DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

Ctenosciara alexanderkoenigi sp. n. (Diptera: Sciaridae), an exotic invader in Germany?

A new species of the genus Ctenosciara Tuomikoski, 1960 is here described based upon a single specimen, obtained from collectings in the garden at Museum Alexander Koenig in Bonn. Ctenosciara alexanderkoenigi sp. n. differs from all other congeneric European species by its striking coloration and distinct male genitalia. However, DNA barcoding reveals associations with two specimens from New Zealand. Therefore a recent migration of Ctenosciara species from the Australasian Region, the likely center of origin of the genus, is discussed. A key to the European species of Ctenosciara is provided. Barcoding results reveale that Ctenosciara exigua is not clearly distinguished from Ctenosciara hyalipennis by its COI sequence (both share the same BIN BOLD:AAH3983) and that its species status may be questionable

Surprisingly high genetic divergence of the mitochondrial DNA barcode fragment (COI) within Central European woodlice species (Crustacea, Isopoda, Oniscidea)

DNA barcoding has become the most popular approach for species identification in recent years. As part of the German Barcode of Life project, the first DNA barcode library for terrestrial and freshwater isopods from Germany is presented. The analyzed barcode library included 38 terrestrial (78% of the documented species of Germany) and five freshwater (63%) species. A total of 513 new barcodes was generated and 518 DNA barcodes were analyzed. This analysis revealed surprisingly high intraspecific genetic distances for numerous species, with a maximum of 29.4% for Platyarthrus hoffmannseggii Brandt, 1833. The number of BINs per species ranged from one (32 species, 68%) to a maximum of six for Trachelipus rathkii (Brandt, 1833). In spite of such high intraspecific variability, interspecific distances with values between 12.6% and 29.8% allowed a valid species assignment of all analyzed isopods. The observed high intraspecific distances presumably result from phylogeographic events, Wolbachia infections, atypical mitochondrial DNAs, heteroplasmy, or various combinations of these factors. Our study represents the first step in generating an extensive reference library of DNA barcodes for terrestrial and freshwater isopods for future molecular biodiversity assessment studies

A new species of Docosia Winnertz from Central Europe, with DNA barcoding based on four gene markers (Diptera, Mycetophilidae)

A new species of Docosia Winnertz, D. dentata sp. n., is described and illustrated, based on a single male specimen collected in Muránska planina National Park in Central Slovakia. DNA sequences (COI, COII, CytB, and ITS2) are included and compared for 13 species of Docosia. There was found only little congruence between the molecular results and previous scarce data about interspecific relationships based on morphology. The COI and CytB gene markers showed the highest interspecific gene distances while ITS2 showed the lowest ones. An updated key to the 23 Central European species of Docosia is also presented

New records of Manota Williston (Diptera: Mycetophilidae) in Europe and North America, including a redescription of Manota unifurcata Lundström and pointers towards the interrelationships among Palaearctic species

Knowledge of the morphology of Manota unifurcata LUNDSTRÖM, 1913 is deepened by a detailed description and illustration of the male genitalia. Based on a study of the characters of the male genitalia in specimens from various countries, it is reconfirmed that M. unifurcata is the only species of Manota WILLISTON in Europe. Its geographic distribution is reviewed, and is augmented by new records from Germany and the first records from Sweden. The relationships of M. unifurcata to other Manota species are discussed. It appears that the species is rather isolated from other Manota in terms of both morphology and geographic distribution. The presence of a male hind tibial organ, which is described here, is a synapomorphous character shown by at least 4 of the 5 East Palaearctic species of Manota. These species and several Oriental Manota, for which presence or absence of the tibial organ is unknown, have several male genitalia characters in common, a fact that indicates their close relationships. Manota ibanezi HIPPA & HUERTA, 2009 from Mexico is shown to occur also in the north-eastern United States (Maine and New York). These are the first records of a named Manota species from the Nearctic Region. Possible explanations for the rarity of Manota in both North America and Europe are discussed

Another crack in the Dark Taxa wall: a custom DNA barcoding protocol for the species-rich and common Eurytomidae (Hymenoptera, Chalcidoidea)

DNA barcodes are a great tool for accelerated species identification and for complementing species delimitation. Furthermore, DNA barcode reference libraries are the decisive backbone feature for any metabarcoding study in biodiversity monitoring, conservation or ecology. However, in some taxa, DNA barcodes cannot be generated with published primers at a satisfying success rate and these groups will consequently be largely missing from any barcoding-based species list. Here, we provide a custom DNA barcoding forward primer for the Eurytomidae (Hymenoptera, Chalcidoidea), elevating the success rate of high-quality DNA barcodes from 33% to 88%. Eurytomidae is a severely understudied, taxonomically challenging, species-rich group of primarily parasitoid wasps. High species numbers, diverse ecological roles and widespread and common presence identify Eurytomidae as one of many crucial families in terrestrial ecosystems. It is now possible to include Eurytomidae when studying and monitoring the terrestrial fauna, highlighting that barcoding-based approaches will need to routinely use different primers to avoid biases in their data and inferences. The new DNA barcoding protocol is also a prerequisite for our integrative taxonomy study of the group, aiming at delimiting and characterising Central European species and filling the GBOL (German Barcode Of Life) DNA barcode reference library with species-named and voucher-linked sequences

