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DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.

Reliable biodiversity data are crucial for environmental research and management. Unfortunately, data paucity prevails for many regions and organismal groups such as aquatic invertebrates. High-throughput DNA-based identification, in particular DNA metabarcoding, has accelerated biodiversity data generation. However, in the process of metabarcoding, specimens are usually destroyed, precluding later specimen-based analyses. Metabarcoding of DNA released into the preservative ethanol has been proposed as a non-destructive alternative, but proof-of-principle studies have yielded ambiguous results, reporting variance in detection probability for various taxa and methodological biases. In this study, we tested the performance of preservative-based metabarcoding of aquatic invertebrates in comparison to a standard morpho-taxonomic assessment based on samples from one of Europe’s last free-flowing rivers, the Vjosa. Multi-habitat samples were collected at 43 sites in two seasons and stored in ethanol, after fixation in formaldehyde for morpho-taxonomic analyses. Preservative-based DNA metabarcoding detected three times more taxa but failed to detect other taxa found using the standard method. In addition to incomplete reference data and primer bias that likely precluded the detection of specific taxa, preservative-based DNA metabarcoding cannot provide accurate abundance estimates. However, the metabarcoding data revealed distinct small-scale and large-scale community patterns in the Vjosa river network, which were also recovered by quantitative data of the standard approach. Overall, our results indicate that preservative-based metabarcoding provides important biodiversity data, which could be further improved by quantitative validation. The method is robust and reliable, even though samples were taken under harsh field-conditions and stored without cooling. Further, our results emphasise the need for reliable DNA barcoding reference libraries. Building those may be supported by preservative-based metabarcoding that maintains intact vouchers for subsequent specimen-based analyses.

In recent years, the decline of insect biodiversity and the imminent loss of provided ecosystem functions and services has received public attention and raised the demand for political action. The complex, multi-causal contributors to insect decline require a broad interdisciplinary and cross-sectoral approach that addresses ecological and social aspects to find sustainable solutions. The project Diversity of Insects in Nature protected Areas (DINA) assesses insect communities in 21 nature reserves in Germany, and considers interactions with plant diversity, pesticide exposure, spatial and climatic factors. The nature reserves border on agricultural land, to investigate impacts on insect diversity. Part of the project is to obtain scientific data from Malaise traps and their surroundings, while another part involves relevant stakeholders to identify opportunities and obstacles to insect diversity conservation. Our results indicate a positive association between insect richness and biomass. Insect richness was negatively related to the number of stationary pesticides (soil and vegetation), pesticides measured in ethanol, the amount of area in agricultural production, and precipitation. Our qualitative survey along with stakeholder interviews show that there is general support for insect conservation, while at the same time the stakeholders expressed the need for more information and data on insect biodiversity, as well as flexible policy options. We conclude that conservation management for insects in protected areas should consider a wider landscape. Local targets of conservation management will have to integrate different stakeholder perspectives. Scientifically informed stakeholder dialogues can mediate conflicts of interests, knowledge, and values to develop mutual conservation scenarios.

Insect declines and biodiversity loss have attracted much attention in recent years, but lack of comprehensive data, conflicting interests among stakeholders and insufficient policy guidance hinder progress in preserving biodiversity. The project DINA (Diversity of Insects in Nature protected Areas) investigates insect communities in 21 nature reserves in Germany. All selected conservation sites border arable land, with agricultural practices assumed to influence insect populations. We taught citizen scientists how to manage Malaise traps for insect collection, and subsequently used a DNA metabarcoding approach for species identification. Vegetation surveys, plant metabarcoding as well as geospatial and ecotoxicological analyses will help to unravel contributing factors for the deterioration of insect communities. As a pioneering research project in this field, DINA includes a transdisciplinary dialogue involving relevant stakeholders such as local authorities, policymakers, and farmers, which aims at a shared understanding of conservation goals and action pathways. Stakeholder engagement combined with scientific results will support the development of sound policy recommendations to improve legal frameworks, landscape planning, land use, and conservation strategies. With this transdisciplinary approach, we aim to provide the background knowledge to implement policy strategies that will halt further decline of insects in German protected areas.

