DNA Metabarcoding Methods for the Study of Marine Benthic Meiofauna

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars 2021.730063/full#supplementary-materialMeiofaunal animals, roughly between 0.045 and 1 mm in size, are ubiquitous and ecologically important inhabitants of benthic marine ecosystems. Their high species richness and rapid response to environmental change make them promising targets for ecological and biomonitoring studies. However, diversity patterns of benthic marine meiofauna remain poorly known due to challenges in species identification using classical morphological methods. DNA metabarcoding is a powerful tool to overcome this limitation. Here, we review DNA metabarcoding approaches used in studies on marine meiobenthos with the aim of facilitating researchers to make informed decisions for the implementation of DNA metabarcoding in meiofaunal biodiversity monitoring. We found that the applied methods vary greatly between researchers and studies, and concluded that further explicit comparisons of protocols are needed to apply DNA metabarcoding as a standard tool for assessing benthic meiofaunal community composition. Key aspects that require additional consideration include: (1) comparability of sample pre-treatment methods; (2) integration of different primers and molecular markers for both the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 18S rRNA genes to maximize taxon recovery; (3) precise and standardized description of sampling methods to allow for comparison and replication; and (4) evaluation and testing of bioinformatic pipelines to enhance comparability between studies. By enhancing comparability between the various approaches currently used for the different aspects of the analyses, DNA metabarcoding will improve the long-term integrative potential for surveying and biomonitoring marine benthic meiofauna.This work was funded by a BEN (Biodiversity-Ecology-Nature) grant (Number T0206/37197/2021/kg) of the Bauer-Hollmann foundation to J-NM

A new tool for faster construction of marine biotechnology collaborative networks.

The increasing and rapid development in technologies, infrastructures, computational power, data availability and information flow has enabled rapid scientific advances. These entail transdisciplinary collaborations that maximize sharing of data and knowledge and, consequently, results, and possible technology transfer. However, in emerging scientific fields it is sometimes difficult to provide all necessary expertise within existing collaborative circles. This is especially true for marine biotechnology that directly addresses global societal challenges. This article describes the creation of a platform dedicated to facilitating the formation of short or mid-term collaborative networks in marine biotechnology. This online platform (https://www.ocean4biotech.eu/map/) enables experts (researchers and members of the marine biotechnology community in general) to have the possibility to showcase their expertise with the aim of being integrated into new collaborations/consortia on the one hand, or to use it as a search tool to complement the expertise in planned/running collaborations, on the other. The platform was created within the Ocean4Biotech (European transdisciplinary networking platform for marine biotechnology) Action, funded under the framework of the European Cooperation in Science and Technology (COST). To build the platform, an inquiry was developed to identify experts in marine biotechnology and its adjunct fields, to define their expertise, to highlight their infrastructures and facilities and to pinpoint the main bottlenecks in this field. The inquiry was open to all experts in the broad field of marine biotechnology, including non-members of the consortium. The inquiry (https://ee.kobotoolbox.org/single/UKVsBNtD) remains open for insertion of additional expertise and the resulting interactive map can be used as a display and search tool for establishing new collaborations

eDNA metabarcoding of rivers: Is all eDNA everywhere, all the time?

Environmental DNA metabarcoding has become a popular tool for the assessment of freshwater biodiversity, but it is largely unclear how sampling time and location influence the assessment of communities. Abiotic factors in rivers can change on small spatial and temporal scale and might greatly influence eDNA metabarcoding results. In this study, we sampled three German rivers at four locations per sampling site: 1. Left river side, surface water 2. Right river side, surface water, 3. Left side, close to the riverbed, 4. Right side, close to the riverbed. For the rivers Ruhr and Möhne, sampling was conducted three times in spring, each sampling one week apart. The Ruhr was again sampled in autumn and the Gillbach was sampled in winter. Sequencing on an Illumina MiSeq with degenerate COI primers Bf2/BR2 revealed diverse communities (6493 Operational taxonomic units, OTUs), which largely differed between rivers. Communities changed significantly over time in the Ruhr, but not in the Möhne. Sampling location influenced recovered communities in the Möhne and in the Ruhr in autumn. Our results have important implications for future eDNA studies, which should take into account that not all eDNA in rivers is everywhere, and not at all times.peerReviewe

