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What If the Animal Is Unknown?

eDNA can flag known lineages well, but unknown or poorly catalogued animals may return broad or unresolved matches.

On this page

  • How DNA reads become species names
  • Why database gaps matter
  • What broad lineage matches can still reveal
Preview for What If the Animal Is Unknown?

Introduction

Environmental DNA (eDNA) can only identify an organism by comparing recovered genetic fragments against reference sequences from organisms that have already been catalogued. This creates an important limitation for cryptozoological investigations: if a lake contains a genuinely unknown animal, there may be no exact sequence in any database to match it. In that situation, eDNA does not simply return a species name. Instead, the result may be assigned to a broader taxonomic group such as a genus, family, or order, or it may remain unclassified altogether. The strength of eDNA is therefore greatest when searching for known lineages, while its ability to recognise truly unknown creatures depends heavily on the completeness and quality of reference libraries.[MDPI]mdpi.comAre Genetic Reference Libraries Sufficient for…by CL Jerde · 2021 · Cited by 30 — However, eDNA metabarcoding's main weakness is t…DNA Databases illustration 1

How DNA Reads Become Species Names

When researchers sequence DNA recovered from lake water, they generate thousands or millions of short genetic reads. Those reads are then compared with large reference collections such as GenBank and the Barcode of Life Data System (BOLD), which contain DNA barcodes from known organisms. BOLD alone now contains millions of barcode records representing more than a million species.[arXiv]arxiv.orgBOLD v4: A Centralized Bioinformatics Platform for DNA-based Biodiversity DataApril 8, 2024…Published: April 8, 2024

The assignment process is essentially a matching exercise. If a recovered sequence closely matches a known species in the reference database, software can assign that read to species level. If no exact match exists, the identification becomes less specific. A sequence might be recognised only as belonging to a particular genus, family, or higher taxonomic group. In some cases, researchers can conclude only that the DNA belongs somewhere within a broad branch of the tree of life.[naturemetrics.com]naturemetrics.comReport Interpretation GuideA species-level assignment will be given if reference databases contain an exact DNA match. A higher taxonomic…

For a lake-monster investigation, this distinction is crucial. Detecting DNA from a known eel, trout, or amphibian is relatively straightforward when reference sequences exist. Detecting an animal never previously sampled by science is much harder because the system has nothing definitive against which to compare the sequence.[MDPI]mdpi.comAre Genetic Reference Libraries Sufficient for…by CL Jerde · 2021 · Cited by 30 — However, eDNA metabarcoding's main weakness is t…

Why Database Gaps Matter

How to Use the NCBI’s Bioinformatics Tools and Databases

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What Would a Truly Unknown Animal Look Like in the Data?

A genuinely undescribed lake-dwelling vertebrate would not necessarily appear in eDNA results as “unknown monster”. Instead, its DNA would likely be interpreted through its nearest known relatives.

Imagine a hypothetical animal closely related to eels but distinct enough to represent an undescribed species. Its DNA might produce results indicating membership within the eel lineage while failing to match any recognised eel species precisely. Researchers could therefore infer that the organism belongs somewhere within that group without being able to assign a formal species name.[naturemetrics.com]naturemetrics.comReport Interpretation GuideA species-level assignment will be given if reference databases contain an exact DNA match. A higher taxonomic…

The further an organism is from known species, the weaker and broader the assignment becomes. Depending on the marker used, the result might be classified only as:<div class="content-enhancement content-enhancement--metric" markdown="1">

  • A species complex
  • A genus[thebiodiversityconsultancy.com]thebiodiversityconsultancy.comFigure 1: Simplified eDNA process www.thebiodiversityconsultancy.com. Reporting…Read more…
  • A family
  • An order
  • An unclassified vertebrate lineage</div>

This is one reason modern eDNA studies increasingly report confidence levels and assignment thresholds rather than treating every match as equally certain.[Archimer]archimer.ifremer.frA Confidence Scoring Procedure for eDNA Metabarcoding…by R Rozanski · 2025 · Cited by 5 — Here, we present a modular procedure…DNA Databases illustration 2

What Broad Lineage Matches Can Still Reveal

Even when species-level identification fails, broad taxonomic matches can still provide useful evidence.

If repeated lake surveys consistently recovered DNA belonging to a vertebrate lineage not otherwise known from the ecosystem, researchers would have a specific biological signal worth investigating. The finding would not prove the existence of a cryptid, but it would narrow the search considerably. Instead of asking whether an unknown animal exists at all, investigators could focus on determining which branch of life the mystery organism belongs to.[PubMed Central]pmc.ncbi.nlm.nih.govPub Med Central Fine-Grained Assignment of Unknown Marine e DNAby S Villon · 2026 — In this study, we propose a deep learning-based approach designed to improve taxonomic assignment at higher level…

This is one of the reasons eDNA remains valuable for lake-monster research despite database limitations. The method can potentially reveal:

  • Whether detected DNA resembles fish, amphibians, reptiles, mammals, or another major group.
  • Whether unusual sequences recur across locations and sampling periods.
  • Whether unexplained signals cluster around a known evolutionary lineage.

