DNAbarcoding and (2006). Oosterbroek,Families TheEuropean Diptera.KNNV-of P. 207pp. the - - Oliverio, A. M., Gan, H., Wickings, K., & Fierer, N. (2018). A DNA - Pape, T. (2009). Palaearctic T. Pape, Palaearctic (2009). Diptera -from In:p. tundradesert. to 121-154 Pape, - Biology operationalbiasLepidoptera. strong barcode trees: gene in European Hausmann,A., ...& Vila, R. (2016). Species-levelpara-and polyphylyin DNA (Diptera: Calliphoridae). phyloge to molecular genomics approach Whiting, M. F., ... & Cameron, S. L. (2012). Beyond barcoding: A mitochondrial Entomology identify forensicallymedically and blowflies. important (Diptera, Empidoidea). Hybotidae family the in diversity for assessing DNA barcodes Using (2013). BiologicalSociety of Journal Linnean the leishmaniasis-endemic areas Peru. of Psychodidae) from (Diptera: species flyof sand identificationbarcoding for Yañez-Trujillano,H. H., Luna-Caipo, V., D. ... & Kato, H. (2015). DNA entomology of review Annual species description:one beyond.step 371 PhilosophicalBiologicalSciencesTransactionsof RoyalSociety B: the offuture taxonomy. 42-50. morphology. the of mediocrity voorVeldbiologie, Uitgeverij. Vereniging Biology and Biochemistry characterizemetabarcoding soil communities.arthropod approach to Tools. Brill T., Meier,Bickel,D. & (eds).R. Diptera Diversity: Status,Challenges and (1702), 20150334 (7 pp). (7pp). 20150334 (1702), , 65 (6), 1024–1040. 1024–1040. (6), , 21 (1), 44–52. 44–52. (1), Frontiers inZoology ZooKeys Gene , 125 , 48 , , 37–43. 37–43. , Molecular Ecology Molecular Resources 511 , (1), 397–423. 397–423. (1), 365 (2), 131–142. 131–142. (2), , 263–278. 263–278. , Acta tropica Acta Systematic Biology , 7(16), 14 pp. pp. 14 , 7(16), , 71 netics and diagnostics of blowflies of blowflies diagnostics neticsand (4), 583–597. 583–597. (4), , 145 , 45–51. 45–51. , , Medical andVeterinary 64 (1), 152–160. 152–160. (1), , 9 (Supplement 1) 9 (Supplement Systematic Systematic Soil , , Accepted Article This article by article rightsprotected This reserved. is All copyright. Pfenninger, M.,Nowak, C., Kley, C., Steinke,D., & Streit,B. (2007). Utility of - of PalaearcticManual a Contribution to (2000b). (eds). Darvas, B. & L. Papp, - of- Manual Darvas, Palaearctic to Papp, a (2000a).L. Contribution & (eds). B. - L. Papp,ManualDarvas,& of Palaearctic to a (1998).Contribution B.(eds). - Papp,L. Darvas, & (1997). B. (eds) Contribution toManual a ofPalaearctic (2011). M.B. Mostovski, & V., Blagoderov, T., Pape, - - Ratnasingham, S., S., & D. Hebert, Ratnasingham, P. The N. ofLife BOLD: (2007). Barcode Data - statistical computing. forA R: environment and language (2018). RCore Team - - Puillandre, N., Lambert, A., Br Potts, S. G., Biesmeijer, J. C., Kremen,C., Neumann, Schweiger,P., O.,& - the for barcodes DNA M. &(2016). Mutanen, J., Kahanpää, L., J. Pohjoismäki, - - Raupach, M. J., Hendrich, L., Küchler,M., S.Deister, F., Morinière, J., & - all for registry DNA-based A (2013). N. D. P. Hebert, & S., Ratnasingham, morphologically cryptic for the barcoding and taxonomy inferenc DNA Herald, Budapest 604 p. Science Appendix. Diptera. Budapest Applied General Vol. 1, Diptera.and p.Budapest ScienceHerald, 880 Vol. . Diptera. Higher 3, Budapest Diptera.Vol. 2, andLower Brachycera. p.592 Science Herald, classificationand survey of taxonomicrichness 1758 and surprises. surprises. and uncertainties ofGermany taxonomic reveals Heteroptera) (Insecta:Hemiptera: of barcode library a DNA bugs M. M. for true Gossner, Building-up (2014). pp). (16 e66213 system. (BIN) Number animal Index species:The Barcode 364. System (http://www.barcodinglife.org). project.org/ . R Foundation for Statistical Vienna, Computing, Austria. URL http://www.R- Ecology AutomaticGap Barcode Discovery primarydelimitation.for species &Evolution inEcology Trends pollinator Global W.Kunin, E. declines:(2010). impacts anddrivers. trends, e0164933. northern European tachinid flies (Diptera: Tachinidae). 16 (9), 1957–1968. 1957–1968. (9), . In: Zhang, Z.-Q. (Ed.) , 21 (8), 1864–1877. 1864–1877. (8), PLoS ONE PLoS , Chironomus Chironomus 9 (9), e106940 pp). (9), (13 , Animal biodiversity: An outline of higher-level higher-level biodiversity:AnimalAn outline of 25 (6), 345–353. 345–353. (6), ouillet, S., &Achaz, G. ABGD,(2012). (Diptera) species. Molecular Ecology Molecular Ecology Notes Dipterology. 978 p. Science Herald, Herald, Science p. 978 Dipterology. e of larval community structure in . Magnolia Press, 222–229. 