The study examined how manganese content affected the corrosion behavior of three Cu–Mn cladding layers fabricated on Q235 steel. The layers contained 10 wt%, 15 wt%, or 20 wt% Mn and were immersed for 180 days in seawater containing Chlorella or diatoms. All three compositions showed low microalgal coverage, but the 20 wt% Mn layer produced the smallest values in both test solutions. The researchers attributed the improved inhibition to the structure of the corrosion-product layer rather than to a difference in the duration of testing: increasing manganese content promoted internal-stress accumulation, which accelerated cracking and flaking of corrosion products.

This mechanism matters because copper alloys are widely used in ships and marine engineering equipment for their antifouling performance. Over long service periods, however, the corrosion-product layer can become denser while microalgae attach to the surface, progressively degrading the material’s ability to resist fouling. In the tested Cu–Mn coatings, the corrosion products instead formed a loose, heavily cracked layer that could readily flake away. As manganese content increased, that layer became more susceptible to cracking and detachment, supporting continued copper-ion release into the surrounding water.

The released copper ions were linked to elevated intracellular reactive oxygen species, or ROS, in attached microalgae. The study’s proposed mechanism is that manganese-induced changes in the corrosion layer enhance copper-ion release, increasing oxidative stress within the organisms. The authors reported that the higher ROS level was associated with extensive apoptosis and effective suppression of attachment. The results therefore connect an observable coating behavior—cracking and flaking of corrosion products—with the biological endpoint of reduced microalgal colonization, although the supplied abstract does not provide the numerical copper-release or ROS measurements needed to quantify those intermediate steps.

The experiment provides a laboratory comparison of manganese content under a defined 180-day immersion period, rather than a direct field trial of coated marine structures. Its findings nevertheless offer a technically focused route for improving durable antifouling metal surfaces: adding more manganese to the Cu–Mn cladding changed the evolving corrosion layer in a way associated with stronger inhibition of two types of microalgae. The 20 wt% Mn composition achieved the best reported outcome, with coverage remaining at only 5.81% for Chlorella and 2.52% for diatoms, but the evidence does not establish how the coatings would perform across other organisms, exposure conditions or real-world service environments.

Subject of Research: Effects of manganese content on corrosion and microalgal attachment in Cu–Mn laser-cladded Q235 steel.

Article Title: Cu–Mn laser cladding layers on Q235 steel: the influence of Mn content on corrosion behavior and long-term microalgal inhibitory performance

Article References: Li, J., Liu, Z., Liu, J., Zeng, G., Wang, X., Yang, H., Ning, H., & Li, M. (2026). Cu–Mn laser cladding layers on Q235 steel: the influence of Mn content on corrosion behavior and long-term microalgal inhibitory performance. Journal of Materials Science, 61(41), 32076-32106. https://doi.org/10.1007/s10853-026-13665-0

Image Credits: AI Generated

DOI: 10.1007/s10853-026-13665-0

Keywords: Cu–Mn coatings, laser cladding, manganese, Q235 steel, marine antifouling, corrosion products, microalgae, Chlorella, diatoms, reactive oxygen species, laser, cladding

Diatoms, single-celled algae encased in ornate silica shells called frustules, are among the most trusted bioindicators in aquatic science. Under the European Water Framework Directive, benthic diatoms are one of five biological elements used to assess the ecological status of surface waters, with diatom indices built on the relative abundance of species and their known sensitivities to pollution and salinity. That system works only if species are identified accurately and consistently, which is precisely where the trouble begins. Traditional identification relies on subtle features of valve shape and stria density, demanding deep taxonomic expertise and often yielding subjective results.

DNA metabarcoding has been proposed as a more objective alternative. The technique amplifies short barcode regions of DNA from an environmental sample, sequences them in bulk on next-generation platforms, and matches the resulting sequences against reference databases to identify the taxa present. Yet studies comparing the two approaches have repeatedly found significant deviations, and the research team behind the new work points to one dominant cause: reference libraries are incomplete, particularly for species from unusual habitats. Soda pans, with their high sodium and hydrogen carbonate content, host rare and halophilic organisms that are exactly the kinds of species missing from genetic databases.

