Metabarcoding and Metagenomics

As human activities on our planet persist, causing widespread and irreversible environmental degradation, the need to biomonitor ecosystems has never been more pressing. These circumstances have required a renewal in monitoring techniques, encouraged by necessity to develop more rapid and accurate tools which will support timely observations of ecosystem structure and function. The World Exposition (from now EXPO2015) hosted in Milan from May to October 2015 was a global event that could be categorized as a mega-event, which can be defined as an acute environmental stressor, possibly generating biodiversity alteration and disturbance. During the six months of EXPO2015, exhibitors from more than 135 countries and 22 million visitors insisted on a 1.1 million square meters area. Faced with such a massive event, we explore the potential of DNA metabarcoding using three molecular markers to improve the understanding of anthropogenic impacts in the area, both considering air and water monitoring. Furthermore, we explore the effectiveness of the taxonomy assignment phase considering different taxonomic levels of analysis and the use of data mining approaches to predict sample origin. Unless the degree of taxa identification still remains open, our results showed that DNA metabarcoding is a powerful genomic-based tool to monitor biodiversity at the microscale, allowing us to capture exact fingerprints of specific event sites and to explore in a comprehensive manner the eukaryotic community alteration. With this work, we aim to disentangle and overcome the crucial issues related to the generalization of DNA metabarcoding in order to support future applications.

AbstractEnvironmental DNA metabarcoding is a powerful tool for biodiversity assessment; however, species identification often relies on sequence similarity searches (BLAST), which are limited by incomplete reference databases and result in frequent misidentifications or unclassified taxa. To overcome these challenges, we designed a novel analytical pipeline, Phylo-MIP, which integrates phylogenetic analysis and species delimitation. Phylo-MIP combines sequence alignment, haplotype thinning, phylogenetic tree construction, and multiple species delimitation methods (bPTP, bPTP-ML, mPTP) to objectively identify Molecular Operational Taxonomic Units (MOTUs) without relying on reference databases. As a validation, we applied Phylo-MIP to previously published eDNA data from river in Kanagawa Prefecture, detecting approximately 28% more species than only BLAST-based methods. We also conducted a eDNA survey in the subalpine zone of Kamikochi, Nagano Prefecture, Japan, where Phylo-MIP revealed distinct community structures associated with specific aquatic environments. Phylo-MIP enables non- invasive and efficient biodiversity monitoring, even in protected areas, and offers a reproducible framework for ecological research. This approach has the potential to become a new standard method in river ecology, taxonomy, conservation biology, and ecosystem management.

Molecular techniques involving environmental DNA (eDNA) are increasingly used for aquatic species detection. Metabarcoding, a widely adapted technique, suffers from primer bias: uneven amplification of species due to primer mismatches. The primer bias can be eliminated by omitting PCR, thereby sequencing all eDNA in a sample. This method, known as metagenomics, offers potential benefits for relative abundance estimates and epigenetic modifications, but is seldom applied to eukaryotic communities and eDNA. This study uses an expanded two-by-two design to compare fish species detection between multi-marker metabarcoding and metagenomics using two filter types (conventional versus high-flow). Environmental DNA was collected in a controlled setup and two field settings, which contained several fish species including European sturgeon (Acipenser sturio). Moreover, we explore methylation patterns obtained from nanopore native sequencing. All species present in the controlled environment were detected using both metabarcoding and metagenomics. In field settings, metagenomics detected more species than metabarcoding. High-flow filters recovered more species across all sequencing datasets, except in metabarcoding of field settings. Relative read counts between metabarcoding and metagenomics illustrate primer bias is present in the used primer sets. Most fish metagenomic sequences were identified as A. sturio across all eDNA samples. We observed three base modifications on the 18S region of A. sturio, where three sites showed different methylation patterns between eDNA samples. Our results demonstrate that metabarcoding and metagenomics function complementary in species detection and metagenomics provides additional insights into base modifications. Moreover, high-flow filters offer strong potential for improved species detection in various environments.