222–229. Press, Magnolia . Order Diptera Linnaeus, Diptera Linnaeus, Order Molecular Ecology Molecular PloS ONE PLoS ONE , 7(3) Molecular , 11 , 355– , 8(7), , 8(7), (11), (11), , Accepted Article This article by article rightsprotected This reserved. is All copyright. - Schlick-Steiner, B. C., B. Schlick-Steiner, - - Reibe, S., Schmitz,Madea, J., (2009). & Molecular identificationB. of - ADNA L. (2018). Hendrich, & J., Morinière, K., M. Hannig, J., Raupach, - ADNA L. (2016). Hendrich, & J., Moriniere, K., M. Hannig, J., Raupach, - Schlick-Steiner, B. C., Arthofer, Arthofer, C., B. Schlick-Steiner, - - Reimann, B., Rulik, B. (2015). (2015). B. Rulik, B., Reimann, - - Riedel, A., Sagata, K., Suhardjono,R., Y. Tänzler, R., & Balke,M. (2013). - of barcoding DNA (2012). J. S. Adamowicz, & J., K., A. Savage, Renaud, Rognes,T., Flouri, T., Nichols, -B., Quince, C.,Mahé, F. (2016).VSEARCH: a - Rivera, J.,& Currie, D.Identification C. (2009). Nearcticof black fliesusing - Rulik, B., Eberle, J., von der Mark, L., Thormann, J., Jung, M., Köhler, F., ... & F., ... M., Köhler, Jung, J., Thormann, L., Mark, der von B., Eberle, J., Rulik, - Testing C., M. Á. &Marcos-García, G., Pérez-Bañón, (2006). S., Rojo,Ståhls, - - Santos, D., D., S., Santos, & Santos, Sampronha, - integrative taxonomy. in conflict resolving from research evolution for challenge!Opportunities biodiversity. exploring to approach A multisource taxonomy: Integrative (2010). H. R. Crozier, 433–444. Parasitology research forensicallyblowfly important species (Diptera: Calliphoridae) from Germany. (Insecta, Carabidae). Coleoptera, genus of the Germany: beetles ground for librarybarcode genus The barcode libraryground for beetles (Insecta, Coleoptera, Carabidae)of Germany: dipterologica lance Germanfly new tothe fauna, byrevealed the GBOL-project. biodiversity research. Integrative taxonomy onthe sustainability fast track-towards more in morphological andmolecularspecies limits. between high correspondence reveals (Diptera) Nearctic Muscidae Northern versatile open source tool for metagenomics. versatilemetagenomics. opensource for tool 236. DNA barcodes (Diptera: Simuliidae). preprocessing for high quality DNA barcodes. qualitypreprocessing barcodes. for high DNA c taxonomic Using F. (2017). Fritzlar, Syrphidae: Paragini). adult morphologyof West Palaearctic molecular barcodes: Invariant mitochondrial DNA sequences vs the larval and Evolution in the21 , st century and extinction rates. Bembidion 8 (12), 1878–1887. 1878–1887. (12), , 21 (2), 283–287. 283–287. (2), Latreille, 1802 allied and taxa. European Journal of Entomology of Journal European Frontiers inZoology Frontiers , Molecular Ecology Molecular Annual Review ofAnnual Entomology 106 Steiner,M., F. Seifert, B.,Stauffer, Christian, C.,& E., (1), 257–261. 257–261. (1), Dasiops calvus Dasiops ZooKeys W., & Steiner, W., &F. Steiner, M. (2014). Take up the C. M. D. (2017). Advances on dipterology dipterology on Advances (2017). D. M. C. Molecular Ecology Resources Molecular Ecology , , onsistency with semiautomated data data semiautomated with onsistency Papéis deZoologia Avulsos , 23 Pandasyopthalmus 759 10 BMC ecologyBMC PeerJ (17), 4192–4194. 4192–4194. (17), (1), 15 (9 pp). pp). 15 (9 (1), , 57–80. 57–80. , Morge (Diptera:Lonchaeidae), a , 4 ZooKeys Methods in in Methods and Ecology , , e2584 (22 pp). pp). (22 e2584 , 55 , 103 , 421–438. 421–438. , , 12 Amara (2), 443. 443. (2), species (Diptera: (Diptera: species (1), 24. 24. (1), , 592 Bonelli, 1810 Bonelli, 1810 , 121–141. 121–141. , , , 9 57 , 224– Studia Studia (33), (33), Accepted Article This article by article rightsprotected This reserved. is All copyright. Schmidt, S., Taeger, A., Morinière, J., Liston, A., - Blank, S. M., Kramp, K., ... & Schmidt, S.,- Schmid-Egger,C.,Morinière, J.,Haszprunar,G., & Hebert, D. P. Šev - - Schumann,H., Bährmann, R., & Stark,A. (1999). Checkliste derDipteren Schmid-Egger,- C., Straka, J., Ljubomirov, T., Blagoev, G. A., Morinière, J., & - Sorg, M., Schwan, H., MüllerA. & W., der Stenmans, Sorg, M., Ermittlung (2013). Schwan, - - C. of Sinclair, Discrimination & S., E. S. (2008). Gresens, - Next-generation M. Gibson,Hajibabaei, & (2012). Spall, Shokralla, J. J., S., - Nachtrag DritterSchumann,H.Dipteren (2010). zur der „Checkliste - Schumann,H. (2004). Zweiter Nachtrag zur „Checkliste derDipteren - H.NachtragSchumann, Erster (2002). derDipteren zur„Checkliste - Serrana, J. M., Miyake, Y., Gamboa, M., & Watanabe, K. (2018). Comparison of Comparison K. &M.,Gamboa,(2018). Watanabe, Y., Miyake, J. M., Serrana, - -Zweiflügler. Diptera 832- p. & J. (2011). Ziegler, H., D. Doczkal, Schumann, - doi: Resources through successes‘Symphyta’) DNAbarcodes: caveats. and of horntails (Hymenoptera, Identification andsawflies Nyman, T. (2017). EcologyResources Molecular identifications for EuropeanCentral years 250 classical of taxonomy: supports largely barcoding DNA N. (2015). Deutschlands. Deutschlands. Palearctic. Crabronidae, Ampulicidae, from Sphecidae) the (Hymenoptera: Western waspof apoid species 99 percent identify barcodes DNA (2019). S. Schmidt, from Central Europe, with DNA barcoding DNA barcoding with Europe, Central from Biomassen flugaktiver Insekten im Naturschutzgebiet Orbroicher Bruch mit mit Bruch Orbroicher Naturschutzgebiet Insekten im flugaktiver Biomassen 98 barcoding. DNA by Chironomidae) (Diptera: 21 sequencing technologies forenvironmental DNA research. Mycetophilidae). Deutschlands“. Deutschlands“. “. Deutschlands “. Deutschlands 1-–354. macroinvertebrate biodiversity stream for morphological identification and metabarcoding DNA 2 932 In: Klausnitzer, B. (ed). , (6), 555–563. 1794–1805. (8), Wirbellose: Insekten č https://doi.org/10.1101/436162 ík, J., Kaspík, J., , Molecular Ecology Resources Molecular Ecology 17 (4), 670–685. 670–685. (4), Entomofauna Germanica 2 Entomofauna Studia ř Studia dipterologica Studia dipterologica ák, D., & Rulik, B. (2016). A new species of ZooKeys

