Robin Schütz

Freshwater biodiversity is declining at a pace that outstrips the capacity of existing monitoring approaches both in temporal and spatial dimensions, highlighting the urgent need for rapid and scalable assessment and attribution of biodiversity states and changes. Here we present a global assessment and unified analysis of riverine fish biodiversity using environmental DNA collected from 1,818 sites across 113 river systems spanning 5 continents. We quantified species richness, functional redundancy, phylogenetic diversity and genetic sequence diversity, and related them to drainage characteristics. Our results showed that environmental DNA effectively captured global patterns of multi-faceted riverine fish biodiversity and disentangled the roles of climate and different aspects of human activities in shaping biodiversity–area relationships. The accumulation of biodiversity with catchment area was consistently enhanced in warmer climates, while human activities weakened this scaling. Further, species richness, functional and genetic sequence diversity exhibited stronger negative responses to human activities in larger catchments. In contrast, phylogenetic diversity was negatively affected by human activities, yet this effect diminished as catchment area increased, highlighting the facet-dependent nature of biodiversity responses to environmental gradients and catchment characteristics. Our findings demonstrate the power of environmental-DNA-based datasets for harmonized, multi-faceted biodiversity assessments, offering a scalable approach for detecting and attributing biodiversity change and informing conservation strategies under accelerating global change.

The analysis of environmental DNA (eDNA) has revolutionized biodiversity assessments in aquatic ecosystems, enabling non‐invasive monitoring of fish communities across diverse regions. However, the global comparability of these eDNA datasets remains ambiguous due to heterogeneous sampling protocols and bioinformatic workflows across studies, making it difficult to assess how robust and comparable the biodiversity patterns inferred from these datasets actually are. Here, we conducted a meta‐analysis of 58 riverine fish eDNA metabarcoding studies, covering 1818 sampling sites worldwide, to evaluate the robustness of eDNA‐derived biodiversity patterns. We found that species richness estimates and metrics of community structure derived under a common bioinformatic workflow were overall consistent with those of original analyses, despite the relatively high variability in bioinformatic analyses in the respective original studies. Contrastingly, congruence of species identity varied more extensively across datasets, mostly reflecting different completeness and regional relevance of reference databases. Restricting taxonomic assignment to basin‐specific species pools improved species identification accuracy, while datasets lacking publicly accessible or well‐curated reference data were more prone to mismatches. Year of sampling had a positive effect on taxonomic congruence, such that more recent studies showed increased robustness, also reflecting improved reference database coverage and enhanced species‐level identification over time and overall method congruence in more recent years. Overall, the suitability and potential of eDNA for global biodiversity monitoring is corroborating overall robust biodiversity estimates, irrespective of the bioinformatic approaches. Our study underlines the effectiveness and need for further harmonization of bioinformatic workflows and strengthened region‐specific reference databases for improved taxonomic resolution and comparability across studies.

Long-term biodiversity data are critical for assessing, understanding, and predicting ecosystem change globally, but are often limited by their availability. We present the longest eDNA metabarcoding time series for freshwater fishes based on archived suspended particulate matter (SPM) from Germany's Environmental Specimen Bank, covering 17 years (2005-2021) across six major European river systems (Rhine, Danube, Elbe, and their tributaries). From 211 annual samples collected according to highly standardized procedures at 13 sites, we detected 63 fish and lamprey species and traced their spatio-temporal trajectories. eDNA metabarcoding captured significantly declining trends at multiple sites with no recoveries. Common and sensitive species declined at nearly half of sites while invasive species spread. Site occupancy and relative read abundance trends were correlated, providing semi-quantitative indicators. Alpha and beta diversity shifted over time and Bayesian hierarchical models revealed that 19% of species showed significant declines in relative read abundance. The eDNA-derived trends showed strong concordance with long-term regulatory fish monitoring data, validating the patterns across methodological approaches. Random forest models and multivariate analyses identified multiple anthropogenic pollutants, measured from the same SPM samples, as drivers of biodiversity patterns. Pesticides, heavy metals, temperature, and fine sediment negatively affected species richness, with site-specific stressor combinations explaining 29% of community variation. Our results demonstrate that archived SPM, originally collected for chemical monitoring, provides a reliable, dual-purpose monitoring matrix integrating pollution and biodiversity trend assessments simultaneously. This approach enables holistic monitoring of global freshwater ecosystem change and can provide an early warning framework supporting evidence-based environmental management.

Environmental DNA (eDNA) metabarcoding has lagged in parasite biodiversity assessments. We implemented this method to examine parasite diversity in sediment and water from 4 physically connected aquatic habitats in coastal South Carolina, USA, as part of a ParasiteBlitz in April 2023. Sediment was collected using a syringe corer, and water was sampled using active filtration and passive collection. Five amplicon libraries, using primers targeting portions of the mitochondrial COI of platyhelminths and 18S ribosomal RNA genes of nematodes, myxozoans, microsporidians, and protists, successfully yielded parasite sequences. Out of >5.8 million sequences, we identified >1,000 parasite amplicon sequence variants (ASVs) corresponding to ~600 parasite operational taxonomic units, from 6 parasite groups. Most diversity was observed among the microsporidians, whose assay demonstrated the highest fidelity. Actively-filtered water samples captured ASVs of all 6 groups, whereas sediment captured only 4, despite yielding 3× as many ASVs. Low DNA yields from passive water samples resulted in fewer, but some unique, ASVs representing 3 parasite groups. The most efficient sampling method varied with respect to parasite group across habitats, and the parasite communities from each habitat were distinct regardless of sampling method. We detected ASVs of 9 named species, 4 of which may represent introductions to the US. The abundance of our results demonstrates the effectiveness and efficiency of eDNA metabarcoding for assessing parasite diversity during short, intensive surveys, and highlights the critical need for more comprehensive sequence databases and the development of primers for those parasite taxa that elude detection using eDNA methods.