Biodiversity loss is a global challenge of the 21st century. Environmental DNA (eDNA)-based metabarcoding offers a cost- and time-efficient alternative to conventional biodiversity surveys, enabling detection of rare, cryptic, and elusive species from environmental samples. However, limited access to genomic technologies restricts the application of eDNA metabarcoding in highly biodiverse remote regions and low- and middle-income countries (LMICs). Here, we directly compared the latest portable nanopore sequencing methods with established Illumina sequencing for vertebrate eDNA metabarcoding of Zambian water samples. Our results show that due to recent improvements in sequencing chemistry and optimized basecalling, nanopore sequencing data can recapitulate or even surpass established protocols, demonstrating the feasibility of in situ biodiversity assessments. eDNA- and camera trap-based species detections had minimal overlap in species detections, suggesting a complementary rather than substituting application of these biodiversity monitoring technologies. We finally demonstrate that our entire eDNA workflow can be successfully implemented in a mobile laboratory under remote field conditions by completing all steps--from sample collection to data analysis--within the Luambe National Park in Zambia. This approach has important implications for capacity building in LMICs and for overcoming limitations associated with sample export.

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

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.

Following the development of high-throughput DNA sequencers, environmental prokaryotic communities were usually described by metabarcoding on short markers of the 16S domain. Among third generation sequencers, that offered the possibility to sequence the full 16s domain, the portable MinION from Oxford Nanopore was undervalued for metabarcoding because of its relatively higher error rate per read. Here we illustrate the limits and benefits of Nanopore sequencing devices by comparing the prokaryotic community structure in a mock community and 52 sediment samples from mangrove sites, inferred from full-length 16S long-reads (16S-FL, ca. 1.5 kpb) on a MinION device, with those inferred from partial 16S short-reads (16S- V4V5, ca. 0.4kpb, 16S-V4V5) on Illumina MiSeq. 16S-V4V5 and 16S-FL retrieved all the bacterial species from the mock, but Nanopore long-reads overestimated their diversity more than twice. Whether these supplementary OTUs were artefactual or not, they only accounted for ca. 10% of the reads. From the sediment samples, with a coverage-based rarefaction of reads and after singletons filtering, Mantel and Procrustean tests of co-inertia showed that bacterial community structures inferred from 16S-V4V5 and 16S- FL were significantly similar, showing both a comparable contrast between sites and a coherent sea-land orientation within sites. In our dataset, 84.7 and 98.8% of the 16S-V4V5 assigned reads were assigned strictly to the same species and genus, respectively, than those detected by 16S-FL. 16S-FL allowed to detect 92.2% of the 309 families and 87.7% of the 448 genera that were detected by the short 16S-V4V5. 16S-FL recorded 973 additional species and 392 genus not detected by 16S-V4V5 (31.5 and 10.4% of the 16S-FL reads, respectively, among which 67.8 and 79.3% were assigned), producted by both primer specificities and diffrent error rates. Thus, our results concluded to an overall similarity between 16S-V4V5 and 16S-FL sequencing strategies for this type of environmental samples.