Euphresco Practibar Workshop: Barcode of Life Data Systems (BOLD)

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The Loch Ness Example and Its Limits

DNA Metabarcoding of eDNA/eRNA (Bioinformatics S14E1)

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The Real Challenge for Future Lake-Monster Searches

The greatest obstacle for eDNA-based cryptozoology may not be collecting DNA from water but interpreting unusual sequences once they are found. Modern sequencing technology can recover enormous amounts of genetic information, yet the value of that information remains tied to the reference libraries available for comparison. Researchers continue to emphasise that database completeness, sequence quality, regional coverage, and taxonomic accuracy are among the most important factors determining what eDNA can reveal.[Wiley Online Library+2Springer]onlinelibrary.wiley.comWiley Online LibraryPersistent Gaps and Errors in Reference Databases…by A De Santiago · 2025 · Cited by 12 — High-throughput environm…

As reference databases grow, the space for genuinely unexplained DNA signals becomes smaller. At the same time, any persistent sequence that cannot be linked to known organisms becomes more scientifically interesting. For future lake-monster investigations, the most persuasive eDNA anomaly would not be a dramatic species name appearing from nowhere, but a reproducible genetic signal that repeatedly falls outside the expectations of an increasingly complete catalogue of life.[Nature+2arXiv]nature.comA taxonomically reliable DNA barcode reference library for…by M Christodoulou · 2025 · Cited by 4 — DNA-based approaches, such a…DNA Databases illustration 3

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Endnotes

1. Source: mdpi.com
Link:https://www.mdpi.com/2073-4441/13/13/1767

<summary>Source snippet</summary><p>Are Genetic Reference Libraries Sufficient for…by CL Jerde · 2021 · Cited by 30 — However, eDNA metabarcoding's main weakness is t…</p>

2. Source: naturemetrics.com
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<summary>Source snippet</summary><p>Report Interpretation GuideA species-level assignment will be given if reference databases contain an exact DNA match. A higher taxonomic…</p>

3. Source: arxiv.org
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Published: April 8, 2024

4. Source: onlinelibrary.wiley.com
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<summary>Source snippet</summary><p>EurekAlert!Incheon National University study evaluates eDNA…6 days ago — The researchers also found that studies using locally curated…</p>

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<summary>Source snippet</summary><p>Wiley Online LibraryIntegrated Reanalysis of Global Riverine Fish eDNA…by Y Zhang · 2026 · Cited by 4 — Restricting taxonomic assignme…</p>

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12. Source: nature.com
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<summary>Source snippet</summary><p>A taxonomically reliable DNA barcode reference library for…by M Christodoulou · 2025 · Cited by 4 — DNA-based approaches, such a…</p>

13. Source: emea.illumina.com
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<summary>Source snippet</summary><p>But it did accurately identify dozens of other species: frogs, toads, ducks, all 13…Read more…</p>

14. Source: link.springer.com
Title: Is the DNA barcode database fit for purpose?
Link:https://link.springer.com/article/10.1007/s11756-025-01999-0

<summary>Source snippet</summary><p>Assessing the…by M Šamulková · 2025 · Cited by 4 — In this study, we assessed the taxonomic coverage and identification accuracy of th…</p>

15. Source: onlinelibrary.wiley.com
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<summary>Source snippet</summary><p>Wiley Online LibraryGenetic Reference Gaps Limit eDNA Metabarcoding and…by EKT Cooper · 2026 — This review reveals major genetic data…</p>

16. Source: onlinelibrary.wiley.com
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<summary>Source snippet</summary><p>PCR bias favours particular template DNA strands from the mixed DNA template. PCR bias can stem from (a) PCR stochasticity – random…Re…</p>

17. Source: biorxiv.org
Title: However, we also
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<summary>Source snippet</summary><p>Maximizing the reliability and the number of species…18 Nov 2022 — With the metabarcoding dataset, the reliability of species assignme…</p>

18. Source: biorxiv.org
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19. Source: pmc.ncbi.nlm.nih.gov
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<summary>Source snippet</summary><p>by S Villon · 2026 — In this study, we propose a deep learning-based approach designed to improve taxonomic assignment at higher level…</p>