Human-induced biodiversity loss and changes in community composition are major challenges of the present time, urgently calling for comprehensive biomonitoring approaches to understand system dynamics and to inform policy-making. In this regard, molecular methods are increasingly applied. They provide tools for fast and high-resolution biodiversity assessments and can also focus on population dynamics or functional diversity. If samples are stored under appropriate conditions, this will enable the analysis of DNA, but also RNA and proteins from tissue or from non-biological substrates such as soil, water, or sediments, so-called environmental DNA (eDNA) or eRNA. Until now, most biodiversity studies using molecular methods rely on recent sampling events, although the benefit of analyzing long-time series is obvious. In this context Environmental Specimen Banks (ESBs) can play a crucial role, supplying diverse and well-documented samples collected in periodically repeated sampling events, and following standardized protocols. Mainly assembled for integrative monitoring of chemical compounds, ESB collections are largely accessible to third parties and can in principle be used for molecular analysis. While ESBs hold great potential for the standardized long-time storage of environmental samples, the cooperation with Biodiversity Biobanks as scientific collections guarantees the long-time storage of nucleotide (DNA, RNA) extracts together with links to analytical results and metadata. The present contribution aims to raise the awareness of the biodiversity research community regarding the high-quality samples accessible through ESBs, encourages ESBs to collect and store samples in DNA-friendly ways, and points out the high potential of combining DNA-based approaches with monitoring chemicals and other environmental stressors.

Environmental DNA (eDNA) metabarcoding is a new, promising, and non-invasive method to detect biodiversity in aquatic environments. So far, it has mainly been used to screen for fish and amphibian diversity and rarely to detect macroinvertebrates. Typically, DNA metabarcoding relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. In comparison to other genes like 16S, COI has a greater taxonomic resolution and availability of an extensive reference database. Benthic stream invertebrates are of critical importance for regulatory biomonitoring, but when using universal primers on eDNA isolated from water, the number of reads and OTUs is “watered down”. This means the target taxa, macroinvertebrates, are underrepresented in comparison to other nontarget taxa, e. g. algae, bacteria, and fungi. The aim of the project was to design an insect-specific primer, which minimizes nontarget amplification. Therefore, data from a time series of 15 months at the Kinzig (Hesse), a silica-rich low-mountain-range stream, which is part of the Rhine‑Main‑Observatory (LTER site) was generated using the universal primers BF2/BR2. With this data we identified the most abundant nontarget taxa and designed a new reverse primer (EPTDr2n) with 3’ ‐ specificity toward benthic invertebrate taxa. Primer specificity was validated in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. 20 eDNA samples from the Kinzig River and its tributaries were then used to test the new primer in situ together with primer fwhF2. The new primer combination showed a much higher amplification of benthic invertebrates, insects in particular, than two other universal primer pairs for both, number of target reads (fwhF2/EPTDr2n: 99.6% versus BF2/BR2: 25.89% and fwhF2/fwhR2n: 39.04%; Fig. 1) and number of target species (fwhF2/EPTDr2n: 305 versus BF2/BR2: 113 and fwhF2/fwhR2n: 185). Additionally, the number of benthic invertebrate species exceeded even the number of 153 species identified by expert taxonomists at nearby sites across two decades of sampling. While several taxa reported, like a few trichopteran genera, flatworms, and some crustaceans, were not found, the primer shows greatly improved results for eDNA metabarcoding of benthic invertebrates(Leese et al. 2021).

We present a unique data set of trait information for 586 insect families in Central Europe, covering the largest known part of described species (over 34,000 species). Life history information and major functional traits were evaluated with fuzzy coding and weighted according to the number of known species in Germany. An overall analysis of the German insect fauna is given and the data set is exemplarily applied to metabarcoding results of malaise trap samples. Due to the high functional and taxonomic diversity in insects, further developments and refinements of traits to be included will be an ongoing process with advancements of upcoming database versions to be subsequently published.