Environmental DNA (eDNA) metabarcoding has become a cornerstone of marine biodiversity monitoring, yet it recovers genetic material irrespective of organism viability and may therefore conflate historical and contemporary community signals. Environmental RNA (eRNA), derived from less stable ribonucleic acid, is hypothesized to be biased toward metabolically active organisms and may provide a more temporally resolved snapshot of living communities. Here we present a paired eDNA/eRNA metabarcoding comparison across a tropical marine seascape, analyzing 19 co-sampled sites spanning coral reefs, mangroves, a seagrass bed, shipwrecks, a cenote, and coastal infrastructure around San Andres Island, Colombia. To our knowledge this is the first in situ, ecosystem-scale paired eDNA/eRNA survey of the broad eukaryotic community across multiple natural habitat types in a tropical marine system, extending mesocosm and freshwater work (e.g., Giroux et al., 2022) to a field setting. Using COI-region amplicon sequencing processed by NatureMetrics, we recovered 1,944 operational taxonomic units (OTUs) across the 19 paired sites. Of these, 1,015 (52.2%) were detected by both approaches, 305 (15.7%) were unique to eDNA, and 624 (32.1%) were unique to eRNA. The eRNA-unique fraction was taxonomically enriched for groups including diatoms (class Bacillariophyceae, phylum Ochrophyta), ciliates, and other protists. Paired Wilcoxon signed-rank tests showed that eRNA recovered significantly higher OTU richness (median 239 vs. 207; W = 36, p = 0.016) and Shannon diversity (median 3.64 vs. 3.38; W = 40, p = 0.026) than eDNA. The mean per-site Jaccard similarity between paired samples was 0.40, indicating substantial turnover in the rare-taxon composition recovered by each method. Principal coordinates analysis of Bray-Curtis dissimilarity showed that habitat type structured abundance-weighted community composition (PERMANOVA F = 2.49, p = 0.001) whereas molecular method did not (F = 1.37, p = 0.107). A PERMDISP test found homogeneous multivariate dispersion between methods (F = 0.01, p = 0.92), reinforcing the absence of a method effect, but significant dispersion heterogeneity among habitats (F = 24.0, p < 0.01), so the habitat result is interpreted with caution. Indicator species analysis identified 73 OTUs significantly associated with one template: eDNA indicators were dominated by dinoflagellates (Dinophyceae) and eRNA indicators by diatoms (Bacillariophyceae) and fungi, consistent with an eRNA bias toward metabolically active microbial eukaryotes. A read-weighted overlap analysis showed that although eRNA-unique OTUs outnumbered eDNA-unique OTUs roughly two to one, the large majority of reads (>95%) fell in shared OTUs, so method-unique detections are predominantly rare taxa. We discuss the complementary value of eRNA for marine monitoring, with the seagrass habitat -- where eRNA reduced masking by terrestrial plant material -- as the clearest use case, and propose, rather than prescribe, the integration of eRNA into routine programs.

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

Knowledge derived from nucleotide sequence data is increasing in importance in the life sciences, as well as decision making (mainly in biodiversity policy). Metadata standards have been established to facilitate sustainable sequence data management according to the FAIR principles (Findability, Accessibility, Interoperability, Reusability). Here, we review the status of metadata available for raw read Illumina amplicon and whole genome shotgun sequencing data derived from ecological metagenomic material that are accessible at the European Nucleotide Archive (ENA), as well as the compliance of the primary sequence data (fastq files) with data submission requirements. While overall basic metadata, such as geographic coordinates, were retrievable in 98% of the cases for this type of sequence data, interoperability was not always ensured and other (mainly conditionally) mandatory parameters were often not provided at all. Metadata standards, such as the Minimum Information about any(x) Sequence (MIxS), were only infrequently used despite a demonstrated positive impact on metadata quality. Furthermore, the sequence data itself did not meet the prescribed requirements in 31 out of 39 studies that were manually inspected. To tackle the most immediate needs to improve FAIR sequence data management, we provide a list of minimal suggestions to researchers, research institutions, funding agencies, reviewers, publishers, and databases, that we believe might have a potentially large positive impact on sequence data and metadata FAIRness, which is crucial for further research and its derived applications.

Ribosomal DNA (rDNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large scale exploration of environmental microbial diversity through metabarcoding approaches have been focused mainly on the V4 and V9 regions of the 18S rDNA gene. The accurate interpretation of such environmental surveys is hampered by technical (e.g., PCR and sequencing errors) and biological biases (e.g., intra-genomic variability). Here we explored the intra-genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra-genomic variability of Nassellaria and Spumellaria is generally low, yet some Spumellaria specimens showed two different copies of the V4 with <97% similarity. From the different sequencing methods, Illumina showed the highest number of contaminations (i.e., environmental DNA, cross-contamination, tag-jumping), revealed by its high sequencing depth; and MinION showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results highlight the requirement for a careful interpretation of Illumina based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full environmental rDNA metabarcoding surveys.

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.

