In GBOL III: Dark Taxa five partners form the consortium

The Zoological Research Museum Alexander Koenig (ZFMK) leads the consortium and has the task of the general coordination and public outreach. The research at ZFMK covers Figitidae and Eurytomidae (Hymenoptera), Psychodidae and Limoniidae (Diptera) and some further taxa. Additionally, ZFMK will apply itself to the methods of “Taxonomics”. Within two further subprojects the aims are to update the databases and develop an OTU platform, as well as develop new and optimized approaches to processing poor-quality samples and to taxonomics

The State Museum of Natural History Stuttgart (SMNS) will focus on the taxa of Platygastroidea and Ceraphronoidea (Hymenoptera), Phoridae and Sciaridae (Diptera) and further taxa. Additional foci are protocols in processing bulk samples and public relations

The Bavarian State Collection of Zoology (ZSM-SNSB) in Munich will study the Ichneumonoidea and Diapriidae (Hymenoptera), the Chironomidae and Cecidomyiidae (Diptera) and further taxa. Further, the ZSM-SNSB will develop an enhanced and accelerated pipeline for scientific publications and the analyses for big data

The application study in the Bavarian Forest, conducted by the Department of Animal Ecology and Tropical Biology of the University of Würzburg will investigate the role of parasitoids during pest outbreaks.

The Entomological Society of Krefeld has the task of public relation and dissimination of results. Additionally they will bring samples (“Krefeld Proben”) to the project that document the insect decline.

Subproject 1 at the Zoological Research Museum Alexander Koenig in Bonn

Like in previous GBOL funding periods, the ZFMK leads and coordinates the consortium. The aims of the work at ZFMK will be embedded in the overall aims of GBOL III: Dark Taxa, i.e., to expand the barcode reference libraries, to increase the knowledge on the selected taxonomic groups and to train a new generation of taxonomists. These goals will be achieved by a dedicated study of the German fauna of these groups using external taxonomic expertise and expertise within the consortium, relying on already available material and newly collected samples, re-evaluating all available information, and using a wide arsenal of state-of-the-art integrative taxonomic methods as well as developing and exploring additional methods.

We will specifically target the German fauna of two groups of parasitoid Hymenoptera and two groups of Diptera that are currently considered dark taxa because any baseline information for these groups – for Germany – is absent, fragmentary, inaccessible or severely error-prone. The targeted taxa at ZFMK are the families Figitidae (Cynipoidea) and Eurytomidae (Chalcidoidea), both parasitoid Hymenoptera, and the dipteran families Psychodidae and Limoniidae. Additional groups are the Ormyridae, the Eupelmidae and the Chalcididae (Hymenotera) as well as the Keroplatidae and Pediciidae (Diptera).

Additional Project at ZFMK I: Extending the GBOL web portal: Becoming an online repository and research environment for Metabarcoding

The web portal: The GBOL web portal provides a reference library for barcodes of plants, animals and fungi from Germany. Besides being a central point for accessing genetic barcodes and using them for barcode comparison and species identification, it provides access and search functionalities for specimen occurrences, images, red list classification, abundance, and taxonomies. It supports citizen scientists and collectors of species by organizing submission of samples and data and by providing advice for specimen collection. Within the first six years of the GBOl project the web portal was established and became an essential component for the communication within the community.

Metabarcoding: An essential tool for accessing biodiversity is metabarcoding. It is supported by barcode reference databases like the Barcode of Life Data Systems (BOLD) and GBOL for the identification of DNA sequences, resulting in a growing demand to make barcoding databases and portals ready for data accompanied with sample collection, barcode analysis and taxon assignment.

However, there is yet no possibility to to include data from metabarcoding, including the derived Operational Taxonomic Unit (OTU) or Amplicon Sequence Variants (ASV) tables. For a comparison of both see: Currently, most metabarcoding projects deposit their raw data (i.e. the sequences produced by the sequencers) in one of the International Nucleotide Sequence Databases like NCBI or ENA and additional information like the ASV/OTU tables in data depositories like Dryad, or as supplemental material to journals. This hampers the re-use of the research data and results, as new samples can not easily be integrated into existing tables, taxonomic assignments can not be updated and no comparison between different tables is possible.