The loss of sodic character is the principal threat facing these habitats, as water management practices can channel lower-salinity water into the pans and dilute them. Accurate identification of species and precise knowledge of their salinity tolerance are therefore essential for judging ecological status. Within the framework of a Hungarian national monitoring project, the researchers sampled 37 soda pans and soda lakes across spring and autumn campaigns in 2019 and 2020, producing 69 samples analysed by light microscopy, of which 26 were also subjected to DNA sequencing. Twenty-two of the water bodies were subsaline and 15 hyposaline, ranging from natural to disturbed conditions.

The team focused on Halamphora, the fourth most common and seventh most species-rich genus among the 72 genera found in their soda samples. Two species in particular, Halamphora dominici and Halamphora veneta, occurred in nearly half of all samples and were frequently dominant, sometimes reaching relative abundances of 40 to 60 percent. The Halamphora veneta group, which also includes H. kevei and H. paraveneta, is notoriously difficult to resolve morphologically, with species boundaries resting on fine differences in stria density and valve outline that can shift with environmental stress, including salinity.

The molecular results delivered a striking revelation. Of 23 short amplicon sequence variants that automated pipelines assigned to H. veneta, phylogenetic analysis and BLAST searches showed they actually belonged to H. dominici. The culprit was a misidentified sequence in the Diat.barcode reference database. Using long-read sequencing of a sample where both species co-occurred, the researchers obtained a full 1547-nucleotide rbcL sequence for H. dominici that showed roughly 2 percent divergence from H. veneta, confirming the two are genuinely distinct species. The newly deposited sequences now supplement the reference library, and the team emphasizes that sequences should only enter databases after precise morpho-taxonomic verification.

Overall, six Halamphora species were detected in the soda habitats. Five were recovered by both methods, while H. oligotraphenta was found only by microscopy because it appeared exclusively in samples that were not sequenced. The comparison between techniques revealed a significant positive correlation between microscopy- and metabarcoding-based relative abundances for H. dominici, with a Pearson correlation coefficient of 0.73. Discrepancies in the other direction were also informative: DNA detected species in some samples where microscopy missed them, likely because sequencing draws on far more cells than the minimum 500 valves counted per slide, while some microscopically observed valves may have belonged to dead cells.

The ecological payoff is clear. Classified as mesohalobous, H. dominici showed a positive correlation with salinity, with an average salinity of 3.1 parts per thousand in the waters where it occurred, compared with 1.6 parts per thousand for the more cosmopolitan H. veneta, which tolerates freshwater to slightly brackish conditions. Given that the ecological status of Hungarian soda pans depends fundamentally on salinity, the correct discrimination between these two species, one a reliable flag of sodic character and the other a generalist, is critical for conservation-relevant assessments.

The study also contributed taxonomic housekeeping. By comparing length-to-width ratios and ultrastructural features, the researchers questioned whether Halamphora elongata is truly distinct from the older H. subcapitata, noting overlapping morphometrics and absent areolar occlusions in their specimens, though formal synonymisation awaits type-material analysis. Meanwhile, H. paraveneta, which lacks any reference sequence in Diat.barcode or GenBank, may have been lurking as an outlier sequence variant assigned to H. veneta, a hypothesis flagged for future work.

As environmental DNA methods face growing pressure to replace traditional microscopy in routine monitoring across Europe, this study offers both a caution and a roadmap. Misidentified dominant species in reference databases can silently distort the results of ecological status assessments, potentially misclassifying protected habitats. But the combination of meticulous morphology and DNA sequencing, applied to extreme habitats that represent environmental endpoints of genetic diversification, can close the gaps. For Hungary’s soda pans, some of Europe’s most distinctive and threatened aquatic ecosystems, that means the microscopic sentinels of their salty character can finally be named with confidence.

Subject of Research: Diatom biodiversity and salinity indicators in Hungarian soda pans assessed by microscopy and rbcL metabarcoding

Article Title:  Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses

Article References: Ács, É., Bíró, T., Boros, E., Dobosy, P., Duleba, M., Földi, A., Kiss, K. T., Levkov, Z., Orgoványi, P., Szén, O. P., Trábert, Z., Vadkerti, E., & Grigorszky, I. (2023). Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses. Metabarcoding and Metagenomics, 7, Article e111679. https://doi.org/10.3897/mbmg.7.111679

Image Credits: AI Generated

DOI: 10.3897/mbmg.7.111679

Keywords: diatoms, metabarcoding, soda pans, Halamphora, salinity, rbcL, ecological status, Water Framework Directive, reference database, Hungary, bioindicators, phylogeny