O_LISmall 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. C_LIO_LIThree 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. C_LIO_LIThe 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. C_LIO_LIWe 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. C_LI

BackgroundBroad-scale monitoring of arthropods is often carried out with passive traps (e.g. Malaise traps) that can collect thousands of specimens per sample. The identification of individual specimens requires time and taxonomic expertise, limiting the geographical and temporal scale of research and monitoring studies. DNA metabarcoding of bulk-sample homogenates is faster and has been found to be efficient and reliable, but is destructive and prevents a posteriori validation of species occurrences and/or relative abundances. Non-destructive DNA metabarcoding from the collection medium has been applied in a limited number of studies, but further tests of efficiency are required in a broader range of circumstances to assess the consistency of the method. MethodsWe quantified the detection rate of arthropod species when applying non-destructive DNA metabarcoding with a short (127-bp) fragment of mitochondrial COI on two types of passive traps and collection media: 1) water with monopropylene glycol (H2O-MPG) used in window-flight traps (WFT, 53 in total); 2) ethanol with monopropylene glycol (EtOH-MPG) used in Malaise traps (MT, 27 in total). We then compared our results with those obtained for the same samples using morphological identification (for WFTs) or destructive metabarcoding of bulk homogenate (for MTs). This comparison was applied as part of a larger study of arthropod species richness in silver fir (Abies alba) stands across a range of climate-induced tree dieback levels and forest management strategies. ResultsOf the 53 H2O-MPG samples from WFTs, 16 produced no metabarcoding results, while the remaining 37 samples yielded 77 arthropod MOTUs in total. None of those MOTUs were shared species with the 389 morphological taxa (343 of which were Coleoptera) obtained from the same traps. Metabarcoding of 26 EtOH-MPG samples from MTs detected more arthropod MOTUs (233) and insect orders (11) than destructive metabarcoding of homogenate (146 MOTUs, 8 orders). Arachnida and Collembola were more diverse in EtOH-MPG samples, but Hymenoptera, Coleoptera and Lepidoptera were less represented than in homogenate. Overall, MOTU richness per trap similar for EtOH-MPG (21.81 MOTUs) than for homogenate (32.4 MOTUs). Arthropod communities from EtOH-MPG and homogenate metabarcoding were relatively distinct, with 162 MOTUs (53%) unique to the collection medium and only 71 MOTUs (23%) present in both treatments. Finally, collection medium did not reveal any significant changes in arthropod richness along a disturbance gradient in silver fir forests. We conclude that DNA metabarcoding of collection medium can be used to complement homogenate metabarcoding in inventories to favour the detection of soft-bodied arthropods like spiders.

The role of seaweeds in the blue bioeconomy has stimulated research efforts around the world but proper species identification and biodiversity assessments, remain a challenge. The South African coast hosts the confluence of the Indian Ocean and Atlantic Ocean, creating a dynamic evolutionary environment that has over time yielded a rich diversity of seaweeds with the highest seaweed diversity occurring along the Agulhas Marine Province. Although South Africa harbours one of the richer seaweed floras in the world, only 24% of the known species are represented by DNA barcodes. We therefore initiated the construction of a taxonomically guided DNA reference library for seaweeds in South Africa with the aim of continuously adding to it in the future. To do this, a seaweed biodiversity survey of the Rhodophyta occurring along a temperate-tropical biogeographic transition zone situated within the Agulhas Marine Province (AMP) in South Africa was carried out. Seaweeds were identified in the field using available field or taxonomic guides and herbarium vouchers were prepared. Subsamples were preserved for DNA analyses and three DNA barcodes (LSU D2-D3; rbcL-3P; COI 5P) were amplified. Sequences were verified on BLAST and preliminary phylogenetic analyses or comparison with the literature were carried out where necessary. A total of 220 barcodes was generated for 88 species and one species variety, including 17 species from or near their type localities and eight generitypes. Novel barcodes were generated for 73 species, nearly half of which were species endemic to Southern Africa. In addition, 21 taxa representing new, potentially new, or reinstated species and at least two new genera were identified as well as one new distribution recorded, all of which require further study. This study significantly adds to the foundational biodiversity knowledge of the South African seaweed flora and highlights new avenues for further research.