20. Source: pubmed.ncbi.nlm.nih.gov
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<summary>Source snippet</summary><p>These are: (i) mislabelling, (ii) sequencing…</p>

21. Source: archimer.ifremer.fr
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<summary>Source snippet</summary><p>A Confidence Scoring Procedure for eDNA Metabarcoding…by R Rozanski · 2025 · Cited by 5 — Here, we present a modular procedure…</p>

22. Source: pmc.ncbi.nlm.nih.gov
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23. Source: pmc.ncbi.nlm.nih.gov
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<summary>Source snippet</summary><p>of DNA barcoding reference databases for marine…by Y Zhou · 2025 · Cited by 12 — By addressing the critical gaps in barcode coverage…</p>

24. Source: pmc.ncbi.nlm.nih.gov
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<summary>Source snippet</summary><p>positive: customized reference databases and new, local…by F Mugnai · 2023 · Cited by 68 — Sequences in reference databases belonging…</p>

25. Source: pmc.ncbi.nlm.nih.gov
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<summary>Source snippet</summary><p>by E Ruiz · 2025 · Cited by 3 — To address these issues, we developed a robust framework to compare the performance of 22 metabarcodes…</p>

Additional References

26. Source: researchgate.net
Link:https://www.researchgate.net/publication/363063003_DNA_Barcode_Gap_Analysis_for_Multiple_Marker_Genes_for_Phytoplankton_Species_Biodiversity_in_Mediterranean_Aquatic_Ecosystems

<summary>Source snippet</summary><p>(PDF) DNA Barcode Gap Analysis for Multiple Marker…Aug 25, 2022 — The main objective of this study was to analyze the current coverage…</p>

27. Source: researchgate.net
Link:https://www.researchgate.net/publication/403137490Guidance_and_best_practices_for_species_identification_using_eDNA_metabarcoding-_When_do_you_call_a_cod_a_cod

<summary>Source snippet</summary><p>Guidance and best practices for species identification…5 Apr 2026 — Here we provide a review and perspective of pitfalls and issues th…</p>

28. Source: researchgate.net
Link:https://www.researchgate.net/publication/393622098_A_taxonomically_reliable_DNA_barcode_reference_library_for_North_Sea_macrobenthos

<summary>Source snippet</summary><p>(PDF) A taxonomically reliable DNA barcode reference…High-throughput sequencing, particularly DNA metabarcoding, offers an alternative…</p>

29. Source: thebiodiversityconsultancy.com
Link:https://www.thebiodiversityconsultancy.com/fileadmin/uploads/tbc/Documents/Resources/eDNA-and-biodiversity-risk-briefing-note.pdf

<summary>Source snippet</summary><p>Figure 1: Simplified eDNA process www.thebiodiversityconsultancy.com. Reporting…Read more…</p>

30. Source: facebook.com
Title: Using e DNA to monitor biodiversity in Loch Rannoche DNA
Link:https://www.facebook.com/groups/rannoch/posts/3688015771334765/

<summary>Source snippet</summary><p>Using eDNA to monitor biodiversity in Loch RannocheDNA - What you were afraid to ask. eDNA studies make use of DNA shed from organisms in…</p>

31. Source: researchgate.net
Title: Gap-analysis for high impact species
Link:https://www.researchgate.net/figure/Gap-analysis-for-high-impact-species-Barcode-coverage-of-each-searched-marker-in_fig3_343613365

<summary>Source snippet</summary><p>Barcode coverage…In this study, we evaluated the completeness of DNA barcode reference data for annelids from the Red Sea, Arabian Gul…</p>

32. Source: youtube.com
Title: How to Use the NCBI’s Bioinformatics Tools and Databases
Link:https://www.youtube.com/watch?v=QLcmEqBayr0

<summary>Source snippet</summary><p>EDNA reference database taxonomic assignment gaps DNA Metabarcoding of eDNA/eRNA (Bioinformatics S14E1) Danny Arends…</p>

33. Source: youtube.com
Link:https://www.youtube.com/watch?v=neIQ3KNBXN8

<summary>Source snippet</summary><p>'DNA barcoding, from theory to practice' organized in the…</p>

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Title: DNA Metabarcoding of e DNA/e RNA (Bioinformatics S14E1)
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<summary>Source snippet</summary><p>EcoLens: AI-Powered eDNA Analysis for Scalable Biodiversity Monitoring and Novel Taxa Discovery…</p>

35. Source: otago.ac.nz
Link:https://www.otago.ac.nz/news/newsroom/first-edna-study-of-loch-ness-points-to-something-fishy

<summary>Source snippet</summary><p>University of OtagoFirst eDNA Study Of Loch Ness Points To Something Fishy5 Sept 2019 — “We found substantial levels of DNA from humans a…</p>

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