The core idea is elegant. In an ideal OTU, every sequence would sit at a dissimilarity smaller than the barcoding gap, the distance threshold that separates sequences of one taxon from those of another, with every read linked to every other read. Real OTUs deviate from this theoretical pattern, and the authors argue that the deviations are not random noise but carry structure of their own. They identify two characteristic ways in which an OTU can go wrong. The first is the composed OTU, an artificial merger of several true units, typically produced by the well-known chaining effect of single linkage clustering, in which sequences A and B are close, B and C are close, but A and C are far beyond the barcoding gap. The second is the OTU with noise, in which a handful of satellite sequences hang loosely off a densely connected core without forming a compact subgroup of their own.

To detect composed OTUs, the method relies on a single statistic computed directly from the array of pairwise dissimilarities within each OTU. The researchers build a graph in which each read is a node and an edge connects two reads whenever their dissimilarity falls below the barcoding gap. They then compute theta, the ratio of missing edges to all possible edges in that graph. For a single OTU, nearly all dissimilarities lie below the gap, so theta is small. For a composed OTU containing two balanced entities, the inter-entity dissimilarities are mostly above the gap, and theta approaches one half. Sorting the OTUs of a sample then becomes a matter of building a smoothed histogram of theta values with a Gaussian kernel and placing the critical threshold at the first local minimum between the first and second modes of the estimated density. Crucially, unlike the breaking phase of the SWARM algorithm, this procedure requires no arbitrarily fixed parameter.

Once composed OTUs have been separated from single ones, a second, supervised step hunts for noise among the single OTUs. The authors fit a two-block Stochastic Block Model to each dissimilarity array, a statistical framework that clusters sequences according to their shared patterns of connectivity and has the unusual plasticity of allowing blocks to be either assortative or dissortative. If an OTU is clean, both blocks are assortative and the diagonal elements of the inferred connectivity matrix are small. If noise reads are present, they are grouped into a dissortative block with large intra-block and inter-block dissimilarities. The two discriminating features, the mean inter-block dissimilarity and the largest mean intra-block dissimilarity, feed a linear Support Vector Machine trained on OTUs labelled by a degree-based expert classifier, which examines the percentage of gap-level connections each read maintains with the rest of the OTU.

Validation came from a rich dataset of diatoms collected in Arcachon Bay on the French Atlantic coast, made available through the Malabar project. Thirty-two samples were balanced across four seasons, four locations and two water columns, pelagic at high tide and benthic, yielding between 19,000 and 36,000 reads per sample of a 312 base pair region of the rbcL chloroplastic marker. Pairwise dissimilarities were computed with Smith-Waterman local alignment scores on a computing cluster, and OTUs were extracted as connected components of the gap-level graph, keeping only units with fifteen reads or more. For the eight samples of the Teychan location, an expert classification was built by visual inspection of heatmaps and community-structured graphs of 654 dissimilarity arrays, and the agreement with the automatic method was striking: only twelve false negatives among 104 expertly composed OTUs and nine false positives among 527 single ones.

Applied to the full dataset of 2,529 OTUs, the method revealed strong statistical links between OTU type and OTU size. A Wilcoxon Mann-Whitney test returned a p-value of roughly 1.5 times ten to the power of minus fifty-five for the composed-versus-single distinction, confirming that composed OTUs are systematically the largest ones. Among single OTUs, the test for noise returned a p-value of about 3.7 times ten to the power of minus twenty-two, showing that noisy units are also significantly larger than clean ones. Cross-validation of the Support Vector Machine on the training set achieved an Area Under Curve of 0.951, and on the 24 test samples the classifier caught every OTU with noise, although it recovered only about 64 percent of the OTUs without noise, a limitation the authors attribute to the gradient of noise intensity that the two SBM features trace simultaneously.

Taxonomic annotation provided an independent check. Among 180 fully annotated OTUs, about 85 percent were monospecific, and almost all single OTUs with noise, 153 of them, were monospecific as well, suggesting that satellite reads often belong to the same species as the core rather than to a different taxon. Only eight fully annotated OTUs were typed as composed, and they fell into three profiles: genuine multi-species mergers with clear two-block structure, monospecific units with loosely connected reads, and monospecific units whose dissimilarity arrays nevertheless split into two blocks, possibly reflecting structured intraspecific molecular diversity. One artefact also surfaced: a single reference sequence of the diatom Rhizosolenia fallax appeared repeatedly across OTUs of several genera and was set aside as a mapping anomaly.

