Metabarcoding and Metagenomics

At times of drastic decrease in biodiversity and loss of species, sometimes referred to as the "sixth mass extinction" or "Holocene extinction", there is a high demand on the development of effective tools for studying and monitoring biodiversity. In the past decade, new promising technologies, such as third generation sequencing (TGS), enabled massive, rapid, and cost-effective data analysis of non-model organisms, accelerating taxonomic identification studies and contributing to conservation applications. Here, we focus on the comparison of the two main TGS providers, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT), for the purpose of DNA barcoding. For ONT, we also tested selected combinations of different types of flow cells and ligation sequencing kits. Out of five tested combinations (PacBio, ONT Flongle flow cell & SQK-LSK110 kit, R9 flow cell & SQK-LSK109 kit, R9 & SQK-LSK100 kit, and R10 flow cell & Q20+ chemistry kit), ONTs Flongle turned out to be most variable in returning the results, but at the same time the most cost efficient. The highest numbers of successfully sequenced samples were achieved with the ONTs R10 & Q20+ chemistry combination. In terms of library preparation time, ONT protocols are the quickest, whereas regarding cost effectiveness - using Sanger pricing per sample as a cut-off - various technologies become affordable depending on the number of samples used. Although both tested platforms are suitable for DNA barcoding, we further discuss their limitations and applicability to different studies, with a special focus on the price and the number of samples. The pipeline we developed, from whole specimens to final DNA barcode consensuses, can aid planning and budgeting biodiversity studies, maximising the number of specimens sequenced in one run and speeding up the sample processing time.

The Neotropical freshwaters of South America host an exceptional level of ichthyofaunal diversity with over 5,160 species, making it the richest continental fauna worldwide. Despite their richness, these freshwater ecosystems face severe threats from human activities, leading to significant declines in fish populations. Traditional fish sampling techniques, such as netting, have been fundamental to ichthyology, offering insights into species richness and abundance. However, the complexity of stream environments limits the effectiveness of conventional sampling tools. As a result, more elusive or niche species are often missed. In recent years, water environmental DNA (eDNA) has emerged as a complementary method to traditional sampling. It allows for detection of aquatic organisms from water samples, expanding the scope of biodiversity assessments. Nevertheless, eDNA filtration faces challenges, especially in turbid waters, including the likelihood of co-extracting inhibitors that can affect amplification and detection processes, as well as the downstream flow of eDNA signals, which means that samples predominantly detect upstream fauna. To address these limitations, the use of bulk samples, such as stomach contents, provides a robust alternative by directly analyzing biological tissues and leveraging the bidirectional mobility of organisms within the stream, enabling the detection of taxa from both upstream and downstream regions. Given these issues, this study combines traditional netting, water eDNA analysis, and dietary metabarcoding to assess the fish biodiversity in three Neotropical streams in the Capim River basin, Para, Brazil. The integration of multiple sampling techniques offers a more accurate picture of biodiversity, helping to overcome the limitations of each individual method and providing essential insights for conservation efforts.

Selecting primers for DNA amplification poses a challenge in environmental DNA (eDNA) studies, particularly in biodiverse tropical ecosystems. Researchers must balance taxonomic coverage, primer specificity, and study feasibility when choosing between a single primer pair and multiple primer pairs. Empirical tests comparing these strategies are essential for improving eDNA protocols, enhancing the detection of aquatic biodiversity, and supporting conservation and environmental monitoring efforts. This study aimed to evaluate how different approaches affect the specificity and efficiency of detecting fish species in eDNA samples from the Teles Pires River basin. We (1) synthesized modified versions of primers commonly used in ichthyofaunal surveys; (2) compared the results generated by these modified primers with the standard primer versions; and (3) evaluated the species detection using two reference sequence databases: a global taxonomic database (GenBank) and a locally constructed taxonomic database containing samples of fish species previously collected in situ. The 12S-V5_mod primers detected the most fish species across the databases (67, 104, and 130 in the Midori2, Local, and joint databases, respectively), followed by the MiFish_mod primers, which detected 47, 101, and 114 species. Finally, the MiFish primers detected 39, 73, and 78 species. The 12S-V5_mod primers also had lower taxonomic specificity of fish species and amplified a broader range of vertebrate taxa than the MiFish_mod primers. The modified primers outperformed the MiFish primers, and detectability nearly doubled when the Local reference database was used. These results speak to the critical need for constructing reference databases of regional biodiversity, and the need to incorporate multiple primer pairs in eDNA studies.