Kommentiere artenlisten der farn- und blütenpflanze and inseketn (Insecta: Diptera, Odonata, Coleopatra: Bupestridae) des GEO-Artentages in Dessau-Roßlau 2009 und vorangegangener jahre mit Erstnachweisen von Brachypeza radiate Jenkinson, 1908 und Trichonta paraterminalis Zaitzev, 1999 (Insecta: Diptera: Mycetophilidae) in der BRD

This study presents results of entomological surveys and vegetation assessment at the further surrounding of the oxbow lake Kuehnauer See at the territory of the city Dessau-Rosslau, Saxony-Anhalt, Germany in 2009. These activities were part of the international action “Tag der Artenviefalt [Day of biodiversity]”, an initiative of the magazine GEO (Gruner + Jahr, Hamburg). The sampling sites examined are briefly described. Records of 55 insect species are listed, including 27 Diptera, 18 Odonata, 10 Buprestidae (Coleoptera) and 198 species of angiosperms and ferns are recorded. Species with particular interest for conservation aw ell as these that were newly recorded from Germany or form the area are discusses. Brachypeza radiate Jenkinson, 1908 and Trichonta paraterminalis Zaitzev, 1999 (Insecta: Diptera: Mycetophilidae) are new records from Germany. The endangered dragonfly species Aeshna isosceles (O.F. Müeller, 1767) (Insecta: Odonata: Aeshnidae) was observed to re-colonize the area. The survey of jewel beetles revealed the presence of several protected species, among them Trachys scrobiculatus Kiesenwetter, 1857 (Coleoptera: Buprestidae). The botanical survey at the site Lobenreite provides a first database update since 1978. Rare species such as Silene otitis (L.) Wibel (Caryophyllaceae) and Vincetoxicum hirundinaria Medik (Asclepiadaceae) were recorded for the first time in the area and the occurrence of a population of Ornithopus perpusillus L. (Fabaceae) could have been confirmed

Testing the Global Malaise Trap Program – How well does the current barcode reference library identify flying insects in Germany?

Biodiversity patterns are inherently complex and difficult to comprehensively assess. Yet, deciphering shifts in species composition through time and space are crucial for efficient and successful management of ecosystem services, as well as for predicting change. To better understand species diversity patterns, Germany participated in the Global Malaise Trap Program, a world-wide collection program for arthropods using this sampling method followed by their DNA barcode analysis. Traps were deployed at two localities: "Nationalpark Bayerischer Wald" in Bavaria, the largest terrestrial Natura 2000 area in Germany, and the nature conservation area Landskrone, an EU habitats directive site in the Rhine Valley. Arthropods were collected from May to September to track shifts in the taxonomic composition and temporal succession at these locations. In total, 37,274 specimens were sorted and DNA barcoded, resulting in 5,301 different genetic clusters (BINs, Barcode Index Numbers, proxy for species) with just 7.6% of their BINs shared. Accumulation curves for the BIN count versus the number of specimens analyzed suggest that about 63% of the potential diversity at these sites was recovered with this single season of sampling. Diversity at both sites rose from May (496 & 565 BINs) to July (1,236 & 1,522 BINs) before decreasing in September (572 & 504 BINs). Unambiguous species names were assigned to 35% of the BINs (1,868) which represented 12,640 specimens. Another 7% of the BINs (386) with 1,988 specimens were assigned to genus, while 26% (1,390) with 12,092 specimens were only placed to a family. These results illustrate how a comprehensive DNA barcode reference library can identify unknown specimens, but also reveal how this potential is constrained by gaps in the quantity and quality of records in BOLD, especially for Hymenoptera and Diptera. As voucher specimens are available for morphological study, we invite taxonomic experts to assist in the identification of unnamed BINs

Into the unknown: the first barcode-assisted checklist of Psocoptera (Insecta, Psocodea) of Georgia with a census on country species richness

This checklist reports 47 species of Psocoptera from 15 families and three suborders from Georgia, of which 31 species are recorded for the first time, increasing the known fauna of the country by more than 65%. Of these, 37 species have been barcoded, representing 210 Barcode Identification Numbers (BINs). An additional 14 species are expected to occur in Georgia but remain undiscovered, meaning that only ≈ 77% of the fauna is currently documented. Barcodes, comments on distributions, and images of voucher specimens are given followed by a map of the sampling sites

Is it time to describe new species without diagnoses? -A comment on Sharkey et al. (2021)

Ahrens, Dirk, Ahyong, Shane T., Ballerio, Alberto, Barclay, Maxwell V. L., Eberle, Jonas, Espeland, Marianne, Huber, Bernhard A., Mengual, Ximo, Pacheco, Thaynara L., Peters, Ralph S., Rulik, Björn, Vaz-De-Mello, Fernando, Wesener, Thomas, Krell, Frank-Thorsten (2021): Is it time to describe new species without diagnoses? -A comment on Sharkey et al. (2021). Zootaxa 5027 (2): 151-159, DOI: https://doi.org/10.11646/zootaxa.5027.2.