Dipterologica . 11.Auflage. AkademischerSpektrum Verlag. , 549 Stresemann, Exkursionsfauna von Deutschland. Vol. Vol. Deutschland. von Exkursionsfauna Stresemann, , 15 assessment and monitoring. andmonitoring. assessment , 127–143. 127–143. , (4), 985–1000. 985–1000. (4), , , , 16 11 9 (2), 437–445. 437–445. (2), bees (Hymenoptera, Apoidea partim). Apoidea partim). bees (Hymenoptera, (1/2), 17–27. (2), 619–630. 619–630. (2), , doi: 10.1111/1755-0998.12963. 10.1111/1755-0998.12963. doi: , . based on four gene markers gene markers (Diptera, onfour based Studia dipterologica Supplement dipterologica Studia Bulletin of entomological research ofentomological Bulletin Molecular Ecology Molecular Molecular Ecology Cricotopus Docosia bioRxiv , 436162. Winnertz species , 2, , 2, , ,

Accepted Article This article by article rightsprotected This reserved. is All copyright. - Stur, E., & Borkent, A. (2014). When DNA barcoding and morphology mesh: mesh: morphology and barcoding DNA When (2014). A. Borkent, &- E., Stur, - E., &Stur, T. stages ofArctic Ekrem, life (2011). Exploring unknown Ssymank,A., Sorg, M., Doczkal, D., Rulik, B., Merkel-Wallner,- G., &Vischer- und Liste Rote F. (2011). & Dziock, K. Rennwald, D., Doczkal, A., - Ssymank, - Bi Oliveira Reip, H.S., J., Spelda, - Völkl, W., Blick, T., Kornacker, P. M., & Martens, H. Quantitativer(2004). Versteirt,V., Nagy,Z. T., Roelants, P.,Denis,L., Breman, F. C., Damiens,D., ... - Insect & to Initiative. Threats Pollinators A. ecosystem an Vanbergen, J., (2013). - Taberlet, Coissac,P., E.,F., Pompanon, Brochmann, C.,&Willerslev, E. (2012). - Wheeler,Q.D., Raven, P.H., or& Taxonomy:Impediment E.O. Wilson, (2004). - N.Lindner, & J., Spelda, P., Oeyen, J. Decker, P., K., Voigtländer, T., Wesener, - Wang,G., Li, C., Guo, X., Xing, D.,Dong, Y., Wang, Z., ... & Zhu, X.- (2012). Verein Krefeld 2013. und 1989 den Jahren in Fallen Malaise Ceratopogonidaediversity in Finnmark,. 27–39. barcoding. Tanytarsini withDNA (Diptera:Chironomidae) Series Naturalis Vol. 1 der InsektenMalaisefallen in Biod für von Anwendung und Hinweise M.zur Empfehlungen Leopold, Praktische Vielfalt Biologische und Naturschutz Gesamtartenliste der Schwebfliegen 115 identifications of andcentipedes millipedes (Chilopoda, Diplopoda). DNA sequence-based to –Fauna Myriapoda acontribution Bavarica: 293–295. 293–295. Überblick über die rezente Fauna von Deutschland. Culicidae) by DNA barcoding. Identification (2015). Bortel, W. Van& 11 service: pressures on pollinators. Ecology Molecular metabarcoding. DNA using assessment biodiversity next-generation Towards expedient? expedient? Geophilomorpha). (Chilopoda, Germanproject: Cryptic lineages in of of Barcode German Life First (2015). (GBOL)– Myriapoda results the ONE, PLoS barcoding. DNA on inChinabased mosquito species themainIdentifying (5), 251–259. , 123–139. 123–139. , 7 (10), e47051. e47051. (10), Science , 1 , 1–5. , 1–5. 303: 285–285. 303: 285–285. , 21 (2018), pp. 1-12. 1-12. pp. (2018), (8), 2045–2050. 2045–2050. (8), Molecular Ecology Resources Molecular Ecology ZooKeys Frontiers in Ecologyand the Environment , ener, U., Melzer, R.R. (2011). Barcoding Barcoding (2011). R.R. Melzer, U., ener, 70 , (3), 13–83. iversitätserfassung und im Monitoring. undimMonitoring. iversitätserfassung 510 (Diptera: Syrphidae) Deutschlands. Syrphidae) (Diptera: Deutschlands. of Belgian mosquito species (Diptera: Stenotaenia linearis Stenotaenia , 15–29. 15–29. , ZooKeys Mitteilungen Entomologischer Natur und Landschaft und Natur , 463 , Zootaxa 15 , 95–131. 95–131. , (Koch, 1835) (2), 449–457. 449–457. (2), , ZooKeys 2743 , 79 (1), (7), (7), , , Accepted Article

This article by article rightsprotected This reserved. is All copyright. - Yu D. W., Ji Y., Emerson B. C., Wang X., Ye C., Yang C., Ding Z. (2012) F.Gebel, Die D.,Raubfliegen Wolff, M.Geller-Grimm, & (2018). - - Whitworth, T. L., Dawson, R. D., Magalon, H., & Baudry, E. (2007). DNA 623. 623. assessment and biomonitoring. biomonitoring. and assessment rapid of for biodiversity metabarcoding soup: Biodiversity arthropods Quelle 339 pp. Deutschlands. Sciences (Diptera: Calliphoridae). genus blowfly the of species identify reliably cannot barcoding , 274 (1619), 1731–1739. Proceedings of the Royal Society B: Biological & Meyer Bestimmungsbücher. Bestimmungsbücher. &Meyer Methods in Methods

Ecology andEvolution Protocalliphora Protocalliphora , 3 (4), 613– Accepted Article Figure 2 Figure 1 Legends This article by article rightsprotected This reserved. is All copyright. (BS). (BS). Table 3 (CDC). candidates diversity cryptic and/or Table 2 is included. each specimensfamily the in of bodysize the average on information Additional database. the reference unnamed species within named and of numbers Table 1 reported numbera family.of species for in and -mm) (in size Figure 3 Muscidae andSyrphidae. Chironomidae, illustra SAL) and & T1-63B sites(Jos, sample

Krona graph within the supplement. the within graph Krona the observation BIN diversity of canbe for each studied within the detailled family A more calcula tree NJ (BIN). Number Index Barcode barcode within the librar Diptera Nemtatocera : All cases of low intraspecific sequence variation at COI - cases of BIN sharing : :

: : : Illustration of between the of relationship percentage “ : Illustration All cases of high intraspecific sequence va sequence highintraspecific of cases All on BIN of reportedinGermany. the on Families and Diptera Information the count, Illstrative c Examples from the metabarcoding results. metabarcoding the from Examples ircular neighbor-joining (NJ) trees for (NJ) for neighbor-joining trees ircular

tions were performed on the tions wereperformed BOLD database. y - eachline in the trees toonecorresponds tiveexamples families Cecidomyiidae, the for riation at COI - cases of multiple BINs multiple of cases - COI at riation Presence-absence overviews for three A) all Brachycera and dark taxa ” and average body” and B) all all Accepted Article https://github.com/GenomicaMicrob/OTUsamples2krona) study. graph (Krona was constr the within families studied of diversity and BIN distribution of observation detailled Enables 2- graph: Krona to Figure file Supplemental S5: Appendix S4: Appendix information. (BS) (CDC), BINsharing Candidates Diversity Cryptic BINs, with S3: Appendix S2: Appendix data information. taxonomy, complete andcollection lengths, sampleIDs, processIDs, GenBank num accession S1: Appendix material Supplementary This article by article rightsprotected This reserved. is All copyright. Results of the metabarcoding analysis. metabarcoding analysis. the of Results metadata collected of all Compendium DNA Metabarcoding laboratory workflow. laboratory workflow. DNA Metabarcoding sizes. family bodyand on performed tests Statistical Information on eachspecies, including a thorough Barcode Gap Analysis, ucted using instructions under under usinginstructions ucted Interactive Krona graph format. html in bers, bers, BIN assignments, CO1-5P sequence on BOLD. on includesThis Excelfile BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER NEMATOCER NEMATOCER BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER BRACHYCERA BRACHYCERA BRACHYCERA INFRAORDER Accepted BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCERA NEMATOCERA BRACHYCERA NEMATOCERA NEMATOCERA BRACHYCERA NEMATOCERA BRACHYCERA Article This article by article rightsprotected This reserved. is All copyright. A A A A

