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Open research questions in Environmental DNA in Biodiversity Studies

29 unresolved questions extracted from the limitations and future-work sections of 223 Environmental DNA in Biodiversity Studies papers in our library. Each links back to the study that raised it.

What the literature leaves open

  • Environmental DNA (eDNA) has the potential to be a powerful tool in blue carbon science for characterizing and quantifying the contribution of marine macrophytes; but its complex, dynamic relationship with bulk biomass is poorly understood.

    Assessing the degradation dynamics of sugar kelp in anaerobic marine sediment using environmental DNA · 2026 · DOI

  • These results suggest that current DFO methods are insufficient to classify drains and, hence, adequately protect sensitive fishes and fish habitat within them.

    Protecting fishes in agricultural drains: evaluating Canadian policy using environmental DNA · 2026 · DOI

  • Several limitations should be acknowledged. While the pre- sent study focuses on collagen-based materials, the work- flow is designed to be adaptable to other types of degraded samples. Its application to different matrices may therefore be feasible, although further validation across diverse sub- strates would be required to fully assess its transferability. The accuracy of species attribution depends on the com- pleteness and phylogenetic coverage of the reference panel. S. Morelli et al. Reads originating from unrepresented or closely related taxa may be preferentially assigned to the most similar available genome or classified as shared, potentially leading to biased or ambiguous taxonomic signals. This reflects a fundamen- tal trade-off between sensitivity and specificity: a restricted reference panel improves interpretability and reduces spuri- ous assignments but may limit the detection of unexpected or unrepresented species, whereas broader panels increase sensitivity at the cost of higher ambiguity. In this study, the reference panel was intentionally restricted to species relevant to collagen-based glue produc- tion, enabling a controlled and application-specific compari- son while prioritizing interpretability over exhaustive taxo- nomic coverage. More generally, reference databases should be carefully curated and tailored to the expected taxonomic composition of the samples. Importantly, the read-ubiquity classifier does not force unambiguous assignment when evidence is insufficient, as reads mapping across multiple genomes are explicitly clas- sified as shared. However, when the true source species is absent from the panel, species-specific assignments should be interpreted with caution. Controlled mixtures with known species proportions would provide a more systematic framework to evaluate performance under varying levels of mixture complexity and cross-mapping and represent an important direction for future work. Although the dataset includes closely related taxa (e.g. bovine, ovine, and caprine species), further valida- tion using controlled mixtures would be necessary to sys- tematically assess the discriminatory power of the workflow. The performance of the workflow may be influenced by fragment length, sequencing depth, and library complex- ity. While fragment length distributions are provided in this study, a systematic evaluation of detection thresholds across varying fragment sizes and coverage levels would represent an important direction for future work. Additionally, uneven sequencing depth or pooling imbalance may influence the absolute number of species-specific reads, although relative patterns remained stable across treatments in this study. Tar- geted capture strategies could enhance sensitivity in some contexts, but their effectiveness depends on prior knowledge of expected taxa and may be less suitable for unknown or heterogeneous samples.

    A damage-aware NGS workflow for conservative species identification from ultra-degraded DNA · 2026 · DOI

  • The detection of rare and invasive fish species in Svalbard waters using eDNA metabarcoding has only been demonstrated at regional scales (e.g., Yarlung Zangbo River). Application-specific validation for Arctic-Atlantic boundary environments requires species-level monitoring protocols tuned to detect boreal fish range expansions in response to Arctic warming and borealization.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • Intraspecific genetic diversity detection using eDNA metabarcoding data in Arctic Calanus and other zooplankton species remains underdeveloped compared to presence-absence community composition. Methods to estimate population-level genetic structure and diversity from amplicon data in the context of Arctic biogeography shifts require validation against mitogenomic reference databases specific to boreal and Arctic Calanidae.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • The spatial and temporal scaling of eDNA metabarcoding from fjord-scale surveys to circumpolar Arctic monitoring frameworks requires standardized sampling protocols and quality assurance procedures. Current efforts to develop circumpolar monitoring programmes (Christensen et al. 2020, Culp et al. 2012) lack integration with eDNA-based biodiversity detection across multiple Arctic regions and seasons.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • Environmental RNA (eRNA) as a complementary approach to eDNA metabarcoding for assessing live versus dead biomass in Arctic marine communities has not been applied to Svalbard waters. Integration of RNA-based biodiversity assessments with traditional eDNA methods could provide metabolic status information for climate-sensitive Arctic fish and zooplankton assemblages.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • Multi-marker eDNA metabarcoding approaches for simultaneous detection of fish, zooplankton, and meroplankton larvae in Arctic waters have not been systematically compared for primer specificity and amplification bias. Optimization of marker-primer combinations for the diverse pelagic communities of Svalbard requires empirical validation of detection sensitivity across multiple taxonomic groups.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • The persistence and degradation rates of environmental DNA in cold Arctic marine systems have not been characterized with the same temporal resolution as temperate waters. Quantitative data on eDNA decay kinetics in Svalbard fjord conditions across different seasons is needed to establish detection thresholds and sampling frequency protocols for reliable biodiversity monitoring.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • Reference databases for eDNA metabarcoding in Arctic marine systems lack comprehensive coverage of polar species, particularly for rare and invasive fish taxa in Svalbard waters. The curation and completeness of GenBank entries for Arctic organisms requires systematic evaluation similar to the marine fish assemblage case study by Claver et al. (2023) to improve species identification accuracy.

