DOI: 10.3897/mbmg.10.188340

Authors: Irene Calderón-Sanou, Finn Piatscheck, Anne-Sophie Benoiston, Jérôme Chave, Fabian J. Fischer, Marilou Hircq, Amaia Iribar, Eliane Louisanna, Frédéric Petitclerc, Jérémy Raynaud, Gaétan Rongier, Uxue Suescun-Darias, Shengli Tao, Valérie Troispoux, Lucie Zinger, Jérôme Murienne, Jérôme Orivel, Heidy Schimann

Abstract: Soil biodiversity represents approximately 60% of all described species on Earth and plays a critical role in terrestrial ecosystem functioning. However, knowledge of tropical soil biota and their roles remains highly fragmentary, especially in hyperdiverse tropical regions that remain largely undersampled relative to their temperate counterparts. Recent non-invasive methods, such as environmental DNA (eDNA) metabarcoding, have enabled large-scale data collection across kingdoms of life and habitats, including understudied compartments such as soils. Here, GUYASOILeDNA is presented as the first large-scale eDNA metabarcoding dataset of tropical soil Bacteria (16S), Eukaryota (18S), Fungi (ITS1), and Insecta (16S) from French Guiana. This dataset describes the distribution of 69,881 operational taxonomic units (OTUs) in 1,410 soil samples collected across a variety of forest types, savannas, pastures, and urban areas. In addition, functional annotations based on trophic guild assignments are provided for 51% of the OTUs. This dataset can be used for meta-analyses, exploring soil taxonomic and functional diversity in relation to environmental factors, studying spatial associations among soil groups, and informing protected area management, among other applications. While this dataset represents an important first step toward understanding soil biodiversity in the region, it also reveals key gaps that may guide future research.

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DOI: 10.3897/mbmg.10.194720

Authors: Gianluca Natta, Samuele Voyron, Erica Lumini, Alex Laini, Angela Roggero, Claudia Palestrini, Antonio Rolando

Abstract: The dung beetle gut microbiota is essential for host health and plays a key role in regulating the biochemical processes underpinning grassland ecosystem functioning. Understanding how its taxonomic and functional composition varies across species may help predict the ecological consequences of changes in dung beetle communities driven by shifts in pastoral practices. Elucidating the mechanisms of microbial acquisition and potential transmission is also crucial. To this end, a multispecies survey of gut-associated prokaryotic and fungal communities was conducted in nine dung beetle species from a valley in the Alps, representing four tribes and three nesting strategies. All individuals were collected from cow dung to minimise dietary variation. Using an amplicon sequence variant (ASV)-based metabarcoding approach targeting the 16S ribosomal RNA (rRNA) gene and the internal transcribed spacer 2 (ITS2) region, taxonomic composition and predicted functional profiles were characterised. Prokaryotic communities exhibited strong host-dependent structuring: both alpha- and beta-diversity analyses revealed significant differentiation among species and tribes, consistent with host phylogeny and suggestive of phylosymbiosis. In contrast, fungal communities were weakly structured by host taxonomy, with extensive overlap among most species and a limited tribal signal, indicating a greater role of environmental acquisition. Despite pronounced taxonomic turnover, predicted functional profiles were relatively consistent across hosts. Fermentation and nitrogen-related pathways dominated prokaryotic communities, whereas fungal communities were largely saprotrophic, with similar trophic distributions among species. This decoupling between taxonomic composition and functional potential suggests substantial functional redundancy in dung beetle gut microbiota, supporting resilient microbial contributions to dung degradation and nutrient cycling in rapidly changing mountain pastoral ecosystems in the Alps.

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DOI: 10.3897/mbmg.10.192801

Authors: Nicole E. White, Mieke van der Heyde, Michelle T. Guzik, Matthew J. Heydenrych, Glenn I. Moore, Morten E. Allentoft

Abstract: Australia has two eyeless cave gudgeons (Milyeringa justitia and M. veritas) which are restricted to subterranean waters and listed as vulnerable and endangered. Despite being a priority conservation target species, they are inadequately monitored as their restricted distribution in groundwater ecosystems impedes conventional surveying methods, constraining effective monitoring and conservation. As part of ongoing efforts to improve monitoring and conservation of these subterranean fish species, we developed and validated a genus-specific eDNA assay for detecting species of Milyeringa. Mitochondrial genomes were sequenced and assembled to inform PCR assay design, which was then validated in-silico, in-vitro, and in-situ. The assay demonstrated high specificity and sensitivity, particularly for M. justitia (Limit of Detection, LOD: 1.36 copies per reaction), and successfully detected M. veritas (LOD: 12.8 copies per reaction) with moderate sensitivity in groundwater samples from Cape Range. No PCR amplification occurred in non-target surface water samples. This assay provides a non-invasive, robust tool for conservation monitoring cryptic subterranean fishes, supporting conservation management in habitats where traditional survey methods are impractical.

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DOI: 10.3897/mbmg.10.189142

Authors: Nina Filippova, Elena Zvyagina, Tagir Ishmanov, Alexandra Mingalimova

Abstract: Where should one look for fungi in a bog? Historically, fungal diversity in peatlands has been studied primarily in peat itself – understandably, given that peat is the defining feature of these ecosystems – using methods such as sporocarp-based observations and microfungal culturing. These approaches suggested relatively modest richness. In this study, we asked whether that picture changes when we sample multiple contrasting substrates beyond peat. We examined fungal diversity, community composition and structure in a raised bog in northern West Siberia by metabarcoding of 266 environmental DNA samples from key substrates: peat, plant litter, wood and mycorrhizal roots, each partitioned by several inner substrate-related parameters. The metabarcoding data were accompanied and verified by sporocarp monitoring for selected substrate groups. This integrated approach allowed us to capture both the visible and the cryptic components of the fungal community, providing a more complete and robust assessment of diversity distribution. Substrate type emerged as the primary driver of community composition (PERMANOVA F = 10.82, p = 0.001), explaining the strongest dissimilarities in beta-diversity. Plant litter harboured the highest total diversity (7,297 SH), followed by peat (3,590 SH), while wood (1,585 SH) and mycorrhizal roots (1,091 SH) were less diverse. Communities in different substrates showed low species overlap (Jaccard index 0.19–0.42), indicating strong substrate partitioning. The highest number of indicator species was found in mycorrhizal roots (85 SH), reflecting the high specialisation of these communities. In contrast, the apparent low indicator status of litter at the aggregate level masked striking host-specific associations: partitioning litter samples by plant host revealed 40–123 indicator SH per host species. Estimated total fungal richness reached ~ 11,500 SH – far exceeding the 208 species recorded by sporocarp-based methods alone. These results reveal that fungal diversity in a raised bog is not concentrated in peat, but distributed across distinct substrate niches, with plant litter being the richest reservoir and mycorrhizal roots harbouring the most specialised communities. This has implications for understanding carbon cycling and decomposition in peatlands. We conclude that only an integrated, substrate-focused approach can uncover the full fungal richness hidden in these unique ecosystems.

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DOI: 10.3897/mbmg.10.194096

Authors: Armando Alibrandi, Julia Plewka, Aurèle Vuillemin, Alexander Bartholomäus, Rolando di Primio, Alexander J. Probst, Jens Kallmeyer

Abstract: Microbial communities influence key processes in oil reservoirs, including souring, corrosion, and hydrocarbon degradation, so characterizing them is important for reservoir management. Reliable characterization depends on obtaining a sample that represents the community living in the subsurface. As nucleic acids are difficult to recover from oil, produced water, the aqueous phase separated from reservoir fluids, is widely used as a convenient proxy for analyzing the reservoir microbiome. However, whether produced water faithfully represents that community, to our knowledge, has never been tested with paired samples. We addressed this question using 16S rRNA gene amplicon and metagenomic sequencing of reservoir fluids and produced water collected from the same production wells across four North Sea oil fields. The two sample types harbored markedly different microbial communities and functional profiles. This difference persisted after accounting for reservoir identity, and it was larger than the differences between reservoirs. Produced water was less diverse, more uniform across fields, and dominated by aerobic genera associated with seawater and aquifers. Reservoir fluids, in contrast, retained the thermophilic, halophilic, and anaerobic taxa characteristic of the deep subsurface. Only 41% of reservoir-fluid genera and 21% of their amplicon sequence variants were also detected in the paired produced water, indicating overlapping broad taxa but largely distinct populations. Notably, the putatively native reservoir community remained detectable in reservoir fluids even after decades of production, while produced water offered only an incomplete, directionally biased view. Reservoir fluids should therefore be sampled wherever feasible to characterize the in-situ microbiome.

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DOI: 10.3897/mbmg.10.191426

Authors: Luís Afonso, Miguel Álvarez-González, Francisco Pascoal, Fátima Sánchez-Barreiro, Verónica Rojo, Camilo Saavedra, Joana Costa, Pablo Covelo, Graham J. Pierce, Ana Mafalda Correia, Catarina Magalhães, Paula Suarez-Bregua

Abstract: Environmental DNA (eDNA) is rapidly becoming a valuable tool for conducting biodiversity research, including studies on marine vertebrates, and metabarcoding of eDNA enables the characterization of biological communities. However, methodological variation across workflow stages can influence results, highlighting the need for standardized and accessible protocols. In this study, two eDNA extraction strategies were evaluated, the performance of two DNA polymerases differing in proofreading capacity was tested, and the Marine Vertebrate eDNA Metabarcoding bioinformatics pipeline (MVeM)—an open-source, adaptable, and reproducible workflow for sequence processing and taxonomic assignment–was developed. For extraction, the standard DNeasy PowerWater Sterivex protocol was applied, and a preliminary bead-beating homogenization step performed on filter material prior to the PowerWater protocol was additionally tested, as was the recovery of free extracellular DNA in controlled seawater samples. Direct extraction from intact filters yielded higher DNA concentrations and amplicon sequence variant (ASV) richness than the bead-beating homogenization method, whereas both approaches effectively captured marine vertebrate diversity. The recovery of free extracellular DNA remained low with both extraction methods. During library preparation and sequencing, the non-proofreading polymerase generated more cetacean ASV reads in mock communities, suggesting that it is a cost-effective option for monitoring this group, whereas the proofreading enzyme improved the amplification of fish taxa. The MVeM pipeline integrates stringent sequence filtering, the Lowest Common Ancestor approach for ambiguous matches, a geographic exclusion list, and contamination control, enabling accurate, biologically realistic, and reproducible taxonomic assignments for marine vertebrate eDNA. Overall, combining appropriate extraction strategies, polymerase selection, and a robust bioinformatics framework provides a reliable, adaptable, and accessible approach for marine vertebrate biodiversity monitoring. Graphical abstract:

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DOI: 10.3897/mbmg.10.185031

Authors: Dan Joseph C. Logronio, Ming-Chih Chiu, Arnelyn D. Doloiras-Laraño, Levente-Péter Kolcsár, Joeselle M. Serrana, Kohei Hamamoto, Kozo Watanabe

Abstract: A longstanding question in ecology and evolutionary biology is which evolutionary mechanisms explain why some species occur frequently while others do not. Previous studies have investigated this phenomenon through computer simulations, empirical studies, and ecological experiments involving a limited number of species. However, community-wide investigations addressing species diversity and underlying genetic diversity in natural settings remain underexplored. To address such species-genetic diversity correlations (SGDC), we employed haplotype-level bulk DNA metabarcoding to infer haplotypes from many aquatic insect species with varying occurrence across 59 sites in the catchment. To examine the relationship between species occurrence and population genetic structure, we assessed the correlations between environmental parameters, female wing length, a proxy for dispersal ability, and haplotype data. We identified 889 haplotypes belonging to 198 taxonomically identified operational taxonomic units (OTUs). While species with higher occurrence in the catchment had greater total genetic diversity across the landscape (ɣ-genetic diversity) and were likely exposed to a wider range of environmental conditions, we found no correlation between species occurrence and genetic differentiation (β-genetic diversity). Furthermore, we observed no significant correlation between local intraspecific genetic diversity and local species diversity (α-SGDC) across all examined OTUs, and only one OTU (Rhyacophila sp.) showed a correlation between genetic differentiation and community divergence (β-SGDC). These results may be explained by the contrasting factors influencing both levels of biodiversity. Notably, gene flow likely offsets genetic differentiation in the metapopulation, while species sorting appears to influence divergence in the metacommunity. Our findings suggest an alternative approach to studying the relationship between species occurrence, population genetic structure, and SGDC from a community-level perspective. They also have important implications for addressing biodiversity loss driven by rapid environmental changes resulting from human activities and climate change.

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DOI: 10.3897/mbmg.10.193334

Authors: Mehrdad Hajibabaei, Florian Leese, Cathryn Abbott, Hitoshi Araki, Donald J. Baird, Pedro Beja, Kristy Deiner, Hideyuki Doi, Nicole Fahner, Kelly D. Goodwin, Margaret E. Hunter, Ryan P. Kelly, Katy Klymus, Michio Kondoh, Ntangenedzeni Olivia Mapholi, Toshifumi Minamoto, Xavier Pochon, Teresita M. Porter, Susanna Theroux, Alejandro Trujillo-González, Alison Watts, Hiroki Yamanaka, Xiaowei Zhang, John Darling, Kristian Meissner

Abstract: Environmental DNA (eDNA) analysis has emerged as a transformative tool for biodiversity assessment and environmental monitoring, offering a non-invasive, cost-effective approach to detecting species and characterizing ecological communities. Despite growing adoption by researchers, regulatory agencies, and industry, the widespread implementation of eDNA methods continues to face challenges, including inconsistent protocols across laboratories and the absence of internationally recognized standards. These barriers have slowed the integration of eDNA into routine monitoring programs and other operational settings, particularly in regulatory contexts. Standardization is widely recognized as an essential prerequisite for building confidence among end users and enabling meaningful comparison of results across studies and jurisdictions. While standards development efforts have begun to emerge at national and regional scales, the lack of international coordination risks producing fragmented or contradictory guidelines that could hinder rather than advance the field. Here, we introduce the International eDNA Standardization Task Force (iESTF; https://iestf.global), an initiative established to facilitate and accelerate the development of inclusive international standards for eDNA-based biodiversity monitoring. The iESTF aims to identify opportunities for new work item proposals within the International Organization for Standardization (ISO) framework, catalog existing standards and best practices, and ensure that standards development is accessible to practitioners in nations historically underrepresented in eDNA research. We outline some key challenges facing eDNA standardization, describe the structure and goals of the iESTF, and present a vision that includes the formation of a truly inclusive international community of practice, the establishment of pipelines for fast-tracking the development of international standards, and support for critical activities such as cross-regional standards testing and validation. The iESTF represents a critical step toward ensuring that eDNA methods achieve their full potential as reliable, globally comparable tools for biodiversity monitoring.

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DOI: 10.3897/mbmg.10.201781

Authors: Martina Lisnerová, Sagar Nayak, Gabrielle Shira van Beest, Monika Bürgerová, Alena Lövy, Hana Pecková, Jiří Kyslík, Pavla Bartošová-Sojková, Ivan Fiala

Abstract: Microsporidians are highly specialised intracellular parasites adapted to an obligate parasitic lifestyle. Although more than 1,500 species have been described to date, their true diversity is likely much higher, particularly in poorly-studied aquatic environments. Here, we applied an optimised tissue-based metabarcoding approach to investigate the diversity and distribution of microsporidians in aquatic invertebrates from three nearby fish ponds in South Bohemia in the Czech Republic. We analysed more than two thousand individuals of chironomids and oligochaetes, which represent important aquatic invertebrate hosts of microsporidians, and compared microsporidian communities across ponds and host morphogroups. In total, 66 microsporidian OTUs were detected across both host groups. Metabarcoding and phylogenetic analyses revealed broad sequence diversity within the studied localities, with parasite sequences clustering into four main clades. The high proportion of OTUs without close matches to described taxa (more than 95%) highlights substantial undocumented diversity of microsporidians in freshwater ecosystems. The occurrence of shared OTUs amongst different invertebrate taxa is consistent with multi-host strategies and/or complex life cycles. Overall, our findings emphasise the ecological complexity of microsporidians in aquatic habitats. Further experimental work, including parasite localisation and transmission studies, is needed to determine whether the observed host associations represent active infections or incidental detection.

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DOI: 10.3897/mbmg.10.199829

Authors: Kwan Wong, Wu Han, Chuming Zhang, Hin Fat Tsang, Zhewei Si, Xiaoli Tong, Mathew Seymour

Abstract: Freshwater biodiversity underpins ecosystem functioning but is rapidly declining due to urbanization and habitat modification, particularly in Southeast Asia where accurate species inventories remain limited. Integrative taxonomy, combining morphological and molecular approaches, provides a robust framework for generating standardized biodiversity data and biomonitoring assessments. We assessed mayfly (Ephemeroptera) biodiversity across a natural-to-urbanized gradient in Hong Kong using nymph morphology and cytochrome c oxidase subunit I (COI) barcoding to establish the first regional DNA barcode reference library. Our integrated taxonomy efforts resulted in 45 unique morphospecies (i.e. gamma-diversity). Subsequently, the Hong Kong mayfly barcode database includes 146 barcode sequences representing 53 Barcode Index Numbers (BINs), of which 67.3% were newly registered in the Barcode of Life Data System (BOLD), including seven putative undescribed lineages and four new regional records. Additionally, we found that mayfly richness declined significantly with increasing urban intensity (β = –0.308, p = 0.007) and conductivity (β = –0.427, p = 0.002), with elevation showing a marginal effect. Indicator species analysis identified Choroterpes elliptica HK01 as potentially indicative of natural sites and Tenuibaetis pseudofrequentus and Caenis lubrica likely corresponding with peri-urban habitats, while no taxa were indicative of urbanized sites. Our results demonstrate both the substantial mayfly diversity in Hong Kong and highlight the detrimental impacts of urbanization on freshwater assemblages. Critically, the barcode library presented here supports future DNA-based biomonitoring applications (e.g. eDNA metabarcoding), providing an important resource for rapid biodiversity assessment of critical habitats and helping to ensure long-term ecosystem health and stability.

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DOI: 10.3897/mbmg.10.183921

Authors: Pavla Urbánková, Jan Štěpka

Abstract: Marker choice is a critical methodological factor in DNA metabarcoding, as it directly influences taxonomic composition and abundance structure of communities. In diatoms, metabarcoding is increasingly evaluated as an alternative to microscopy-based assessments, particularly for ecological status evaluation under the Water Framework Directive (WFD). Here, we compared two widely used rbcL markers (263 bp and 331 bp long) using identical benthic diatom samples to evaluate their suitability for ecological assessment. Both markers produced highly consistent community patterns and IPS-based ecological classifications. Differences between datasets were mainly restricted to low-abundance taxa, while dominant taxa—driving most of the IPS signal—were consistently detected. Decomposition of IPS differences showed that variation was primarily driven by changes in the relative abundance of shared taxa and inconsistencies in species-level assignment, whereas marker-specific taxa had negligible influence. Overall, the 331-bp marker provided higher taxonomic resolution for abundant taxa, and its lower detection of rare taxa did not compromise ecological assessment. It is therefore well suited for routine biomonitoring under the WFD. Although marker-specific calibration may further improve quantitative accuracy, our results indicate that the 331-bp marker can be reliably applied without major methodological adjustments.