FAMILY rmfnia 0 Cremifaniidae 1 0% N/A 1.5-2.6 N/A N/A 0 Coenomyiidae 1 0% 14.0-20.0 N/A N/A N/A Clusiidae 9 6 Chloropidae 198 101 67% 2 1.5-8.0 51% 7 Chaoboridae 76% 97 3 1.0-5.0 7 29% 59 131 101 0 0 43%2.0-10.0 58% 0% 128 1.0-5.0 332Ceratopogonidae 39% 2 0% Carnidae 11 7 2 9 Canacidae 2 64% 1.2-2.5 1 450% 1.0-2.5 0 Camillidae 4 33% 0% 7 7 1.6-5.0 0% 1 0 100% 1 Braulidae 9 0 11% 2.0-3.5 0% 3 0% 0 N/A N/A 7.5-10.0 N/A 3 5 60% 1.0-3.0 3 Athericidae 5 1.3-4.5 N/A 43% N/A Asteiidae 7 3 36% N/A 5 Anthomyzidae 14 2.5-20.0 0% 0 Acroceridae 11 Conopidae Conopidae Coelopidae Chyromyidae Chironomidae Chamaemyiidae Cecidomyiidae Canthyloscelidae Campichoetidae Calliphoridae Bombyliidae Blephariceridae Aulacigastridae Atelestidae Asilidae Anthomyiidae Agromyzidae Acartophthalmidae iinde( lcia) 1 25%201. 02 20% 2 7 54% 10 13 2.0-15.0 4.0-7.0 57% 64% 12 14 Bolitophilidae 22 21 Bibionidae (&Pleciidae) Mycetobiidae) (& Anisopodidae

GERMANY IN REPORTED SPECIES 29 836 1 3 62 40 7 2 1 29% 3 7 81 4.0-12.0 227 88% 8 7 552 2 52 2 5 696

BINS 0 3 18 188 218 1 9 0 2 455 17 927 0 0 35 6 2

0% 100% 22% 83% 39% 50% 17% 0% 40% 65% 59% 111% 0% 0% 56% 15% 29% / SPECIES BARCODED % RATIO

2.0-5.0 1.5-3.5 8.0-20.0 4.0-12.0 1.0-6.0 1.0-2.5 5.0-15.0 2.5-9.0 0.5-8.0 1.0-10.0 1.0-5.0 0.5-3.0 2.5-9.0 2.5-4.0 4.0-16.0 1.0-20.0 3.0-15.0 SIZE (MM)

BARCODE OF TAXA /W TOTAL NUMBER 0 3 18 178 214 2 9 N/A 2 438 17 926 N/A N/A 39 6 2

BARCODE /W UNNAMED 0 0 6 64 149 0 0 N/A 0 286 13 882 N/A N/A 6 1 1

TAXA DARK % OF N/A 0% 33% 36% 70% 0% 0% N/A 0% 65% 76% 95% N/A N/A 15% 17% 50%

BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCERA NEMATOCERA BRACHYCERA NEMATOCERA NEMATOCERA NEMATOCERA BRACHYCERA Accepted BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER BRACHYCERA NEMATOCER BRACHYCERA BRACHYCERA Article This article by article rightsprotected This reserved. is All copyright. A A A A

Culicidae 46 8 17% 3.0-9.0 0 7 0% dnia %2050NAN/A N/A N/A N/A N/A N/A 2.0-5.0 N/A 66 N/A 1.5-7.0 40% 0% 0 0% 0% N/A 9 Odiniidae 0 167 0 9 1.5-3.0 2.0-18.0 1 Neottiophilidae 56% 1 55% 0% 11 Muscidae 317 6.0-9.0 174 44% N/A N/A 16 0 100% 6 Microphoridae 3.0-6.0 1 25 14% 1 N/A Megamerinidae 1 34% 2.0-7.0 10.0-22 16 Lonchaeidae 47 7 37% 0% 25 N/A 67 Lauxaniidae 2.5-10.0 N/A 0 Hypodermatidae 5 58% N/A 13 7 30% Hippoboscidae 12 6.0-11.0 44 0% 0 2.0-5.0 3 Helcomyzidae 82% Fanniidae 56 46 Brachystomatidae) (& Empididae 2 Dryomyzidae 3 67% 1 5.0-18.050% 112 52% Dolichopodidae 356 58 2 31% 1.0-9.0 112 1 Ditomyiidae 4 25% 3 0 6.0-8.0 Diadocidiidae 4 0% 1 75% 0 0% 3-4.5.0 3 Nycteribiidae Nycteribiidae Mycetophilidae Milichiidae Micropezidae Lonchopteridae Limoniidae Keroplatidae Hybotidae Hilarimorphidae Heteromyzidae) (& Heleomyzidae Gasterophilidae Eginiidae Drosophilidae Dixidae Cylindrotomidae Cryptochetidae eprnde1 %4060NAN/A N/A N/A 4.0-6.0 0% 0 1 Hesperinidae 383 161 42% 1.0-12.0 107 161 66% 9 280 60 229 2 74 4 177 1 59 16 6 4 1 8 573 13 13 130 0 28 4 8 1 0 0 306 17 5 5 96 30 140 0 58 0 73% 0% 47% 25% 133% 25% 0% 0% 53% 131% 38% 56% 34% 50% 61% 0% 78% 0% 1.0-11.0 2.0-18.0 1.5-7.0 3.0-5.5 2.5-4.0 11.0-16.0 2.0-4.0 1.5-5.0 2.0-15.0 1.0-6.0 3.0-16.0 2.0-5.0 2.0-11.0 4.0-15.0 1.0-6.0 2.0-7.0 1.2-12.0 9.0-16.0 132 N/A 28 4 8 1 N/A N/A 301 16 4 6 91 30 139 N/A 55 N/A 16 N/A 5 1 2 0 N/A N/A 89 9 1 0 50 12 83 N/A 26 N/A 12% N/A 18% 25% 25% 0% N/A N/A 30% 56% 25% 0% 55% 40% 60% N/A 47% N/A BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER NEMATOCER BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA Accepted BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCER BRACHYCERA BRACHYCERA NEMATOCER BRACHYCERA NEMATOCER Article This article by article rightsprotected This reserved. is All copyright. A A A A A

Opetiidae 1 1 100% 2.0-5.0 0 1 0% ehiia 1 82%251. 75 19% 76 27 36% 24 2.5-10.0 211 27% 6 8% 25% 2.0-20.0 Tephritidae 110 297 28 22 43% 3.5-35.0 Tachinidae 494 214 4 22% 2.0-25.0 N/A 55% N/A 440Syrphidae 242 32% N/A 0 18 21 66 N/A 2.0-14.0 14% 1 2.0-7.0 0 24% N/A 19 Sciomyzidae 78 3.0-12.0 0% 7 N/A 0 3 Scenopinidae 0% 2.0-11.0 N/A 0 57 Scatophagidae 90% 8.0-9.0 9 10 Rhinophoridae 0% 0 0% Pyrgotidae 1 0 1 1 1.7-2.5 100% Pseudopomyzidae 1 4 Platypezidae 23 17% 0 1.5-6.00% 12 Piophilidae 12 4 2 100% Phaeomyiidae 3 0% 0 67% 3.0-11.0 1.5-7.0 2 4 12 Pediciidae 36 33% 13 Otitidae 26 36% 0 5.0-35.0 3 13 0% 23% 2.5-11.0 N/A N/A N/A Tethinidae Tethinidae Tanypezidae Tabanidae Strongylophthalmyiidae Sphaeroceridae Sepsidae Sciaridae Scatopsidae Sarcophagidae Rhagionidae Ptychopteridae Psilidae Platystomatidae Pipunculidae Phoridae Periscelididae Pallopteridae Opomyzidae Oestridae Simuliidae 50 19 38% 1.2-6.0 18 9 9 50% 18 1.2-6.0 38% Simuliidae 50 19 25 50% 50 2.0-6.0 36% 51 Psychodidae 143 31 342 47 130 35 8 30 3 111 364 6 16 15 6 10 1 58 1 137 12 2 42 289 1 8 4 0 0 1 46 0 79 15 310 30 49 20 0 40% 67% 38% 79% 17% 50% 27% 0% 0% 100% 79% 0% 58% 48% 91% 64% 38% 57% 0% 2.5-10.0 3.0-11.0 2.0-12.0 0.5-6.0 1.0-5.0 2.5-7.0 2.0-5.0 9.0-18.0 1.5-3.5 5.0-8.0 6.0-30.0 3.0-5.5 0.7-5.5 2.0-6.0 1.0-6.0 0.5-4.0 3.0-22.0 2.0-20.0 7.0-15.0 12 2 40 276 1 7 4 N/A N/A 1 45 N/A 77 13 284 30 49 20 N/A 8 0 7 166 0 0 1 N/A N/A 0 3 N/A 31 1 81 24 17 10 N/A 67% 0% 18% 60% 0% 0% 25% N/A N/A 0% 7% N/A 40% 8% 29% 80% 35% 50% N/A Accepted Article BRACHYCERA BRACHYCERA BRACHYCERA BRACHYCERA NEMATOCERA NEMATOCERA BRACHYCERA BRACHYCERA NEMATOCERA