CA extern Due to increasing human transformation of virtually all habitats on Earth, setting global priorities for conservation is essential. The emerging disciplines of macroecology and macroevolution (MEE) can provide a global perspective and information for such prioritization but remain relatively separated from conservation prioritization, partly because MEE researchers are unaware of the requirements for effective conservation prioritization and existing approaches implementing these. Indeed, existing approaches for conservation prioritization at large scale are scattered across literature. Here, we systematically review this literature and provide a guideline for researchers in MEE as to which of approaches might be suitable for their needs. From >11 000 scientific publications, we identified 134 methods suitable for commonly used MEE data and geographic scale. Furthermore, we use a ‘trait matrix' to identify families of similar approaches and to design a guideline to select the most suitable method, given a user-defined set of data and analysis scope. The guidelines are freely available via the conserveR R-package (). Finally, we used our review to identify increasing scalability, continuous monitoring and the integration of molecular, remote sensing and animal movement data as key areas for future impacts of MEE in conservation prioritization. We anticipate that our review can serve as a guide to MEE researchers interested in linking their data to conservation as well as for conservation scientists interested in using MEE approaches.

Background Small and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient. Methods Three Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored. Results The small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample. Conclusion We demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80–90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.

Metabarcoding is increasingly used to assess species diversity by high-throughput sequencing where millions of sequences can be generated in parallel and multiple samples can be analysed in one sequencing run. Generating amplified fragments with a unique sequence identifier ('tag') for each sample is crucial, as it allows assigning sequences to the original samples. The tagging through so-called fusion primers is a fast and cheap alternative to commercially produced ligation-based kits. However, little is known about potential bias and inconsistencies introduced by the long nucleotide tail attached to those primers, which could lead to deficient reports of community composition in metabarcoding studies. We therefore tested the consistency and taxa detection efficiency of fusion primers in (1) a one-step and (2) two-step PCR protocol as well as (3) a commercially manufactured Illumina kit using mock communities of known composition. The Illumina kit delivered the most consistent results and detected the highest number of taxa. However, success of the two-step PCR approach was only marginally lower compared to the kit with the additional advantage of a much more competitive price per library. While most taxa were also detected with the one-step PCR approach, the consistency between replicates including read abundance was substantially lower. Our results highlight that method choice depends on the precision needed for analysis as well as on economic considerations and recommend the Illumina kit to obtain most accurate results and the two-step PCR approach as a much cheaper yet very robust alternative.

Species occurrence records from online databases are an indispensable resource in ecological, biogeographical and palaeontological research. However, issues with data quality, especially incorrect geo-referencing or dating, can diminish their usefulness. Manual cleaning is time-consuming, error prone, difficult to reproduce and limited to known geographical areas and taxonomic groups, making it impractical for datasets with thousands or millions of records. Here, we present CoordinateCleaner, an r-package to scan datasets of species occurrence records for geo-referencing and dating imprecisions and data entry errors in a standardized and reproducible way. CoordinateCleaner is tailored to problems common in biological and palaeontological databases and can handle datasets with millions of records. The software includes (a) functions to flag potentially problematic coordinate records based on geographical gazetteers, (b) a global database of 9,691 geo-referenced biodiversity institutions to identify records that are likely from horticulture or captivity, (c) novel algorithms to identify datasets with rasterized data, conversion errors and strong decimal rounding and (d) spatio-temporal tests for fossils. We describe the individual functions available in CoordinateCleaner and demonstrate them on more than 90 million occurrences of flowering plants from the Global Biodiversity Information Facility (GBIF) and 19,000 fossil occurrences from the Palaeobiology Database (PBDB). We find that in GBIF more than 3.4 million records (3.7%) are potentially problematic and that 179 of the tested contributing datasets (18.5%) might be biased by rasterized coordinates. In PBDB, 1205 records (6.3%) are potentially problematic. All cleaning functions and the biodiversity institution database are open-source and available within the CoordinateCleaner r-package.

