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      Taming the Wild West of Molecular Tools Application in Aquatic Research and Biomonitoring

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          Abstract

          Modern high-throughput sequencing technologies are becoming a game changer in many fields of aquatic research and biomonitoring. To unfold their full potential, however, the independent development of approaches has to be streamlined. This discussion must be fuelled by stakeholders and practitioners and, scientific results collaboratively filtered to identify the most promising avenues. Furthermore, aspects such as time, budget, skills and the application context have to be considered, finally communicating good practice strategies to target audiences.  Since 2016, the EU COST Action DNAqua-Net is taming the wild west of molecular tools application in aquatic research and biomonitoring. After nucleating available knowledge by the formation of a highly international and transdisciplinary network of scientists, stakeholders, practitioners and enterprises, fields of high methodological diversity were identified. Relevant aspects are currently ground truthed, thereby reducing the plethora of pipelines, parameters and protocols to a subset of good practices or standardisations. To effectively bridge the science-application interface, the very same network is exploited for the dissemination of results (Leese et al. 2018). The internal working group structure of DNAqua-Net is used to provide an overview of existing methodological fields of diversity in DNA-based aquatic biomonitoring: WG1 - DNA Barcode References: Different marker systems are targeted for the same organism group. Even in case the same molecular marker is investigated, different primer pairs are frequently applied for DNA metabarcoding. Both aspects challenge the further development of high-quality and complete DNA barcode reference libraries (Weigand et al. 2019). WG2 - Biotic Indices & Metrics: Index systems are developed from molecular data in various ways: from the estimation of species' biomass (as a proxy for abundance) from sequence reads, to the correlation of presence/absence data of molecular operational taxonomic units (MOTUs) with environmental parameters (Pawlowski et al. 2018). WG3 - Field & Lab Protocols: Using environmental DNA (eDNA) metabarcoding as an example, diverse sampling techniques based on varying water volumes, different filter systems and collection devices as well as a multitude of laboratory protocols for PCR, replication and sequencing are considered. WG4 - Data Analysis & Storage: During the process of MOTU identification, varying threshold values and conceptually different pipelines are used, potentially impacting the final list of MOTUs or species retrieved. Furthermore, routine storage concepts for big biodiversity data are only in development and some sample types (e.g. eDNA) have no sophisticated metadata descriptions. WG5 - Implementation Strategy & Legal Issues: The working group picks up collaboratively filtered good practice strategies and generates room for discussions at the science-policy interface (Hering et al. 2018). The CEN working group WG28 "DNA methods" has been initiated and the development of standardisations is fostered.

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          Most cited references 4

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          The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems.

          The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.
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            DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

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              Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive.

              Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to the WFD that may be required to facilitate the transition to molecular data.
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                Author and article information

                Journal
                Biodiversity Information Science and Standards
                BISS
                Pensoft Publishers
                2535-0897
                June 19 2019
                June 19 2019
                : 3
                Article
                10.3897/biss.3.37215
                © 2019

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