Supplementary material 1 from: Macher J, Macher T, Leese F (2017) Combining NCBI and BOLD databases for OTU assignment in metabarcoding and metagenomic datasets: The BOLD_NCBI _Merger. Metabarcoding and Metagenomics 1: e22262. https://doi.org/10.3897/mbmg.1.22262

Metabarcoding and metagenomic approaches are becoming routine techniquesfor use in biodiversity assessment and in ecological studies. The assignment of taxonomic information to millions of sequences obtained via high-throughput sequencing is challenging, as many DNA reference libraries are lacking information on certain taxonomic groups and can contain erroneous sequences. Combining different reference databases is therefore a promising approach for maximising taxonomic coverage and reliability of results. The "BOLD_NCBI_Merger" bash script is introduced, which combines sequence data obtained from the National Centre for Biotechnology Information (NCBI) GenBank and the Barcode of Life Database (BOLD) and prepares it for taxonomic assignment with the software MEGAN

Combining NCBI and BOLD databases for OTU assignment in metabarcoding and metagenomic datasets: The BOLD_NCBI _Merger

Metabarcoding and metagenomic approaches are becoming routine techniquesfor use in biodiversity assessment and in ecological studies. The assignment of taxonomic information to millions of sequences obtained via high-throughput sequencing is challenging, as many DNA reference libraries are lacking information on certain taxonomic groups and can contain erroneous sequences. Combining different reference databases is therefore a promising approach for maximising taxonomic coverage and reliability of results. The "BOLD_NCBI_Merger" bash script is introduced, which combines sequence data obtained from the National Centre for Biotechnology Information (NCBI) GenBank and the Barcode of Life Database (BOLD) and prepares it for taxonomic assignment with the software MEGAN

A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy

The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (<1 mm) in size and difficult to identify based on morphology. Metabarcoding approaches are promising for the study of these environments and taxa, but it is yet unclear if commonly applied metabarcoding primers and replication strategies can be used. In this study, we took sediment cores from two near natural upstream (NNU) and two ecologically improved downstream (EID) sites in the Boye catchment (Emscher River, Germany), metabarcoding their meiofaunal communities. We evaluated the usability of a commonly used, highly degenerate COI primer pair (BF2/BR2) and tested how sequencing three PCR replicates per sample and removing MOTUs present in only one out of three replicates impacts the inferred community composition. A total of 22,514 MOTUs were detected, of which only 263 were identified as Metazoa. Our results highlight the gaps in reference databases for meiofaunal taxa and the potential problems of using highly degenerate primers for studying samples containing a high number of non-metazoan taxa. Alpha diversity was higher in EID sites and showed higher community similarity when compared to NNU sites. Beta diversity analyses showed that removing MOTUs detected in only one out of three replicates per site greatly increased community similarity in samples. Sequencing three sample replicates and removing rare MOTUs is seen as a good compromise between retaining too many false-positives and introducing too many false-negatives. We conclude that metabarcoding hyporheic communities using highly degenerate COI primers can provide valuable first insights into the diversity of these ecosystems and highlight some potential application scenarios

Comparative mitogenomics of native European and alien Ponto-Caspian amphipods

European inland surface waters are home to a rich diversity of native amphipod crustaceans, many of which face threats from invasive Ponto-Caspian counterparts. In this study, we analyse mitochondrial genomes to deduce phylogenetic relationships and compare gene order and nucleotide composition between representative native European and invasive Ponto-Caspian taxa across five families, ten genera and 20 species (with 13 newly sequenced herein). We observe various gene rearrangement patterns in the phylogenetically diverse native species pool. Pallaseopsis quadrispinosa and Synurella ambulans exhibit notable deviations from the typical organisation, featuring extensive translocations of tRNAs and the nad1 gene, as well as a tRNA-F polarity switch in the latter. The monophyletic invasive Ponto-Caspian gammarids display a conserved gene order, primarily differing from native species by a tRNA-E and tRNA-R translocation, which reinforces previous findings. However, Chaetogammarus warpachowskyi shows extensive rearrangement with translocations of six tRNAs. The invasive corophiid, Chelicorophium curvispinum, maintains a highly conserved gene order despite its distant phylogenetic position. We also discover that native species have a significantly higher GC and lower AT content compared to invasive species. The mitogenomic differences observed between native and invasive amphipods warrant further investigation and could provide insights into the mechanisms underlying invasion success