Mushroom forays present unparalleled opportunities to record macrofungal biodiversity in a place and time. The 2024 NAMA annual foray at the Cispus Center near Randle, Washington was the first mass barcoding activity performed by the NAMA DNA Sequencing Committee (DNAMA). The workflows and methods utilized are presented along with planned improvements. Ultimately, 803 ITS barcodes were generated (83.7% of those attempted) for 513 unique taxa belonging to 209 Genera. Of these, 18 novel putative species-level operational taxonomic units (OTUs) were discovered as well as 28 new distribution reports at state level or above. Methods and suggestions for replicating and improving mass barcoding at forays are discussed.

Quantifying in-situ environmental DNA (eDNA) concentrations is increasingly used to infer fish abundance and biomass in freshwater ecosystems. While various sampling strategies (i.e. the combination of sample collection, eDNA preservation and extraction protocols) have been proposed to collect eDNA, little efforts have been undertaken to evaluate how these different strategies affect taxon-specific eDNA recovery and the subsequent estimates of in-situ eDNA concentrations. In this study, we compared a point (i.e. eDNA collection from relatively small and spatially separated water samples filtered using low capacity filter units) and a spatially integrated sampling strategy (i.e. eDNA collected from spatially integrated larger water volumes filtered using high capacity filter units) in two natural lakes. Through quantitative analyses of total DNA, total fish eDNA and species-specific eDNA, we assessed the performance of both strategies to infer in-situ eDNA concentrations. Our results showed that the integrated strategy led to a reduced recovery of fish eDNA and a subsequent underestimation of in-situ eDNA concentrations compared to the point sampling strategy. While the exact mechanisms underlying this pattern require further investigation, our findings highlight the importance of carefully selecting sampling strategies according to study objectives.

Monitoring freshwater biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution models (SDM). Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared a triad of approaches in order to understand assessment of aquatic macroinvertebrate biodiversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species distribution model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species distribution model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the two approaches, particularly at local scale. Overall, the more specific set of primers, namely fwhF2/EPTDr2n, was most efficient for the detection of target species and for characterizing the diversity of EPT. Moreover, we found that the species richness measured by eDNA was poorly correlated to the richness measured by kicknet sampling and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the statistical model. Overall, however, neither eDNA nor the traditional approach had strong links to the predictive models, indicating inherent limitations in upscaling species richness estimates. Future challenges include improving the accuracy and sensitivity of each approach individually yet also acknowledge their respective limitations, in order to best meet stakeholder demands addressing the biodiversity crisis we are facing.

Effective management of biodiversity requires regular surveillance of multiple species. Analysis of environmental DNA by metabarcoding (eDNA) holds promise to achieve this relatively easily. However, taxonomic inquiries into eDNA data need suitable molecular reference data, which are often lacking. We evaluate the impact of this reference data void in a case study of fish diversity in the remote fiords of New Zealand. We compared eDNA-derived species identifications against Baited Remote Underwater Video (BRUV) data collected at the same time and locations as the eDNA data. Furthermore, we cross referenced both eDNA and BRUV data against species lists for the same region obtained from literature surveys and the Ocean Biodiversity Information System (OBIS). From all four data sources, we obtained a total of 116 species records (106 ray-finned fishes, 10 cartilaginous fishes; 59 from literature, 44 from eDNA, 25 from BRUV, 25 from OBIS). Concordance of taxonomies between the data sources dissolved with lowering taxonomic levels, most decisively so for eDNA data. BRUV agreed with local biodiversity information much better and fared better in detecting regional biodiversity dissimilarities. We provide evidence that eDNA metabarcoding will remain a powerful but impaired tool for species-level biodiversity management without locally generated reference data.

Environmental DNA (eDNA) analysis has gained traction as a precise and cost effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for a given environmental condition. Author SummaryThe use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent years. Though, due to differences in the ecology of the environment studied and the novelty of the technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide a concise interpretation of best practices given different project constraints. For the conditions of the San Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support for managers to decide their standards ahead of project submission not only for estuarine conditions but for other waterine conditions alike.