We will extend the functionalities of the web portal for the requirements of metabarcoding within the database project of GBOL phase III “Dark Taxa”:

Registry service for ASV sequences: WP 1 will set up a repository for ASV/OTU sequences and ASV/OTU tables. This will decrease the threshold for re-analyzing metabarcoding data. Starting from the raw data the current GBOL reference database will be extended to register and ASV sequences, assign unique and stable IDs to each sequence based on their genetic locus, as well as to allow for the storage, dissemination and updating of ASV tables, including extended search functionalities.

With these extensions to the GBOL web portal, it will be the first tool available which allows the users/scientists to store ASV tables instead of raw data and, thereby, increase the reproducibility of the results from metabarcoding studies.

Dynamic Taxon annotation: A special focus is on the development of tools for dynamic taxon annotation and OTU classification. These will allow to make OTU sequences and tables comparable over the time and to track changes on OTU assignment while the information attached to OTUs can be held up to date. The application and stable identifiers (e. g. DOIs) and a versioning system will make the ASV/OTU tables and sample data findable and citable in the long term. Advanced search and analysis tools that incorporate data from external databases like BOLD, ENA will enhance the functionalities to compare and analyse sequences and visualize occurrence data in the GBOL webportal.

Additional Project at ZFMK II: New and optimized approaches to poor-quality samples and taxonomics

In GBOL III: Dark Taxa we will inevitably face two problems when studying, handling and sequencing our target taxa: 1) for some species, we will have only few, small and possibly degraded samples at hand; 2) some taxonomic and species delimitation issues will not be possible to solve with morphology and DNA barcode data.

In both cases, target DNA enrichment may help us. Target DNA enrichment uses oligonucleotide probes or ‘baits’, followed by high throughput sequencing, to acquire DNA sequences from target genes. It was originally designed to specifically sequence disease-associated markers in human samples and has frequently been used to compile phylogenomic datasets but is now also increasingly used to get DNA barcoding data in metabarcoding approaches to environmental samples and in studies using ancient and museum DNA. In GBOL III: Dark Taxa, we will use it to access the available DNA of species for which fresh specimens cannot be collected in the field. DNA barcodes from dry-pinned museum specimens and other suboptimal material will in many cases be the only possibility to add reference sequences of species to the GBOL database. We aim at doing this in a non-destructive manner also for precious (e.g., type) specimens.

Furthermore, target DNA enrichment can be used to enrich and sequence hundreds of genes for so-called taxonomics approaches. These will be applied to selected taxonomically challenging taxa. The ZFMK is currently developing a universal taxonomics approach (; Eberle et al. 2020 DOI: 10.1016/j.tree.2019.12.003), and GBOL III: Dark Taxa can participate in its theoretical and practical framework, trying to solve our difficult cases. 

The GBOL III: Dark Taxa project will build a dedicated pipeline specialized on DNA barcoding of poor-quality material, based on extensive testing of available and modified protocols, and will also help generating taxonomics datasets in an optimized way.

Subproject 2 at the State Museum of Natural History Stuttgart

The State Museum of Natural History Stuttgart (SMNS) will focus on the German diversity of four Dark Taxa of Hymenoptera (Ceraphronoidea, Platygastroidea) and Diptera (Sciaridae, Phoridae). The aims are to expand the barcode libraries for these taxa, train a new generation of  taxonomic  experts,  describe  new  species,  explore  species  trees  and  speciation  patterns, investigate the ecological diversity of species, document new morphological character sets, develop identification keys and disseminate results through scientific and popular channels. Additionally work on groups from previous phases of GBOL within Hymenoptera (Mymaridae, Perilampidae, Pteromalidae, Torymidae, Trichogrammatidae) will continue, to further expand the existing database. In Diptera, the families of Tachinidae and Sarcophagidae will also be targeted and a voucher collection of Dipteran larval stages is will be built, utilizing DNA barcoding and morphology.

The SMNS will process material from the previous phases of GBOL and recent monitoring projects in SW Germany amongst the shared material within the consortium for their projects. Additional targeted sampling carried out in Baden-Württemberg using Malaise, yellow pan, pitfall and baited traps will also allow comprehensive coverage of soil- associated species and targeted data collection on habitat preferences. Samples will be sorted into small and large fractions using semi-automated sieving. The SMNS will collate and analyze information on workflows and methods from all GBOL I, II and III projects, developing written protocols for mass processing of samples and voucher specimens for biodiversity discovery initiatives on hyperdiverse taxa.