Environmental DNA (eDNA) metabarcoding has become a prominent method in aquatic biodiversity research and monitoring. A widely underexplored sample type is suspended particulate matter (SPM), composed of small organic and inorganic particles that contain eDNA. SPM is typically collected with sedimentation boxes, for instance by the German Environmental Specimen Bank for chemical monitoring of rivers. Its standardized collection and storage make SPM a potentially ideal source for eDNA time series analysis, linking environmental pressures and responses. This study evaluated key steps in the laboratory workflow allowing for the implementation of SPM in biodiversity research and monitoring. After validating an in‐house extraction protocol, we extracted eDNA from three 12‐year‐old SPM samples representing three river sites. To quantify replicate variation in species detection, each sample was processed with three subsamples, four extraction replicates per subsample, and four PCR replicates per extraction replicate (48 replicates per SPM sample). Additionally, mechanically ground samples were investigated to test for decreased inter‐sample variation. Our analysis revealed extensive, and plausible taxa lists for fish and aquatic invertebrates with significant differences between the sites. Within sites, we found high stochasticity in species detection of all replicates with the highest similarity between PCR replicates. In agreement with this, a Bayesian occupancy model demonstrated that species detection probability decreases in the PCR, while replicates earlier in the workflow were more effective in increasing species richness detection compared to PCR replicates or further increasing sequencing depth. Generally, 4–8 replicates were sufficient to recover 50% of all species, which contributed over 90% of the reads. Based on our results, we recommend at least eight replicates for biodiversity assessments using SPM. These should include at least one subsample or extraction replicate to maximize species richness. Our study supports the future use of SPM samples in biodiversity monitoring and provides a framework for assessing other eDNA matrices for robust species detection.

Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for methodological standardization. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.

Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. The demand for methodological standardization and the implementation of eDNA-based assessments into the regulatory monitoring (e.g., Water Framework Directive) are imminent. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for a comprehensive fish monitoring is the choice of suitable primer pairs to accurately depict the present fish fauna. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial community composed of DNA from 45 Central European fish species and examined the discriminatory power and reproducibility of five fish primer pairs. Our study highlights the effect of the primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair proved to be best choice for eDNA metabarcoding of Central European freshwater fish. Here, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs also displayed good discriminatory power and reproducibility. However, more general primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.

Forest canopies are highly diverse ecosystems, but despite several decades of intense research, there remain substantial gaps in our knowledge of their biodiversity and ecological interactions. One fundamental challenge in canopy research is the limited accessibility of the ecosystem. Consequently, previous studies have relied on the application of either highly invasive methods such as chemical knockdown, or on time‐consuming and expensive setups such as canopy walkways or cranes. Therefore, time‐ and cost‐efficient, ideally minimally invasive yet comprehensive applications are required to help close this knowledge gap. High‐throughput metabarcoding of environmental DNA (eDNA) collected from water, soil, or air provides a minimally invasive method for biodiversity assessment, yet its potential for canopy biodiversity monitoring has not been explored. Herein, we conducted metabarcoding of eDNA washed off the canopy via rainwater to explore its potential for biodiversity monitoring and ecological research. We placed four 1 m² rain samplers beneath the canopies of four different trees (beech, oak, larch, and pine) prior to a major rain event, filtered eDNA from the collected rainwater, and performed cytochrome c oxidase subunit I (COI) gene metabarcoding to profile the invertebrate community. Additionally, we collected and identified all specimens present in the rainwater to assess if eDNA only came from specimens physically present in the rainwater. We detected 50 invertebrate species by eDNA metabarcoding, of which 43 were not physically present in the water sample, thus likely representing true canopy biodiversity signals. Furthermore, we observed distinct species occurrence patterns corresponding to the four trees, suggesting that ecological patterns such as host specificity can potentially be assessed using the method. In conclusion, our study provides a proof of principle that rainwash eDNA metabarcoding offers a minimally invasive and comprehensive method for biodiversity monitoring in tree canopies.

Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can collect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., when drinking). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2 km stretch of the river Mulde (Germany), we collected 18 1-L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 22.2%, and 7.4% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 24.8% more detected fish and lamprey species, mammal, and bird species richness increased disproportionately by 68.9% and 77.3%, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called ‘bycatch’. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy is chosen carefully. With the increased use of eDNA metabarcoding as part of national fish bioassessment and monitoring programs, the complimentary information provided on bycatch can be used for biodiversity monitoring and conservation on a much broader scale.

Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because freshwater ecosystems act as sinks in the landscape, they also collect traces of terrestrial species via surface runoff or when specimens come into direct contact with water (e.g., for drinking purposes). Thus, fish eDNA metabarcoding data can provide information on fish but also on other, even terrestrial vertebrate species that live in riparian habitats. This data become available and may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2-km stretch of the river Mulde (Germany), we collected 18 1L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 24% and 7% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 25 % more detected fish and lamprey species, mammal and bird species richness increased disproportionately by 69 % and 84 %, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true, when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called ‘bycatch’. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy regarding biological replication is chosen carefully. With the consideration of frequent eDNA metabarcoding as part of national biomonitoring programs, the additional information provided by the bycatch can be used to further investigate the state of the environment and its biodiversity on a much broader scale.