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DOI: 10.3897/mbmg.10.196568

Authors: Nikolas J. Willmott, Jack L. Scanlan, Mark J. Blacket, Brendan C. Rodoni, Narelle Nancarrow, Therésa M. Jones, Francesco Martoni

Abstract: Understanding insect predator–prey interactions is critical for crop protection, particularly within biological control and integrated pest management frameworks. Ladybeetles (Coleoptera, Coccinellidae) are widely used biocontrol predators of aphids (Hemiptera, Aphididae), which are among the most economically damaging pests of grains worldwide. However, post-release dietary assessment of ladybeetles remains challenging because of their polyphagous feeding behaviour. DNA metabarcoding provides a powerful molecular approach for resolving arthropod community composition and trophic interactions through simultaneous amplification and identification of multiple DNA fragments from mixed samples. Here, we applied a widely used insect surveillance metabarcoding workflow, modified through development and implementation of aphid-specific primers, to enhance targeted detection of aphid prey consumed by ladybeetles. We evaluated performance of this prey-targeted metabarcoding approach for dietary analysis and quantified aphid DNA detectability under controlled feeding and starvation regimes. We hypothesised that aphid DNA could be reliably recovered from whole ladybeetle samples using standard surveillance metabarcoding protocols, enabling detection of prey taxa present exclusively as gut contents, which may impact surveillance and diagnostics programs. Aphid DNA from multiple species was detected up to seventy-two hours post-ingestion and up to one hundred hours following cessation of ad libitum feeding, although detectability varied among aphid species and pre-feeding starvation treatments. These results demonstrate that prey-targeted metabarcoding increases sensitivity for dietary inference in predatory insects and provides a robust framework for detecting cryptic trophic interactions. Our findings further indicate that unintended prey detection may be common in arthropod metabarcoding datasets, with important implications for interpretation in biosurveillance and biosecurity monitoring.

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DOI: 10.3897/mbmg.10.168096

Authors: Bassam Abubaker, Csilla Stenger-Kovács, Frederic Rimet, Kálmán Tapolczai

Abstract: DNA metabarcoding of diatoms is a next-generation biomonitoring tool, utilizing high-throughput sequencing technology for the identification and assessment of diatom biodiversity in aquatic ecosystems. Diatom DNA metabarcoding has been widely recognized as a powerful biomonitoring approach in comparison with the traditional morphological identification, which requires high expertise, is more costly and needs more time. This review provides a comprehensive overview of the methodological development, various applications, and challenges in this rapidly emerging field. A systematic search on the Web of Science database resulted in 128 papers directly related to diatom metabarcoding. Manual data extraction showed that elements of the metabarcoding workflow such as sampling, DNA extraction, PCR amplification, choice of barcode, high-throughput sequencing, and bioinformatic analyses have a direct influence on the results. This review highlights the influence of each workflow step on the reliability of the results. Despite its considerable advantages, this novel approach faces persistent challenges, including incomplete reference libraries, abundance quantification, and the diversity of bioinformatics analyses that can influence the outcomes. Nevertheless, diatom DNA metabarcoding rapidly developed into an efficient tool, fundamentally transforming our understanding of aquatic ecosystems and enhancing global biomonitoring capabilities.

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DOI: 10.3897/mbmg.10.177614

Authors: Satsuki Tsuji, Naoki Shibata, Tetsu Yatsuyanagi, Yusuke Fuke

Abstract: Environmental DNA (eDNA) analysis is increasingly recognised as a valuable method for assessing genetic diversity. However, its resolution and applicability are limited by the short length of sequences that can be analysed (typically < 400 bp) and high analytical costs. This study developed a practical, low-cost long-fragment eDNA analysis method using commercial full-length plasmid sequencing via a nanopore platform and evaluated its effectiveness in assessing population genetic structure. 1 L of surface water was collected from 52 sites across Hokkaido, Japan, targeting Barbatula oreas. Two mitochondrial regions (ND5 and cyt b; approximately 1,000 bp each) were species-specifically amplified and sequenced. Amplicon preparation (for sequencing) took 2.5 hours, with a total cost per sample of 4,390 JPY (≈25.55 EUR, ≈29.87 USD). High-quality reads were obtained from 34 samples, allowing for the reconstruction of multiple haplotypes per region through haplotype phasing. The eDNA concentration required to achieve a 50% sequencing success was within a range easily attainable for common species. Phylogenetic analysis using 62 concatenated haplotypes (1,968 bp) obtained from each sample identified two clades and multiple regional subgroups, providing higher-resolution phylogeographic information than the previous study. Furthermore, the differentiation of each clade and group was suggested to reflect geological and climatic events. These results demonstrate the feasibility and utility of long-fragment eDNA analysis for evaluating genetic diversity, and its broad application is anticipated in ecological research, conservation management, and environmental policy formulation.

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DOI: 10.3897/mbmg.10.185622

Authors: Georgia Thomson-Laing, Charlotte Robson, Konstanze Steiner, Lena Schallenberg, Anastasija Zaiko, Susanna A. Wood

Abstract: Biodiversity is declining in freshwater ecosystems, increasing the need for efficient monitoring tools. Environmental DNA (eDNA) is a powerful approach for detecting aquatic species, but sampling methodology can influence detection outcomes. This study evaluated eDNA sampling methods for detecting shortfin eel (Anguilla australis) and longfin eel (Anguilla dieffenbachii) in a turbid lowland lake in Aotearoa New Zealand. The sampling methods compared included active pump filtration, passive samplers, tow nets and sediment sampling. The spatial distribution of eDNA was assessed, and occupancy modelling was used to optimise sampling strategies. Active pump filtration of 3 L water samples produced the most consistent detections of eel eDNA. Passive samplers deployed for 13 h yielded the highest eel eDNA concentrations, although they exhibited high inter-replicate variability. Sediment samples and short-duration passive samplers performed poorly, and collection issues with tow-net sampling led to its exclusion from further analysis. Eel eDNA concentrations were higher nearshore, particularly near the lake’s outflow, potentially reflecting eel activity. Detection rates were higher for A. australis than for A. dieffenbachii, likely due to species-specific differences in abundance, distribution and eDNA dynamics. Occupancy models showed detection probability was driven by sampling method for A. australis and by both sampling method and location for A. dieffenbachii. Pump filtration and long-duration passive sampling provided the highest detection probabilities, with three replicates per site sufficient for reliable detection, although only nearshore for A. dieffenbachii. These findings highlight the importance of sampling method and location when designing eDNA surveys for fish in lakes, with broader relevance across eDNA applications, including single-species detection and metabarcoding.

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DOI: 10.3897/mbmg.10.174467

Authors: Megan R. Shaffer, Elizabeth Andruszkiewicz Allan, Ryan P. Kelly, Nicolaus G. Adams, Stephanie K. Moore, Elizabeth Brasseale, Pedro F. P. Brandão‐Dias, Olivia M. Scott, Andrew O. Shelton, Kim M. Parsons

Abstract: Environmental DNA (eDNA) samples capture mixtures of DNA from a wide range of organisms and often yield metabarcoding datasets that include taxa beyond the intended scope of the primer set used. The presence of this non-target DNA (termed “molecular bycatch”) can make it difficult to interpret trends in the primary targets. Here, we present an example in which including non-target detections alters the ecological interpretation of a metabarcoding time series and highlight a quantitative approach that can account for molecular bycatch in a biologically meaningful way. We collected high-resolution temporal samples from an estuary (Hood Canal, Washington, USA), using a surface-stationed autonomous sampler over a 48-hour period. We metabarcoded samples with a primer set that targets fish, but also amplifies (non-target) marine mammals with lower efficiency. We then transformed sequence read proportions into absolute DNA concentrations using a mock community–based bias correction and single-species concentrations via droplet digital PCR (ddPCR). We found that non-target mammals were substantially under-represented in the raw metabarcoding data relative to their biased-corrected proportions. Cross-validation of this quantitative metabarcoding approach showed that predicted DNA concentrations closely matched independent ddPCR measurements for total 12S DNA and two single-species concentrations (Atlantic bottlenose dolphin and Pacific herring) in both mock communities and environmental samples. After estimating absolute species concentrations for all species, we revealed species-specific patterns that were not apparent in the observed read counts alone. Our results underscore the importance of correcting for species-specific amplification bias when evaluating community composition from metabarcoding data, particularly in the context of non-target detections.

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DOI: 10.3897/mbmg.10.183786

Authors: Carla Schifino Robles Huber, Luciano Köche Huber, Laura Roberta Pinto Utz

Abstract: This study investigates the taxonomic composition, richness and diversity of microbial and metazoan communities in two adjacent ecosystems — a preserved Native Forest and a Highland Grassland — within a well-conserved area of the Atlantic Forest located in the southernmost state of Brazil. Topsoil was analysed using high-throughput sequencing and metabarcoding to assess bacterial, archaeal and eukaryotic communities, divided into microeukaryotes and metazoans, therefore representing four groups of organisms. Bacterial communities were largely composed of Pseudomonadota, Acidobacteriota and Actinomycetota, while archaeal communities were dominated by Thermoproteota. Unicellular eukaryotes showed habitat-specific dominance patterns, with Cercozoa prevailing in Native Forest and Apicomplexa in Highland Grassland; metazoans were represented mainly by Annelida and Nematoda. Alpha diversity analysis revealed differences in richness and in two diversity indices (Shannon and Simpson), which were significantly higher in the Native Forest, with the ordination analyses showing clear spatial structure. Zeta diversity analyses revealed taxon-specific turnover dynamics and indicated that drivers of community structure varied across organism categories. Co-occurrence networks amongst eukaryotes showed well-structured associations within habitats. These results underscore the ecological complexity of even closely-situated habitats and emphasise the importance of long-term conservation of these environments. They also highlight the value of multi-site biodiversity frameworks, such as zeta diversity in capturing fine-scale community differentiation across ecosystems.

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DOI: 10.3897/mbmg.10.186778

Authors: Maria Kahlert, Demetrio Mora, Wolf-Henning Kusber, Nélida Abarca, Jonas Zimmermann

Abstract: Introduction: DNA metabarcoding is currently under intense scrutiny for its future implementation in biodiversity monitoring and ecological assessment, particularly within the EU Water Framework Directive (WFD). However, before metabarcoding can be deployed for WFD reporting, challenges such as the incompleteness of reference databases need to be addressed. In this context, this study presents the results of a national barcoding project (FRESHBAR) focusing on benthic freshwater diatoms of Sweden, a key organism group regarding both ecology and environmental assessment. One major goal of the project was the publication and vouchering of all data, material, and results. Methods: A total of 312 diatom cultures were established, with a focus on oligotrophic and acid habitats. The cultures were sequenced for two barcodes (rbcL and 18SV4) and identified using light microscopy; selected strains were also studied by scanning electron microscopy. All data, including sampling metadata, barcode sequences, images, and voucher material, were published following the FAIR principles. Some cultures were archived in diatom culture collections. Results: Almost all strains were successfully sequenced and identified as 51 taxa across 17 genera, with the highest diversity in the genera Eunotia and Fragilaria. An attempt was also made to identify all taxa at the species level. Approximately half of the taxa were new to Diat.barcode, a curated diatom reference database. Conclusion: FRESHBAR represents the first large-scale effort to generate barcode reference sequences for Nordic benthic freshwater diatoms. As all data are publicly available, the added sequences and morphological information will help identify all taxa properly to the species level, unravel taxon relationships within diatoms, and improve reference databases for environmental monitoring and research.

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DOI: 10.3897/mbmg.10.183268

Authors: Elisavet Kaitetzidou, Piotr Gadawski, William P. Goodall-Copestake, Gabriela Dankova, Konstantinos Gkagkavouzis, Sylwia Holak, Tomasz Rewicz, Karolina Bącela-Spychalska, Tomasz Mamos, Kasia Fantoni, Aleksandra Jabłońska, Grzegorz Tończyk, Łukasz Trębicki, Filippos A. Aravanopoulos, Claudia Bruschini, Georgi Bonchev, Magda Bou Dagher Kharrat, Fedor Čiampor, Filipe O. Costa, Leonardo Dapporto, Torbjørn Ekrem, Sónia Ferreira, Matthias Geiger, Axel Hausmann, Paul D. N. Hebert, Belma Kalamujić Stroil, Stefaniya Kamenova, Ivona Kautmanova, Emre Keskin, Mladen Kučinić, Tatsiana Lipinskaya, Marko Mutanen, Spiros Papakostas, Ben Price, Rina Ramírez, Rodolphe Rougerie, Björn Rulik, Nikolaus Szucsich, Michelle Van Der Bank, Alexandros Triantafyllidis, Peter M. Hollingsworth, Michał Grabowski

Abstract: DNA barcoding has become a cornerstone for species identification and biodiversity monitoring, enabling applications from ecological research to conservation and environmental policy. The International Barcode of Life (iBOL) provides global coordination, but national nodes are essential for implementing barcoding at scale, building local capacity and translating scientific advances into practice. This paper synthesises experiences from 20 countries (17 in Europe), drawing on a survey and a workshop conducted under the Horizon Europe Biodiversity Genomics Europe project. We examine how national nodes are initiated, governed and sustained and identify common challenges, such as defining scope, securing funding, harmonising methods and engaging stakeholders. Most nodes were initiated by research communities and operate as informal networks with heterogeneous governance and staffing models. Key priorities include constructing comprehensive DNA barcode reference libraries, aligning activities with biomonitoring needs and promoting FAIR and CARE data principles. We highlight strategies for capacity building, methodological standardisation and stakeholder engagement, alongside approaches for diversifying funding and strengthening communication. Based on these insights, we present ten practical recommendations to guide the establishment and long-term success of national DNA barcoding nodes. Strengthening these infrastructures will enhance Europe’s ability to deliver robust DNA-based biodiversity monitoring, underpin metabarcoding and metagenomic studies and contribute to global efforts in species discovery, conservation and environmental management.

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DOI: 10.3897/mbmg.10.183708

Authors: Sabrina Gurten, Daniela Sint, Michael Traugott, Oskar Ragnar Rennstam Rubbmark

Abstract: Environmental DNA (eDNA) metabarcoding of floral samples has emerged as a valuable tool for characterizing pollinator communities and plant–pollinator interactions. However, commonly used universal arthropod primers often fail to consistently recover bee (Apiformes) DNA from mixed-template samples. To overcome this limitation, a new COI metabarcoding primer pair specifically designed for bees was developed: BeePrimeF and BeePrimeR. In silico evaluations and in vitro assays demonstrated high target specificity, with only limited amplification of non-target taxa. When applied to floral eDNA from Austrian flower strips, the BeePrime primers doubled the number of samples testing positive for bees and substantially increased the detected bee diversity. Individual floral samples typically yielded multiple bee species rather than single detections, and across all samples, BeePrime detected more than four times as many distinct bee species as a commonly used universal primer. In addition, BeePrime produced significantly higher proportions of bee reads per sample, thereby improving sequencing efficiency and data quality. All molecular identifications were corroborated by specimen-based surveys conducted at the same sites and validated through morphological identification. Overall, BeePrime overcomes a key methodological bottleneck in pollinator eDNA metabarcoding and substantially strengthens the molecular toolkit available for monitoring bee diversity and resolving plant–pollinator networks.

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DOI: 10.3897/mbmg.10.182039

Authors: Daniël van Berkel, Niels W. P. Brevé, Mark de Boer, Emmanuel G. Reynaud, Reindert Nijland

Abstract: Molecular techniques involving environmental DNA (eDNA) are increasingly used for aquatic species detection. Metabarcoding, a widely adopted technique, suffers from primer bias: uneven amplification of species due to primer mismatches. Primer bias can be eliminated by sequencing all eDNA in a sample directly. This method, known as (PCR-free) metagenomics, offers potential benefits for relative abundance estimates, but is seldom applied to fish communities and eDNA. This study used an expanded two-by-two design to compare fish species detection between multi-marker Oxford Nanopore Technologies’ (ONT) metabarcoding and ONT metagenomics (native sequencing) using two filter types (conventional versus coarse). Moreover, we explored methylation patterns obtained from ONT metagenomics. Environmental DNA was collected in a controlled setup and two field settings, one representing a capture-release scenario. All species present in the controlled environment were detected by both metabarcoding and metagenomics. In field settings, metagenomics detected more species than metabarcoding. Coarse filters recovered more species across all sequencing datasets, except in metabarcoding of field settings. Relative read counts between metabarcoding and metagenomics illustrated that primer bias was present in the used primer sets. Most fish metagenomic sequences were identified as European sturgeon (Acipenser sturio) across all eDNA samples. We observed three base modifications on the 18S region of A. sturio, where three nucleotide positions showed different methylation patterns between eDNA samples. Our results demonstrate that metabarcoding and metagenomics function complementarily in biodiversity estimates with metagenomics providing additional insights into base modifications. Furthermore, coarse filters offer strong potential for improved species detection in various environments.

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DOI: 10.3897/mbmg.10.191385

Authors: Michelle Scriver, Anastasija Zaiko, Rūta Gudaitytė, Aurelija Samuilovienė

Abstract: Microbial communities underpin the safety, consistency, and functional properties of fermented foods; however, traditional culture-based methods can result in poorly characterized communities across manufacturers and batches. Molecular approaches enable rapid, cultivation-independent profiling of microbial community composition and function. To gain deeper insights into microbial communities, high-throughput metabarcoding was applied to characterize bacterial and fungal communities in fermented plant-based drink concentrates from seven European manufacturers. Samples were collected at two time points representing independent batches. Bacterial and fungal communities were profiled using 16S rRNA gene and internal transcribed spacer 2 (ITS2) amplicon sequencing, respectively. Taxonomic profiles were complemented by functional assessment to flag beneficial taxa associated with fermentation and taxa with potential spoilage or safety relevance. Bacterial communities were dominated by lactic acid bacteria, including Lactobacillus spp. (e.g., L. acidophilus), Leuconostoc spp., and Lactococcus spp., alongside probiotic genera such as Bifidobacterium spp. Low-abundance sequences assigned to taxa with potential safety relevance, including Clostridium perfringens, were detected. Fungal communities were dominated by the fermentative yeast Saccharomyces spp., alongside environmental taxa such as Fusarium, some of which may have mycotoxin-producing potential. Community composition varied by manufacturer and batch for both bacteria and fungi; however, manufacturer effects were substantially stronger for bacterial communities, explaining 74.8% of bacterial variance compared to 35.4% for fungi. These findings indicate that metabarcoding with functional assessment can serve as an initial flagging system for routine microbial monitoring and enables tracking of community composition and consistency across batches and markets, thereby improving quality insights in fermented food production.

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DOI: 10.3897/mbmg.10.185694

Authors: Michaela Šamulková, Pavel Beracko, Patrik Macko, Kornélia Tuhrinová, Zuzana Čiamporová-Zaťovičová, Fedor Čiampor Jr

Abstract: Freshwater biomonitoring under the EU Water Framework Directive (WFD) relies on multiple biological quality elements, with macrozoobenthos being one of the most widely used, although still constrained by time-consuming morphological identification and the continued decline in taxonomic expertise. These constraints, together with ongoing rapid biodiversity loss, create an urgent need for high-resolution, scalable assessment methods such as DNA metabarcoding. Here, we evaluated this molecular approach as a WFD-compatible method and compared the performance of bulk and environmental DNA (eDNA) sampling for ecological quality classification in Slovakia. During spring 2022–2023, we sampled 34 WFD long-term monitoring sites using (i) bulk macroinvertebrate kick-samples, and (ii) water and (iii) sediment eDNA. A ~420 bp fragment of the mitochondrial COI gene was amplified using the BF3/BR2 primers, and sequenced. Filtered reads were clustered into OTUs based on 97% identity and taxonomically assigned using the BOLD database. Raw read counts were used as abundance proxies to calculate 12 ASTERICS metrics and multimetric Ecological Quality Ratios (EQRs). Sequencing yielded > 32.1 million reads and 1,205 target OTUs, of which 702 corresponded to 558 Linnaean species. Bulk samples captured the highest richness, followed by water and then sediment eDNA. Bulk samples classified 22 sites as very good Ecological Quality Class (EQC), whereas water and sediment samples frequently reduced EQC by two to three classes. EQC differences were driven mainly by presence/absence metrics (EPT taxa and BMWP), while several abundance-dependent metrics remained stable. Bulk metabarcoding provides a comprehensive signal but still inflates presence/absence metrics, highlighting the need for recalibration and optimised eDNA sampling, including primer selection, to enable comparable non-invasive assessments.