The taxonomy of fish in the northeast Pacific area has been recently revised using molecular genetic methods, including the development of a reference library of DNA fragments for species identification. Such libraries are the basis for the development of non-invasive, high-throughput methods for monitoring biodiversity using environmental DNA (eDNA). In order to validate this approach, we used a water eDNA metabarcoding technique based on 12S rRNA and COI mitochondrial fragments and assessed the composition of the twenty northeast Pacific fish species held in a fish tank at the Primorsky Aquarium (Vladivostok, Russia). Only the 12S fragment revealed data on fish-related operational taxonomic units (OTUs). Approximately 68% of the reads were classified into two species of the genus Oncorhynchus, whose shredded fillet is used for feeding. According to the taxonomic identification for the rest of the reads, 8 out of 20 fish species in the tank (40%) were identified unambiguously, while two species could not be identified. Ten taxa can be considered conditionally identifiable since they might be concealed behind a conflicting taxonomic identification at the genus or family level. In this case, an improvement of the reference library would provide resolution. We detected contamination, which may be related to both intra-laboratory contaminants occurring during DNA extraction and water intake supplying the fish tank.

Marine sponges have recently been recognised as natural samplers of environmental DNA (eDNA) due to their effective water filtration and their ubiquitous, sessile and regenerative nature. However, laboratory workflows for metabarcoding of sponge tissue have not been optimised to ensure that these natural samplers achieve their full potential for community survey. We used a phased approach to investigate the influence of DNA isolation procedures on the biodiversity information recovered from sponges. In Phase 1, we compared three treatments of residual ethanol preservative in sponge tissue alongside five DNA extraction protocols. The results of Phase 1 informed which ethanol treatment and DNA extraction protocol should be used in Phase 2, where we assessed the effect of starting tissue mass on extraction success and whether homogenisation of sponge tissue is required. Phase 1 results indicated that ethanol preservative may contain unique and/or additional biodiversity information to that present in sponge tissue, but blotting tissue dry generally recovered more taxa and generated more sequence reads from the wild sponge species. Tissue extraction protocols performed best in terms of DNA concentration, taxon richness and proportional read counts, but the non-commercial tissue protocol was selected for Phase 2 due to cost-efficiency and greater recovery of target taxa. In Phase 2 overall, we found that homogenisation may not be required for sponge tissue and more starting material does not necessarily improve taxon detection. These results combined provide an optimised DNA isolation procedure for sponges to enhance marine biodiversity assessment using natural sampler DNA metabarcoding.

As global biodiversity declines continue, conservation efforts are increasingly important in megadiverse areas such as the Neotropics where biodiversity is especially imperiled. The accurate identification of specimens is critical to successful conservation plans. However, in groups such as freshwater fishes, different identification methodologies have documented challenges. Using a biodiversity survey of fishes from the Barra del Colorado Wildlife Refuge in northeastern Costa Rica, we compared: (1) morphological identifications in the field, (2) morphological identifications in the lab by experts, (3) DNA barcode-based identifications, and (4) identifications based on an integrative approach. Our results suggest that both barcode-based identifications and field morphological identifications provided fewer correct species identifications than lab identifications performed by experts using morphology. We attribute shortfalls of DNA barcoding in this case to the misidentification of reference material, the use of outdated taxonomy for references sequences, and the non-uniform representation of groups in public databases across taxa. We suggest the use of an integrative approach to identify freshwater fishes in Costa Rica and other megadiverse areas of the Neotropics where similar issues with public barcode reference libraries exist. We also recommend the creation of regional curated barcode reference libraries to aid in the identification of traditionally difficult to identify species/specimens. We also provide the most up to date species list for the ichthyofauna of the Barra del Colorado Wildlife Refuge identifying 51 species from 42 genera, 21 families, and 17 orders. Generating accurate species lists for protected areas and areas of importance will provide conservation practitioners with effective tools for tracking diversity changes over time.

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.

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) provide a non-invasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D-loop region were contained in the sample and amplified by two-step tailed PCR. The 15 PCR replicates indexed different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes were compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes were further reduced. The approach proposed in this study successfully eliminated most of false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.