    Tracking biodiversity in changing Arctic waters: insights from eDNA metabarcoding in Svalbard · 2026 · DOI

  • Uncertain delimitations and identifications due to either limited information of diagnostic characters or conflicting taxonomy were further investigated using DNA barcode information.

    A DNA barcode-assisted annotated checklist of the spider (Arachnida, Araneae) communities associated to white oak woodlands in Spanish National Parks · 2018 · DOI

  • For the vast majority of publications including DNA barcodes, the generation and publication of ancillary data associated with the barcoded material is seldom highlighted and often disregarded, and the analysis of those data sets to uncover new distribution patterns of species has rarely been explored, even though many BOLD records represent new and/or significant discoveries.

    Streamlining the use of BOLD specimen data to record species distributions: a case study with ten Nearctic species of Microgastrinae (Hymenoptera: Braconidae) · 2014 · DOI

  • The Caribbean is a recognized biodiversity hotspot; however, on islands such as Jamaica, many faunal groups of ecological and forensic importance, including flesh flies, remain poorly characterized, particularly at the genetic level.

    DNA Barcoding of Flesh Flies (Diptera: Sarcophagidae) in Jamaica: A Preliminary Assessment · 2026 · DOI

  • We investigated bdelloid rotifer diversity and biogeographical patterns in small aquatic, moss-associated habitats across South-Western Greenland, a poorly explored and geographically isolated region.

    Cryptic diversity constrains biogeographical inference in microscopic animals: evidence from bdelloid rotifers in Greenland · 2026 · DOI

  • We identified mixed results when examining the correlation between eDNA catfish read composition and historic or contemporary netting data, with significant correlations corresponding to random combinations of both filter size and eDNA assay type.

    Environmental <scp>DNA</scp> as a Detection and Quantitative Tool for Brown Bullhead Catfish ( <scp> <i>Ameiurus nebulosus</i> </scp> ): A Comparison With Conventional Netting · 2026 · DOI

  • Aedes aegypti and Culex quinquefasciatus are important vectors of dengue, Zika, chikungunya, West Nile virus, and other arboviruses, yet their feeding patterns remain poorly characterized in many tropical regions.

    Bloodmeal metabarcoding reveals host feeding patterns for Aedes aegypti and Culex quinquefasciatus in Jutiapa, Guatemala and Texas, USA · 2026 · DOI

  • Insufficient knowledge of sampler coverage area for the different organism types was identified as a limitation to the deployment of aerial monitoring networks.

    Towards the reliable use of aerial eDNA for ecosystem monitoring · 2026 · DOI

  • However, there is no standard protocol that can guarantee sufficient detection rates and repeatability, despite companies offering an extensive range of analyses.

    Variable performances of commercial eDNA inventories challenge their use for surveying stream fish communities · 2026 · DOI

  • Abstract Distinguishing native from non-native species is complicated by cryptogenic taxa, whose native or non-native status remains uncertain, and cryptic species, which are genetically distinct yet morphologically indistinguishable taxa.

    When cryptogenic species are also cryptic: reframing biogeographic uncertainty in the environmental DNA era · 2026 · DOI

  • Nevertheless, the limited uptake of eDNA beyond scientific contexts suggests that the characteristics of the innovation alone are insufficient to ensure its adoption.

    Widespread adoption of environmental DNA (eDNA)-based biodiversity monitoring in Canada will depend on trust more than on education: insights from a SWOT analysis · 2026 · DOI

  • mansoni</ns3:italic> DNA purity as adaptive sampling alone is insufficient for enrichment.

    How useful is Nanopore adaptive sampling for sequencing Schistosoma mansoni miracidia? · 2025 · DOI

  • Its scope thereafter broadened, uncovering previously unrecorded biodiversity via metabarcoding in both well-studied and understudied ecosystems across all taxonomic groups.

    Environmental <scp>DNA</scp>: The next chapter · 2024 · DOI

  • In the case of the vast and remote ocean twilight zone (OTZ), management is particularly challenged by the lack of data.

    Applying environmental DNA approaches to inform marine biodiversity conservation: The case of the Ocean Twilight Zone · 2024 · DOI

  • While strategies exist for optimizing sample and sequence library preparation, best practices for bioinformatic processing of amplicon sequence data are lacking in animal diet studies.

    A total crapshoot? Evaluating bioinformatic decisions in animal diet metabarcoding analyses · 2020 · DOI

  • Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested.

    Sorting things out: Assessing effects of unequal specimen biomass on <scp>DNA</scp> metabarcoding · 2017 · DOI

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29 open questions have been extracted from the limitations and future-work passages of 223 Environmental DNA in Biodiversity Studies papers in our library. Each one below links back to the study that raised it, so you can read the original claim in context.

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