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DOI: 10.3897/mbmg.10.173083

Authors: Alejandro Rodríguez-Gijón, Víctor López-Maroto, Mireia Mestre, Salvador Sánchez-Carrillo, Rafael Laso-Pérez

Abstract: Wetland ecosystems are of particular environmental importance due to their role as biodiversity shelters. However, the intense anthropization of wetland landscapes have propelled a decline in water quality and quantity globally during the last few decades. Under this context, microbial communities offer an accurate approach to better understand the conservation state of these water bodies. With the goal of contributing to environmental monitoring of wetlands of global significance, we present here a newly-generated 16S rRNA amplicon dataset derived from Las Tablas de Daimiel National Park, a wetland system in Spain designated as a Biosphere Reserve by UNESCO and a Ramsar site of international importance. Samples were collected across four different stations in the region, covering both the water column and the sediments, and environmental metadata were measured. In total, our dataset compiles 1,766 archaeal ASVs and 19,855 bacterial ASVs covering 11 and 64 phylum-level groups, respectively. This dataset offers a valuable resource for future monitoring work at the Las Tablas de Daimiel National Park to improve its health and conservation.

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DOI: 10.3897/mbmg.10.184566

Authors: Marina Chauvet, Paul Terzian, Anne Moné, Didier Debroas, Gisèle Bronner, Cécile Lepère

Abstract: Microbial eukaryotes play a vital role in biogeochemical cycles and aquatic food webs. Over past decades, their taxonomic diversity has been investigated using short-read DNA metabarcoding, which, while effective, is hampered by the limited resolution of the targeted regions. In this study, we utilised both short-read (Illumina) and long-read (PacBio) metabarcoding approaches to analyse eukaryotic diversity in a lake ecosystem over one year, allowing for a direct comparison of the results obtained from each method. Our metabarcoding analysis revealed a high degree of congruence between long-read and short-read data at broad taxonomic levels. However, at finer taxonomic scales, such as the genus level, long-read sequencing achieved higher resolution, enabling more precise identification of microbial eukaryotes. This enhanced taxonomic resolution proved especially valuable for tracking seasonal dynamics within key groups, including ciliates and chytrid fungi. The resolution offered by long-read sequencing provides a more detailed picture of the microbial eukaryotic community, thereby facilitating the exploration of ecological interactions at a finer scale. For example, this approach allowed us to monitor the dynamics of the chytrid genus Zygorhizidium in relation to various diatom genera, which are primary targets for chytrid parasitism.

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DOI: 10.3897/mbmg.10.162895

Authors: Gen Ito, Daisuke Sasaki, Masayuki K. Sakata, Seigo Kawase, Toshifumi Minamoto, Hiroki Yamanaka

Abstract: To enhance taxonomic resolution in environmental DNA (eDNA) metabarcoding, we evaluated the utility of the internal transcribed spacer 2 (ITS2) region. We developed ITS2 metabarcoding primers and constructed an ITS2 reference database for Japanese freshwater fishes by sequencing an additional 61 species and compiling existing sequences. We then compared the taxonomic resolution of ITS2 metabarcoding with that of the widely used mitochondrial 12S rRNA (MiFish) marker. Our results demonstrated that ITS2 provided higher resolution, particularly for species in Oncorhynchus and Rhinogobius, which are often difficult to distinguish using MiFish. The improved resolution in Oncorhynchus highlights ITS2’s potential for monitoring economically and ecologically important salmonid species. Additionally, ITS2 resolved some closely related species that MiFish could not differentiate, underscoring its value in biodiversity assessments. Our findings suggest that ITS2 can complement MiFish in eDNA-based fish biodiversity monitoring, particularly for taxa where mitochondrial markers have limited resolution. Future studies should further explore the applicability of ITS2 across diverse freshwater ecosystems to optimize its integration into multi-marker metabarcoding approaches.

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DOI: 10.3897/mbmg.10.176285

Authors: Yuya Ishihara, Kazumi Aoki, Ayumi Sakata, Masayuki K. Sakata, Kyohei Hamano, Qianqian Wu, Masaki Miya, Toshifumi Minamoto

Abstract: As mammalian diversity declines, effective biodiversity monitoring tools are urgently needed. To address this issue, we assessed the detectability of mammalian environmental DNA (eDNA) by applying eDNA metabarcoding to stemflow samples. We collected stemflow samples six times from June 2024 to January 2025 from 11 trees in the Oji Zoo, Kobe, Japan. One tree was inside the cage and thus in direct contact with the captive mammals, whereas the others were outside the cages, i.e., could not be directly contacted by captive mammals. We amplified eDNA extracted from the stemflow samples with MiMammal primers targeting the mitochondrial 12S rRNA gene, and the PCR products were then sequenced. We detected eDNA of 22 mammal species, 14 of which were zoo species and eight of which were not. Environmental DNA of Japanese squirrels (Sciurus lis), which could directly contact the target tree, was detected at all six sampling time points. In addition, we found a significant negative relationship between eDNA-based detection and the distance from each mammal cage to each tree, as well as significant differences in the composition of the detected species between the trees inside and outside the cages. In conclusion, our pioneering analysis shows that stemflow eDNA metabarcoding can be useful for detecting and monitoring mammals. The potential for its application in natural settings will need to be explored in subsequent studies.

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DOI: 10.3897/mbmg.10.177451

Authors: Hiroki Yamanaka, Takaya Hirohara, Marshal S. Hoy, Dorothy M. Chase, Jeffrey J. Duda, Carl O. Ostberg

Abstract: Environmental DNA (eDNA) analysis has significantly contributed to biodiversity monitoring due to its sampling simplicity and inherent high sensitivity. While the technique has matured considerably for species detection and characterising community compositions via metabarcoding, its applications in population and landscape genetics remain underexplored, primarily because these fields require the extraction of high-quality genetic information from samples. To facilitate further technical development in this domain, we investigated the availability of intact fish cells in water — a source expected to yield high-quality DNA templates — by performing a five-day replicated tank experiment using live and dead Rainbow Trout (Oncorhynchus mykiss) as experimental subjects for eDNA production. Propidium monoazide (PMA), a photoreactive intercalating dye which is permeable to disrupted biological membranes and thereby inhibits PCR amplification of DNA, was employed to differentially quantify PMA-resistant eDNA as a proxy for eDNA derived from intact versus compromised cells relative to the total eDNA pool, based on whether fish were living or dead. Our results showed significantly higher concentrations of total eDNA released by dead fish compared to live fish on Day 2 (12.9×) and Day 3 (7.4×). Furthermore, the amount of PMA-resistant eDNA was significantly elevated in the dead fish treatments on Day 2 (29.3×), Day 3 (31.3×) and Day 5 (5.2×). Collectively, these findings indicate that dead fish release significantly greater amount of both total eDNA and PMA-resistant eDNA, which likely represents eDNA from intact cells, into the surrounding environment compared to live fish. Our established methodology for assessing the availability of intact cells in water and the resulting quantitative data provide crucial insight for advancing environmental genetics approaches within the eDNA framework.

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DOI: 10.3897/mbmg.10.174834

Authors: Torrey W. Rodgers, Arianna Basto, Raider Castro, Erin Marcela Rivera Groves, Charles C. Y. Xu, Elena Hernández, Karen E. Mock, Adrian Forsyth

Abstract: Metabarcoding of vertebrate DNA derived from invertebrates (iDNA) is a promising method for monitoring of mammal assemblages. However, optimization of field and laboratory methods for efficient and economical sampling and analysis is needed. We used metabarcoding of carrion flies to survey for mammals at Los Amigos Biological Station in the Peruvian Amazon. We used an extraction-free, direct PCR approach to metabarcode vertebrate DNA, whereby buffer from tubes that contained flies for 3-5 hours was used directly in PCR, negating the need for labor-intensive DNA extraction. We also examined two different pooling methods to investigate the efficacy of pooling multiple fly samples to economize sequencing of large numbers of samples. Metabarcoding detected 27 mammalian taxa from 9 orders and 18 families plus four anuran taxa from three families from 240 carrion flies collected over 3 days. Pooling of buffer from multiple flies prior to PCR resulted in failure to detect many species compared to sequencing of individual flies. However, pooling of PCR product after an initial PCR from individual flies, but prior to indexing, resulted in species detection nearly identical to sequencing of individual flies. These methodological findings will contribute to the feasibility of iDNA metabarcoding for vertebrate monitoring and biodiversity conservation.

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DOI: 10.3897/mbmg.10.174450

Authors: Alison W. Watts, Zachary Gold, Nastassia V. Patin, Nicolaus G. Adams, Jacoby D. Baker, Fouad El Baidouri, Erin Grey, Ann E. Holmes, Kym Jacobson, Sean Jungbluth, Tadashi Kajita, Anders Kiledal, Matthew A. Lemay, Enrique Montes, Frank E. Muller-Karger, Jeffrey Miller, Matthew B. Ogburn, Kathleen J. Pitz, Katherine Silliman, Andrew Thompson, Luke R. Thompson

Abstract: With the rapid uptake of environmental DNA (eDNA) metabarcoding for a wide range of conservation and management uses, there is a growing need for guidance and best practices for species identification. A range of methodological and interpretive decisions influences taxa assignment. Here we provide a review and perspective of pitfalls and issues that can impact accurate taxonomic assignment across the full eDNA metabarcoding workflow: primer selection, lab handling, bioinformatics, taxonomic assignment, and communication of results. This paper addresses the complexities and challenges of the metabarcoding-based species identification process, offering recommendations for robust workflows and effective communication of findings. Accurate interpretation and communication of eDNA metabarcoding results requires an acknowledgment of methodological limitations such as incomplete reference databases, contamination risks, ambiguous sequences, and detection biases. Thus, we argue that transparency of methods and limitations, alongside proactive alignment of decisions with project objectives are critical for the successful application of eDNA metabarcoding in conservation and management decisions. We provide guidance for developing protocols that support species identification from eDNA metabarcoding sequences, and give recommendations on communication strategies for stakeholders and end users. To support these recommendations, we outline steps in the workflow that can impact species identification, with a discussion of the strengths and weaknesses at each stage. Ultimately, this guidance can improve the accuracy, reliability, and usability of eDNA and other metabarcoding and amplicon sequencing approaches to species identification while fostering trust and understanding among diverse end users.

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DOI: 10.3897/mbmg.10.173612

Authors: Ioannis Kavakiotis, Peter Woollard, Camila Babo, Elianne Egge, Dawid Krawczyk, Tiina Laamanen, Joana Paupério, Karolina Bacela-Spychalska, Nicolas Pade, Filipe O. Costa, Michal Grabowski, Veera Norros, Kristian Meissner, Spiros Papakostas

Abstract: Environmental DNA (eDNA) has emerged as a transformative tool for monitoring aquatic biodiversity, offering a non-invasive and highly sensitive approach to detecting organisms across diverse ecosystems. However, its effective downstream application across Europe in environmental management is hindered by inconsistencies in data standardisation, metadata reporting, and accessibility. This perspective comprehensively evaluates current data repositories, data submission workflows, and standardisation efforts within the European aquatic eDNA landscape. By employing a multi-method approach, including an inventory of eDNA databases, a metadata assessment, a stakeholder questionnaire, and a generative Artificial Intelligence (AI)-driven analysis of scientific literature, our findings reveal substantial variability in metadata reporting practices, with several areas misaligned with Findable, Accessible, Interoperable, and Reusable (FAIR) principles. While some repositories demonstrate strong data curation and accessibility, others lack essential metadata descriptors, limiting interoperability. We identify critical gaps in metadata submission, particularly concerning sampling methods and wet lab workflows, which heavily impact data reusability. The use of generative AI in this study further enabled large-scale identification of recurring reporting weaknesses, highlighting structural challenges that extend beyond individual studies. Addressing these gaps and leveraging advanced computational approaches through international standards and harmonised guidelines represents a clear way forward, as articulated in the recent “Making eDNA FAIR” paper by Takahashi et al. (2025), which is based on the use of Darwin Core (DwC) and Genomics Standards Consortium (GSC) MIxS standards, as well as Global Biodiversity Information Facility (GBIF)’s “Publishing DNA-derived data through biodiversity data platforms” guidelines. Furthermore, additional complementary principles strengthen this framework. The Collective benefit, Authority to control, Responsibility, Ethics (CARE) principles emphasise Indigenous data governance and responsible sample stewardship, while the Transparency, Responsibility, User focus, Sustainability, Technology (TRUST) principles provide criteria for repository reliability and long-term digital preservation. Together, the combined application of FAIR, CARE, and TRUST principles provides a structured foundation for ensuring robust, interoperable, and ethically managed eDNA data that support aquatic biodiversity research, management, and conservation across Europe.

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DOI: 10.3897/mbmg.10.181378

Authors: Nicole D. Juby, William G. R. Crampton, Nathan R. Lovejoy

Abstract: DNA barcodes are instrumental for identifying species from tissues and environmental samples. Metabarcoding-based assessments of biodiversity depend on taxonomically accurate barcode sequence libraries that can be queried to determine which species are present in a sample. The mitochondrial 12S ribosomal RNA gene has been shown to function as a robust barcode for differentiating taxa and identifying species using environmental DNA (eDNA), but complete 12S reference barcodes remain scarce in public databases. We assembled a library of 12S rDNA reference barcodes for 152 species of Neotropical electric fishes from the order Gymnotiformes (Teleostei, Ostariophysi). These fishes are an abundant and diverse component of Neotropical freshwater ecosystems, but are often difficult to collect through conventional sampling approaches because of their nocturnal activity and association with dense substrates or deep-water habitats. This makes eDNA metabarcoding an especially suitable approach for their detection. We compiled a dataset comprising all gymnotiform families, with most sequences covering almost the entire 12S gene. Across the gene and within two widely used 12S eDNA metabarcoding loci (MiFish and teleo), our sequences show sufficient interspecific divergence to discriminate the majority of knifefish species included in our analyses. This dataset, therefore, provides a valuable new set of resources for differentiating amongst gymnotiform species and identifying them in environmental DNA samples.

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DOI: 10.3897/mbmg.10.172476

Authors: Agathe Pirog, Juliette Lavarec, Sabine Rousselot, Cécile Kaerle, Christophe Duchamp, Guillaume Queney

Abstract: Accurate species identification is essential in metabarcoding diet studies, notably to distinguish domestic from wild species, as they have markedly different ecological and socio-economic implications. This is particularly true for the genus Ovis, where domestic sheep (Ovis aries) and European mouflon (O. musimon) often co-exist, but are indistinguishable with common mitochondrial markers due to insufficient variation. Misidentification of these taxa can skew carnivore predation assessments, notably for species like the grey wolf (Canis lupus), impacting wildlife conservation and management efforts. Here, we developed a diagnostic D-loop fragment that effectively differentiates O. aries and O. musimon haplotypes in diet metabarcoding studies. Using reference samples from 46 European mouflons and 46 domestic sheep collected in the French Alps, we identified a 178 bp fragment (OvisCR) containing 43 SNPs, distinguishing four mouflon haplotypes and 35 sheep haplotypes. No haplotypes were shared between the two species, though one O. musimon haplotype was closely related to two O. aries haplotypes. The performance of the OvisCR fragment was tested using 115 wolf scat samples. We sequenced a general metabarcoding marker (a 60–140 bp amplicon of the 12S region) alongside the new amplicon on the Ion Torrent platform to assess the ability of the latter to refine Ovis identification. Following sequencing, two bioinformatic pipelines (DADA2 and OBITools) were employed to compare their effectiveness in resolving Ovis operational taxonomic units (OTUs) or amplicon sequence variants (ASVs). The two pipelines demonstrated similar accuracy, with the OvisCR fragment achieving Ovis species-level assignment for more than 81% of scats with Ovis reads detected, regardless of the pipeline used. This new marker provides a robust and practical tool for metabarcoding applications, improving ecological inference in carnivore trophic studies and supporting biodiversity conservation management strategies. While effective in the French context, its broader geographic applicability requires prior validation using region-specific reference datasets that encompass the diversity of domestic sheep, European mouflon and other Ovis species.

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DOI: 10.3897/mbmg.10.174947

Authors: Boris A. Levin, Marina A. Levina, Oleg N. Artaev, Aleksey A. Bolotovskiy, Alexander A. Gandlin, Ilya S. Turbanov, Aleksandra S. Komarova, Nikolai S. Mugue, Ivan V. Pozdeev, Alexander B. Ruchin, Kirill V. Litvinov, Stepan A. Podolyako, Evgeniy Simonov

Abstract: This study represents the first thorough genetic assessment of freshwater fishes from the Volga River Basin – the longest (3,690 km) river in Europe. DNA barcode sequences (COI) were studied in 84 species (approx. 85% of all diversity) belonging to 57 genera and 25 families. In total, 1,327 DNA barcodes were used to: (i) build a DNA barcode reference library and check for the presence of a barcoding gap; ii) assess the genetic and taxonomic diversity of Volga fishes and iii) clarify the biogeography of Volga fishes. DNA barcodes are reliable means for the identification of over 95% of analysed species showing barcode gaps. An overlap between intra- and interspecific genetic distances was present in four species of genera Leuciscus and Rutilus. DNA barcoding identified seven new species for the Volga. Amongst these, two are aggressive alien species (Hemiculter leucisculus and Pseudorasbora parva), while five are indigenous to the region and belong to the genera Salmo, Leuciscus, Gobio and Cobitis from neighbouring basins, which naturally or human-aided colonised the Volga Basin. Moreover, three new phylogenetic lineages were discovered in the genera Leucaspius, Phoxinus and Proterorhinus. While the fishes from sub-basins of the Volga usually do not demonstrate sorting of the haplotypes, the more ancient Kama displays regional endemism for rheophilic species (e.g. Barbatula, Cottus). Our results suggest the Kama might serve as refuge during Pleistocene climate oscillations. In summary, the generated COI dataset fills the existing gap in COI database of fishes inhabiting freshwaters of Eastern Europe.

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DOI: 10.3897/mbmg.10.173793

Authors: Lukas Damm, Sahar Khodami, Verena Rubel, Massimiliano Molari, Annemiek Vink, Thorsten Stoeck, Pedro Martinez Arbizu

Abstract: Metabarcoding offers a powerful approach for assessing benthic biodiversity in remote and understudied deep-sea environments. However, the methodological performance of different DNA extraction strategies remains insufficiently evaluated, especially for benthic meiofaunal communities. In this study, we compared two extraction strategies—community DNA (ComDNA) from isolated benthic metazoans and sedimentary environmental DNA (SedDNA) from whole sediment—to assess benthic metazoan diversity in the Clarion-Clipperton Fracture Zone (CCZ), an area targeted for future polymetallic nodule mining. Using the 18S V1–V2 rRNA marker, we analyzed alpha and beta diversity, taxonomic composition, and the number of metazoan operational taxonomic units (OTUs) recovered by each strategy. ComDNA extractions yielded substantially higher benthic metazoan OTU richness (2,145 OTUs) than SedDNA (392 OTUs), with only 1.2% of OTUs shared between them. Community composition also differed significantly, driven by strategy-specific detection biases. To evaluate the effectiveness of SedDNA for biodiversity monitoring, we modeled the sediment volume required to recover OTU richness comparable to ComDNA samples. Depending on sequencing depth and statistical approach, we estimated that the processing of 27–82 mL of sediment is necessary to match ComDNA-derived richness. Our findings underscore the superior taxonomic resolution of ComDNA extractions but also highlight the potential of optimized SedDNA protocols for scalable biodiversity monitoring. We recommend sediment homogenization, increased sample volume, and a higher sequencing depth of at least 100,000 reads per sample for improving SedDNA-based assessments, particularly in heterogeneous deep-sea environments. These results provide critical methodological guidance for the development of standardized, efficient monitoring strategies in the context of deep-sea mining impact assessments.