The recovery of amplifiable DNA from formaldehyde{square}fixed (FF) zooplankton samples has long been considered unfeasible. Nevertheless, advances in DNA sequencing and methods for retrieving highly degraded genetic material have demonstrated that even million{square}year{square}old samples and FF museum specimens can yield usable DNA. To access the biological information preserved in long{square}term zooplankton time series, we assessed methodologies for extracting amplifiable DNA from community samples stored for up to 28 years in formaldehyde at room temperature. On one hand, we report the failure of a method previously described as successful for FF zooplankton samples, likely due to the cold{square}storage conditions (4{square}{degrees}C) used in the original study. On the other hand, by adapting two extraction protocols designed for FF museum specimens--representing harsher and softer alternatives (HHA and HPC, respectively)--we successfully amplified and sequenced a subset of FF zooplankton samples. As expected, DNA integrity and sample pH were inversely related to preservation time, and only short DNA fragments were recovered, ruling out the use of commonly employed [≥]300{square}bp metabarcoding markers. While DNA integrity appeared to be a better predictor than DNA yield for amplification success, the presence of a gel band of the expected size did not always guarantee congruence with microscopy{square}based assessments. Although amplifiable DNA was recovered from most samples, including some of the oldest, community compositions concordant with microscopy were consistently recovered only from samples preserved for up to two years. Beyond this point, the HHA and HPC methods produced divergent results, reflecting a trade{square}off between the removal of formaldehyde{square}induced cross{square}linkages and the avoidance of additional DNA damage. Among the small universal markers tested ([~]120-170{square}bp), including one nuclear rRNA marker and two mitochondrial markers, only the 18S rRNA V9 region consistently amplified. We conclude by providing a set of recommendations aimed at improving the methods presented here.

The Oxford Nanopore Technologies (ONT) sequencing platform is compact and efficient, making it suitable for rapid biodiversity assessments in remote areas. Despite its long reads, ONT has a higher error rate compared to other platforms, necessitating high-quality reference databases for accurate taxonomic assignments. However, the absence of targeted databases for underexplored habitats, such as the seafloor, limits ONTs broader applicability for exploratory analysis. To address this, we propose an approach for building environmentally-targeted databases to improve 16S rRNA gene (16S) analysis using Oxford Nanopore Technologies (ONT), using seafloor sediment samples from the Norwegian coast as an example. We started by using Illumina short-read data to create a database of full-length or near full-length 16S sequences from seafloor samples. Initially, amplicons are mapped to the SILVA database, with matches added to our database. Unmatched amplicons are reconstructed using METASEED and Barrnap methodologies with amplicon and metagenome data. Finally, if the previous strategies did not succeed, we included the short-read sequences in the database. This resulted in AQUAeD-DB, which contains 14 545 16S sequences clustered at 95% identity. Comparative database analysis reveal that AQUAeD-DB provides consistent results for both Illumina and Nanopore read assignments (median correlation coefficient: 0.50), whereas a standard database showed a substantially weaker correlation. These findings also emphasize its potential to recognize both high and low-abundance taxa, which could be key indicators in environmental studies. This work highlights the necessity of targeted databases for environmental analysis, especially for ONT-based studies, and lays foundations for future extension of the database.

Large commercial ports facilitate the introduction of non-indigenous species (NIS), while smaller harbours and marinas contribute to their regional spread. Harbour networks are thus important drivers of introductions. Despite extensive research effort on NIS in recent years, no study has yet assessed genetic connectivity among harbours considering whole-community composition. Here, we analysed spatio-temporal patterns of metazoan communities over one year in four medium-size harbours along the NW Mediterranean coast sampled by deploying standardised biological collectors. Using cytochrome c oxidase subunit I (COI) metabarcoding, we identified 1,770 metazoan molecular operational taxonomic units (MOTUs), of which 82 were classified as NIS based on a custom database of Mediterranean NIS. Despite their lower species count compared to natives, NIS accounted for 34-70% of reads in harbours. The southernmost harbour had the highest NIS number of reads, likely due to its proximity to aquaculture facilities. While we observed some variation in the spatial structure of metazoan communities across harbours, NIS showed consistently low differentiation values, sharing significantly more MOTUs among sites. Seasonal patterns influenced both NIS and the rest of the community. Haplotype diversity was significantly higher in NIS, which also exhibited lower genetic differentiation across harbours compared to native species, indicating NIS spread via local boating and likely recurrent introductions. These findings highlight distinct dynamics between NIS and native species in artificial environments, emphasising the importance of continued monitoring in harbour networks to manage coastal NIS proliferation. HIGHLIGHTSCOI metabarcoding of standardised collectors detected over 1,700 MOTUs of marine metazoans in ports over a year. Less than 4% were NIS MOTUs, but they comprised 34-71 % of the reads. NIS were more homogeneously distributed among ports than other MOTUs. NIS showed higher genetic variability but lower genetic differentiation than native species. Different dynamics underpin NIS and native assemblages in port communities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/688838v1_ufig1.gif" ALT="Figure 1000"> View larger version (32K): org.highwire.dtl.DTLVardef@fa0a5corg.highwire.dtl.DTLVardef@1be29ccorg.highwire.dtl.DTLVardef@1aa4e85org.highwire.dtl.DTLVardef@91842_HPS_FORMAT_FIGEXP M_FIG C_FIG