Uncovering bacterial and functional diversity in macroinvertebrate mitochondrial-metagenomic datasets by diferential centrifugation

PCR-free techniques such as meta-mitogenomics (MMG) can recover taxonomic composition of macroinvertebrate communities, but sufer from low efciency, as >90% of sequencing data is mostly uninformative due to the great abundance of nuclear DNA that cannot be identifed with current reference databases. Current MMG studies do not routinely check data for information on macroinvertebrate-associated bacteria and gene functions. However, this could greatly increase the efficiency of MMG studies by revealing yet overlooked diversity within ecosystems and making currently unused data available for ecological studies. By analysing six ‘mock’ communities, each containing three macroinvertebrate taxa, we tested whether this additional data on bacterial taxa and functional potential of communities can be extracted from MMG datasets. Further, we tested whether diferential centrifugation, which is known to greatly increase efciency of macroinvertebrate MMG studies by enriching for mitochondria, impacts on the inferred bacterial community composition. Our results show that macroinvertebrate MMG datasets contain a high number of mostly endosymbiont bacterial taxa and associated gene functions. Centrifugation reduced both the absolute and relative abundance of highly abundant Gammaproteobacteria, thereby facilitating detection of rare taxa and functions. When analysing both taxa and gene functions, the number of features obtained from the MMG dataset increased 31-fold (‘enriched’) respectively 234-fold (‘not enriched’). We conclude that analysing MMG datasets for bacteria and gene functions greatly increases the amount of information available and facilitates the use of shotgun metagenomic techniques for future studies on biodiversity

Assessing the phylogeographic history of the montane caddisfly Thremma gallicum using mitochondrial and restriction-site-associated DNA (RAD) markers

Repeated Quaternary glaciations have significantly shaped the present distribution and diversity of several European species in aquatic and terrestrial habitats. To study the phylogeography of freshwater invertebrates, patterns of intraspecific variation have been examined primarily using mitochondrial DNA markers that may yield results unrepresentative of the true species history. Here, population genetic parameters were inferred for a montane aquatic caddisfly, T hremma gallicum , by sequencing a 658‐bp fragment of the mitochondrial CO 1 gene, and 12,514 nuclear RAD loci. T . gallicum has a highly disjunct distribution in southern and central Europe, with known populations in the Cantabrian Mountains, Pyrenees, Massif Central, and Black Forest. Both datasets represented rangewide sampling of T. gallicum . For the CO 1 dataset, this included 352 specimens from 26 populations, and for the RAD dataset, 17 specimens from eight populations. We tested 20 competing phylogeographic scenarios using approximate Bayesian computation (ABC ) and estimated genetic diversity patterns. Support for phylogeographic scenarios and diversity estimates differed between datasets with the RAD data favouring a southern origin of extant populations and indicating the Cantabrian Mountains and Massif Central populations to represent highly diverse populations as compared with the Pyrenees and Black Forest populations. The CO 1 data supported a vicariance scenario (north–south) and yielded inconsistent diversity estimates. Permutation tests suggest that a few hundred polymorphic RAD SNP s are necessary for reliable parameter estimates. Our results highlight the potential of RAD and ABC‐based hypothesis testing to complement phylogeographic studies on non‐model species

The mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus

Aquatic macroinvertebrates are often identified, based on morphology, but molecular approaches like DNA barcoding, metabarcoding and metagenomics are increasingly used for species identification. These approaches require the availability of DNA references deposited in public databases. Here we report the mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus, a European Union island country in the Mediterranean. Only three species could be provisionally assigned to a binomial species name, highlighting the current lack of molecular references for aquatic macroinvertebrates from Cyprus. Graphical Abstrac