Multiple stressors diversely and often adversely affect stream ecosystems around the globe. Therefore, understanding multiple stressor effects on different organisms is essential for a better ecosystem understanding, an accurate water quality assessment and improved ecosystem management. However, while multiple stressor effects should be assessed at species level this taxonomic resolution is often not achieved e.g. for stream macroinvertebrates. Due to their high abundance and diversity, species-level identification is often not feasible with morphology‑based approaches. DNA metabarcoding represents an alternative approach for studying multiple stressor interactions at species level. In an outdoor experiment over 10,000 specimens from the insect orders Ephemeroptera, Plecoptera and Trichoptera (EPT), which are routinely used as bioindicators, and their responses to stressors were studied. In the experiment salinity, fine sediment deposition and flow velocity were manipulated in a full‑factorial design in 64 mesocosms with two microhabitats each (streambed and leaf litter), resulting in eight replicates per treatment. DNA metabarcoding revealed 122 EPT Operational Taxonomic Units (OTUs), from which the most abundant 27 alone showed 14 different response patterns to the applied stressors. The high taxonomic resolution achieved by DNA metabarcoding revealed species specific stressor responses that were hidden at a lower taxonomic resolution. As a prominent example, Rhithrogena semicolorata responded negatively to fine sediment deposition and flow velocity reduction, while Ecdyonurus torrentis (both Heptageniidae, Ephemeroptera) was insensitive to experimental manipulation, highlighting different stressor responses among species within the same family (Fig. 1, Beermann et al. 2020). Even for well-studied organisms such as EPT taxa, this study shows that DNA metabarcoding has the potential to depict response patterns at species or OTU level despite high specimen abundance. Consequently, DNA metabarcoding promises to be a rewarding method when investigating and assessing multiple stressor effects on stream water quality.

Worldwide, multiple stressors affect stream ecosystems and frequently lead to complex and non-linear biological responses. These combined stressor effects on ecologically diverse and functionally important macroinvertebrate communities are often difficult to assess, in particular species-specific responses across many species and effects of different stressors and stressor levels in concert. A central limitation in many studies is the taxonomic resolution applied for specimen identification. DNA metabarcoding can resolve taxonomy and provide greater insights into multiple stressor effects. This was detailed by results of a recent multiple stressor mesocosm experiment, where only for the dipteran family Chironomidae 183 Operational Taxonomic Units (OTUs) could be distinguished. Numerous OTUs showed very different response patterns to multiple stressors. In this study, we applied DNA metabarcoding to assess multiple stressor effects on all non-chironomid invertebrates from the same experiment. In the experiment, we applied three stressors (increased salinity, deposited fine sediment, reduced flow velocity) in a full-factorial design. We compared stressor responses inferred through DNA metabarcoding of the mitochondrial COI gene to responses based on morphotaxonomic taxa lists. We identified 435 OTUs, of which 122 OTUs were assigned to EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa. The most common 35 OTUs alone showed 15 different response patterns to the experimental manipulation, ranging from insensitivity to any applied stressor to sensitivity to single and multiple stressors. These response patterns even comprised differences within one family. The species-specific taxonomic resolution and the inferred response patterns to stressors highlights the potential of DNA metabarcoding in the context of multiple stressor research, even for well-known taxa such as EPT species.

Streams and rivers represent hotspots of biodiversity in their natural state. This biodiversity is declining worldwide due to pollution, exploitation and hydromorphological degradation of these systems. One of the last big, natural rivers in Europe is the Vjosa in the Balkan region. The catchment is characterized by natural flow dynamics, resulting in high habitat diversity and turnover, and hosts several sensitive and endemic species (e.g. Isoperla vjosae). DNA metabarcoding represents a promising approach to characterize this biodiversity but methodological drawbacks such as primer bias or incomplete reference databases limit the application, particularly in taxonomically underexplored regions. Here, we assessed stream biodiversity with a focus on macrozoobenthic (MZB) taxa via a non-destructive, voucher-preserving DNA metabarcoding protocol. In this approach, ethanol used for preservation of multi-habitat samples in the field was used as DNA template, allowing to retain the integrity of the original sample and further comparison of molecular and morphological taxa lists. Samples were taken in spring and autumn 2018 at 48 sites allocated over the Vjosa catchment. The preservative ethanol was filtered through 0.43 um nitrocellulose membranes from which DNA was extracted. Subsequently, a 421 bp fragment of the COI gene was amplified with the primer pair BF2/BR2 and Illumina sequenced. After filtering for sequences with similarity to reference entries >85%, 4,123 OTUs were obtained, of which 921 were identified as MZB taxa. Dipterans and ephemeropterans were most abundant, followed by plecopterans. Some taxa (e.g. molluscs) were not identified due to a known primer bias and >7,000 OTUs could not be assigned above 85 % similarity. Using the here presented voucher-preserving approach allowed us to identify the pitfalls of DNA metabarcoding as tool for biodiversity assessment in taxonomically unexplored regions such as the Vjosa catchment. However, the comparison of specimen abundance data and molecular data showed the power of non-destructive fixative metabarcoding for detecting MZB communities with highly increased taxonomic resolution.