Zooplankton are key players in marine ecosystems, linking primary production to higher trophic levels. The high abundance and high taxonomic diversity renders zooplankton ideal for biodiversity monitoring. However, taxonomic identification of the zooplankton assemblage is challenging due to its high diversity, subtle morphological differences and the presence of many meroplanktonic species, especially in coastal seas. Molecular techniques such as metabarcoding can help with rapid processing and identification of taxa in complex samples, and are therefore promising tools for identifying zooplankton communities. In this study, we applied metabarcoding of the mitochondrial cytochrome c oxidase I gene to zooplankton samples collected along a latitudinal transect in the North Sea, a shelf sea of the Atlantic Ocean. Northern regions of the North Sea are influenced by inflow of oceanic Atlantic waters, whereas the southern parts are characterised by more coastal waters. Our metabarcoding results indicated strong differences in zooplankton community composition between northern and southern areas of the North Sea, particularly in the classes Copepoda, Actinopterygii (ray-finned fishes) and Polychaeta. We compared these results to the known distributions of species reported in previous studies, and by comparing the abundance of copepods to data obtained from the Continuous Plankton Recorder (CPR). We found that our metabarcoding results are mostly congruent with the reported distribution and abundance patterns of zooplankton species in the North Sea. Our results highlight the power of metabarcoding to rapidly assess complex zooplankton samples, and we suggest that the technique could be used in future monitoring campaigns and biodiversity assessments. HighlightsO_LIZooplankton communities are different in northern and southern areas of the North Sea C_LIO_LIMetabarcoding results are consistent with known species distributions and abundance C_LIO_LIMetabarcoding allows for fast identification of meroplanktonic species C_LI

Traditional detection of aquatic invasive species, via morphological identification is often time-consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina-based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and to substantially decrease sample turnaround times. However, nanopore-sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves. We sampled water from sites with pre-existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. We extracted, amplified and sequenced eDNA with a turnaround of 3.5 days. The majority of processed reads were [≥] 99 % identical to reference sequences. There were no taxa detected other than those known to occur. The lack of detections of some species at some sites could be explained by their known low abundances. This is the first reported use of MinION to detect aquatic invasive species from eDNA samples. The approach can be easily adapted for other metabarcoding applications, such as biodiversity assessment, ecosystem health assessment and diet studies.

Human impacts have been eroding marine ecosystems in such a way that biodiversity patterns are changing. Therefore, policies and science-based solutions are indispensable for monitoring threats to the most impacted species. In such effort, the analysis of elasmobranchs environmental traces via eDNA metabarcoding represent a candidate tool for effective monitoring and conservation that is often advocated to be cost-effective and easily replicated. Here, we tested a realistic approach to monitor future changes through elasmobranchs metabarcoding with published primers, in which, elasmobranch diversity from the coastal waters of the Fernando de Noronha Archipelago (Brazil) was studied here. We detected a total of three elasmobranch species, namely Hypanus berthalutzae, Ginglymostoma cirratum, and Prionace glauca among numerous other fish species. Even though the technique proved to be a useful tool, some practical constraints were identified, and primarily caused by currently published environmental primers. In order to ensure the broad application of the method, we pointed out feasible adjustments to the problematic parameters based on our survey and other elasmobranch metabarcoding studies. The current drawbacks of the approach need to be considered by managers, conservation actors, and researchers, who are considering this methodology in order to avoid unrealistic promises for the cost incurred.

