The glacial lakes in the Tatra Mountains (Western Carpathians, Slovakia) and their invertebrate fauna have been attracting scientific attention for over a century. This relatively small area with numerous steep valleys, stretching across vegetation zones over an altitudinal gradient, offers a promising basis to study the impact of changing environmental conditions on species composition and distribution. A typical example documented throughout the last decades is acidification and subsequent recovery of the lakes upon switching to less polluting industrial practices. The ongoing climate change poses yet another challenge to the unique biota inhabiting these pristine alpine ecosystems. Thus, their biodiversity requires further monitoring, which can take advantage of DNA metabarcoding as a means to circumvent the drawbacks of morphological identification and manual sorting of samples. Here, we present outcomes of such study, focusing on Tatra Mountain valleys where lakes are dispersed throughout the altitudinal gradient. Littoral benthos was sampled from 17 lakes by the kicking technique in September 2017, 2019–2022. Organisms were picked out from each sample fixed with ethanol, dried, homogenised in a bead mill, subjected to spin-column DNA extraction, the COI marker amplified using BF3/BR2 PCR primers, and sequenced on the Illumina MiSeq platform. The sequences were processed using the Cutadapt, PEAR, VSEARCH, LULU and BOLDigger algorithms, obtaining taxonomic identification for the resulting OTUs (operational taxonomic units, clustered at the species-level 97% sequence identity). The patterns in community composition were analysed through TaxonTableTools, R packages Vegan and indicspecies, also relying on a rich dataset of the lakes’ chemical and physical parameters. Upon revision and abundance filtering, the dataset contained 537 invertebrate OTUs, where 199 aquatic species were identified. Altitude was reflected considerably well in the metabarcoding ordination diagrams, whose first two axes explained 17.5% of variability in the overall distance matrix, or up to 55% of variability per individual valleys. However, several lakes broke the altitudinal order, rather reflecting the importance of overall lake conditions and nutrient content (P, organic C and N). Possible indicator species were highlighted both for the alpine cluster (OTUs identified as Pseudodiamesa nivosa, Micropsectra radialis, Cernosvitoviella pusilla, Daphnia pulex) and for sub-alpine lakes (M. notescens, Prodiamesa olivacea, Cricotopus sp., Agabus bipustulatus, Nemoura cinerea). The approach proved to be remarkably efficient for ecosystem assessment through simultaneous detection of a broad range of taxa including benthic macroinvertebrates, planktonic crustaceans, meiofauna, and numerous terrestrial groups. This study was supported by the project VEGA 2/0084/21.

With the establishment of the Water Framework Directive (WFD), the European Union has set the goal to achieve good ecological status for all water bodies in Europe. To assess the biological quality, biodiversity assessments are carried out with established morphology-based methods. However, traditional biomonitoring remains cost- and time-consuming. Here, DNA-based methods, such as DNA metabarcoding, have the potential as new complementary and efficient tools for regulatory biomonitoring. However, while DNA-based methods have been long established in the scientific community, a validation as a method for regulatory biomonitoring has to be conducted yet.

Therefore, the GeDNA project was initiated to leap the gap between the scientific community and the applied sector. One aim of the project is to evaluate DNA metabarcoding for the assessment of macroinvertebrates and the comparison to traditional methods. Therefore, macroinvertebrate bulk samples were collected in two WFD sampling campaigns (2020 and 2021) across Germany. All samples were morphologically identified by taxonomic experts and subsequently processed with DNA metabarcoding.

Results revealed high concordance in species composition for benthic invertebrates. DNA metabarcoding identified significantly more non-biting midge taxa and exhibited similar proportions of the important bioindicator ‘EPT’ taxa (mayflies, stoneflies, caddisflies: Ephemeroptera, Plecoptera, Trichoptera). Ecological status classes derived from invertebrate assessments showed high congruence between the two methods (Spearman rho = 0.86). The choice of abundance or presence/absence data had minimal influence on the assigned status classes for both BQEs.

In conclusion, this pilot study coordinated via federal, and state environmental agencies demonstrated high similarity between the methods for the stream types analysed, suggesting suitability of DNA metabarcoding of benthic invertebrates for WFD monitoring without major modifications.

In the current era of rapid ecosystem change, long-term biodiversity data pose the basis for quantifying trends, predicting consequences, and supporting management actions. However, long-term biodiversity data are scarcely available. A long-term sample collection that has been little explored so far is the German Environmental Specimen Bank (ESB), which contains terrestrial, freshwater, and marine samples collected for decades in a highly standardised manner. Samples are collected monthly or annually using standard operating procedures and are then stored in aliquots in cryotanks at -150°C. The ultra-cold storage condition preserves environmental DNA (eDNA) present in the samples, which is an ideal source for holistic biodiversity assessments. The TrendDNA project aims to test, if eDNA-based methods such as DNA metabarcoding and metagenomics are suitable for studying biodiversity change using the ESB samples. One of the ESB sample types is suspended particulate matter (SPM), collected from 13 different sites in the Rhine, Saar, Danube, Elbe, Mulde, and Saale rivers, dating back to 2005. SPM is collected monthly using sedimentation traps, which are then pooled into yearly homogenates. In the project, we are analysing 211 SPM samples from 6 rivers and 17 years using eDNA metabarcoding targeting fish and invertebrates. We highlight that eDNA data from the TrendDNA project provides comprehensive and plausible taxa lists, shows evidence of the introduction of invasive species, and unveils significant patterns of faunal community changes across years and sites. These results can aid in understanding biodiversity patterns, species turnover, tracking the invasion of species, assessing the progress of endangered species protection, and predicting future change. We provide an outlook on further project results and highlight the potential that the ESB can play in complementing long-term biodiversity monitoring in Germany.

Routine biomonitoring of freshwater ecosystems still follows a conventional methodology based on the primary morphological identification of the five biological quality elements (phytoplankton, macrophytes, phytobenthos, benthic invertebrate fauna and fish). In addition to the time and financial demands or often complicated morphological determination especially in megadiverse benthic invertebrates, this approach can also be problematic due to the methodical processing of the collected bulk samples. For example, the subsampling method causes the final analysis to be based on only a limited sample volume. Consequently, important bioindicator taxa may be overlooked, and the face of true biodiversity may be distorted, leading to an inaccurate ecological status assessment. Therefore, this study aimed to test the applicability of DNA metabarcoding at long-term monitored sites, aiming to streamline the process, remove the aforementioned shortcomings and thus increase the efficiency and scale of freshwater biomonitoring. 

Samples of benthic invertebrates were collected at 12 sites from different categories of water bodies. Without time-consuming bulk processing and morphological determination, the obtained final taxa lists based on metabarcoding data confirmed a 14% increase in identified species within the five target benthic classes (Insecta, Clitellata, Malacostraca, Bivalvia and Turbellaria) compared to previous conventional (WFD) sampling. Biological indices included in the multimetric evaluation of the ecological status were calculated using the program ASTERICS, and the same or higher ecological quality class was identified compared to previous assessments. More detailed indices analysis showed that the ecological quality class changed more significantly in 5 localities (from 2 to 1), mainly due to the influence of the BMWP and EPT taxa indices, which indicates a higher sensitivity of metabarcoding approach in detecting the present macroinvertebrates species spectrum. The results of this study provide useful data on the effectiveness of DNA metabarcoding and have a great potential to contribute to the implementation of DNA approaches in routine biomonitoring of freshwater ecosystems.