Diatoms are single-celled algae encased in intricate silica shells, and they are among the most important players in freshwater ecosystems. They respond rapidly and predictably to changes in their environment, making them essential indicators for assessing ecological status under the European Union’s Water Framework Directive. Yet traditional identification of these organisms requires extensive training, is time-consuming, and often produces inconsistent results between laboratories. DNA metabarcoding offers a way forward, but it depends on complete reference libraries that link genetic sequences to species names.

The problem, until now, has been stark. Only approximately 15 percent of all European diatom species are represented in DNA reference databases. Abundant species are covered more frequently, but rare and geographically restricted species are missing entirely. Most of the curated databases were developed in Central Europe, meaning that many species observed in Sweden—especially those thriving in oligotrophic and acidic habitats—are absent. This gap can lead to misclassification of ecological status when metabarcoding data are used to calculate diatom indices for environmental assessment.

To address this, the national barcoding project FRESHBAR was conducted between 2019 and 2023. Researchers established 312 diatom cultures, focusing on oligotrophic and acid habitats that are poorly represented in existing databases. Most isolates came from fresh samples taken in coordination with the Swedish national monitoring program, with additional material from enrichment cultures and earlier pilot projects. The team used the micropipette method to establish 273 monoclonal cultures from single cells, growing them in specialized media at room temperature.

The cultures were sequenced for two genetic markers: rbcL and 18SV4. The rbcL marker has been highlighted as the most promising barcode for diatom metabarcoding because it better separates species and poses fewer practical problems than 18SV4. Sequencing was successful for almost all clonal cultures, with 301 strains producing rbcL sequences and 299 producing 18SV4 sequences. For 294 strains, both markers were generated simultaneously.

The morphological identification work, carried out using light microscopy and scanning electron microscopy, revealed 51 different taxa across 17 genera. The genera Eunotia and Fragilaria showed the highest diversity, with Achnanthidium containing the most sequences overall. Approximately half of the taxa were new to Diat.barcode, the curated diatom reference database. The 301 rbcL sequences enlarged the number of reference sequences available from Nordic countries by 200 percent.

Among the highlights was the conspicuous species Eunotia myrmica, which had not previously been represented in any database. The relatively high proportion of Eunotia taxa, along with Tabellaria and Brachysira sequences, is significant because these genera prefer oligotrophic and acidic conditions and occur frequently in Swedish freshwaters but are poorly represented in diatom databases. The researchers also documented colony formation, auxospore development, and ultrastructural details through electron microscopy, providing morphological characteristics that are comparatively rare in diatom barcoding projects.

All data, including sampling metadata, barcode sequences, images, and voucher material, were published following FAIR principles. Physical vouchers are stored in the Herbarium Berolinense in Berlin, with each strain linked to a unique stable identifier. Sequences were submitted to GenBank, and the rbcL sequences will be incorporated into the next version of Diat.barcode. Additionally, 36 clonal cultures were deposited at the BCCM/DCG Culture Collection in Ghent, Belgium, and 32 at the UTEX Culture Collection of Algae in the United States, ensuring long-term preservation of living material.

The project represents a critical step toward operationalizing metabarcoding for environmental assessment under the Water Framework Directive. With more sequences available for Nordic taxa, scientists can now use metabarcoding with greater certainty, and the increased coverage will help identify taxa properly to the species level, unravel taxon relationships within diatoms, and improve reference databases for monitoring and research across understudied regions.

The significance of the FRESHBAR approach lies partly in its reliance on clonal cultures rather than environmental samples. When DNA sequences are generated directly from mixed biofilm communities, linking a sequence to a particular species can be ambiguous, especially when closely related taxa coexist in the same sample. By isolating single cells and growing them into unialgal cultures, the researchers ensured that every barcode sequence could be unambiguously assigned to a morphologically identified organism. This voucher-linked strategy is considered the gold standard for building reference libraries, because it anchors genetic data to physical specimens that can be re-examined as taxonomic understanding evolves.