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, the species was recently rediscovered in one location of the river Dee in Wales after 22 years of absence. As many species belonging to the Perlodidae, this species can be a bio-indicator, utilised for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling for example, is an invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the river Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone - resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the big discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.

O_LIMetabarcoding is the most widely applied method for studying plant communities using environmental DNA, with shotgun sequencing and capture probes being alternative methods. Any methods ability to detect and correctly identify plant taxa may vary with DNA preservation, DNA reference library and the size of the local flora, making it difficult to compare results from different environments. C_LIO_LIHere we compare these methods using lake surface-sediments from Northern Fennoscandia with the PhyloNorway genome skim reference library (1500 taxa) that includes almost all species of the regional flora. We also undertook vegetation surveys from around the lakes to estimate the true positive detection rate, identify false positive detections, and provide optimal filtering cut-off thresholds for the three methods. C_LIO_LIOptimal filter cutoffs were one PCR replicate and 3 reads for metabarcoding, and 3.2x10e-6 and 5.6x10e-6 of queried reads, respectively, for the shotgun and capture probe data. Applying these thresholds, the rate of false positives was too high for reliable identification at species level based on shotgun (49%) and capture probes (62%), whereas it was low for metabarcoding (5-12%). All methods were reliable at genus and family levels after applying the optimal filtering thresholds (<4% false positives). Our results show that metabarcoding on average detects 2.1 times as many true positive taxa as shotgun sequencing, and 6.4 times as many taxa as capture probes. Proportions of filtered metabarcoding and shotgun reads were significantly related to abundance categories from vegetation surveys, but this was not the case for capture probe data. C_LIO_LIWe expect the false positive rate of shotgun sequencing to decrease with increasing genome completeness in the reference libraries, and be advantageous for highly degraded DNA with fragments too short for metabarcoding. At present, metabarcoding provides the highest detectability and taxonomic resolution for correct identification of vascular plants. C_LI

AO_SCPLOWBSTRACTC_SCPLOWThe two most commonly used approaches to study the composition of environmental protist communities are marker gene metabarcoding and whole genome analysis through metagenomics. Raw metabarcoding data are usually processed into Operational Taxonomic Units (OTUs) or amplicon sequence variants (ASVs) through clustering or denoising approaches, respectively. Analogous approaches have been developed to assemble metagenomic sequence reads into metagenome-assembled genomes (MAGs). Understanding the correspondence between the data produced by these two approaches can help to integrate information between the datasets and to explain how metabarcoding OTUs and MAGs are related with the underlying biological entities they are hypothesised to represent. Due to the nature of their construction, MAGs do not contain the most commonly used barcoding loci, meaning that sequence homology approaches cannot be used to match OTUs and MAGs. We made an attempt to match V9 metabarcoding OTUs from the 18S rRNA gene (V9 OTUs) and MAGs from the Tara Oceans expedition (2009-2013) based on the correspondence of their relative abundances across the same set of samples. We evaluated the performance of several methods for detecting correspondence between features in these two compositional datasets and developed a series of controls to filter artefacts of data structure and processing. After selecting the best-performing correspondence metrics, ranking the V9 OTU/MAG matches by their proportionality/correlation coefficients and applying a set of selection criteria, we identified candidate matches between V9 OTUs and MAGs. In a subset of cases, V9 OTUs and MAGs could be successfully matched with one another with a one-to-one correspondence, implying that they likely represent the same underlying biological entity. More generally, matches we observed could be classified into 4 scenarios: Scenario I - one V9 OTU matches more than one MAG; Scenario II - more than one V9 OTU matches more than one MAG; Scenario III - more than one V9 OTU matches one MAG; Scenario IV - one V9 OTU matches one MAG. These diverse scenarios for V9 OTU-MAG matches illustrate the complex nature of the OTU/MAG relationship. Notably, we found some instances in which different OTU-MAG matches from the same taxonomic group were not classified in the same scenario, with all four scenarios possible even within the same taxonomic group, illustrating that factors beyond taxonomic lineage influence the relationship between OTUs and MAGs. Overall, each scenario produces a different interpretation of V9 OTUs, MAGs and how they compare in terms of the genomic and ecological diversity that they represent.

