Introduction
The Water Framework Directive (WFD) outlines the requirements of the European Commission for regular assessment of European surface waters (Bengtsson et al., 2012), as a measure to monitor and ensure that water bodies of the member states have a high ecological quality. A large number of different protocols applicable to the assessment of water bodies by the member states are based on a diverse array of morphologically based descriptions of macroinvertebrates to plant life combined with chemical/physical parameters are currently implemented for assessment (Birk et al., 2012). This diversity in methodology has shown the need for cross comparison and standardisation to obey central directives. Furthermore, current assessment protocols rely heavily on invasive sampling and fauna identification by taxonomic experts, which is both time-consuming and costly (Leese et al., 2016). One example of stream ecosystem assessment protocols is the Danish Stream Fauna Index (DSFI) (Skriver, Friberg, & Kirkegaard, 2000). This method uses macroinvertebrate diversity as Biological Quality Elements (BQE) (Agouridis, Wesley, Sanderson, & Newton, 2015; Elbrecht & Leese, 2017). The water quality of the stream ecosystems are categorised into one of seven groups, ranging from poor (1), to very good (7) (Skriver et al., 2000). The DSFI categories translate to the ecological status classes as described by the WFD (Baattrup-Pedersen et al., 2004).
It has been proposed that bioassessment methods can be improved through the use of molecular techniques such as metabarcoding (Blackman et al., 2019; Pawlowski et al., 2018). Next generation sequencing (NGS) has become an increasingly mainstream and convenient approach to perform analysis of diverse ecosystems, The use of standardised DNA barcoding based on universal genetic markers allows for the identification of species through sequence data (Leese et al., 2016). Previous studies have yielded generalised protocols for sampling and DNA extraction from marine sediments (Aylagas, Mendibil, Borja, & Rodríguez-Ezpeleta, 2016), as well as broad range primer sets targeting invertebrate biomarkers (Elbrecht & Leese, 2017), laying the foundation for high-throughput assay development. Recently, an amplicon sequencing approach based on targeting the cytochrome c oxidase I (COI ) gene was developed and successfully applied as an alternative method of performing water quality assessments in streams (Kuntke, de Jonge, Hesselsøe, & Nielsen, 2020).
The drawback of molecular approaches for bioassessment is that design of barcoding targets can be problematic. Often not all of the desired diversity can be captured in a single barcode (i.e. one DNA amplification). Therefore, no consensus on genes and specific barcodes has been reached so far (Leese et al., 2016). Samples collected for fauna indexing according to water quality assessments contain DNA from all materials present within the ecosystem, including soil, water, plants and organisms inhabiting the sampling site. By choosing a universal approach that captures the majority of diversity present in a sample, it could potentially be possible to report on the state and dynamics of entire ecosystem, rather than focusing on a single source of DNA in the sample. Microbial communities of stream water ecosystems have previously been shown to correlate with land usage, and the status of the stream environment (Lear et al., 2013). Furthermore, quality assessment of ground and stream water via detection of selected microbial indicators such as Escherichia coli andClostridium perfringens has also shown promising results (Francy, Helsel, & Nally, 2000). Moreover, the superior size of bacterial and archaeal diversities have previously been linked to changes in ecosystem quality after an oil spill (Urakawa, Garcia, Barreto, Molina, & Barreto, 2012). Collectively, these findings suggest that microbial community studies may present a promising approach for fast, accurate and cost effective bioassessment of freshwater ecosystems.
In the present study we characterise the complete biome profile of 53 bulk samples collected from freshwater streams across Denmark. The results are aligned with the pre-determined ecological quality status based on conventional water quality assessment using macroinvertebrate composition as BQE. A high-throughput amplicon sequencing approach using a universal primer set that targets all three domains (Bacteria ,Archaea and Eukaryota ) was chosen for this purpose. The potential of whole biome analysis for ecosystem quality determination is assessed. Furthermore, taxa from all three domains with potential as indicators for ecological status were explored.