The dual-marker strategy adopted by the project reflects a deliberate compromise within the diatom metabarcoding community. The rbcL plastid marker offers finer species-level resolution, which matters greatly in a group where cryptic and pseudocryptic diversity has repeatedly been uncovered within what light microscopy once treated as single morphospecies. The 18SV4 ribosomal marker, by contrast, resolves fewer closely related diatom species but has the advantage of being amplifiable across a broad range of eukaryotic lineages. This makes it possible to run parallel metabarcoding of diatoms alongside other organism groups in the same assay, a prospect that appeals to monitoring agencies seeking more holistic assessments of ecosystem condition. By sequencing both markers for nearly every strain, FRESHBAR produced a dataset that can serve either purpose.

The ecological focus on oligotrophic and acidic waters deserves emphasis. Nutrient-poor, low-pH habitats dominate much of the Nordic landscape, shaped by bedrock geology, thin soils, and historical acidification from atmospheric deposition. The diatom flora of these waters—rich in genera such as Eunotia, Tabellararia, and Brachysira—differs markedly from that of the calcareous and eutrophic waters of Central Europe, where most existing reference data were compiled. When such Nordic taxa are absent from a reference library, metabarcoding pipelines may either fail to assign their sequences or misassign them to the nearest available relative, skewing the diatom indices used to grade ecological status. The underrepresentation documented in this study therefore has direct consequences for how Swedish and other boreal waters would be scored under DNA-based monitoring.

The morphological dimension of the project adds value that purely sequence-based efforts often lack. Colony formation, auxospore development, and fine ultrastructural details captured by scanning electron microscopy document features that are difficult or impossible to observe in degraded environmental material. Auxospore formation, in particular, is tied to the cell size cycle of diatoms, in which cells shrink with each division and are restored to full size through sexual reproduction; documenting this process in culture helps clarify species boundaries and life-history traits. Such observations, paired with sequences from the same strain, provide the integrative evidence that modern diatom taxonomy increasingly demands, especially as species complexes in genera like Fragilaria and Eunotia are dissected into separate taxa.

The commitment to FAIR data principles—making findings findable, accessible, interoperable, and reusable—addresses a persistent weakness in barcoding literature, where sequences are deposited but the associated material and metadata are scattered or lost. By linking each strain to a herbarium voucher in Berlin, living cultures in Ghent and Texas, photographs, sampling metadata, and sequences in public repositories, the project created a traceable chain from a specific Swedish waterbody to a specific genetic sequence. This traceability allows future researchers to re-verify identifications, extract additional markers, or apply new analytical methods to the same material years or decades later.

Looking ahead, the study illustrates a model that other geographically underrepresented regions could follow. Roughly half of the 51 taxa documented were new to Diat.barcode, suggesting that even a nationally scoped effort can substantially close regional gaps in a short time. The remaining challenges for operational metabarcoding under the Water Framework Directive—standardized laboratory protocols, harmonized bioinformatics pipelines, and continued database curation—remain substantial, but reference library completeness is the prerequisite on which everything else depends. As national monitoring programs across Europe transition toward DNA-based methods, projects like FRESHBAR demonstrate that targeted culturing campaigns in poorly covered habitats and regions can deliver the foundational data needed to make molecular assessment of freshwater quality reliable, reproducible, and ecologically meaningful.

Subject of Research: New DNA barcode reference data of freshwater diatoms ( Bacillariophyceae ) from Sweden: old acquaintances and new taxa

Article Title: New DNA barcode reference data of freshwater diatoms ( Bacillariophyceae ) from Sweden: old acquaintances and new taxa

Article References: Kahlert, M., Mora, D., Kusber, W.-H., Abarca, N., & Zimmermann, J. (2026). New DNA barcode reference data of freshwater diatoms (Bacillariophyceae) from Sweden: old acquaintances and new taxa. Metabarcoding and Metagenomics, 10, Article e186778. https://doi.org/10.3897/mbmg.10.186778

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.186778

Keywords: barcode, reference, data, freshwater, diatoms, Bacillariophyceae, Sweden, acquaintances, taxa, scientific research