This article by article rightsprotected This reserved. is All copyright. huaede1 13 Thaumaleidae 15 8% 1 87% 3.0-5.0 13 yohgde4 1 Xylophagidae 4 25% 0 0% 5.0-11.0 1 4 Ulidiidae 9 24 Trichoceridae 18 225% N/A N/A 0 133% 17 N/A 1.5-7.0 71% 0% 3.0-9.0 2. Thyreophoridae 2 24 Xylomyidae Trixoscelididae Tipulidae Therevidae 3 4 123 32 1 0 46 4 33% 0% 37% 13% 6.0-20.0 2.0-4.0 7.0-35.0 2.5-15.0 -109 44% 4 5-11.0 9 1 N/A 46 4 0 N/A 15 1 0% N/A 33% 25% Accepted Article BOLITOPHILIDAE BIBIONIDAE ANTHOMYIIDAE AGROMYZIDAE CDC SPECIES FAMILY RANK This article by article rightsprotected This reserved. is All copyright. CERATOPOGONIDAE Brachypogon sociabilis sociabilis Brachypogon nigriventris Bibio clavipes Bibio ranunculi Phytomyza continua Phytomyza cichorii Napomyza (2) CDC austriaca Bolitophila (2) CDC (2) CDC winthemi Pegomya (2) CDC solennis Pegomya (2) CDC (2) CDC flavifrons Pegomya lancifer Hydrophoria (2) CDC Delia nuda liturata Anthomyia (2) CDC (2) CDC (2) CDC CDC (2) (2) CDC (2) CDC (2) CDC BOLD:ACE8195 BOLD:ACU5368 BOLD:ACR0881 1.06 1.87 BOLD:ACJ0544 1.06 1.87 BOLD:ACE4539 1.98 0.87 BOLD:ACL2003 BOLD:AAY2701 BOLD:AAX3741 1.27 2.18 BOLD:AAG4863 1.27 2.18 BOLD:AAG1783 0.54 5.53 BOLD:ACD8686 0.85 2.67 2.5 8.83 BOLD:AAG2479 BOLD:AAG2460 0.61 3.04 3.26 2.84 2.47 1.24 1 1.2 MEAN INTRA-SP 6.43 5.44 3.71 2.31 3.13 2.46 MAX INTRA-SP BOLD:ACE4540 BOLD:ACE4540 BOLD:AAY3895 BOLD:AAM6330 BOLD:AAP2990 BIN BOLD:ABW3958 BOLD:ABW3958 BOLD:ACI5612 BOLD:ABX1732 BOLD:ACC6151 BOLD:ABA6845 BOLD:ACM6225 BOLD:AAG6754 BOLD:ADC1814 BOLD:ACJ0545 Accepted Article CHIRONOMIDAE This article by article rightsprotected This reserved. is All copyright. Smittia reissi reissi Smittia Polypedilum convictum Paratanytarsus laccophilus Paraphaenocladius impensus 4ES sp. Forcipomyia grandiforceps Ceratopogon CDC (3) Paraphaenocladius (2) exagitans CDC (2) CDC stylatus Parametriocnemus (2) CDC fuscimanus Orthocladius (2) CDC flexuella Mesosmittia (2) CDC 4SW sp. Limnophyes natalensis Limnophyes (2) CDC (2) CDC (2) CDC brumalis Gymnometriocnemus bicinctus Cricotopus Brillia bifida (2) CDC (2) CDC CDC (2) CDC (2) CDC (2) CDC (4) CDC BOLD:ACQ8860 BOLD:ACP4327 BOLD:ACT9278 BOLD:ACF2457 BOLD:ACU4175 BOLD:ACT2714 BOLD:ACT8523

1.51 2.89 BOLD:AAB7361 1.51 2.89 0.5 2.41 BOLD:ACD4501 BOLD:AAI6018 1.86 3.2 BOLD:AAD7726 6.93 2.31 2.54 5.88 BOLD:AAE3719 2.54 5.88 BOLD:AAI2687 0.76 2.03 2 2.66 BOLD:AAV5075 BOLD:ADE7569 0.79 2.02 BOLD:ACR9428 1.49 4.03 2.18 2.63 1.72 2.45 2.09 6.85 5.98 3.94 3.47 4.61 3.14 11.99 BOLD:ACT1270 BOLD:ACT1270 BOLD:ACU9207 BOLD:AAT9677 BOLD:ADI4999 BOLD:AAM6200 BOLD:ABW3984 BOLD:ACS9748 BOLD:AAW4661 BOLD:AAC8842 BOLD:ACT5784 BOLD:AAC4200 BOLD:ACQ4724 BOLD:ACT9205 BOLD:ACX3046 BOLD:ACU4856 BOLD:ACU4225 Accepted Article EPHYDRIDAE EMPIDIDAE DOLICHOPODIDAE CONOPIDAE This article by article rightsprotected This reserved. is All copyright. Scatophila signata signata Scatophila Scatella obsoleta duplosetosa Polytrichophora flavipes Philygria flaviceps Hydrellia salinarius Halmopota Ditrichophora palliditarsis fuscella Ditrichophora laterale Allotrichoma holosericeus Microphor anomalus Microphor

CDC (2) (2) CDC Kowarzia tenella (2) CDC barbatula Kowarzia adulatoria Hemerodromia (2) CDC testacea Myopa (2) CDC (2) CDC CDC (2) (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC BOLD:ACH6989 BOLD:ACH9043 BOLD:ACU4112 BOLD:ABA7494 BOLD:ABA8628 BOLD:ACK3229 BOLD:ABV8173 BOLD:ABA7827 BOLD:ABA8748 BOLD:ABA8606 BOLD:ACF1575 7.21 10.71 BOLD:ACJ6935 BOLD:ACJ6728 8.52 8.52 BOLD:AAK8836 3.72 3.72 5.39 10.8 BOLD:ACJ6935 5.39 10.8 4.06 5.47 3.3 3.3 1.25 2.05 1.19 4.22 2.43 3.87 3.81 6.44 12.7 11 3.3 3.3 2.5 4.11 2.03 6.33 3.81 6.57 7.62 6.44 BOLD:ACJ7236 BOLD:ACJ6729 BOLD:ACB6469 BOLD:ACH9042 BOLD:AAK8838 BOLD:ABA7651 BOLD:ABA7493 BOLD:ABA8627 BOLD:ABA8663 BOLD:ABA8652 BOLD:ABA7826 BOLD:AAX8675 BOLD:ABA8605 BOLD:ABA8753 BOLD:ACJ7236 Accepted Article MYCETOPHILIDAE MUSCIDAE MILICHIIDAE LIMONIIDAE LAUXANIIDAE HELEOMYZIDAE FANNIIDAE This article by article rightsprotected This reserved. is All copyright. Boletina dispecta Helina evecta meigenii Euphylidorea lutescens Chionea Heleomyza serrata