Genetic diversity is the most basal level of biodiversity and determines the evolutionary capacity of species to adapt to changing environments, yet it is typically neglected in routine biomonitoring and stressor impact assessment. For a comprehensive analysis of stressor impacts on genetic diversity, it is necessary to assess genetic variants simultaneously in many individuals and species. Such an assessment is not as straightforward and usually limited to one or few focal species. However, nowadays species diversity can be assessed by analysing thousands of individuals of a community simultaneously with DNA metabarcoding. Recent bioinformatic advances also allow for the extraction of exact sequence variants (ESVs or haplotypes) in addition to Operational Taxonomic Units (OTUs). By using this new capability, we here evaluated if the analysis of intraspecific mitochondrial diversity in addition to species diversity can provide insights into responses of stream macrozoobenthic communities to environmental stressors. For this purpose, we analysed macroinvertebrate bulk samples of three German river systems with different stressor levels using DNA metabarcoding. While OTU and haplotype number were negatively correlated with stressor impact, this association was not as clear when studying haplotype diversity across all taxa. However, stressor responses were found for sensitive EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa and those exceedingly resistant to organic stress. An increase in haplotype number per OTU and haplotype diversity of sensitive taxa was observed with an increase in ecosystem quality and stability, while the opposite pattern was detected for pollution resistant taxa. However, this pattern was less prominent than expected based on the strong differences in stressor intensity between sites. To compare genetic diversity among communities in river systems, we focussed on OTUs, which were present in all systems. As OTU composition differed strongly between rivers, this led to the exclusion of a high number of OTUs, especially in diverse river systems of good quality, which potentially diminished the increase in intraspecific diversity. To better understand responses of intraspecific genetic diversity to environmental stressors, for example in river ecosystems, it would be important to increase OTU overlap between compared sites, e.g. by sampling a narrower stressor gradient, and to perform calibrated studies controlling for the number of individuals and their haplotypes. However, this pioneer study shows that the extraction of haplotypes from DNA metabarcoding datasets is a promising source of information to simultaneously assess intraspecific diversity changes in response to environmental impacts for a metacommunity.

Small and rare specimens can remain undetected when metabarcoding bulk samples with a high size heterogeneity of specimens. This is especially critical for malaise trap samples, where most of the biodiversity is often contributed by small specimens. How to size sort and in which proportions to pool these samples has not been widely explored. We set out to find a size sorting strategy that maximizes taxonomic recovery but remains highly scalable and time efficient.Three 3 malaise trap samples where size sorted into 4 size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate samples never sorted, pooled in equal proportions and in 4 different proportions favoring the small size fractions. DNA from the pooled fractions as well as the individual size classes were extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally wet sieving strategies were explored.The small size fractions harbored the highest diversity, and were best represented when pooling in favor of small specimens. Not size sorting a sample leads to a 45-77% decrease in taxon recovery compared to size sorted samples. A size separation into only 2 fractions (below 4 mm and above) can already double taxon recovery compared to not sorting. However, increasing the sequencing depth 3-4 fold can also increase taxon recovery to comparable levels, but remains biased toward biomass rich taxa in the sample.We demonstrate that size fractionizing bulk malaise samples can increase taxon recovery. The most practical approach is wet sieving into two size fractions, and proportional pooling of the lysates in favor of the small size fraction (80-90% volume). However, in large projects with time constraints, increasing sequencing depth can also be an alternative solution.

DNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically “watered down”, i.e. diluted, compared to whole specimen ‘bulk samples’ due to greater co-amplification of abundant non-target taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3’-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.

DNA metabarcoding of macroinvertebrates is increasingly used for aquatic bioassessment and -monitoring. A major strength of metabarcoding is the high taxonomic resolution provided while the inability to deliver reliable abundance data is regarded a main drawback. Data on the potential of metabarcoding to disentangle site-specific and seasonal variation with presence/absence data have been poorly explored. In addition, the robustness of ecological status assessments compliant with the European Water Framework Directive (Directive 2000/60/EG) have only been compared in few studies, none of which adressed changes across seasons. Therefore, we here investigated seasonal as well as site-specific effects on macroinvertebrate communities at six river sites in a near-natural (Sieg) and urban (Emscher) German river through metabarcoding. Furthermore, we evaluated the usability of the method for ecological status assessments. Our data showed distinct seasonal effects on OTU composition at the near-natural river Sieg. However, ecological status assessment was constant through seasons and comparable to assessments based on available morphological identification. In contrast to the river Sieg, we found strong site and stressor-specific impacts in the heterogeneous urban river Emscher. Here, ecological status assessment varied between sampling sites ranging from good to bad status but was largely consistent between seasons. Our study demonstrates the ability of presence/absence metabarcoding data to reliably assess invertebrate community composition in streams and infer environmental (natural or anthropogenic) impacts. Together with the technical robustness, our study encourages the wider adoption of the technique in stream bioassessment and -monitoring.

DNA (meta)barcoding is applied to study biodiversity and is available for standardized biodiversity assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries. The technique of “mitogenome-skimming” or “mitogenomics,” in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99% of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification. Here, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six ‘mock’-communities—comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each—we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis. The enrichment protocol will speed up building reference libraries for whole mitochondrial genomes, as dozens of species can be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments, using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8–7 taxa) but differences in read numbers assigned to taxa of interest (33.8%–93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.

Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates. Copyright Rosetta C. Blackman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Traditionally, taxonomic characterisation of organisms has relied on their morphology; however, molecular methods are increasingly used to monitor and assess biodiversity and ecosystem health. Approaches such as DNA amplicon diversity assessments are a particularly useful tool when morphology-based taxonomy is difficult or taxa are morphologically ambiguous, for example for freshwater bacteria and fungi as well as many freshwater invertebrate species. DNA metabarcoding provides the ability to distinguish cryptic taxa (which can differ markedly in their ecological requirements and tolerances) and in addition it can provide valuable insights into the genetic and ecological diversity of taxa and ecosystems. While DNA metabarcoding has been used mostly on tissue of sampled specimens, recent years have seen an increased use of metabarcoding on environmental DNA samples: DNA extracted not from sampled specimens, but from the surrounding soil or water. However, the ability of metabarcoding of specimens and metabarcoding of environmental DNA (eDNA) to assess biodiversity and the impact of anthropogenic stressors on freshwater ecosystems is largely understudied. In this talk, several studies that document the advantages and still open challenges of (e)DNA metabarcoding for assessing impacts of environmental stressors on aquatic ecosystems will be presented. These studies, performed in Europe and New Zealand, integrate impacts across different biotic groups, i.e. look at stressor effects on bacterial, protist, fungal and macroinvertebrate communities. Specifically, we use various case studies from freshwater ecosystems to address the following questions: whether eDNA samples, which can be relatively quickly obtained from the water, can act as reliable proxies for catchment-level stressor impacts by comparing these to DNA obtained from local bulk samples, and whether DNA metabarcoding data can also provide quantitative information rather than only presence-absence data. In view of the case studies presented, a perspective on the urgent next steps that need to be taken in order to include genetic tools in routine biomonitoring will be derived and linked to the vision of the international network DNAqua-Net.