The SMNS is also going to host a training workshop on integrative taxonomy and nomenclature for all students involved in GBOL III: Dark Taxa projects. Additionally, a species conference summarizing the results of all Dark Taxa work packages and offering the opportunity for all PhD and MSc students to present their work will be organized at the end of the projects runtime.

Subproject 3 at the Bavarian Natural History Collections

Throughout GBOL III: Dark Taxa, the ZSM-SNSB in Munich will focus on (1) generating high numbers of DNA barcodes of the selected dark taxa, (2) creating protocols and, inter alia, applying metabarcoding approaches to efficiently screen and recover rare or target species from bulk samples, (3) applying big data analyses for biomonitoring and biodiversity studies, and (4) establishing an innovative open access web publishing workflow. In total, three dipteran and five hymenopteran dark taxa will be handled and revised via integrative taxonomy. 12 national and international specialists will be involved in the project to provide identifications and to improve the quality of taxonomic training offered to the PhD students.

Subproject 4 at the Department of Animal Ecology and Tropical Biology, University of Würzburg

The regular outbreaks of the Gypsy Moth (Lymantria dispar) cause severe defoliation in oak and mixed-oak forests. For this reason large forest areas are treated with insecticides to control their propagation. In 2019 and 2020, ww collected more than 20.000 caterpillars via canopy fogging of whole oak trees in oak-mixed forest sites with high and low densities of Gypsy Moths. Furthermore, half of the forest sites were sprayed with the insecticid mimic. We want to identify the community cryptic (hyper-) parasitoids in the caterpillars via metabarcoding to measure the role of the parasitoids during an outbreak of the Gypsy moth. Additionally, we want to calculate caterpillar – parasitoid networks and how they react to the Gypsy Moth density and to the treatment with the insecticde.

Raupe eines Geometriden mit Parasitoiden

Subproject 5 of the Entomological Society Krefeld e.V.

Location and structure: The EVK – founded in 1905 – is a registered association with an impressive tradition of scientific entomological research. The EVK is one of the typical scientifically oriented societies founded especially in Germany in the period between 1880-1920.  The headquarters as well as the collections and the archive of the society are located in Krefeld. Throughout society’s history, members have produced well over two thousand publications on entomological topics. To date, three honorary doctorates and numerous scientific prizes have been awarded to EVK entomologists.

Focus: Research on Systematics and Phylogeny, Ecology and Behaviour, entomological Methodology, outstanding taxonomic expertise and unique experience with long-term monitoring using malaise traps and the standardization of the trap technology.

Size of Institution: Currently about 70 entomologists as members and additional about 30 external entomologists in the working groups. The EVK is responsible for the development and curatorial preservation of one of the important museal reference collections on entomology in Germany (Entomological Collections Krefeld).

Previous experience: The members of the EVK are specialists in the biology and systematics of insects. The EVK has many years of expertise in insect monitoring and the creation of species inventories, habitat profiles, mapping, nutritional representation, behaviour and general biology of insects, as well as the creation of identification keys. The association has been working for decades in cooperation with diverse Universities and scientific institutes without geographical limitation and as a contractor for lower and higher landscape authorities, regional environmental offices and foundations. This also affects the field of work at the interface between area-related entomological species inventories and nature conservation and development planning. With regard to the collection and evaluation of biodiversity data about insects of various orders, the Entomological Society Krefeld is currently dealing with a R&D project on biodiversity loss in FFH Annex I habitat types and the changes in biodiversity of selected higher taxa in long-term comparison. Of particular relevance and a result of this project is the evaluation published 2017 of  one of the best monitoring dataset on insects. The EVK is the only institution that has been using malaise traps in a strictly standardized methodology over decades. There is no comparable data and long-term experience anywhere else in Europe.

Participation within GBOLIII: Dark Taxa: More than 30 entomologists with many years of practical experience in the methodology and for various insect taxa including coordination by: Dr. Martin Sorg & Thomas Hörren.