Phronia obtusa CDC (2) CDC (2) CDC obtusa Phronia sericoma Brevicornu griseicolle Brevicornu (2) CDC humeralis Mydaea (2) CDC equitans Phyllomyza (2) CDC longipennis Minettia (2) CDC postica Fannia CDC (2) (2) CDC (2) CDC (2) CDC (3) CDC (3) CDC BOLD:ADD1050 BOLD:ACV1127 BOLD:ABA7652 CDC(2) 9.06 13.6 BOLD:ACU9474 BOLD:AAY5580 BOLD:ACB3279 BOLD:ACU9122 0.96 1.45 BOLD:ACR0546 0.96 1.45 BOLD:ABW2012 7.03 2.35 0.83 1.18 BOLD:AAY8505 0.83 1.18 BOLD:AAY6368 1.99 4.58 BOLD:AAE0058 1.95 5.84 BOLD:ACB3455 1.39 4.05 1.91 1.1 0.37 9.01 1.83 4.88 1.1 3.54 11.2 4.27 BOLD:ABV4905 BOLD:ABV5195 BOLD:ACR0548 BOLD:ABX8716 BOLD:ACG3518 BOLD:ACJ2989 BOLD:ABA1564 BOLD:ABA1563 BOLD:AAY5581 BOLD:AAY5579 BOLD:ACD1934 BOLD:ADB5997 BOLD:AAE3133 BOLD:ACD3072 Accepted Article PHORIDAE PALLOPTERIDAE This article by article rightsprotected This reserved. is All copyright. Megaselia pulicaria complex Megaselia longicostalis Megaselia glabrifrons consetigera Megaselia valida Zygomyia Stigmatomeria crassicornis CDC (2) CDC Stigmatomeria crassicornis Megaselia subpalpalis CDC (2) CDC CDC (2) Megaselia subpalpalis (2) CDC (2) CDC Megaselia sepulchralis Megaselia ruficornis rufa Megaselia (3) CDC (2) CDC Megaselia nigriceps lutea Megaselia CDC (2) Toxoneuraaff. modesta CDC (3) Zygomyia angusta CDC (2) (2) CDC (2) CDC (2) CDC (3) CDC (3) CDC BOLD:ACX1476 BOLD:ABW0168 BOLD:ABW0168 BOLD:AAP4698 BOLD:ACF7950 BOLD:ADA4916 BOLD:ACI6910 3.41 5.13 BOLD:ACB4053 3.41 5.13 3.29 14.88 BOLD:AAY5526 BOLD:AAY6370 0.56 1.86 1.05 2.17 BOLD:AAL9083 1.05 2.17 BOLD:ACF7622 2.27 4.27 5.46 17.53 BOLD:ACF7708 BOLD:ACD9573 1.83 8.31 BOLD:AAG7022 0.76 7.16 BOLD:AAG3351 2.14 6.46 0.66 0.65 9.51 5.85 1.32 1.78 2.63 14.5 11.96 5.72 BOLD:ACG3433 BOLD:ACG3433 BOLD:ACG2938 BOLD:ACV1580 BOLD:AAY5526 BOLD:AAY5527 BOLD:ACU7541 BOLD:ACZ9853 BOLD:ACZ9853 BOLD:ACG4585 BOLD:ACD9606 BOLD:AAU8534 BOLD:AAL9073 BOLD:AAY6384 BOLD:ACG3608 BOLD:AAG7025 BOLD:AAG3263 Accepted Article SCIARIDAE RHINOPHORIDAE PSYCHODIDAE This article by article rightsprotected This reserved. is All copyright. Corynoptera tetrachaeta tetrachaeta Corynoptera subtilis Corynoptera polana Corynoptera luteofusca Corynoptera grothae Corynoptera vittata Bradysiopsis Bradysia trivittata Bradysia tilicola regularis Bradysia Bradysia praecox inusitata Bradysia brevispina Bradysia albipennis nr. Psychoda (2) CDC sarcophagina Rhinomorinia (3) CDC tarsella Megaselia CDC (2) (2) CDC (2) CDC (2) CDC CDC (2) (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC BOLD:ACH4332 BOLD:ACI5443 BOLD:ACN5049 BOLD:ACT9420 BOLD:ACF7764 BOLD:ACQ8494 BOLD:ACO7236 BOLD:ACR0949 BOLD:ACB1143 BOLD:ACP0919 BOLD:ACQ7807 BOLD:ACU9870 0.75 1.78 BOLD:ACD9526 0.75 1.78 BOLD:ACE0332 5.61 0.45 6.61 2.86 1.55 4.16 2.91 1.95 8.16 4.75 2.24 0.57 2.87 0.1 1.09 6.61 8.4 3.45 4.16 6.26 3.81 11.8 9.36 4.62 3.57 6.03 1.67 2.35 BOLD:ACE7273 BOLD:ACE7273 BOLD:ACE4845 BOLD:ACG3259 BOLD:ABA0876 BOLD:ACF7226 BOLD:ACZ7449 BOLD:ACG5327 BOLD:ACG5327 BOLD:ACD5314 BOLD:ACF6941 BOLD:ACJ1951 BOLD:ACK0158 BOLD:ACC1999 BOLD:AAH3947 BOLD:AAN6444 BOLD:ACC1391 BOLD:ACF3561 Accepted Article SYRPHIDAE SPHAEROCERIDAE SIMULIIDAE This article by article rightsprotected This reserved. is All copyright. chrysocoma albipila Cheilosia cryophilum Simulium scutellata Leptosciarella Leptosciarella fuscipalpa Epidapus atomarius tridentata Corynoptera

(2) CDC (2) CDC clunipes Spelobia coxata Opacifrons (2) CDC (2) CDC morio Trichosia CDC (2) hemerobioides Sciara (2) CDC Scatopsciara subciliata (2) CDC neglecta Scatopsciara degener Pnyxiopsis (3)CDC (2) CDC (2) CDC (2) CDC CDC (2) (2) CDC (2) CDC (2) CDC BOLD:ACG4078 BOLD:ACQ8733 BOLD:ACX3063 BOLD:ACJ9791 BOLD:ACL4032 BOLD:ACJ5068 BOLD:AAZ1026 BOLD:AAU1818

0.53 1.78 BOLD:ACC7986 0.53 1.78 BOLD:ACE2293 5.17 1.83 2.89 6.93 BOLD:AAG7312 2.89 6.93 BOLD:ACP2618 6.41 14 BOLD:ACD5342 0.78 3.99 BOLD:ACQ8933 4.1 4.1 BOLD:AAH4004 4.32 1.93 4.84 5.24 0.07 9.95 3.69 2.51 1.49 7.98 9.24 3.98 9.95 3.69 6.88 3.14 BOLD:ACF9729 BOLD:ACI9623 BOLD:ACD6061 BOLD:ACE2641 BOLD:ACD4767 BOLD:ACJ1561 BOLD:ABY6892 BOLD:AAW3610 BOLD:ACF9400 BOLD:ACP5793 BOLD:ACU9243 BOLD:ACO9950 BOLD:ACR4627 BOLD:ACA8369 BOLD:ACQ2637 Accepted Article