Expectations are high regarding the potential of eDNA metabarcoding for diversity monitoring. To make this approach suitable for this purpose, the completeness and accuracy of reference databases used for taxonomic assignment of eDNA sequences are among the challenges to be tackled. Yet, despite ongoing efforts to increase coverage of reference databases, sequences for key species are lacking, and incorrect records in widely used repositories such as GenBank have been reported. This compromises eDNA metabarcoding studies, especially for high diverse groups such as marine fishes. Here, we have developed a workflow that evaluates the completeness and accuracy of GenBank. For a given combination of species and barcodes a gap analysis is performed, and potentially erroneous sequences are identified. Our gap analysis based on the four most used genes (cytochrome c oxidase subunit 1, 12S rRNA, 16S rRNA and cytochrome b) for fish eDNA metabarcoding found that COI, the universal choice for metazoans, is the gene covering the highest number of Northeast Atlantic marine fishes (70%), while 12S rRNA, the preferred region for fish-targeting studies, only covered about 50% of the species. The presence of too close and too distant barcode sequences as expected by their taxonomic classification confirms presence of erroneous sequences in GenBank that our workflow can detect and eliminate. Comparing taxonomic assignments of real marine eDNA samples with raw and clean reference databases for the most used 12S rRNA barcodes (teleo and MiFish), we found that both barcodes perform differently, and demonstrated that the application of the database cleaning workflow can result in drastic changes in community composition. Besides providing an automated tool for reference database curation, this study confirms the need to increase 12S rRNA reference sequences for European marine fishes, encourages the use of a multi-marker approach for better community composition assessment, and evidences the dangers of taxonomic assignments by directly querying GenBank.

Large-scale visual surveys are an integral part of waterfowl conservation and management programs. In this paper we explore the possibility of using environmental DNA-based surveys as a cost-efficient, complementary tool to estimate populations of North American waterfowl species. To achieve this, we first evaluated the performance of all currently available avian metabarcoding primers and compared them to newly designed primers targeting the mitochondrial ND2 gene within the Anatidae tribes of North America. All the existing avian assays showed strong cross-priming amplification with other vertebrates. In contrast, in-silico analyses of our waterfowl targeted assays showed a high degree (>90%) of avian specificity, encompassing all the 132 Anatidae species sequenced thus far. We used this targeted metabarcoding approach to track the temporal variation in the relative abundance of waterfowl species during the fall migration at Montezuma National Wildlife Refuge, New York, a major resting area for waterfowl on their journey to and from North American nesting areas. We compared eDNA results with visual surveys conducted by us and from those reported on eBird. Our results showed that eDNA detected all waterfowl species (n= 25) during fall migration. Positive correlations existed between standardized eDNA read counts and the relative abundance of waterfowl species as reported in eBird on the day of sampling and up to five days prior. However, this approach did not provide a good metric for absolute abundance of waterfowl species: only 8 out of 25 waterfowl species showed significant correlations between the number of eDNA reads and the total abundance of birds. Overall, while eDNA-targeted metabarcoding has not yet been applied to study bird communities extensively, our results demonstrate that this technique can be used as an effective complementary tool for assessing species composition of waterfowl communities and estimating relative abundance of species within those communities.

Monitoring fish species through ichthyoplankton surveys provides important information for fish stock assessment and management. To test the effectiveness of DNA metabarcoding to identify fish species and to capture seasonal variations in local ichthyofauna, monthly ichthyoplankton and 2 L water samples were collected over 13 months in the lower section of the Guadiana River Estuary in southeast Portugal. Both sample types underwent high-throughput sequencing for three genetic markers (COI, 12S, 16S), with morphological identification also performed for ichthyoplankton. Bulk and water samples identified a total of 131 fish species throughout the year. DNA metabarcoding demonstrated higher taxonomic resolution and diversity detection, with 115 species recovered, while morphology identified only 23 species. Ichthyoplankton metabarcoding also detected 40% more fish than water eDNA, recovering almost the double of species despite the fact that both approaches used the same metabarcoding primers. The integration of multiple molecular markers was crucial to maximize diversity detection in both DNA-based methods. In addition, DNA metabarcoding was able to identify ichthyofaunal spawning periods and captured significant seasonal variations in fish community, with higher diversity observed during the warmer months. With this strategy, around 66% of the historically recorded ichthyoplankton taxa in the region were identified, along with several new records. The findings demonstrated the capability of (e)DNA metabarcoding to uncover seasonal variations in the regional fish community, provided new insights on the ichthyofauna of the Guadiana Estuary, and revealed the need for more in-depth studies to improve the efficiency of multiple sampling methods for fish species identification.