Metabarcoding is a powerful tool for biodiversity assessment and has become increasingly popular in recent years. Although its reliability and applicability have been proven in numerous scientific studies, metabarcoding still suffers from some drawbacks. One is the usually mandatory destruction of specimens before DNA extraction, which is problematic because it does not allow a later taxonomic evaluation of the results. Additionally, metabarcoding often implements a time-consuming step, where specimens need to be separated from substrate or sorted in different size classes. A non-destructive protocol, excluding any sorting step, where the extraction of DNA is conducted from a samples fixative (ethanol) could serve as an alternative. We test an innovative protocol, where the sample preserving ethanol is filtered and DNA extracted from the filter for subsequent DNA metabarcoding. We first tested the general functionality of this approach on 15 mock communities comprising one individual of eight different macroinvertebrate taxa each and tried to increase DNA yield through different treatments (ultrasonic irradiation, shaking, freezing). Application of the method was successful for most of the samples and taxa, but showed weaknesses in detecting mollusc taxa. In a second step, the community composition detected in DNA from ethanol was compared to conventional bulk sample metabarcoding of complex environmental samples. We found that especially taxa with pronounced exoskeleton or shells (Coleoptera, Isopoda) and small taxa (Trombidiformes) were underrepresented in ethanol samples regarding taxa diversity and read numbers. However, read numbers of Diptera (mainly chironomids) and Haplotaxida were higher in ethanol derived DNA samples, which might indicate the detection of stomach content, which would be an additional advantage of this approach. Concerning EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa which are decisive for the determination of ecological statuses, both methods had 46 OTUs in common with 4 unique to the ethanol samples and 10 to the bulk samples. Results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. For a comprehensive picture on total biodiversity, the method might however not be sufficient and conventional bulk sample metabarcoding should be applied.

Background: Chironomids, or non-biting midges, often dominate stream invertebrate communities in terms of biomass, abundance, and species richness and play an important role in riverine food webs. Despite these clear facts, the insect family Chironomidae is often treated as a single family in ecological studies or bioassessments given the difficulty to determine specimens further. We investigated stressor responses of single chironomid operational taxonomic units (OTUs) to three globally important stressors (increased salinity, fine sediment and reduced water flow velocity) in a highly replicated mesocosm experiment using a full-factorial design (eight treatment combinations with eight replicates each). Results: In total, 183 chironomid OTUs (97% similarity) were obtained by applying a quantitative DNA metabarcoding approach. Whereas on the typically applied family level, chironomids responded positively to added fine sediment and reduced water velocity in the streambed and negatively to reduced velocity in the leaf litter, an OTU-level analysis revealed a total of 15 different response patterns among the 35 most common OTUs only. The response patterns ranged from (a) insensitivity to any experimental manipulation over (b) highly specific sensitivities to only one stressor to (c) additive multiple-stressor effects and even (d) complex interactions. Conclusion: Even though most OTUs (> 85%) could not be assigned to a formally described species due to a lack of accurate reference data bases at present, the results indicate increased explanatory power with higher taxonomic resolution. Thus, our results highlight the potential of DNA-based approaches when studying environmental impacts, especially for this ecologically important taxon and in the context of multiple stressors. CA Beermann

DNA (meta)barcoding is used to study biodiversity and is available for standardised assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries.The technique of ‘mitogenome-skimming’ or ‘mitogenomics’, in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99 % of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification.Here, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six ‘mock’- communities – comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each – we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis.The presented protocol will greatly speed up building reference libraries for whole mitochondrial genomes, as dozens of species could be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

Phylogenetic analyses of nuclear and mitochondrial genomes have shown that polar bears captured the mitochondrial genome of brown bears some 160,00 years ago. This hybridization event likely led to an extinction of the original polar bear mitochondrial genome. However, parts of the mitochondrial DNA occasionally integrates into the nuclear genome, forming pseudogenes called numts (nuclear mitochondrial integrations). Screening the polar bear genome for numts, we identified only 13 such integrations. Analyses of whole-genome sequences from additional polar bears, brown and American black bears as well as the giant panda indicates that the discovered numts entered the bear lineage before the initial ursid radiation some 14 million years ago. Our findings suggests a low integration rate of numts in the bear lineage and a complete loss of the original polar bear mitochondrial genome.