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DOI: 10.3897/mbmg.10.164232

Authors: Adrián Gómez-Repollés, Eva Egelyng Sigsgaard, Mads Reinholdt Jensen, Philip Francis Thomsen

Abstract: Environmental DNA (eDNA) is increasingly used in aquatic research and applied biodiversity monitoring, mainly through a metabarcoding approach. This allows detailed analyses of specific taxa of interest but involves amplification by PCR, which can lead to significant taxonomic bias. On the other hand, broad tree-of-life approaches such as shotgun sequencing are easily dominated by prokaryotic DNA, failing to detect less abundant taxa such as metazoans. However, the proportion of eukaryotic DNA could potentially be increased through water filtration with larger pore sizes. In this study, we investigate how filter pore size influences the taxonomic composition of retained aquatic eDNA. We conducted high-throughput shotgun sequencing and 18S rDNA metabarcoding on 15 seawater eDNA samples collected at the same locality but filtered with different pore sizes spanning from 0.2 to 8.0 µm. We could assign only 0.78% of the total shotgun reads to the superkingdom level, but smaller pore sizes (0.2 and 1.2 μm) yielded significantly higher proportions of bacterial reads compared to eukaryotic reads (63 vs. 28%), while larger pore sizes (5.0 and 8.0 μm) yielded significantly higher proportions of eukaryotic reads compared to bacterial reads (49 vs. 31%). Of the 19 metazoan phyla detected with shotgun sequencing, all but one were more abundant in larger pore sizes. Most of the eukaryotic phyla detected (39 of 54) were shared between shotgun and metabarcoding analyses, indicating similar performance of the methods regarding presence/absence estimation at high taxonomic levels. Finally, we compared the reliability of genus-level detections between the two methods for selected groups of metazoans to evaluate the potential of shotgun sequencing for biomonitoring. Our study suggests that the prokaryotic dominance in shotgun sequencing data from aquatic eDNA can be reduced by increasing filter pore size. As reference databases expand, the taxonomic coverage and resolution of shotgun sequencing should improve, likely enhancing the potential of shotgun sequencing for future eDNA research.

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DOI: 10.3897/mbmg.10.173634

Authors: Aimee L. van der Reis, Mary A. Sewell, Rachael M. Wade, Wendy A. Nelson

Abstract: The geniculate coralline algal genus Corallina L. (Rhodophyta, Corallinales) presents longstanding challenges in taxonomy and nomenclature. Recent studies have uncovered previously unrecognised diversity within the genus, highlighting the importance of revisiting historical names and type specimens. In this study, we resolve the taxonomic identity of Corallina armata through DNA sequencing of the rbcL and COI gene regions and designate an epitype to stabilise its nomenclature. Phylogenetic analyses consistently grouped Corallina sequences into species-specific clades across both markers. Using population genetic approaches, single nucleotide polymorphism data revealed clear genetic separation of C. armata from related species, supported by hierarchical clustering, based on genetic distance and principal component analysis of allele frequencies. A pairwise permutational analysis of variance confirmed significant genetic differentiation amongst species (p < 0.05). To support broader taxonomic efforts and utilised in this study, we developed modular universal rbcL primer suites for macroalgae, which performed well in silico across the GenBank database, indicating strong potential for international application (inclusive of some microalgae). Taxonomic identification results from the short-read sequences for rbcL (Ochrophyta and Rhodophyta) and COI showed both primer sets enabled species-level identification, although the rbcL primer sets captured less genetic variation than COI. Nonetheless, each gene revealed distinct interspecific relationships, underscoring the value of multi-locus approaches for robust species delimitation in future barcoding and metabarcoding studies.

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DOI: 10.3897/mbmg.10.139674

Authors: Sage Handler, Katerina Coveny, Thomas W. A. Braukmann, Nigel E. Raine, Dirk Steinke

Abstract: Essential ecosystem services are provided by many interactions, including plant-pollinator, predator-prey, and host-parasitoid. These services support food and natural systems through pollination and pest control, however they are challenging to qualify, and previous observational studies may underestimate their complexity. The cavity nesting Hymenoptera are a good example showing all these three interactions and they can be monitored using trap nests. For this study, trap nests were installed at schools across Canada by community scientists to investigate cavity-nesting bee and wasp distributions and interactions. DNA metabarcoding was used to identify the occupants and their food sources. New bee and wasp distributions were found that might be the result of previous under-sampling or recent range expansions. Detailed bipartite and tripartite networks describing species interactions suggest some novel bee, wasp, and parasite associations. These results encourage further investigation into these interactions using molecular methods as detailed range maps and networks provide information to natural historians and conservationists alike.

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DOI: 10.3897/mbmg.10.174578

Authors: Chloe Victoria Robinson, Brittany Catrina Visona-Kelly, Emma June Laqua, Gary James Sutton, Colleen M. Talty, Michael William Dallas Judson

Abstract: Globally, vessel strikes, entanglements, and ocean pollution negatively impact cetaceans by compromising skin integrity and health through direct injury or disease and by increasing susceptibility to chronic bacterial, viral, or fungal infections. Characterizing skin microbial assemblages in free-living cetaceans offers a means to evaluate and establish baseline trends in skin conditions. Currently, skin microbial data are collected only via biopsy sampling, which is a mildly invasive approach that represents only a small surface area of skin. In this study, we employed non-invasive environmental DNA (eDNA) flukeprint sampling of West Coast Transient killer whales in California and British Columbia to evaluate the method’s effectiveness in detecting unique skin-associated bacteria. Using a paired approach, we found a greater number of bacterial exact sequence variants and bacterial genera unique to flukeprints compared with seawater controls. Functional pathways of unique flukeprint bacterial exact sequence variants were dominated by predicted functions likely related to DNA replication and repair, cell signaling, and protein folding. We compared bacterial results with visual skin condition assessments and other factors, finding that flukeprint bacteria are explained by a combination of geographic region, sampling month, whale age, whale sex, and the percentage of skin lesions visible in lateral photographs. This work demonstrates the applicability of eDNA flukeprint sampling for improving understanding of cetacean skin health, with implications for studying endangered or surface-elusive cetaceans that are not good candidates for biopsy sampling.

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DOI: 10.3897/mbmg.9.169630

Authors: Sahar Khodami, Alexandra Ostmann, Jana Packmor, Kai Horst George, Pedro Martínez Arbizu

Abstract: Understanding how meiofaunal communities respond to spatial protection measures is essential for evaluating the effectiveness of marine conservation strategies, particularly in heavily impacted regions such as the Baltic Sea. Here, we present one of the first integrated assessments of meiofaunal diversity across marine protected areas (MPAs) and adjacent reference areas (REFs) in the Baltic Sea. Our study combines whole-community metabarcoding of two genetic markers—mitochondrial COI and ribosomal 18S—with classical morphological identification of harpacticoid copepods. Sampling was conducted in the Fehmarn Belt and Oderbank regions as part of the DAM pilot mission MGF Ostsee, prior to the exclusion of mobile bottom-contact fishing. We provide a valuable baseline of community composition under current conditions. Results highlight strong complementarity between the genetic markers: 18S offered broader taxonomic coverage and higher alpha diversity, particularly for Nematoda and Platyhelminthes, while COI enabled more precise species-level resolution, especially for Copepoda. Spatial analyses revealed distinct assemblages between Fehmarn Belt and Oderbank, with notably higher copepod diversity in Fehmarn Belt. Significant differences between MPAs and REFs were detected only in Oderbank. Morphological and molecular datasets showed substantial but incomplete overlap, with each method capturing unique taxa, demonstrating their combined value for comprehensive biodiversity assessments. Our findings suggest that meiofauna communities reflect both regional environmental gradients and high small-scale patchiness. This study underscores the utility of multi-marker metabarcoding alongside traditional taxonomy for monitoring meiobenthic diversity and highlights the importance of incorporating meiofauna into long-term ecological assessments. The dataset provides a crucial reference point for evaluating future recovery and management outcomes following trawling exclusion.

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DOI: 10.3897/mbmg.9.156215

Authors: Julia K. Nuy, Till L. V. Bornemann, Daniela Beisser, Alexander J. Probst, Jens Boenigk

Abstract: Lake ecosystems are hotspots for important ecosystem functions, yet linking their microbiome to physicochemical parameters remains a challenge. Here, we compare 16S rRNA gene-based amplicon sequencing, metagenomics, and metatranscriptomics across 21 European lakes using three strategies: (i) mapping shotgun reads to amplicon-derived OTUs, (ii) marker-specific profiling (rpS3 for metagenomes, rRNA reads for metatranscriptomes), and (iii) recovery of 16S rRNA genes from shotgun assemblies. Strategy (iii) proved unfeasible due to chimeric and highly variable assemblies and was excluded from further analyses. Both strategies (i) and (ii) revealed systematic methodological constraints. Amplicons yielded significantly lower richness and Shannon diversity than metagenomes and metatranscriptomes, while marker-based profiling highlighted broader detection of rare and active taxa. Despite these differences, all lakes showed the same relative ranking of diversity (metatranscriptomes > metagenomes > amplicons), indicating consistent methodological signatures across ecosystems. Beta-diversity analyses confirmed stronger concordance between metagenomes and metatranscriptomes than between either of these and amplicons. Differential abundance analyses further revealed method-specific detection biases, particularly for Proteobacteria and Bacteroidetes, that persisted even after correcting for 16S rRNA gene copy number and primer bias. Linking communities to physicochemical parameters, Mantel and Procrustes analyses showed the strongest global associations for metatranscriptomes, while metagenomes yielded the most stable explanatory OTUs in dbRDA and BioEnv models. Our results demonstrate that the sequencing approach is not merely a technical choice but represents an analytical dimension that fundamentally influences how microbiome–environment interactions are detected and interpreted.

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DOI: 10.3897/mbmg.9.167231

Authors: Stefan Shilev, Mariana Petkova, Vanya Popova, Ivelina Neykova, Ivan Rangelov

Abstract: Integrating intercropping with green manure within crop rotation offers a sustainable pathway to enhance soil microbial diversity and overall agroecosystem health. This study evaluated the effects of diverse fertilisation practices on the soil microbiome in a barley (Hordeum vulgare L.) cropping system following the incorporation of an oat-vetch (Avena sativa and Vicia sativa) and green manure incorporation versus single cropping of oats. The field experiment took place at the Agricultural University of Plovdiv, Bulgaria. Using high-throughput metagenomic sequencing, we assessed the structure and functions of the soil microbial community. The fertilisation treatments included mineral fertiliser, vermicompost, a combination of vermicompost and mineral fertiliser, and biochar, along with a non-fertilised control. The investigation compared two preceding cropping systems before barley cultivation: an oat-vetch intercrop used as green manure, incorporated into the soil at the ripening stage, and a conventional approach where barley was sown directly without prior green manuring. All treatments were conducted in triplicate. Sequencing allows for detailed taxonomic and functional profiling. The most abundant bacterial phyla identified were Actinobacteriota, Proteobacteria, and Bacteroidota, with Actinobacteriota showing increased abundance under fertilised conditions. Soils treated with compost and compost-mineral mixtures exhibited notably higher alpha diversity. Beta diversity analyses (PCA, PCoA, UPGMA) revealed clear differences in microbial community structure among the treatments. Certain genera, such as Sphingomonas, Noviherspirillum, and Agromyces, were particularly enriched in soils receiving vermicompost or green manure. Their presence suggests enhanced microbial functionality and nutrient cycling in those plots. Overall, the combination of green manure and organic inputs supported more diverse and functionally active microbial communities, contributing to the resilience and sustainability of barley-based cropping systems. This study focuses on the bacterial component of the soil microbiome; nutrient cycling and soil health are governed by the combined interactions among bacteria, fungi, and protozoa.

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DOI: 10.3897/mbmg.9.161359

Authors: Mathis Ventura, Nicolas Mazuras, Shanèze Noël, Lise Dupont, Virginie Roy

Abstract: DNA metabarcoding is a powerful approach for assessing biodiversity and unravelling trophic interactions, particularly in the context of biological invasions where introduced predators may impact native communities. However, the analysis of predator digestive contents with DNA metabarcoding poses technical challenges including the specificity of primers used to amplify the target DNA, amplification bias and contamination issues, which must be carefully considered when working on new taxa. This study addresses these challenges by developing and validating a methodology for assessing the diversity of terrestrial gastropods predated by the exotic flatworm Obama nungara. Specifically, an optimized primer pair providing taxonomic resolution for at least 40 gastropod species and genetic lineages, including taxonomically complex and diverse slug genera such as Arion and Deroceras is proposed. The primer pair was optimized through in silico predictions, laboratory controls, and field testing. Mock communities, laboratory-fed individuals, and technical replicates from the field were used as metabarcoding controls to calibrate filter thresholds for Operational Taxonomic Units. Preliminary field results revealed significant dietary diversity in O. nungara, suggesting a broad feeding spectrum. Beyond O. nungara, the primers and the assay developed here can also be applied to study the diets of other terrestrial predators or to detect gastropods in environmental DNA samples, providing a useful tool for ecological and conservation research.

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DOI: 10.3897/mbmg.9.151675

Authors: Thomas Baudry, Valentin Vasselon, Carine Delaunay, Alexandre Arqué, Fabian Rateau, Géraldine Lala, Claire Maurice-Madelon, Frédéric Grandjean

Abstract: Environmental DNA (eDNA) metabarcoding revolutionized the biodiversity monitoring in aquatic ecosystems, giving access to taxonomic lists in a non-disruptive way. Although the method has limits, such as reduced taxonomic resolution for certain groups and difficulties in estimating species abundance, it has proven its effectiveness in many contexts. In Martinique, a Caribbean island, traditional methods like electrofishing (TEF) are known to be stressful for organisms, non-selective and disruptive for the ecosystem, and have been progressively abandoned for routine monitoring. The aim of this project was to explore the possibility of using the eDNA-based metabarcoding method for the detection of fish and decapods in Martinique streams, by first validating it with TEF. We selected 14 stations, a representative panel of the river diversity, and performed TEF and eDNA-based monitoring to compare both, based on the species richness. Then, from eDNA taxonomic inventories, we assessed the ecological state of the studied stations, using Simpson index and investigated how stations abiotic characteristics shape assemblages. Here, we confirmed the eDNA metabarcoding method is a reliable tool for monitoring fish and decapods, confirming most of the taxa caught by TEF and revealing the presence of additional (native and/or invasive) species. We faced some issues in discriminating some genetically close species (e.g. Sicydium sp.) potentially leading to under-representation in community assemblages, but not in functional diversity. Additional efforts are needed to raise standardized protocols, but we encourage stakeholders to join such an initiative to shed light on the rich biodiversity in sometimes poorly studied regions and to face invasions.

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DOI: 10.3897/mbmg.9.161301

Authors: Saiesha Harpal, Johan Groeneveld, Sandi Willows-Munro, Jenny Huggett, Ashrenee Govender

Abstract: DNA metabarcoding of zooplankton samples is well suited to surveys of marine pelagic ecosystems; however, consistent amplification across zooplankton groups cannot be assumed when using “universal” miniCOI primers. Under-representation of some groups may result from primer bias or inadequate coverage in barcode reference libraries. We designed taxon-specific mini-barcode primers to improve the representation of copepods, euphausiids, chaetognaths, and hydrozoans in metabarcoding outputs of mixed zooplankton samples from the Southwest (SW) Indian Ocean. In silico analyses of downloaded sequences per group identified the most informative mini-barcode region, and barcode gap analyses confirmed that the selected regions could distinguish among species within each group. In vitro analyses of DNA extracts from individual specimens per group showed that the new mini-barcode primers outperformed the standard and universal miniCOI primers by consistently recovering higher Phred quality scores. Metabarcoding of four in situ mixed zooplankton samples collected with plankton tow nets and processed with a combination of taxon-specific and universal primers identified 220 species. Use of the primer cocktails increased the proportionate representation of the target groups by three- to five-fold compared to a previous study. Read counts were dominated by copepods and euphausiids, implying that they had the highest relative biomass in samples. We conclude that a combination of universal and taxon-specific primers in metabarcoding assays will achieve a more comprehensive assessment of biodiversity by enhancing species richness estimates across different groups.

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DOI: 10.3897/mbmg.9.159944

Authors: Zheyuan Liu, Xiaoru Du, Zehua Zhang, Yawen Mu, Jianghua Yang, Jiaxin Yang, Xiaowei Zhang

Abstract: Invasive species pose a significant threat to global biodiversity and the stability of ecosystems. Although environmental DNA (eDNA)-based quantitative PCR is considered effective, its limited multiplexing capacity makes it impractical for large-scale monitoring of invasive species. To address this limitation, we developed a novel and efficient approach for invasive species monitoring by combining multiplex PCR amplification with high-throughput sequencing. In this study, we screened 46 aquatic invasive species of major concern in China. We have integrated and designed 91 pairs of primers that can simultaneously amplify these species in a single PCR system. The validated method was applied to field monitoring in the Pearl River Basin to evaluate its practical performance. Multiplex PCR sequencing successfully detected 28 invasive species, with over 90% of environmental samples testing positive for invasive species DNA, demonstrating the method’s high sensitivity and broad applicability. Furthermore, all 11 invasive species identified through metabarcoding were also consistently detected by multiplex PCR sequencing, showing a strong positive correlation and high concordance across all monitoring sites. In conclusion, multiplex PCR sequencing represents a powerful and cost-effective tool for simultaneously detecting multiple aquatic invasive species in the early stages of invasion. It significantly improves detection efficiency, reduces monitoring costs and provides a solid foundation for developing a scientific and scalable monitoring system for aquatic invasive species.

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DOI: 10.3897/mbmg.9.165436

Authors: Erin Hill, Liz Milla, Francisco Encinas-Viso, James O'Dwyer, Ben Gooden, Mariana Hopper, John Roberts

Abstract: Genetic sampling from European honeybee (Apis mellifera) hives is a promising repository of terrestrial environmental nucleic acids (eNA) from a range of taxonomic sources. eNA samples from honeybee hives can be used to monitor environments via vegetation assessments and plant pathogen detection, while simultaneously assessing honeybee hive health through the identification of honeybee pests and pathogens. However, there is still limited comparison across the different types of hive samples commonly used for biomonitoring. There is also a need for less invasive sampling methods to facilitate adoption of hive eNA surveillance at scale. The aim of this study was to compare the effectiveness of using non-invasive swabbing of hive entrances with common sample types collected from honeybee hives (honey, pollen, and bees) for eNA detection of various taxa. Using a DNA metabarcoding approach for bacteria (16S), animal (COI), fungi (ITS1), and plant (ITS2) identification, and metatranscriptomic sequencing for virus detection, swabbing was found to provide comparable or better detection of taxa compared with more invasive sampling methods. Swabs were most informative for identifying bacterial and fungal communities, while plant detections were similar between swabs and honey samples. Detection of RNA viral diversity was highest in honey samples, however, swabs also showed good recovery of plant and insect viruses. A minimum of two swab replicates was recommended to increase taxa yield for DNA metabarcoding. Our study suggests that swabbing of hive entrances is a highly effective biomonitoring method for non-invasively collecting hive eNA and delivering comprehensive surveillance for animal, plant and microbial communities in the environment.