This article by article rightsprotected This reserved. is All copyright. proxima Cheilosia personata Cheilosia mutabilis Cheilosia lenis Cheilosia impressa Cheilosia flavipes Cheilosia derasa Cheilosia eioyalpoa CDC (2) (2) CDC (2) CDC Sericomyia lappona albimanus CDC (2) noctiluca Pipiza CDC (2) (2) CDC Parasyrphus punctulatus Paragus pecchiolii (2) CDC (2) rufus CDC Merodon nuda (2) CDC nitens vernalis-agg. Cheilosia CDC (3) (3) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC (2) CDC

BOLD:ACX0819 BOLD:AAY9747 BOLD:AAY8875 BOLD:AAW3615 BOLD:AAY9045 BOLD:AAW3649 BOLD:AAW3651 .639 BOLD:AAB1553 2.06 3.9 BOLD:AAL7898 0.37 3.01 BOLD:AAL4100 1.54 3.92 BOLD:AAZ4514 1.11 2.65 BOLD:ABA3664 0.96 4.86 BOLD:ADI8358 0.68 1.09 BOLD:AAY8880 0.81 2.44 BOLD:AAB2384 3.97 3.97 BOLD:ACF0974 2.07 3.84 3.28 1.35 1.94 3.85 1.95 8.79 0.58

6.91 1.88 2.74 7.86 5.74 8.79 3.47

BOLD:AAW3607 BOLD:ACH1700 BOLD:AAY9746 BOLD:AAY8876 BOLD:AAW3651 BOLD:AAW3610 BOLD:AAY9044 BOLD:ACJ4919 BOLD:ACG4983 BOLD:ACG4772 BOLD:ACG8255 BOLD:AAQ1380 BOLD:ACH5745 BOLD:ACH1529 BOLD:ACJ5218 BOLD:ABY8734 Accepted Article TACHINIDAE TABANIDAE This article by article rightsprotected This reserved. is All copyright. glaucopis Tabanus bromius Tabanus Phorinia aurifrons CDC (2) CDC CDC (2) (2) CDC aurifrons Phorinia Peribaea (2) CDC fissicornis (2) CDC moerens Kirbya (2) CDC Cyzenis albicans selecta Bessa dubitata Actia (2) CDC

BOLD:AAF3859 BOLD:ACJ5745 1.22 1.86 BOLD:ACJ2730 1.22 1.86 BOLD:ACB0896 1.18 2.18 BOLD:ADK1760 1.45 2.38 BOLD:ACP3766 2.36 2.36 3.76 11.2 BOLD:ADK4076 3.76 11.2 BOLD:ACH1961 2.22 8.17 3.27 2.04

4.43 2.93

BOLD:ACM9631 BOLD:AAW3422 BOLD:ACH1972 BOLD:AAF3858 BOLD:AAF3864 BOLD:ACH1641 BOLD:ACB0795 BOLD:ACJ2910 BOLD:ACB0261 Accepted Article EPHYDRIDAE EMPIDIDAE E DOLICHOPODIDA CALLIPHORIDAE ANTHOMYIIDAE BS RANK SPECIES FAMILY This article by article rightsprotected This reserved. is All copyright. Hydrellia subalbiceps subalbiceps Hydrellia (3) BS nigricans Hydrellia Ephydra riparia murina Ephydra macellaria Ephydra BS (4) schumanni Allotrichoma Allotrichoma laterale Allotrichoma filiforme Allotrichoma bezzii Kowarzia tenella Kowarzia barbatula Kowarzia tenella madicola Kowarzia BS (2) hygrophilus Sphyrotarsus argyrostomus Sphyrotarsus BS (2) truncorum (3) BS petrophiloides Medetera illustris Lucilia BS (2) caesarillustris Lucilia caesar Lucilia vicina Calliphora Calliphora loewi vagans Hylemya nigrimana Hylemya N/A N/A 0 0.37 1.58 0.31 0.31 2.83 0.46 2.83 6.44 6.44 BS (2) BS (2) BS (2) BS (2) 0.35 1.22 BOLD:ACA1124 BOLD:ACA1124 1.22 0.35 BOLD:AAA7470 3.07 0.95 BOLD:AAB6579 1.07 1.07 / BOLD:AAG2729 0N/A BOLD:ACF1575 0.310.13 10.8 5.39 10.8 5.39 BOLD:ADB6106 1.37 0.91 0.7 0.84 0.34 0.23 0.23 N/A 0 0.08 4.8 0 N/A N/A MEAN INTRA-SP 2.43 2.59 0.52 0.31 0 0 0.15 11.3 0 0 0 MAX INTRA-SP BOLD:ABA6492 BIN BOLD:ABA8624 BOLD:ACJ6935 BOLD:ACJ7236 Accepted Article MYCETOPHILIDAE MUSCIDAE E LONCHOPTERIDA GROUP ITEAPHILA- This article by article rightsprotected This reserved. is All copyright. Notiphila cinerea cinerea Notiphila Zygomyia valida Zygomyia angusta Zygomyia valida Zygomyia angusta flava Mycetophila distigma Mycetophila groenlandica Boletina (2) BS Boletina gripha similis Hydrotaea dentipes Hydrotaea nitidifrons Lonchoptera (2) BS lutea Lonchoptera (2) BS sp. 2 Iteaphila 1 sp. Iteaphila 4 sp. Anthepiscopus (2) BS 1 sp. Anthepiscopus 1 sp. Anthepiscopus indet. Anthepiscopus nitidula Psilopa compta Psilopa punctatonervosa Philygria Philygria flavipes subnigra Notiphila riparia Notiphila graecula Notiphila 0.38 0.38 0.15 0.77 0.41 0.15 0 0.62 0 14.5 14.5 14.5 0.19 14.5 N/A 0.19 0 N/A 0 BS (2) BS (2) BS (2) BS (2) BS (2) BS (2) BS (2) BS (2) BS (2) 11.3 11.3 BOLD:ACJ7111 11.311.3 BOLD:ACD9492 0.480.14 2.15 9.782.15 BOLD:AAZ9882 BOLD:ACD3033 0.150.07 0.91 0.91 11.3 0.08 1.19 0.16 0.26 4.6 4.6 N/A 0.52 0 0.39 4.49 1.58 11.3 0.16 2.03 0.35 0.46 15.4 15.4 0 0.9 0 1.09 9.77 BOLD:AAG6948 BOLD:ACK3229 BOLD:AAX5585 BOLD:ABA7513 8 BOLD:ABW016 BOLD:AAY5526 BOLD:AAY8340 BOLD:AAF6783 BOLD:ABX0277 Accepted Article SYRPHIDAE SIMULIIDAE SARCOPHAGIDAE PHORIDAE This article by article rightsprotected This reserved. is All copyright. Triphleba bicornuta BS (2) (2) BS bicornuta Triphleba hioi rsiea BS (6) impudens Cheilosia Cheilosia crassiseta vernalis-agg. Cheilosia Cheilosia sp. BOLDACF0974 ruficollis Cheilosia melanura Cheilosia BS (8) fraterna Cheilosia Cheilosia chlorus-group Cheilosia chlorus Cheilosia chloris Cheilosia sp. BOLDAAW3615 impressa Cheilosia barbata Cheilosia flavipes Cheilosia albipila Cheilosia valgus Brachypalpus laphriformis Brachypalpus Baccha sp.BOLDABA3006 obscuripennis Baccha s.s. elongata Baccha elongata Baccha (2) BS equinum Simulium balcanicum Simulium haemorrhoa Sarcophaga depressifrons Sarcophaga sp. BOLD:ACF0365 Triphleba 0 0 0.47 0.7 0.47 0.47 0.06 0.71 N/A 0.2 0 1.95 8.79 5.74 N/A 8.79 N/A 0 0 BS (6) BS (2) BS (3) BS (2) / BOLD:AAM403 0N/A BOLD:ACF0365 0 N/A / BOLD:AAW364 0 N/A BOLD:ACF0974 1.420.57 1.23 1.23 N/A 1.59 0 0.66 N/A N/A 2.07 N/A 0.55 0.12 0 0.1 2.51 0.56 2.02 0 2.66 0 1.22 0 3.84 0 0.87 0.18 0 0.3 6.88 1.54 6 7 BOLD:ABA3006 BOLD:ABV4597 5 BOLD:AAW361 0 BOLD:AAW361 BOLD:AAY9039 Accepted Article

