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.

Facing the infinity: tackling large samples of challenging Chironomidae (Diptera) with an integrative approach.

Background: Integrative taxonomy is becoming ever more significant in biodiversity research as scientists are tackling increasingly taxonomically challenging groups. Implementing a combined approach not only guarantees more accurate species identification, but also helps overcome limitations that each method presents when applied on its own. In this study, we present one application of integrative taxonomy for the highly abundant and particularly diverse fly taxon Chironomidae (Diptera). Although non-biting midges are key organisms in merolimnic systems, they are often cast aside in ecological surveys because they are very challenging to identify and extremely abundant. Methods: Here, we demonstrate one way of applying integrative methods to tackle this highly diverse taxon. We present a three-level subsampling method to drastically reduce the workload of bulk sample processing, then apply morphological and molecular identification methods in parallel to evaluate species diversity and to examine inconsistencies across methods. Results: Our results suggest that using our subsampling approach, identifying less than 10% of a sample's contents can reliably detect >90% of its diversity. However, despite reducing the processing workload drastically, the performance of our taxonomist was affected by mistakes, caused by large amounts of material. We conducted misidentifications for 9% of vouchers, which may not have been recovered had we not applied a second identification method. On the other hand, we were able to provide species information in cases where molecular methods could not, which was the case for 14% of vouchers. Therefore, we conclude that when wanting to implement non-biting midges into ecological frameworks, it is imperative to use an integrative approach.

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.

From DNA barcodes to ecology: Meta-analysis of central European beetles reveal link with species ecology but also to data pattern and gaps.

DNA barcoding has been used worldwide to identify biological specimens and to delimit species. It represents a cost-effective, fast, and efficient way to assess biodiversity with help of the public Barcode of Life Database (BOLD) accounting for more than 236,000 animal species and more than 10 million barcode sequences. Here, we performed a meta-analysis of available barcode data of central European Coleoptera to detect intraspecific genetic patterns among ecological groups in relation to geographic distance with the aim to investigate a possible link between infraspecific variation and species ecology. We collected information regarding feeding style, body size, as well as habitat and biotope preferences. Mantel tests and two variants of Procrustes analysis, both involving the Principal Coordinates Neighborhood Matrices (PCNM) approach, were applied on genetic and geographic distance matrices. However, significance levels were too low to further use the outcome for further trait investigation: these were in mean for all ecological guilds only 7.5, 9.4, or 15.6% for PCNM + PCA, NMDS + PCA, and Mantel test, respectively, or at best 28% for a single guild. Our study confirmed that certain ecological traits were associated with higher species diversity and foster stronger genetic differentiation. Results suggest that increased numbers of species, sampling localities, and specimens for a chosen area of interest may give new insights to explore barcode data and species ecology for the scope of conservation on a larger scale. We performed a meta-analysis of available barcode data of central European beetles to detect intraspecific genetic patterns among ecological groups in relation to geographic distance, regarding feeding style, body size, as well as habitat and biotope preferences. Our study confirmed that certain ecological traits were associated with higher species diversity and foster stronger genetic differentiation. However, significance levels were too low to further use the outcome for further trait investigation.

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 Platyarthrushoffmannseggii Brandt, 1833. The number of BINs per species ranged from one (32 species, 68%) to a maximum of six for Trachelipusrathkii (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.

The dark side of pseudoscorpion diversity: The German Barcode of Life campaign reveals high levels of undocumented diversity in European false scorpions.

DNA barcoding is particularly useful for identification and species delimitation in taxa with conserved morphology. Pseudoscorpions are arachnids with high prevalence of morphological crypsis. Here, we present the first comprehensive DNA barcode library for Central European Pseudoscorpiones, covering 70% of the German pseudoscorpion fauna (35 out of 50 species). For 21 species, we provide the first publicly available COI barcodes, including the rare Anthrenochernes stellae Lohmander, a species protected by the FFH Habitats Directive. The pattern of intraspecific COI variation and interspecific COI variation (i.e., presence of a barcode gap) generally allows application of the DNA barcoding approach, but revision of current taxonomic designations is indicated in several taxa. Sequences of 36 morphospecies were assigned to 74 BINs (barcode index numbers). This unusually high number of intraspecific BINs can be explained by the presence of overlooked cryptic species and by the accelerated substitution rate in the mitochondrial genome of pseudoscorpions, as known from previous studies. Therefore, BINs may not be an appropriate proxy for species numbers in pseudoscorpions, while partitions built with the ASAP algorithm (Assemble Species by Automatic Partitioning) correspond well with putative species. ASAP delineated 51 taxonomic units from our data, an increase of 42% compared with the present taxonomy. The Neobisium carcionoides complex, currently considered a polymorphic species, represents an outstanding example of cryptic diversity: 154 sequences from our dataset were allocated to 23 BINs and 12 ASAP units.

Coverage and quality of DNA barcode references for Central and Northern European Odonata.

BACKGROUND: Dragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches. METHODS: This study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD. RESULTS: Over 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

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.

A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding-based biomonitoring.

This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species-level assignment, so called "dark taxa." Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the "taxonomic impediment"; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species-rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.

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

BACKGROUND: 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. NEW INFORMATION: 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.

Towards a DNA Barcode Reference Database for Spiders and Harvestmen of Germany.

As part of the German Barcode of Life campaign, over 3500 arachnid specimens have been collected and analyzed: ca. 3300 Araneae and 200 Opiliones, belonging to almost 600 species (median: 4 individuals/species). This covers about 60% of the spider fauna and more than 70% of the harvestmen fauna recorded for Germany. The overwhelming majority of species could be readily identified through DNA barcoding: median distances between closest species lay around 9% in spiders and 13% in harvestmen, while in 95% of the cases, intraspecific distances were below 2.5% and 8% respectively, with intraspecific medians at 0.3% and 0.2%. However, almost 20 spider species, most notably in the family Lycosidae, could not be separated through DNA barcoding (although many of them present discrete morphological differences). Conspicuously high interspecific distances were found in even more cases, hinting at cryptic species in some instances. A new program is presented: DiStats calculates the statistics needed to meet DNA barcode release criteria. Furthermore, new generic COI primers useful for a wide range of taxa (also other than arachnids) are introduced.

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.

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, Docosia 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.