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DOI: 10.3897/mbmg.9.154548

Authors: Thomas Reinhart, Armando Espinosa Prieto, Thomas Begoc, Hugues Tinguy, Francis Bick, Etienne Chanez, Jean-Nicolas Beisel, Laurent Hardion

Abstract: Despite their ecological importance, mosses remain under-represented in ecological studies due to their challenging detection in field surveys and morphological identification, exacerbated by the lack of expert botanists. In this study, we optimise an environmental DNA method for the detection of bryophytes from river water samples, with the aim of facilitating their inclusion in biodiversity assessments. We compared three different methods in terms of species detection and community dissimilarity at seven sites along a river. The methods include (i) visual transect surveys conducted by bryologists based on macro- and micro-morphology, (ii) multi-marker PCR metabarcoding of the rbcL and the ITS2 markers with newly designed primers targeting bryophytes, and (iii) hybridisation capture (HC) for the same markers. We found that PCR metabarcoding recovered more than 50% (n = 37) of the species observed in the field, while hybridization capture detected only 16% (n = 11). PCR metabarcoding identified the most species, 101 species compared to 68 observed in the field and 27 with HC. Both the PCR and HC metabarcoding approaches identified bryophyte species not recorded in field surveys but expected in the catchment. Molecular methods, particularly PCR metabarcoding, recovered rare and elusive species difficult to observe in the field and occurring outside our transect. The two markers used in the molecular approaches contributed uniquely to species detection, making a multi-marker approach necessary to study this group. Environmental DNA and field surveys represent integrative methods that collectively enhance detection of inconspicuous species and yield the most comprehensive species inventory.

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DOI: 10.3897/mbmg.9.163750

Authors: Małgorzata Chwalińska, Michał Karlicki, Sarah Romac, Fabrice Not, Anna Karnkowska

Abstract: In the last decades, environmental metabarcoding has revolutionised biodiversity research, particularly for microbial organisms such as protists, enabling large-scale assessments of diversity and ecological patterns across time and space. With the advent of long-read sequencing, Nanopore-based metabarcoding represents a promising alternative to short-read approaches. Due to the limited number of available studies, the effectiveness of Nanopore sequencing - alone or in combination with short-read data - for assessing the biodiversity and ecological patterns of protists in different ecosystems is not yet sufficiently explored. Here, we present BaNaNA (Barcoding Nanopore Neat Annotator), a pipeline designed to generate high-quality OTUs and abundance estimates from Nanopore sequencing data. The performance of the pipeline was evaluated using a mock community as well as on marine and freshwater environmental samples to demonstrate its relevance for protist biodiversity and ecological studies. Our results show that BaNaNA generates high-quality full-length 18S rDNA OTUs from Nanopore long reads that are directly comparable to short-read V4-18S rDNA ASVs, supporting their synergistic use in long-term biodiversity studies. While both approaches reveal similar overall community diversity, long-read OTUs provide greater taxonomic resolution, richer phylogenetic information enabling the discovery of new clades and yield fewer false positives. These advantages make long-read Nanopore metabarcoding not only a powerful cost effective complement, but also a reliable replacement to short-read methods. By providing a pipeline for processing Nanopore data, BaNaNA paves the way for a broader application of long-read Nanopore sequencing in protist ecology and biodiversity research.

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DOI: 10.3897/mbmg.9.163640

Authors: Michaela Šamulková, Pavel Beracko, Patrik Macko, Ondrej Vargovčík, Kornélia Tuhrinová, Zuzana Čiamporová-Zaťovičová, Margita Lešťáková, Emília Mišíková Elexová, Fedor Čiampor Jr

Abstract: Routine biomonitoring under the Water Framework Directive (WFD) follows a conventional methodology primarily based on the morphological identification of taxa within the five biological quality elements (phytoplankton, phytobenthos, macrozoobenthos, macrophytes and fish). This identification is particularly challenging for macrozoobenthos due to their high taxonomic diversity. Moreover, this approach is time-consuming and resource intensive. Our current study aims to: (i) evaluate the implementation of DNA metabarcoding in long-term monitored sites to assess the effectiveness of three sample types (bulk samples of macrozoobenthos, and eDNA from water and sediments), (ii) evaluate the ecological status of water bodies using metabarcoding data, and (iii) compare ecological metrics values using conventional and molecular approaches. Sampling was conducted at 17 localities in Slovakia, covering eight stream types. DNA metabarcoding detected 30% more species than conventional monitoring performed over 15 years without needing time-intensive sample processing and morphological identification. At the same time, the bulk samples captured more macrozoobenthos taxa than the other sample types. On average, the bulk samples detected 85% of the taxa captured by DNA metabarcoding. The differences in values of ecological metrics obtained within the conventional approach and between the conventional and metabarcoding approaches were relatively small, typically corresponding to differences within one ecological quality class. However, more pronounced differences were observed in non-abundance-based metrics, such as EPT and BMWP, indicating higher sensitivity of species detection with DNA metabarcoding. This study demonstrates the effectiveness of DNA metabarcoding across multiple metrics and highlights its potential to enhance routine biomonitoring.

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DOI: 10.3897/mbmg.9.154667

Authors: Igor Stanković, Sára Shemesh, Nikola Hanžek, Mirela Šušnjara, Marija Gligora Udovič, Keve T. Kiss, Zsuzsa Trábert, Zsolt Udvari, Tibor Bíró, Edit Vadkerti, Éva Ács

Abstract: This study presents the first occurrences of the diatom species Discostella asterocostata in Europe’s second largest river, the Danube River and the earliest occurrence in European freshwater ecosystems. Samples collected between 2016 and 2022 in Croatia and Hungary were analysed using light and scanning electron microscopy (SEM) along with molecular techniques to confirm the presence of the species. Morphometric comparisons with known populations from Asia and the USA revealed no significant morphological differences. Molecular analyses facilitated the detection of D. asterocostata even in samples with low abundance and confirmed the genetic similarity with already sequenced populations. Environmental correlations showed that D. asterocostata had a significant positive correlation with temperature and a negative correlation with total organic carbon and nitrogen forms, suggesting that it favours warmer waters with lower nutrient forms. Given the ongoing increase in water temperatures due to climate change, the occurrence and potential spread of this species in European rivers may be related to changing environmental conditions. Our results emphasise the importance of integrating traditional and molecular approaches for the early detection of non-indigenous diatom species. The establishment of D. asterocostata in Europe raises questions regarding its ecological role and long-term impact on native phytoplankton communities and emphasises the need for continuous monitoring and research on the responses of large river ecosystems to global environmental change.

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DOI: 10.3897/mbmg.9.148923

Authors: Mandy Sander, Arne J. Beermann, Denis Brömmling, Dominik Buchner, Martina Weiss, Florian Leese

Abstract: Environmental DNA (eDNA) metabarcoding of streams can deliver information on biological communities with minimally invasive efforts and at low costs. However, the captured eDNA may not necessarily originate only from organisms physically present at a local site but also come via drift from sources upstream. Therefore, data on community composition can be inherently different to specimens occurring at a certain site, limiting direct comparisons to site-based assessment data. To increase the local signal, two possible ways were recently proposed: First, targeting eRNA collected from water samples, which degrades faster than eDNA, and second, using eDNA from water, in which organisms from the stream were shortly incubated in a water container. Based on these two options, we here derived and tested three strategies to obtain a more localized signal of biodiversity: 1. eRNA metabarcoding of a direct stream water sample, 2. eDNA and eRNA metabarcoding of water, in which animals collected via kicknet multi-habitat sampling were shortly incubated, and 3. eDNA and eRNA metabarcoding of water, in which natural substrate exposures (NSEs) that were actively colonised by invertebrates in the stream for four weeks were shortly incubated. For comparison, we used eDNA directly isolated from the stream as the more regional signal. As the local point-based sample reference, we used the animal bulk sample that was incubated. While our results support that eRNA collected from the stream (strategy 1) does not improve the local signal, both incubation strategies increased the proportion of species identified by both metabarcoding of eDNA/eRNA and the locally collected bulk sample by about 20%. Interestingly, we detected 20% more species in the NSEs (strategy 3) than in the kicknet samples (strategy 2), highlighting the potential of this less invasive invertebrate sampling approach. In summary, our study demonstrates that incubating collected animals in stream water before filtering the water increases the local community signal and provides an animal-friendly strategy for biomonitoring.

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DOI: 10.3897/mbmg.9.150825

Authors: Ondrej Vargovčík, Kornélia Tuhrinová, Pavel Beracko, Jiří Kopáček, Zuzana Čiamporová-Zaťovičová, Patrik Macko, Michaela Šamulková, Fedor Čiampor

Abstract: Monitoring changes in freshwater fauna requires timely, efficient, and comprehensive data collection, needs that are increasingly met by molecular methods. This is especially important in remote regions along environmental gradients, such as the Tatra Mountain lakes in Slovakia. In this study, DNA metabarcoding was applied to littoral benthic invertebrates collected from lakes in three valleys, spanning a gradient from the subalpine to alpine zones. Ordination analyses grouped the lakes based on community composition, and these groups were evaluated in relation to altitude, physico-chemical properties, indicator species, and previous data. To assess their additional value, surface sediment samples from the littoral zone were also analyzed using metabarcoding. The results revealed two distinct lake groups, primarily differentiated by altitude, alkalinity, and other correlated environmental parameters. The placement of specific lakes within these groups corresponded with known patterns of recovery from acidification, catchment characteristics, and potentially climate change or lake connectivity. Metabarcoding-based indicator species analysis successfully identified even taxa that had been previously overlooked or misidentified in morphology-based studies. While sediment metabarcoding provided limited supplementary information, its ordination patterns aligned with those from invertebrate analyses. Overall, our study of an alpine lake system supports broader trends: species richness declines with increasing elevation; community composition reflects environmental gradients; and DNA metabarcoding offers high taxonomic resolution and valuable insights into freshwater invertebrate biodiversity.

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DOI: 10.3897/mbmg.9.152554

Authors: Cátia Alves, Lara Baptista, Patrícia Madeira, Nuno Vaz Álvaro, António Santos, Harald Meimberg, Sérgio P. Ávila, Manuel Curto

Abstract: DNA barcode databases for marine invertebrates faces significant challenges, with several species revealing taxonomic issues like misidentification, ambiguity or shared barcodes. This highlights the importance of the availability of robust genetic data to resolve taxonomic uncertainties and improve species identification, which in many groups of invertebrates, like crustaceans, is lacking. Based on molecular data from the Cytochrome c oxidase I 5′ gene region (COI) for barcoding, this study highlights some of these challenges by addressing existing taxonomic ambiguities and highlighting the scarcity of genetic data for several of the shore crab genus Pachygrapsus Randall, 1840. As expected, molecular data availability is highly skewed towards taxa more prevalent in the Northern Hemisphere. Our analysis reveals a clear barcoding gap amongst Pachygrapsus species, with average interspecific p-distances of 18.75%, exceeding previously reported values for crustaceans. Furthermore, high intraspecific variation (0.70% average) suggests potential taxonomic issues, particularly within P. gracilis (de Saussure, 1857). East and west Atlantic P. gracilis populations exhibit substantial divergence (3.6% p-distance), raising the possibility of ongoing speciation due to limited gene flow across the Atlantic Ocean. Similarly, populations of P. crassipes in the east and west North Pacific exhibit substantial divergence (3.3% p-distance), which suggests possibly another speciation process. COI barcoding proves effective for species identification within Pachygrapsus and in unravelling intraspecific patterns indicative of cryptic diversity guiding further taxonomic revisions integrating morphological, genetic and ecological data.

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DOI: 10.3897/mbmg.9.152948

Authors: Jeerisuda Promprao, Nuttha Sanevas, Ekaphan Kraichak, Santi Saraphol, Piriya Klankeo, Saowalak Kaewmee, Chonlada Mano, Baby Kyi Soe, Anchittha Rujeerapaiboon, Kanok Preativatanyou, Padet Siriyasatien, Catherine Walton, Narissara Jariyapan

Abstract: Aedes aegypti poses a significant threat by transmitting diseases like dengue, chikungunya and Zika, leading to high morbidity and mortality worldwide. In recent years, the role of microalgae in mosquito larvicidal activity has gained attention due to their role as a key mosquito nutritional resource. Understanding microalgal communities in water from breeding sites and Ae. aegypti larval guts might be beneficial for implementing mosquito control strategies. The purpose of this work was to investigate the diversity of microalgae in the guts and breeding sites of Ae. aegypti larvae in Nakhon Si Thammarat, Thailand, using light microscopy and metabarcoding. Results showed a total of 27 algal genera from this study. Ten genera were identified using light microscopy and an additional 21 genera were identified through 23S metabarcoding. Four genera, Tetradesmus, Nannochloropsis, Monoraphidium and Leptolyngbya, were detected by both approaches. Two genera, Tetradesmus and Leptolyngbya, were present in all guts and water samples. Spearman’s correlation analysis, alpha diversity and beta diversity demonstrated a high degree of compositional similarity between gut samples and water samples collected from breeding sites. The results indicated that Ae. aegypti larvae could consume nearly all the microalgae in their breeding habitat. Information provided in this study would help choose microalgae that could potentially be a biocontrol agent for further development of eco-friendly products for the future management of mosquito larvae.

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DOI: 10.3897/mbmg.9.144935

Authors: Nastassia V. Patin, Kathleen J. Pitz, Jacoby D. Baker, Francisco P. Chavez, Kelly D. Goodwin

Abstract: Marker gene sequencing (“metabarcoding”) is the primary sequencing approach currently used for molecular biodiversity surveys, but this approach is taxonomically limited and hampered by amplification biases. In contrast, shotgun metagenomes avoid extensive PCR amplification and can theoretically capture the full taxonomic breadth of the eDNA pool. However, eukaryotic DNA is often a small component of metagenomes; consequently they have seen limited use in metazoan biodiversity surveys. Here, we compare metabarcoding and shotgun metagenomes on a large (>200 sample size) set of marine water column eDNA samples and show that metagenomes can provide biodiversity information comparable to that of metabarcoding surveys. They provide more consistent and even detections of important phytoplankton like chlorophytes and haptophytes and can detect metazoan taxa of management and conservation interest, such as the humpback whale. However, genus- and species-level taxonomic assignments are often inaccurate for metagenomic data and require more manual annotation relative to metabarcoding taxonomy. Reference database gaps remain an obstacle to accurate and comprehensive biodiversity surveys for both metabarcoding and shotgun metagenomes. We provide examples of taxa that may benefit from one approach over another and highlight cases of metagenomic utility.

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DOI: 10.3897/mbmg.9.144371

Authors: Nerea Vallejo, Miren Aldasoro, Lander Olasagasti, Joxerra Aihartza, Inazio Garin

Abstract: Human-driven landscape transformations are the primary factors driving biodiversity changes in the present century. For insectivorous bats, land-use modifications and intensification of agriculture negatively impact bat foraging activity and, consequently, the potential ecosystem services they can provide. However, little is known about specific dietary niche adaptations to anthropogenic land changes. Here, we analysed the influence of land cover and agricultural practices, especially livestock, on the diet of the notch-eared bat. We collected faecal samples from sixteen maternity colonies in the Iberian Peninsula and southern France during two different sampling periods, May and July of 2020. We analysed 318 faecal samples using DNA metabarcoding, resulting in 241 identified prey species. Spiders were the most consumed prey item, followed by livestock pest flies (Muscidae). Dietary diversity was higher in May, while the weighted percentage of occurrence (wPOO) of spiders was higher in July. Consumption of harmful livestock pests was more likely in areas where livestock were prevalent near the colony; however, other landscape characteristics such as forest cover and urban areas had a greater influence on the wPOO values of pests. Our results highlight the consistency of the dietary composition of the notch-eared bat across large geographical areas, and its reliance on gleaning for hunting motionless prey. The interaction between livestock farming densities, livestock management types and land use around the colony will influence the densities of pest flies and other arthropods, ultimately shaping the trophic niche of M. emarginatus.

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DOI: 10.3897/mbmg.9.144128

Authors: John K. Pearman, Jack Sissons, Joseph Kanyi Kihika, Georgia Thomson-Laing, Susanna A. Wood

Abstract: Molecular-based techniques provide the potential for novel insights into the functioning of ecosystems, especially those that are globally rare such as coastal dune lakes. In the surface sediments of lakes, microbial communities play a vital role in biogeochemical cycling and techniques such as metagenomics can provide information on the roles play in these ecosystems. The current study aimed to investigate the taxonomic and functional composition of six coastal dune lakes on Chatham Island using sediment DNA approaches. The use of metabarcoding (16S rRNA gene) and metagenomics showed that there were distinct differences in the microbial community composition and functional potential amongst the lakes, especially in the lakes with higher salinity. Investigation of metabolic potential with metagenomics showed that the abundance of genes involved in nitrogen cycling were related to the nitrogen:phosphorus ratio while assimilatory sulfate reduction was correlated with sulfur and organic matter concentrations. Analysis showed differences in the carbon fixation strategies amongst the lakes. The lake with the highest salinity levels also had elevated levels of osmoprotectants and related transporters. The sequencing of sediment DNA enables the investigation of the composition and functioning of lake environments providing a basis for the increased understanding of the processes occurring within lakes.

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DOI: 10.3897/mbmg.9.141904

Authors: Alexander Edwards, Birgit Gemeinholzer

Abstract: 1. Insect decline is threatening ecosystem stability, making information about foraging preferences of pollinators a vital piece of information to acquire. A powerful emerging tool to study pollinator foraging behaviour is pollen-metabarcoding. This usually involves lethal sampling of insects. 2. Here, we propose a new, non-lethal way of sampling DNA for the analysis of pollen loads of bumble bees as well as the pollinator. The new methodology does not significantly harm the insect and is easy to implement in a wide range of study designs. The tool is cheap and easy to acquire, can easily be used in the field and has the potential to become a powerful tool in studying plant-pollinator interactions. 3. To test its feasibility, plant-pollinator networks were analysed using metabarcoding of the ITS2 region. Plants flowering at the time of collection were also recorded as a reference comparison. 4. Bumble bees with ambiguous morphology were additionally identified, based on COI barcoding. 5. With the workflow developed here, it is possible to gain knowledge about plants and their pollinators in a non-lethal way without reducing population sizes. This makes this method particularly suitable for endangered and protected species.

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DOI: 10.3897/mbmg.9.137772

Authors: Paula Gauvin, David Eme, Isabelle Domaizon, Frédéric Rimet

Abstract: Freshwater ecosystems are increasingly threatened by human activities, leading to biodiversity loss and ecosystem degradation. Effective biodiversity monitoring, particularly through the use of aquatic macroinvertebrates as bioindicators, is crucial for assessing ecological health. While traditional morphological methods face limitations, DNA metabarcoding offers higher accuracy and efficiency in species identification using environmental DNA. However, the success of metabarcoding is contingent on the quality of reference libraries, which are often incomplete or biased. This study aimed to construct and share a comprehensive COI-based DNA barcode library for freshwater macroinvertebrates in France, specifically targeting short gene regions amplified with fwhF2/fwhR2N primers, suitable for degraded DNA. A list of species occurring in French freshwater ecosystems was established from official national checklists and Alpine lake surveys. The resulting library was analysed for taxonomic completeness, barcode coverage and cryptic diversity. The checklist consisted of 2,841 species across 10 phyla, for which 56% had at least one COI-5P sequence available in the Barcode of Life Data System (BOLD). The analysis of cryptic diversity, based on Barcode Index Numbers (BINs) highlighted a potential high rate of cryptic diversity, although it might have been overestimated due to the wide geographic origin of the sequences. Alignment challenges with the primers were identified for certain taxa, particularly amongst Coleoptera, Diptera and Malacostraca. The genetic diversity approached by the number of haplotypes per species highlighted that most of the species have limited diversity, with only three species having more than 100 haplotypes. Finally, this study showed that a total of 57 haplotypes were shared amongst 116 distinct species. This work emphasises the need for expanded sequencing efforts to improve barcode coverage and highlighted the pitfalls associated with the use of these primers for further biodiversity assessment of macroinvertebrates with DNA.