This article by article rightsprotected This reserved. is All copyright. Cheilosia nigripes nigripes Cheilosia Melanostoma sp. A Melanostoma scalare Melanostoma mellinum-agg. Melanostoma mellinum Melanostoma dubium Melanostoma Melanogaster hirtella Melanogaster aerosa Eupeodes sp. BOLDAAB2384 BS (5) nitens Eupeodes nielseni Eupeodes Dasysyrphus sp. BOLDAAB2865 pinastri Dasysyrphus (3) BS Dasysyrphus lenensis venustus Dasysyrphus laskai Dasysyrphus BS (2) hilaris Dasysyrphus Chrysotoxum festivum BS (2) bicinctum Chrysotoxum lenis Cheilosia (2) BS Cheilosia carbonaria montana Cheilosia (2) BS canicularis Cheilosia BS (2) pubera Cheilosia grisella Cheilosia nivalis Cheilosia Cheilosia faucis antiqua Cheilosia grisella aff. Cheilosia Cheilosia sp. BIOUG17085-G07 N/A N/A N/A 0 0 0.49 0.49 0.58 1.3 0.26 1.21 0.53 0.7 3.97 1.05 3.97 1.25 N/A 2.1 0

BS (7) BS (2) BS (3) 0.37 0.370.37 BOLD:AAY8876 BOLD:ACI2500 0.380.08 0.18 0.18BOLD:AAW361 0.7 0.88 BOLD:AAY8874 1.14 3.13 BOLD:AAB2384 BOLD:AAB2384 3.13 1.14 BOLD:AAA7375 0.52 0.35 BOLD:AAJ0967 20.86 3.85 3.85 N/A 0.49 0 N/A 0.75 0 N/A 0 N/A 0.39 0.15 0.12 0.58 0.3 0 7.86 0 0.87 0 0 1.94 0 0 0 0 1.03 0.37 0.17 0.58 0.3 0 9

BOLD:AAB2866 BOLD:AAQ4015 BOLD:AAB2865 Accepted Article

This article by article rightsprotected This reserved. is All copyright. Melanostoma sp.Melanostoma B Platycheirus scutatus BS (3) (3) BS scutatus-group Platycheirus scutatus Platycheirus peltatus-group Platycheirus peltatus Platycheirus nielseni Platycheirus Platycheirus tatricus Platycheirus melanopsis BOLDAAA9506 sp. Platycheirus Platycheirus perpallidus (5) BS Platycheirus occultus fulviventris Platycheirus clypeatus Platycheirus BOLDACF4733 sp. Platycheirus Platycheirus europaeus Platycheirus angustatus Pipiza sp. BOLDAAL4100 noctiluca-agg. Pipiza nocticula Pipiza bimaculata Pipiza vittiger Parasyrphus lineola Parasyrphus Paragus pecchiolii Paragus majoranae Paragus sp. BOLDABZ4619 BS (5) BS (2) Paragus aff. haemorrhous avidus B Merodon avidus Merodon sp.Melanostoma BOLDAAB2866 0.63 0.63 0.96 1.44 N/A 4.86 0.26 0 0.87 0.11 0.16 0.24 0.24 N/A 0.72 0.9 0 0.51 2.03 1.04 1.95 0.55 1.95 N/A 1.65 0 BS (2) BS (2) BS (3) BS (2) BS (3) BS (4) / BOLD:ABZ4619 0N/A BOLD:AAQ1379 0 N/A 0.05 0.190.05 BOLD:AAG4665 BOLD:AAA9506 0.88 0.38 0.19 0.19 0.87 0.07 N/A 0.55 0.63 0.44 0.44 N/A 0 0.25 N/A 1.04 0.21 0.84 N/A N/A 0.39 0.87 0.37 0 1.03 2.69 0.71 0 0 0.62 0 1.04 1.15 2.02 0 0 BOLD:ACE7140 BOLD:ABA3664 BOLD:AAC6630 BOLD:AAP0412 BOLD:ACF4733 BOLD:AAL4100 Accepted Article TACHINIDAE STRATIOMYIDAE This article by article rightsprotected This reserved. is All copyright. Platycheirus splendidus Lydella stabulans BS (2) (2) BS melania Medina thompsoni Lydella stabulans Lydella morrisii Beris geniculata BS (2) (2) BS Temnostoma meridionale Temnostoma apiforme sibirica montana Sphegina virgata taeniata Sphaerophoria BOLDAAA7374 sp. Sphaerophoria rueppellii Sphaerophoria philanthus Sphaerophoria interrupta-group Sphaerophoria interrupta Sphaerophoria infuscata Sphaerophoria Sphaerophoria bankowskae Sericomyia silentis Sericomyia lappona dignota Scaeva pyrastri Scaeva pyrastri dignota Scaeva eialcus BS (3) separata Medina Medina luctuosa N/A N/A 0.49 0 0.24 0.75 0.05 0.38 N/A 0.24 0.25 0 N/A 0.91 0 0.48 1.47 N/A 0

BS (9) BS (2) BS (2) BS (2) BS (2) 0.12 0.44 BOLD:AAP8653 BOLD:AAP8653 0.44 0.12 BOLD:AAV6543 0.52 0.52 BOLD:ABX4867 0N/A 1.35 1.351.35 BOLD:AAG6902 N/A N/A 0 N/A 2.06 0.25 N/A 0.68 N/A 0.35 0.4 N/A 0.31 0 0 0 3.9 0.91 0 1.31 0 0.52 0.41 0 6.54 BOLD:AAA7374 BOLD:AAB1553 BOLD:AAF2374 BOLD:AAF2374 4 BOLD:AAW338 Accepted Article

This article by article rightsprotected This reserved. is All copyright.

Accepted Article This article by article rightsprotected This reserved. is All copyright.

Accepted Article

This article by article rightsprotected This reserved. is All copyright.