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DOI: 10.3897/mbmg.9.144340

Authors: Ayumi Sakata, Toshifumi Minamoto, Tetsuya Sado, Ryo O. Gotoh, Masaki Miya

Abstract: Environmental DNA (eDNA) techniques are increasingly employed in biodiversity monitoring of aquatic and terrestrial animals, plants, and fungi, holding great potential to revolutionize biodiversity assessments. However, sampling and basic laboratory protocols still require refinement to optimize eDNA metabarcoding performance. Homogenization as a pre-treatment for eDNA extraction is known to enhance the concentration and quality of extracted eDNA for some groups of organisms. We previously developed a simple and efficient method for capturing arboreal biodiversity using stemflow as a source of eDNA; however, its performance with or without homogenization had not yet been compared. In this study, we evaluated the performance of two different homogenization methods using eDNA metabarcoding and qPCR assays. Metabarcoding analyses revealed that the method without homogenization detected the fewest species, while nearly identical and higher numbers of species were detected in samples subjected to bead-beating and frozen bead-beating homogenization. Similarly, qPCR analyses revealed that the method without homogenization yielded the lowest DNA concentration, while nearly identical and higher DNA yields were observed for bead-beating and frozen bead-beating homogenization. These findings suggest that, considering cost and effort, the bead-beating method without freezing is the most advantageous.

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DOI: 10.3897/mbmg.9.138172

Authors: Gabriela Jijón, Isabella M. Errigo, Jessica Wicks, Natalie N. Goldston, Lillian Davis, Daniel Davis, Samantha Standring, John M. Chaston, Paul B. Frandsen, Blanca Rios-Touma

Abstract: As unimpacted freshwater environments decline worldwide, developing effective and accurate biomonitoring approaches is critical for detecting ecosystem deterioration and facilitating conservation efforts. Freshwater macroinvertebrate communities comprise several phyla and thus a broad taxonomic expertise is required to identify these precisely. Molecular approaches for characterizing invertebrate communities have the potential to be more accurate, time-effective, and thus less costly than traditional morphological methods. However, evaluations comparing the two methods for macroinvertebrates are lacking in the Neotropics, where rapid deployment and increased accuracy are critical due to the escalating deterioration of freshwater environments. In this study, we compared the performance of DNA bulk tissue metabarcoding using an eDNA primer pair (fwhF2 & EPTDr2n) and morphological methodologies for identifying macroinvertebrate communities. Our aim was to evaluate the suitability of metabarcoding for assessing the biological quality of Ecuadorian streams along an altitudinal gradient. We found low taxonomic overlap between methods at family and genus ranks, though many molecular OTUs were unclassified at family and genus levels. Morphological identification resulted in higher Andean-Amazonian Biotic Index (AAMBI) scores for each site, with only three sites classified into the same biological category for both methodologies. One reason is that only family-level information is used for AAMBI. Existing DNA barcode reference libraries contained incomplete coverage of Ecuadorian taxa for specimens determined with the morphological approach, indicating a need to improve the representation of Ecuadorian taxa in these libraries to generate more accurate and complete metabarcoding determinations. Our study suggests that metabarcoding requires more development for freshwater biomonitoring in the Neotropics and should, at the moment, be used complementary to traditional identification approaches.

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DOI: 10.3897/mbmg.9.132076

Authors: Susanna Theroux, Adam Sepulveda, Cathryn L. Abbott, Zachary Gold, Alison W. Watts, Margaret E. Hunter, Katy E. Klymus, Shana Lee Hirsch, Joseph M. Craine, Devin N. Jones, Rachel J. Brown, Joshua A. Steele, Miwa Takahashi, Rachel T. Noble, John A. Darling

Abstract: The rapid advancement of environmental DNA (eDNA) science in the past two decades has inspired a concomitant growth in the development of eDNA sampling and analytical methods. However, these methods are often developed by individual laboratories or institutions, which can isolate protocols within programmes, agencies or regions and prevent the beneficial exchange of data and ideas. Recent efforts to advance national and international coordination have resulted in a groundswell of standardisation efforts, but there is still considerable confusion around the role of formal standards for regulatory or research applications. With this commentary, we hope to provide clarity on the terminology used in standardisation discussions, including the differences between formal standards and best practice guidelines. Additionally, we discuss how eDNA method choice may be informed by environmental management scenarios and review examples of formal eDNA method standards being used to inform management action. The eDNA community now has an opportunity to develop a roadmap for method development to help close standardisation gaps, advance eDNA method adoption and accelerate our ability to monitor biological life at the scales our current environmental challenges demand.

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DOI: 10.3897/mbmg.9.130834

Authors: Tiina Laamanen, Veera Norros, Petteri Vihervaara, Jacqueline Jerney, Pirkko Kortelainen, Katharina Kujala, Stefan Lambert, Janne Mäyrä, Lilja Nikula, Ida Palmroos, Mikko Tolkkinen, Kristiina Vuorio, Kristian Meissner

Abstract: Human activities are causing rapid biodiversity loss across ecosystems, affecting human well-being and crucial ecosystem services. Traditional biodiversity monitoring tools cannot keep up with the increasing demands of monitoring due to their limited spatial or temporal coverage, high costs, and lack of taxonomic expertise. Thus, implementation of novel molecular monitoring methods such as environmental DNA (eDNA) and DNA metabarcoding, are necessary. Molecular monitoring methods offer significant benefits for biodiversity monitoring and environmental assessment: high sensitivity and accuracy, non-invasive sampling, broad taxonomic range and cost and time efficiency. However, the diverse methodological approaches lead to poor comparability between studies and surveys, highlighting the need for standardised assessments. We used the Technology Readiness Level (TRL) framework to evaluate the maturity of molecular monitoring methods, providing a structured assessment of their readiness for routine use. In a systematic literature review, 420 articles fulfilling the study criteria were assessed and both individual studies and method categories ranked according to the TRL scale. The findings revealed a growing number of studies, particularly in aquatic environments, with most studies validating molecular technologies on a small scale but lacking large-scale system demonstrations. Aquatic eDNA-based methods targeting fish showed overall higher technology readiness compared to other sample types and taxa and applications of molecular monitoring methods ranked into the highest TRL were predominantly freshwater studies. Key barriers to the broader implementation of molecular methods to monitoring include the need for international standards, better quantitative estimates and comprehensive reference libraries. National and international cooperation is crucial for establishing common standards, ensuring reliable and comparable results and expediting the routine use of molecular methods in biodiversity monitoring. Recent efforts towards international standardisation are encouraging, but further coordinated actions are necessary for the global implementation and acceptance of these methods.

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DOI: 10.3897/mbmg.9.133264

Authors: Valentin Vasselon, Sinziana F. Rivera, Éva Ács, Salomé F.P. Almeida, Karl B. Andree, Laure Apothéloz-Perret-Gentil, Bonnie Bailet, Ana Baričević, Kevin K. Beentjes, Juliane Bettig, Agnès Bouchez, Camilla Capelli, Cécile Chardon, Mónika Duleba, Tina Elersek, Clémence Genthon, Maša Jablonska, Louis Jacas, Maria Kahlert, Martyn G. Kelly, Jan-Niklas Macher, Federica Mauri, Marina Moletta-Denat, Andreia Mortágua, Jan Pawlowski, Javier Pérez-Burillo, Martin Pfannkuchen, Erik Pilgrim, Panayiota Pissaridou, Frédéric Rimet, Karmen Stanic, Kálmán Tapolczai, Susanna Theroux, Rosa Trobajo, Berry Van der Hoorn, Marlen I. Vasquez, Marie Vidal, David Wanless, Jonathan Warren, Jonas Zimmermann, Benoît Paix

Abstract: DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an “all for one but prove them all” strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.

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DOI: 10.3897/mbmg.9.128235

Authors: Lauren Kelly Rodriguez, Lorenzo De Bonis, Jack McKee, James A. McKenna, Teddy Urvois, Eleonora Barbaccia, Eileen Dillane, Caterina Lanfredi, Helene Hjellnes, Armelle Jung, Enrico Villa, Arianna Azzellino, Jon-Ivar Westgaard, Erwan Quéméré, Bettina Thalinger

Abstract: The comparability of methods applied to environmental DNA (eDNA) samples across laboratories remains a significant challenge for biodiversity monitoring on a global scale. Performance differences between protocols can jeopardize effective conservation strategies across regions and focal species. To address potential discrepancies amongst four international partners within a collaborative eDNA initiative, an inter-laboratory comparison (i.e., ring test) was conducted to compare efficiencies of established DNA extraction methodologies based on 39 eDNA samples. Each laboratory contributed eight to eleven samples collected throughout the North-East Atlantic and the Mediterranean Sea near sperm whales, porbeagle sharks, basking sharks, bottlenose dolphins and common dolphins. After lysis, aliquots were exchanged between laboratories before subsequent DNA extraction using each facility’s preferred method. Extracts were returned to the lysates’ respective laboratories of origin for measurements of total DNA concentration, as well as quantitative PCR using three novel species-specific assays for marine megafauna. Our findings revealed similar concentrations of total DNA, yet a significant reduction in extraction performance for targeted qPCR reactions by one laboratory, who has therefore modified their extraction method to be used for the remainder of this project. Overall, detection success differed based on the target taxa with sharks being less often detected (and at lower concentrations) than marine mammals. Significant interaction effects were found between combinations of laboratories and species, suggesting a link between extraction protocols and variable environmental conditions. Our study serves as a foundational step towards establishing reproducible practices that are crucial for the success of multinational eDNA projects to enable comparable results.

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DOI: 10.3897/mbmg.8.128689

Authors: Katy E. Klymus, Jacoby D. Baker, Cathryn L. Abbott, Rachel J. Brown, Joseph M. Craine, Zachary Gold, Margaret E. Hunter, Mark D. Johnson, Devin N. Jones, Michelle J. Jungbluth, Sean P. Jungbluth, Yer Lor, Aaron P. Maloy, Christopher M. Merkes, Rachel Noble, Nastassia V. Patin, Adam J. Sepulveda, Stephen F. Spear, Joshua A. Steele, Miwa Takahashi, Alison W. Watts, Susanna Theroux

Abstract: Environmental DNA (eDNA) and RNA (eRNA) metabarcoding has become a popular tool for assessing biodiversity from environmental samples, but inconsistent documentation of methods, data and metadata makes results difficult to reproduce and synthesise. A working group of scientists have collaborated to produce a set of minimum reporting guidelines for the constituent steps of metabarcoding workflows, from the physical layout of laboratories through to data archiving. We emphasise how reporting the suite of data and metadata should adhere to findable, accessible, interoperable and reproducible (FAIR) data standards, thereby providing context for evaluating and understanding study results. An overview of the documentation considerations for each workflow step is presented and then summarised in a checklist that can accompany a published study or report. Ensuring workflows are transparent and documented is critical to reproducible research and should allow for more efficient uptake of metabarcoding data into management decision-making.

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DOI: 10.3897/mbmg.8.135461

Authors: Shin-ichiro Oka, Tetsuya Sado, Masayuki Ushio, Masaki Miya

Abstract: Environmental DNA (eDNA) analysis is a valuable tool for detecting species presence in coastal environments. However, its effectiveness can be influenced by factors such as diffusion and degradation after being released by organisms. Understanding eDNA dynamics in response to tidal currents is pivotal, especially in the intertidal zone where water movement is pronounced. This study assessed the impact of tidal movements on fish eDNA metabarcoding across two regions separated by a 10-degree latitude difference: the temperate Boso Peninsula (35°N) and the tropical Motobu Peninsula (25°N). Over two days, we conducted four rounds of water sampling at three sites within 20 km of each Peninsula’s coast during substantial tidal differences (1.5–2 m), covering both outgoing and incoming tides. Fish eDNA metabarcoding analysis revealed notable differences in fish community compositions between the two Peninsulas, with statistically significant variations amongst the three sites within each Peninsula. At each site, comparison between outgoing and incoming tides showed significant differences at five of the six sites. However, variation partitioning analysis suggested that the impact of tidal changes was limited compared to other factors in both regions. These findings suggest that tidal movements may not be a critical factor in designing sampling plans for comparing fish community structures through eDNA metabarcoding at this spatial and temporal scale. Additionally, they highlight the need for empirical studies to explore the underlying mechanisms driving differences in eDNA-based fish community composition related to tidal changes.

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DOI: 10.3897/mbmg.8.129814

Authors: Lisa Wolany, Julian Enss, Florian Leese, Dominik Buchner

Abstract: The rapid advancement of molecular biodiversity monitoring tools, particularly DNA metabarcoding, has improved specimen identification in bulk samples, such as those from Malaise traps, where traditional morphological identification is impractical. While not yet standardized, a typical first step in insect bulk sample analysis is the extraction of DNA from homogenized specimens. While this step yields reliable metabarcoding results, it destroys the specimens, preventing further use in monitoring and taxonomic analysis. Non-destructive lysis, which preserves specimen integrity, is still being evaluated for its effectiveness in accurately assessing bulk sample biodiversity. In this study, we assessed the suitability of non-destructive lysis for Malaise trap samples and compared its performance with homogenization using an established metabarcoding workflow. Five bulk samples were collected with Malaise traps. Samples were first incubated in a lysis buffer containing Proteinase K (non-destructive lysis) and then homogenized. DNA was extracted from both treatments and metabarcoding was performed to compare OTU richness, accumulation, and beta diversity. On average, homogenized samples yielded 3.8% more OTUs than non-destructive lysis samples. Although homogenization provides a more comprehensive and cost-effective assessment of Malaise trap bulk samples, non-destructive lysis still recovered at least 80% of the OTUs identified through homogenization and revealed similar patterns of community change. Even though our results show that both methods yield comparable data on insect biodiversity and can be used for monitoring, we consider non-destructive lysis as not suitable for integration into automated workflows or large-scale biomonitoring due to the much higher costs. Nonetheless, this method remains important in cases where morphological integrity needs to be preserved and additional sampling is not possible.

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DOI: 10.3897/mbmg.8.135082

Authors: Heba Mohamad, Katherina Schimani, Maitham Al-Shaheen, Nélida Abarca, Regine Jahn, Adil Al-Handal, Wolf-Henning Kusber, Jonas Zimmermann

Abstract: The Euphrates-Tigris catchment area is one of the major drainage basins in the Middle East but climate change, overuse of waters and subsequent salinization has brought this region to the brink of ecological devastation. The aim of this study was to investigate the biodiversity of epiphytic diatoms in Iraq, for the first time using a combination of morphological and metabarcoding methods. 47 samples were collected at six sampling sites along the Rivers Tigris and Euphrates in northern Basra, Southern Iraq, during the summer of 2019 (dry season) and winter of 2020 (rainy season). The composition of the epiphytic diatoms in each sample was compared with both methods. The microscopic-morphological analysis of the environmental samples resulted in the identification of 284 infrageneric taxa in 59 genera. For the metabarcoding analysis the V4 region of the 18S marker gene was used and resulted in 1454 ASVs (Amplicon sequence variants) comprising 54 genera and 108 species with several ASVs belonging to the same taxa. The non-metric multidimensional scaling (NMDS) plots for morphology showed a clear seasonal effect in the diatom community composition, while the NMDS plots for 18SV4 analyses did not show a seasonal effect in community composition for the same samples. The incomplete taxonomic reference database for the studied sites turned out to be the major limitation of the molecular approach. Nevertheless, the combination of morphological and molecular methods increased the detection and identification of the diatom assemblages and laid the foundation for understanding their biodiversity in this region.

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DOI: 10.3897/mbmg.8.127959

Authors: Danielle Botha, Sandra Barnard, Morné Du Plessis, Mushal Allam, Kai Behn, Arshad Ismail, Anja Linstädter, Malesela Vincent Mokoka, Zamantungwa Thobeka Happiness Mnisi, Frances Siebert

Abstract: Increasing livestock densities and more severe drought events challenge sustainable management in South Africa’s semi-arid savannas. Effective mitigation strategies require accurate assessments of livestock foraging behaviour. By utilising high-throughput sequencing technology, this study evaluated the use of a dual-locus metabarcoding approach (trnL and rbcL) together with study-area-specific reference libraries, to analyse cattle diets in two bioregions of the eastern semi-arid South African savanna. Both markers demonstrated the ability to identify various plant families, but trnL exhibited a higher diversity in terms of family and genus identification at both sampling sites. Forbs, although comprising a diverse component of savanna plant communities, have relatively small above-ground biomass, but can still serve as crucial forage items, especially during dry periods. Our study underscores the significant role of forbs in cattle diets, demonstrating a shift in cattle foraging preferences from grass-based diets to higher inclusions of forbs and woody taxa during the drier season. Although grasses, such as Setaria, were still prevalent, forbs, belonging to the genera Malvastrum, Asparagus, Pollichia and Ipomoea were also important food items for cattle as well as woody taxa belonging to Fabaceae, Combretaceae, Ebenaceae, and Malvaceae with a selection of food items from trees and shrubs from genera Albizia, Combretum, Euclea and Vachellia. Furthermore, our study highlights the value of a dual-locus metabarcoding approach for understanding herbivorous diets. Using trnL and rbcL markers, with study-area-specific reference libraries, improves taxonomic resolution for accurately reconstructing cattle diets in semi-arid savannas. This study may improve biodiversity estimates and inform sustainable rangeland management strategies in semi-arid African savanna ecosystems.

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DOI: 10.3897/mbmg.8.125095

Authors: Kristen M. Westfall, Gregory A. C. Singer, Muneesh Kaushal, Scott R. Gilmore, Nicole Fahner, Mehrdad Hajibabaei, Cathryn L. Abbott

Abstract: Applied eDNA metabarcoding is increasingly being considered as a tool to inform management decisions, regulations, or policy development. Because these downstream considerations are coming to the forefront of eDNA applications, optimizing workflow elements is essential to increasing standardization, efficiency, and competency of metabarcoding results. Reference DNA sequences are critical workflow elements that currently lack consistent approaches to generating, curating, or publishing. We present a complete mitochondrial genome and nuclear ribosomal DNA cistron reference DNA sequence library for 92% of the freshwater fish species of British Columbia, Canada. This resource is published as the Novel Applied eDNA Metabarcoding Reference Sequences (NAMERS) repository (https://namers.ca), a user-friendly and interactive website for specialists and non-specialists alike to explore and generate custom reference libraries for taxa and genes of interest. We demonstrate the power of NAMERS for optimization of applied eDNA metabarcoding study design by analyzing the number of primer mismatches and species resolution power of existing metabarcoding markers. NAMERS demonstrates that high quality curated genomic information is within a reasonable reach to meet the increasing demand for actionable eDNA metabarcoding applications. The framework used here incorporating the pillars of accuracy, completeness and accessibility can be applied for new iterations of other reference sequence databases to bring DNA-based monitoring into a new era.

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DOI: 10.3897/mbmg.8.130531

Authors: Francesco Martoni, James Buxton, Kathryn S. Sparks, Tongda Li, Reannon L. Smith, Lea Rako, Mark J. Blacket

Abstract: Ants are often the most ubiquitous and ecologically influential components of terrestrial systems, exhibiting an exceptionally high level of endemic diversity. Simultaneously, exotic ants can be amongst the most environmentally and economically devastating biological invaders. Distinguishing between exotic and related natives is essential for early detection and to reduce the chance of establishment, but at the species-level there remains a great deal of uncertainty among several of the most diverse, widespread and ecologically dominant genera. This taxonomic impediment negatively impacts the use of molecular techniques relying on reference databases, due to the poor state of ant DNA sequences in publicly available repositories, with many species not represented at all and others incorrectly identified. As a result, biosecurity screening for targeted exotic ants and assigning ant species-level identifications for biodiversity remain two separate areas of focus in Australia. Here we propose a workflow for the identification of ants that enables simultaneous processing of large numbers of “bulk” ant samples each containing many individuals, increasing resolution for taxonomic assignment, and generating a curated database linked to voucher specimens. We use a non-destructive DNA extraction method and compare two high throughput sequencing (HTS) metabarcoding platforms – MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) – processing up to 180 bulk mixed ant samples per run. This approach allowed for the acquisition of curated DNA sequences from voucher specimens morphologically examined by expert taxonomists. This work highlights the advantages and current limitations of DNA-based identifications, the needs for standardisation, as well as the importance of a taxonomy-based curation of DNA database for both biodiversity and biosecurity.

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DOI: 10.3897/mbmg.8.128646

Authors: João T. Fontes, Kazutaka Katoh, Rui Pires, Pedro Soares, Filipe O. Costa

Abstract: Environmental DNA (eDNA) metabarcoding is revolutionising the study of aquatic ecosystems, enabling high-throughput analysis of biodiversity with minimal disturbance. Despite its potential to support fisheries management, species identification and downstream analysis reliability are hindered by the lack of standardisation in DNA fragment choice. This study compares the species discrimination power of three markers used in marine fish (e)DNA (meta)barcoding – 12S rRNA, 16S rRNA and cytochrome oxidase subunit I (COI) – as well as two amplicons for each. We analysed sequences from NCBI GenBank for 10 orders of Actinopterygii (ray-finned fishes), including mitochondrial genomes. We assessed species discrimination by determining the percentage of monophyletic species in Neighbour-Joining trees and calculating congeneric divergences for two datasets: one with genomic regions extracted from mitochondrial genomes (771 species) and another with independent sequences for each region (3,879 species). Amongst (meta)barcoding amplicons in the mitochondrial genomes’ dataset, the COI Folmer and Leray-Lobo regions had the highest discriminatory power, with 89.2% and 87.0% monophyletic species, respectively, while the 12S Teleo region had the lowest at 71.6%. Conversely, using independent sequences of these amplicons, Folmer and Leray-Lobo had the lowest percentages of monophyletic species, at 64.8% and 63.5%, respectively, while Actinopterygii 16S (Ac16S) had the highest at 83.0%. Species discrimination is influenced by the marker’s evolutionary rate, fragment length, target fish order and the quality of reference sequence data. We recommend considering species discriminatory power differences for amplicon selection, especially for species-level identifications. We advise a standard multi-marker approach under certain scenarios, particularly when the presence of close congeneric species is expected.

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DOI: 10.3897/mbmg.8.124564

Authors: Christian Bräunig, Sandra Meid, Björn Quast, Vera Rduch, Peter Grobe

Abstract: Despite the effectiveness of DNA metabarcoding for gaining insights into biodiversity and environmental species composition, a centralised management and storage option including easy accessibility of already-published data is lacking. Since most data are published as supplementary material or in private repositories, DNA metabarcoding has a huge untapped potential to be used for analyses across multiple taxa, sample locations or multiple research projects. We developed a platform to register, manage and identify amplicon sequence variants (ASVs) or zero-radius OTUs (ZOTUs), respectively, against several barcode reference datasets. Moreover, ASV tables can be uploaded, managed, versioned and published with DOIs thus contributing to the full research Data Life Cycle.

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DOI: 10.3897/mbmg.8.126058

Authors: Shana Hirsch, Neha Acharya-Patel, Phyllis Akua Amamoo, Giomar H. Borrero-Pérez, Ni Kadek Dita Cahyani, Joape G. M. Ginigini, Kaleonani K. C. Hurley, Manuel Lopes-Lima, Mark Louie Lopez, Ntanganedzeni Mapholi, Koffi Nouho Ouattara, Diana A. Pazmiño, Yoshimi Rii, Fabiano Thompson, Sophie von der Heyden, Mrinalini Watsa, Vanessa Yepes-Narvaez, Elizabeth Andruszkiewicz Allan, Ryan Kelly

Abstract: Environmental DNA (eDNA) includes a set of rapidly emerging technologies that have the potential to support environmental monitoring and biodiversity conservation through novel, non-invasive, cost-effective and democratic methods and tools. Meanwhile, eDNA researchers are developing international standards for eDNA technologies, methods and data outputs. For eDNA technologies to be accessible, useful and appropriate, we must ensure that any standards developed include a broad conception of users from around the world, a diversity of ecological contexts and locations and, most importantly, a realistic outlook on research capacities and infrastructure. In this article, we assemble perspectives on international standardisation of eDNA from a diverse and global group of users and experts from Africa, South America and the Pacific Islands. The authors of this article collaborated by answering and discussing a set of open-ended questions aimed at eliciting hopes, concerns and experiences regarding eDNA standards. The result is a set of emergent themes and a generative consensus to highlight the need for the creation of adaptable standards, the development of regional capacity, increased sensitising to data sovereignty and the viewing of standardisation as a global capacity-building activity.

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DOI: 10.3897/mbmg.8.122375

Authors: Devin N. Jones, Ben C. Augustine, Patrick Hutchins, James M. Birch, Kevan M. Yamahara, Scott Jensen, Rodney Richardson, Regina Trott, James R. Campbell, Elliott Barnhart, Adam J. Sepulveda

Abstract: Unprecedented rates of biodiversity loss and ecosystem function necessitate the use of rapid, efficacious, and cost-effective biomonitoring tools. The combination of autonomous samplers and high throughput sequencing (i.e., “metabarcoding”) of environmental DNA (eDNA) samples enables characterization of entire communities at high frequency and can be an important tool for conservation and management, allowing researchers to track fluctuations in biodiversity. We deployed two autonomous samplers at two U.S. Geological Survey streamgage sites in the upper Snake River (Wyoming and Idaho, USA) to collect eDNA samples from July-September 2021 and 2022 to characterize fish diversity. We used a probabilistic approach to evaluate the effects of water temperature, water discharge, filter pore size, water volume filtered, number of samples collected, timing, and primers on the probability of detecting eDNA from fish species known to be present. We detected eDNA from 13/15 species present in these areas of the Snake River. Overall, we did not find evidence that filter pore size, water volume filtered, water discharge, and water temperature affected the probability of detecting fish species’ eDNA. By contrast, primers and sampling day affected fish detection probabilities, indicating that primer choice and sampling day can either over- or under- estimate species diversity. These results indicate that users would ideally consider sampling on non-consecutive days and which primer set will maximize species detections.

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DOI: 10.3897/mbmg.8.127688

Authors: Iryna Kapshyna, Gritta Veit-Köhler, Leon Hoffman, Sahar Khodami

Abstract: Sand nourishment is a common and often applied coastal protection measure along the beaches of the German Baltic Sea coast. At the tourist beach of Ahrenshoop, large-scale sand nourishments are completed every five years along a 4-km stretch. The nourishment in question was executed between the end of October 2021 and mid-February 2022. Meiofaunal communities at the beach-water interface were monitored at six stations before (September 2021) and for one year after the impact (March and September 2022, March 2023). To assess the effects of sand nourishment on meiofauna communities, two complementary approaches were employed: whole community metabarcoding and morphological determination and counting of the animals. Both methods produced consistent results regarding community response to disturbance and subsequent recovery. During the period from autumn 2021 to spring 2023, drastic changes in the composition of communities were observed. Directly after the impact, the taxa Acari and Annelida nearly disappeared, abundances of Copepoda decreased substantially, and Platyhelminthes increased notably. The fact that the studied beach held a low diversity may have facilitated that metabarcoding and morphological determination followed the same trend. After initial studies comprising both methods, efficient assessments using only metabarcoding may be considered for comparable sites and situations.

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DOI: 10.3897/mbmg.8.121060

Authors: Beatriz Santos, Luís Afonso, Filipe Alves, Ana Dinis, Rita Ferreira, Ana M. Correia, Raul Valente, Ágatha Gil, Luis Filipe C. Castro, Isabel Sousa-Pinto, Massimiliano Rosso, Cinzia Centelleghe, Sandro Mazzariol, Catarina Magalhães, Maria Paola Tomasino

Abstract: Cetaceans are key sentinel species of marine ecosystems and ocean health, being a strategic taxonomic group that evaluates the well-being of aquatic habitats and detects harmful environmental trends. Respiratory diseases are amongst the main causes of death in these animals, so identifying the microbiome community in their exhaled breath condensates (EBC), i.e. blow, has been proposed as a key biomarker for assessing respiratory health. Yet, to characterise microbiomes related to these animals’ respiratory tract and use them as a proxy for health status, it is necessary to develop baseline data on the microorganisms associated with cetaceans. Here, the short-finned pilot whale (SFPW, Globicephala macrorhynchus) was used as a case study to validate the most suitable primer set to explore the prokaryotic diversity of the cetaceans’ respiratory tract. DNA extracted from blow samples (n = 12) of animals off Madeira Island was sequenced to amplify both V3-V4 and V4-V5 hypervariable regions of the 16S rRNA gene, using the same sequencing platform (Illumina MiSeq). Independently of the primer set used, all blows shared Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria phyla in their composition. V3-V4 resulted in a higher diversity of taxa with relative abundance above 1%, whereas the V4-V5 primers captured a higher number of microbial Amplicon Sequence Variants, detecting the rare microbial biosphere with pathogen potential. Additionally, it captured the core microbiome more efficiently. Thus, this study provides a detailed characterisation of SFPW respiratory-associated microbial communities, strengthening the idea of sociality influencing microbiome composition in the respiratory tract. Moreover, it supports the use of blow as a relevant biomarker for the physiological state of the airways in free-ranging cetaceans.

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DOI: 10.3897/mbmg.8.129227

Authors: Jonathan Warren, Sean Butler, Nick Evens, Laura Hunt, Martyn Kelly, Lindsay Newbold, Daniel S. Read, Joe D. Taylor, Kerry Walsh

Abstract: DNA sequencing of diatom assemblages from biofilms has already been used to assess the ecological status of freshwater in the UK. However, recent work using DNA data from these biofilms suggests that alternate metrics that capture the broader taxonomic and functional information to demonstrate importance of microbial biofilms could be useful. Exploring this potential requires large numbers of samples over time and space to be analysed. Sample archives could be used to meet this need, but the compositional stability of microbial communities in stored biofilm samples for more than one year is uncertain. This study compared changes in diatom assemblage structure using metabarcoding analysis of river biofilm samples before and after storage at -20 °C in an RNAlater-based nucleic acid preservative. We found minimal changes in the diatom assemblages in the samples when stored for up to three years. Slight differences in certain groups observed resulted in four samples changing ecological status. However, the overall differences were not significant across replicates, suggesting any genuine differences in assemblages are likely masked by sub-sampling, PCR, or primer biases. These findings are similar to those observed in other studies looking at variations between analysts and sequencing instruments. This indicates that the diatom assemblages in the archived biofilm samples are stable. This will give greater confidence that archived samples can be used for further research, including exploring broader microbial taxa and their responses to environmental change, potentially leading to the development of reliable microbial metrics for integration into biomonitoring programs.

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DOI: 10.3897/mbmg.8.115512

Authors: Yujin Kang, Seungwoo Han, Youngkeun Song

Abstract: Significant advancements in environmental DNA (eDNA) analysis technology have led to its widespread adoption for species monitoring. However, the low DNA concentration in eDNA often raises concerns about PCR bias, which is a primary issue in enhancing the reliability of eDNA survey results. This study aims to identify the suitable eDNA analysis method and detection strategy for obtaining the presence data of Eurasian otters in an urban stream and to investigate the seasonal distribution patterns of Eurasian otters and their potential prey species. The eDNA survey revealed the presence of six out of the 13 mammal species recorded in fieldwork and literature reviews, including Canis lupus, Felis silvestris and Mustela sibirica. Notably, Eurasian otters were not detected in the eDNA metabarcoding conducted in April and October, despite traditional surveys confirming their presence. In contrast, qPCR assays successfully amplified Eurasian otters from the same samples that were analysed using metabarcoding. When evaluating detection criteria, based on the number of positive samples in repetitions, Eurasian otters were detected at 42.9% to 85.7% of the sampling sites in April and at all sampling sites in October. This suggests a higher detection probability of Eurasian otters in October, indicating a potential expansion of their home range during that season compared to April. Metabarcoding results revealed similar findings regarding fish species as traditional surveys, with Cyprinidae accounting for the largest proportion of fish species at the family level (April, 54.57%; October, 43.58%), followed by Gobiidae (April, 16.90%; October, 22.88%). At the species level, P. parva was the dominant fish species in Saetgang, constituting 5.68% and 6.35% of relative abundance in April and October, respectively. This implies that Eurasian otters as opportunistic predators, may increasingly take advantage of the availability of species within the family Cyprinidae, notably Pseudorasbora parva, as a food source within the study area during the months of April and October. This study highlights that qPCR is the more effective approach in urban areas to offer insights into otters’ distribution patterns, while metabarcoding is useful to provide the properties of the biological environment. Furthermore, this study indicated that it is necessary to determine the suitable eDNA analysis methods depending on the research purpose to obtain detection results effectively.

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DOI: 10.3897/mbmg.8.104655

Authors: Graham S. Sellers, Christopher L. Jerde, Lynsey R. Harper, Marco Benucci, Cristina Di Muri, Jianlong Li, Graeme Peirson, Kerry Walsh, Tristan Hatton-Ellis, Willie Duncan, Alistair Duguid, Dave Ottewell, Nigel Willby, Alan Law, Colin W. Bean, Ian J. Winfield, Daniel S. Read, Lori Lawson Handley, Bernd Hänfling

Abstract: Environmental DNA (eDNA) metabarcoding is transforming biodiversity monitoring in aquatic environments. Such an approach has been developed and deployed for monitoring lake fish communities in Great Britain, where the method has repeatedly shown a comparable or better performance than conventional approaches. Previous analyses indicated that 20 water samples per lake are sufficient to reliably estimate fish species richness, but it is unclear how reduced eDNA sampling effort affects richness, or other biodiversity estimates and metrics. As the number of samples strongly influences the cost of monitoring programmes, it is essential that sampling effort is optimised for a specific monitoring objective. The aim of this project was to explore the effect of reduced eDNA sampling effort on biodiversity metrics (namely species richness and community composition) using algorithmic and statistical resampling techniques of a data set from 101 lakes, covering a wide spectrum of lake types and ecological quality. The results showed that reliable estimation of lake fish species richness could, in fact, usually be achieved with a much lower number of samples. For example, in almost 90% of lakes, 95% of complete fish richness could be detected with only 10 water samples, regardless of lake area. Similarly, other measures of alpha and beta-diversity were not greatly affected by a reduction in sample size from 20 to 10 samples. We also found that there is no significant difference in detected species richness between shoreline and offshore sampling transects, allowing for simplified field logistics. This could potentially allow the effective sampling of a larger number of lakes within a given monitoring budget. However, rare species were more often missed with fewer samples, with potential implications for monitoring of invasive or endangered species. These results should inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals.

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DOI: 10.3897/mbmg.8.123387

Authors: Eli M. S. Gendron, Clemen J. Oliveira, Johan Desaeger, Dorota L. Porazinska

Abstract: Florida’s strawberry production provides significant economic benefit for the State; however, plant-parasitic nematodes (PPNs) pose a significant barrier to production. A better understanding of the distribution of nematode diversity in these fields could help to evaluate the potential risk to crops in agricultural fields and support more sustainable PPN management, but accurate analysis of constituent nematode species is key. The use of targeted mitometagenomics (mtMG) to identify nematode species has shown promise with nematode mock communities, but it remains unclear how it compares in natural agricultural settings and to the more traditional morphology-based approach. In this study, we performed a diversity survey of nematode communities across four different strawberry fields at four depths in the State of Florida using both mtMG and morphological methods. We observed significant differences in nematode community richness and composition between the two methods. Both methods failed to detect taxa recovered by the other method, due to method-specific biases resulting from differential detection of trophic groups. Importantly, both methods did agree on the detection and distribution of Meloidogyne, the most abundant PPNs with the added benefit of the mtMG precisely describing specific species. Despite significant community differences, both methods pointed to the important role of both field and depth in shaping these communities and provided evidence of PPN migration across the soil profile. In conclusion, our findings support the complementary use of multiple detection/identification methods when evaluating nematode diversity, particularly for PPNs.

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DOI: 10.3897/mbmg.8.119614

Authors: Ashrenee Govender, Sandi Willows-Munro, Sohana P. Singh, Johan C. Groeneveld

Abstract: DNA metabarcoding requires only a single DNA fragment to detect a species in mixed zooplankton samples, compared to morphology-based methods that rely on the presence of intact specimens. However, metabarcoding protocols have not yet been fully standardised, thus hindering data comparability between studies. To converge on standardised metabarcoding protocols, we used an experimental field-sampling approach to compare the effects of sampling gear (ring-, Manta- and WP2 nets), day and night (DN) sampling and tow duration (5-, 10- and 15-minute tows) on marine zooplankton composition. High-throughput sequencing of the cytochrome c oxidase subunit I (COI) gene region with different primers and taxonomic assignment of amplicon sequence variants at 97% similarity to barcode records were used to identify species. Metabarcoding detected a total of 224 species, of which 92% matched prior occurrence records from the region. Malacostraca (crabs, hermit crabs, lobsters, prawns and shrimps) was the best-represented class (49%), followed by Copepoda (21%), Actinopterygii (ray-finned fishes; 21%), and Gastropoda (9%). Species counts ranged from 9–61 species per tow, with high proportions of unique species in replicate tows. Mean species counts did not differ significantly between net types, DN samples or tow durations, respectively. Proportionate representation amongst taxonomic classes remained within a narrow range, except when sampling deeper habitats with a smaller mesh size. DN samples showed no evidence of daily vertical migration of zooplankton. Consistent inferred species composition across net types, tow duration and DN sampling treatments reflects high detection sensitivity of individual-based sampling, allowing for greater flexibility in planning of zooplankton sampling regimes.

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DOI: 10.3897/mbmg.8.121817

Authors: Thorsten Stoeck, Sven Nicolai Katzenmeier, Hans-Werner Breiner, Verena Rubel

Abstract: Oxford Nanopore Technologies has recently launched a duplex sequencing strategy and announced an improved error rate which is in a similar order of magnitude as Illumina sequencing. We therefore conducted a pilot study to assess whether Nanopore duplex sequencing has potential to be used as a technology in eDNA-based marine biomonitoring. Specifically, we investigated bacterial communities of sediment samples collected from Atlantic salmon (Salmo salar) aquaculture installations and compared the ecological trends obtained from short Illumina (V3-V4 region of the 16S rRNA gene) and long Nanopore (full length 16S rRNA gene) sequence reads. The obtained duplex rate of Nanopore amplicon reads with a Phred score ≥ 30 was 36%, notably higher compared to previous reports from bacterial genome sequencing. When inferring alpha- and beta-diversity from Illumina ASVs and Nanopore OTUs, we found highly congruent ecological patterns. Only when collating ASVs and OTUs across taxonomic ranks, beta-diversity analyses of Illumina-data slightly changed, due to the difficulties to assign a taxonomy to short sequence reads. While on family rank, both sequence datasets had good agreement, genus-assignments of Illumina data were critical, resulting in higher disagreement between the two protocols. Our data provide evidence that eDNA-based monitoring of aquaculture-related environmental impacts could equally well be conducted with the improved Nanopore duplex sequencing. We discuss to what extent eDNA-based biomonitoring could benefit from long-read information.

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DOI: 10.3897/mbmg.8.108649

Authors: Marie-Josée Cros, Jean-Marc Frigerio, Nathalie Peyrard, Alain Franc

Abstract: An accurate and complete taxonomic description of the diversity present in an environmental sample is out of reach at this time. Instead, metabarcoding is used today and it is expected that OTUs represent a category relevant for biodiversity inventories on a molecular basis. However, artefacts in the production of OTUs can occur at different stages and may impact ecological conclusions. We propose to evaluate the quality of OTUs in a sample by characterising the deviation of each OTU’s dissimilarity array from that of an ideal OTU where all sequences are at distances smaller than the barcoding gap. We consider two deviations: the creation of composed OTUs, corresponding to the artificial merging of several OTUs and the creation of noisy OTUs that contain some sequences that are loosely associated with the core sequence of the OTUs and that do not form a compact subgroup. We propose a simple and automatic 2-step method that successively categorises the OTUs of a sample as composed or single and then identifies OTUs with noise amongst the single ones. The associated code is available at https://forgemia.inra.fr/alain.franc/otu_shape. We applied the method on 32 samples of diatoms from Arcachon Bay (France) that represent contrasted environmental conditions and we obtained good agreement with expert categorisation of OTUs. We suggest that single OTUs without noise can be used as such for further ecological studies. Composed OTUs should be post-treated with classical clustering or community detection tools. The quality of single OTUs with noise remains to be further tested via supplementary studies on a diversity of organisms.

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DOI: 10.3897/mbmg.8.111307

Authors: Mohammed Ahmed, Dieter Slos, Oleksandr Holovachov

Abstract: The Store Mosse National Park in the south of Sweden was surveyed for nematode diversity and distribution using DNA metabarcoding. Fifty samples were collected from five vegetation types in the park across a range of habitats (e.g. soil, litter, lichens, sphagnum and roots). The other habitats, aside from soil and litter, were sampled in order to capture the diversity of nematodes that may be uniquely associated with them. Nematodes were characterised using the V7-V8 variable domain (~ 350 bp) of the 18S ribosomal RNA gene. We identified 46 families, 76 genera (21 new to Swedish fauna) and 60 species (31 new to Swedish fauna). Some nematodes showed strong associations with their habitats, especially at the species level. Although soil and litter supported the most diverse nematode communities, our results support a strong justification for sampling across different media types to quantify nematode diversity accurately. Soil and litter communities showed high levels of stability with balanced distribution of all the various trophic and coloniser-persister groups.

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DOI: 10.3897/mbmg.7.111679

Authors: Éva Ács, Tibor Bíró, Emil Boros, Péter Dobosy, Mónika Duleba, Angéla Földi, Keve T. Kiss, Zlatko Levkov, Péter Orgoványi, Orsolya Pálné Szén, Zsuzsa Trábert, Edit Vadkerti, István Grigorszky

Abstract: The research presented investigates whether DNA-based metabarcoding can replace the morphology-based identification of diatom taxa in the ecological status assessments of aquatic habitats. When comparing data obtained with microscopy and metabarcoding, significant deviations have been noticed. One of the main reasons includes the incompleteness of the reference database used for taxonomic annotation of sequences. The database library should be complemented with species inhabiting unique habitats and having specific environmental requirements representing environmental endpoints for genetic diversification. Soda pans and soda lakes are examples of an extreme habitat with the loss of sodic character as the main threat; thus, accurate identification of species and exact information on their salinity tolerance is essential for adequate ecological status assessment. In the present study, by using microscopy and metabarcoding, we investigated taxa of the genus Halamphora that are common in soda pans and soda lakes. We detected six species of which Halamphora dominici and H. veneta occurred frequently and often in high abundance (it was often dominant having relative abundance higher than 5%). Analyses of DNA data confirmed the separation of the two species; as a result, the reference database library has been supplemented with sequences of H. dominici. Furthermore, we have confirmed that this species, which is a significant indicator of sodic character, shows a positive correlation with salinity.

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DOI: 10.3897/mbmg.7.111467

Authors: Lena A. Schallenberg, Georgia Thomson-Laing, David Kelly, John K. Pearman, Jamie D. Howarth, Marcus J. Vandergoes, Jonathan Puddick, Sean Fitzsimons, Andrew Rees, Susanna A. Wood

Abstract: Introduced trout can induce trophic cascades, however, a lack of pre-introduction data limits knowledge on their impact in many lakes. Traditional paleolimnological approaches have been used to study historic species changes, but until recently these have been restricted to taxa with preservable body-parts. To explore the ecosystem effects of Salmo trutta (brown trout) introduction on an oligotrophic lake in Aotearoa-New Zealand, we used a multi-marker sedimentary environmental DNA (sedDNA) approach coupled with pigments to detect changes across multiple trophic levels. DNA was extracted from core depths capturing approximately 100 years before and after the expected arrival of S. trutta, and metabarcoding was undertaken with four primer sets targeting the 12S rRNA (fish), 18S rRNA (eukaryotes) and cytochrome c oxidase (COI; eukaryotes) genes. The earliest detection of S. trutta eDNA was 1906 (1892–1919 CE with 95% high probability density function) suggesting their introduction was shortly before this. Native fish diversity (12S and 18S rRNA) decreased after the detection of S. trutta, albeit the data was patchy. A shift in overall eukaryotic and algal communities (18S rRNA and COI) was observed around 1856 (1841–1871 CE) to 1891 (1877–1904 CE), which aligns with the expected S. trutta introduction. However, taxonomy could not be assigned to many of the 18S rRNA and COI sequences. Pigment concentrations did not change markedly after S. trutta introduction. SedDNA provides a new tool for understanding the impact of disturbances such as the introduction of non-native species; however, there are still several methodological challenges to overcome.

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DOI: 10.3897/mbmg.7.110194

Authors: Katherina Schimani, Nélida Abarca, Oliver Skibbe, Heba Mohamad, Regine Jahn, Wolf-Henning Kusber, Gabriela Laura Campana, Jonas Zimmermann

Abstract: Polar regions are among the most extreme habitats on Earth. However, diatom biodiversity in those regions is much more extensive and ecologically diverse than previously thought. The objective of this study was to add knowledge to benthic diatom biodiversity in Western Antarctic coastal zones via identification by means of morphology, DNA metabarcoding and cultured isolates. In addition, a taxonomically validated reference library for Antarctic benthic diatoms was established with comprehensive information on habitat, morphology and DNA barcodes (rbcL and 18SV4). Benthic samples from marine, brackish and freshwater habitats were taken at the Antarctic Peninsula. A total of 162 clonal cultures were established, resulting in the identification of 60 taxa. The combination of total morphological richness of 174 taxa, including the clones, with an additional 73 taxa just assigned by metabarcoding resulted in 247 infrageneric taxa. Of those taxa, 33 were retrieved by all three methods and 111 only by morphology. The barcode reference library of Antarctic species with the new references obtained through culturing allowed the assignment of 47 taxa in the metabarcoding analyses, which would have been left unassigned because no matching reference sequences were available before. Non–metric multidimensional scaling analyses of morphological as well as molecular data showed a clear separation of diatom communities according to water and substratum types. Many species, especially marine taxa, still have no record in reference databases. This highlights the need for a more comprehensive reference library to further improve routine diatom metabarcoding. Overall, a combination of morphological and molecular methods, along with culturing, provides complementary information on the biodiversity of benthic diatoms in the region.

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DOI: 10.3897/mbmg.7.112290

Authors: Wiebke Sickel, Vera Zizka, Alice Scherges, Sarah J. Bourlat, Petra Dieker

Abstract: Biodiversity is declining at alarming rates worldwide and large-scale monitoring is urgently needed to understand changes and their drivers. While classical taxonomic identification of species is time and labour intensive, the combination with DNA-based methods could upscale monitoring activities to achieve larger spatial coverage and increased sampling effort. However, challenges remain for DNA-based methods when the number of individuals per species and/or biomass estimates are required. Several methodological advancements exist to improve the potential of DNA metabarcoding for abundance analysis, which however need further evaluation. Here, we discuss laboratory, as well as some bioinformatic adjustments to DNA metabarcoding workflows regarding their potential to achieve species abundance estimation from arthropod community samples. Our review includes pre-laboratory processing methods such as specimen photography, laboratory methods such as the use of spike-in DNA as an internal standard and bioinformatic advancements like correction factors. We conclude that specimen photography coupled with DNA metabarcoding currently promises the greatest potential to achieve estimates of the number of individuals per species and biomass estimates, but that approaches such as spike-ins and correction factors are promising methods to pursue further.

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DOI: 10.3897/mbmg.7.109085

Authors: Luisa Paez-Triana, Giovanny Herrera, Laura Vega, Diego García-Corredor, Martin Orlando Pulido Medellín, Alberto Paniz-Mondolfi, Marina Muñoz, Juan David Ramírez

Abstract: Rhipicephalus sanguineus sensu lato (s.l.), commonly known as the domestic dog tick, is a globally distributed tick. This species plays a significant role in human and animal health, as well as in economy due to its ability to infest livestock. The use of shotgun metagenomics has proven valuable in identifying tick-borne pathogens and key members of the tick microbiome, including endosymbionts. However, the application of shotgun metagenomics in R. sanguineus s.l. ticks in Latin America remains largely unexplored. Therefore, our objective aimed to explore and further analyze the metagenome of the tropical lineage of R. sanguineus s.l. ticks in Colombia. Through our analysis, we identified the three most prevalent pathogens harbored by these ticks, namely: Anaplasma phagocytophilum, Francisella tularensis, and Theileria equi. However, the most abundant microorganism detected was not a pathogen but the endosymbiont Coxiella mudrowiae. Interestingly, Coxiella mudrowiae exhibited significant negative correlations with several pathogens and other endosymbionts. Furthermore, we successfully constructed 27 medium-quality metagenome-assembled genomes (MAGs) for this microorganism, enabling us to conduct a pangenome analysis by comparing them with available genomes and identifying proteins of interest, such as those involved in vitamin B synthesis. This study represents the first implementation of shotgun metagenomics as a methodology to expand our understanding of pathogens and endosymbionts in the circulating tropical lineage of R. sanguineus s.l. ticks in Colombia. The findings of this research serve as a foundation for the development of prevention and mitigation strategies against pathogens transmitted by this tick species. Information gained from this study can contribute to the improvement of public health measures and veterinary practices aimed at controlling the impact of tick-borne diseases.

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DOI: 10.3897/mbmg.7.109389

Authors: Aman Deep, Dana Bludau, Marius Welzel, Sandra Clemens, Dominik Heider, Jens Boenigk, Daniela Beisser

Abstract: Sequencing of amplified DNA is the first step towards the generation of Amplicon Sequence Variants (ASVs) or Operational Taxonomic Units (OTUs) for biodiversity assessment and comparative analyses of environmental communities and microbiomes. Notably, the rapid advancements in sequencing technologies have paved the way for the growing utilization of third-generation long-read approaches in recent years. These sequence data imply increasing read lengths, higher error rates, and altered sequencing chemistry. Likewise, methods for amplicon classification and reference databases have progressed, leading to the expansion of taxonomic application areas and higher classification accuracy. With Natrix, a user-friendly and reducible workflow solution, processing of prokaryotic and eukaryotic environmental Illumina sequences using 16S or 18S is possible. Here, we present an updated version of the pipeline, Natrix2, which incorporates VSEARCH as an alternative clustering method with better performance for 16S metabarcoding approaches and mothur for taxonomic classification on further databases, including PR2, UNITE and SILVA. Additionally, Natrix2 includes the handling of Nanopore reads, which entails initial error correction and refinement of reads using Medaka and Racon to subsequently determine their taxonomic classification.

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DOI: 10.3897/mbmg.7.96780

Authors: Tamar Beridze, Bella Japoshvili, Tamar Edisherashvili, Cort Anderson, Levan Mumladze

Abstract: Due to anthropogenic influences, habitat degradation and a continuous loss of biodiversity in freshwater ecosystems are occurring on a large scale, while these ecosystems constitute invaluable natural resources. Therefore, it is essential to study and monitor freshwater ecosystems to guide conservation efforts. Freshwater ecosystems are one of the less-studied fields in Georgia. Studies about the species distribution of many taxa and/or regions carried out during the last century have not been updated for decades. Here, we report the results of an environmental DNA (eDNA) metabarcoding exercise, based on samples collected from the Rioni River, a tributary to the Black Sea and a crucial aquatic ecosystem regionally and globally. The only comprehensive review of the fish of the Rioni River dates back to 1956. We compared the eDNA-based taxonomic composition to the known faunal composition within the Rioni River and found that the eDNA-based taxonomic coverage approached 75% of the expected total fish fauna. A number of new species occurrences were also found, including the first detection of three invasive alien species (Carassius gibelio, Pseudorasbora parva, Rhinogobius lindbergi) in the Rionis River Basin and a new country record of the ninespine stickleback (genus Pungitius) for Georgia. In spite of the usefulness of the eDNA metabarcoding approach, the sparsity of the fish DNA barcode reference library for the region emerged as a limitation to this study. However, our findings still represent a great leap forward in updating fish status on the Rioni River and testing the effectiveness of the eDNA sampling for aquatic species.

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DOI: 10.3897/mbmg.7.103856

Authors: Till-Hendrik Macher, Robin Schütz, Atakan Yildiz, Arne J. Beermann, Florian Leese

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

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DOI: 10.3897/mbmg.7.103279

Authors: Valerie Levesque-Beaudin, Dirk Steinke, Mieke Böcker, Bettina Thalinger

Abstract: Bird nests are fascinating microcosms harboring a wide range of arthropods parasitizing the nesting birds or feeding on prey remains, feces, and the nest material. Studies of these communities have been entirely based on emergence traps which collect live organisms out of the nests. The analysis of nest contents and environmental DNA (eDNA) via metabarcoding could expand our knowledge and identify prey, exuviae, and other animal remains in bird nests. Here, we investigated the potential of arthropod remains, nest dust, and feathers to better describe arthropod diversity accumulated in 20 bird nests collected in Guelph (Canada). We used subsampling strategies and tested two extraction approaches to investigate the distribution of DNA in nests, account for low-quality DNA, and the presence of inhibitory substances. In total, 103 taxa were detected via metabarcoding. Arthropod remains delivered the highest number of taxa (n = 67), followed by nest dust (n = 29). Extractions with the PowerSoil kit outperformed DNeasy extractions coupled with PowerClean Pro inhibitor removal. Per nest, on average 5.5% taxonomic overlap between arthropod remains of different size classes was detected and subsamples of nest dust extracted with the PowerSoil kit showed 47.3% taxonomic overlap indicating a heterogeneous eDNA distribution in nests. Most detected species were either feeding in the nest, i.e., herbivorous / predatory, or bird food. We also detected molecular traces of 25 bird species, whose feathers were likely used as nest material. Consequently, the metabarcoding of bird nest materials provides a more complete picture of nest communities, which can enable future studies on functional diversity and better comparisons between nesting species.

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DOI: 10.3897/mbmg.7.99979

Authors: Bastien Parisy, Niels M. Schmidt, Helena Wirta, Laerke Stewart, Loic Pellissier, William E. Holben, Sam Pannoni, Panu Somervuo, Mirkka M. Jones, Jukka Siren, Eero Vesterinen, Otso Ovaskainen, Tomas Roslin

Abstract: Understanding how different taxa respond to abiotic characteristics of the environment is of key interest for understanding the assembly of communities. Yet, whether eDNA data will suffice to accurately capture environmental imprints has been the topic of some debate. In this study, we characterised patterns of species occurrences and co-occurrences in Zackenberg in northeast Greenland using environmental DNA. To explore the potential for extracting ecological signals from eDNA data alone, we compared two approaches (visual vegetation surveys and soil eDNA metabarcoding) to describing plant communities and their responses to abiotic conditions. We then examined plant associations with microbes using a joint species distribution model. We found that most (68%) of plant genera were detectable by both vegetation surveys and eDNA signatures. Species-specific occurrence data revealed how plants, bacteria and fungi responded to their abiotic environment – with plants, bacteria and fungi all responding similarly to soil moisture. Nonetheless, a large proportion of fungi decreased in occurrences with increasing soil temperature. Regarding biotic associations, the nature and proportion of the plant-microbe associations detected were consistent between plant data identified via vegetation surveys and eDNA. Of pairs of plants and microbe genera showing statistically supported associations (while accounting for joint responses to the environment), plants and bacteria mainly showed negative associations, whereas plants and fungi mainly showed positive associations. Ample ecological signals detected by both vegetation surveys and by eDNA-based methods and a general correspondence in biotic associations inferred by both methods, suggested that purely eDNA-based approaches constitute a promising and easily applicable tool for studying plant-soil microbial associations in the Arctic and elsewhere.

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DOI: 10.3897/mbmg.7.106844

Authors: Antoniya Hubancheva, Vedran Bozicevic, Jérôme Morinière, Holger R. Goerlitz

Abstract: A comprehensive understanding of trophic interactions in terrestrial ecosystems is crucial for ecological research and conservation. Recent advances in non-invasive methods, such as environmental DNA (eDNA) metabarcoding, have enabled researchers to collect vast amounts of data on wild animal diets. However, sharing this data and metadata effectively and transparently presents new challenges. To address this, a new type of scholarly journal publication has emerged that aims to describe datasets rather than report research investigations. In this paper, we present a dataset of consumed prey species and parasites based on the metabarcoding of 113 faecal samples from the greater and lesser mouse-eared bats (Myotis myotis and Myotis blythii), along with a detailed description of the data sampling, laboratory analysis, and bioinformatics pipeline. Our dataset comprises 1018 unique Barcode Index Numbers (BINs) from 12 Classes and 43 Orders. In addition, we provide interactive Krona charts to visually summarize the taxonomic relationships and relative read abundance of the consumed prey species and parasites. This data can be used for meta-analysis, exploring new predator-prey and host-parasite interactions, studying inter- and intraspecific ecological interactions, and informing protected area management, among other applications. By sharing this dataset, we hope to encourage other researchers to use it to answer additional ecological questions and advance our understanding of trophic interactions in terrestrial ecosystems.

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DOI: 10.3897/mbmg.7.102455

Authors: Nina Röder, Klaus Schwenk

Abstract: Aquatic emergent insect communities form an important link between aquatic and terrestrial ecosystems, yet studying them is costly and time-consuming as they are usually diverse and superabundant. Metabarcoding is a valuable tool to investigate arthropod community compositions, however high-throughput applications, such as for biomonitoring, require cost-effective and user-friendly procedures. To investigate if the time-consuming and labour-intensive DNA extraction step can be omitted in metabarcoding, we studied the difference in detection rates and individual read abundance using standard DNA extraction versus direct PCR protocols. Metabarcoding with and without DNA extraction was performed with artificially created communities of known composition as well as on natural communities both of the dipteran family Chironomidae to compare detection rates, individual read abundances and presence-absence community composition. We found that the novel approach of direct PCR metabarcoding presented here did not alter detection rates and had a minor effect on individual read abundances in artificially created communities. Furthermore, presence-absence community compositions of natural chironomid communities were highly comparable using both approaches. In conclusion, we showed that direct PCR protocols can be applied in chironomid metabarcoding approaches, with possible application for a wider range of arthropod taxa, enabling us to study communities more efficiently in the future.

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DOI: 10.3897/mbmg.7.86883

Authors: Andreas Kolter, Martin Husemann, Lars Podsiadlowski, Birgit Gemeinholzer

Abstract: Landscape changes, over time, lead to changes of floral resources available to pollinators, which in turn may result in the disappearance of ecologically specialized species. Here, we use pollen metabarcoding to infer historic and recent interactions between plants and bumblebees (Bombus). Bumblebees from Cuxhaven (Germany) were sampled from historical museum collections (1968/69) and in the field (2019). Pollen attached to their bodies was barcoded using multiple plant markers (ITS1, ITS2 and trnL-P6 loop). Our results show shifts in foraging habits between the historic and recent sampling periods, mostly determined by fewer Fabaceae interactions in 2019. The successful implementation of scalable molecular techniques for the analysis of historical pollen samples underscores the value of museum collections as a resource for biodiversity research. This study provides proof of concept of a comparative analysis of recent and historical pollination data. However, to ensure the robustness of our results, it is crucial to consider the broader methodology used. Our study found variation in the efficacy of the three plant barcoding markers. The ITS1 marker exhibited the highest species-level identification success, while the trnL-P6 loop demonstrated utility in amplifying degraded DNA across diverse plant families.

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