Perhaps the most provocative result concerns the environment itself. The proportions of composed, noisy and clean OTUs varied from sample to sample, and a Wilcoxon rank test on the fractions of composed OTUs in benthic versus pelagic samples returned a p-value of 1.6 times ten to the power of minus four. Benthic samples carried a mean fraction of 0.19 composed OTUs against 0.10 in pelagic ones, strong evidence that the molecular diversity of the benthic diatom flora is structured differently, with species distances globally smaller than in the pelagic realm. The authors hypothesise that habitat may influence the alternation of sexual and asexual reproduction and thereby shape patterns of DNA diversity, though the chloroplastic rbcL marker makes any such influence indirect. No difference emerged for the fraction of noisy OTUs among single units.

The practical guidance that follows is a gradient of trust. Cliques, in which every read connects to every other, are the rarest and most reliable units, though only four were found across all 32 samples. Single OTUs without noise, whose SBM parameters stay below eight, below the barcoding gap of nine, can be used as such for ecological studies. Composed OTUs, which are very likely spurious mergers reflecting the inadequacy of a single barcoding gap for the largest clusters, should be post-treated with classical clustering or community detection tools to be reshaped into smaller, more reliable units. Single OTUs with noise, whose quality remains to be tested across a wider diversity of organisms, sit in between and deserve further scrutiny.

In an era when sequencing pipelines stream millions of reads faster than any expert can inspect them one by one, the appeal of this approach lies in its automation and its transparency. Every step, from the theta statistic to the SBM features and the SVM frontier, is elementary enough to be implemented in Python or R, and the associated code is openly available on the forgemia platform of INRAE. Used as a quality filter within a pipeline, the method promises to strengthen the foundations of molecular ecology, letting researchers know which OTUs can carry the weight of a biodiversity inventory and which ones need reshaping before any ecological conclusion is drawn from them.

Subject of Research: An automatic method for evaluating the quality of Operational Taxonomic Units in metabarcoding data based on dissimilarity arrays

Article Title: Simple approaches for evaluation of OTU quality based on dissimilarity arrays

Article References: Cros, M.-J., Frigerio, J.-M., Peyrard, N., & Franc, A. (2024). Simple approaches for evaluation of OTU quality based on dissimilarity arrays. Metabarcoding and Metagenomics, 8, Article e108649. https://doi.org/10.3897/mbmg.8.108649

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.108649

Keywords: metabarcoding, OTU quality, dissimilarity arrays, diatoms, Arcachon Bay, barcoding gap, Stochastic Block Model, Support Vector Machine, chaining effect, biodiversity, rbcL marker, molecular ecology

The research, led by David Jenkins and colleagues, tackles the question through beta diversity, the measure of how dissimilar two communities are in their species composition. The logic is elegant: if the clearest possible separation between adjacent communities occurs at a particular spatial resolution, then that resolution represents the empirical size of a community. Maximal beta diversity, in other words, marks the scale at which nature draws its sharpest boundaries between assemblages of species.

To test this, the team assembled point data for ants, birds, diatoms, and trees, four taxa chosen for their radically different life histories, dispersal abilities, and data origins. Ant records spanned two centuries of literature synthesis, totaling nearly 25,000 samples and 699 species. Bird data came from the eBird community science platform, with over 810,000 cleaned reports from January and July between 2010 and 2015, standardized as counts per person-hour. Diatoms were sampled from the top five centimeters of lake sediment across 1,070 lakes in a single year, yielding 1,957 species. Tree data, covering stems wider than five centimeters at breast height, came from the US Forest Service’s Forest Inventory Analysis across four years spaced five years apart, totaling nearly 40,000 samples and 403 species.

The analytical framework organized all georeferenced observations into hexagonal polygons, with cell size defining the spatial grain. At each grain, ranging from one square kilometer to one million square kilometers, the researchers calculated mean pairwise beta diversity between each central hexagon and its immediate neighbors. This neighborhood-based approach, borrowed conceptually from remote sensing research on optimized spatial resolution, controls for sample size across grains because hexagonal grids are constrained to fewer than six neighbors.

A critical methodological decision involved how beta diversity was calculated. The team used a relatively new deviation from a null model based on spatial heterogeneity, called beta NBD for its underlying negative binomial distribution. This measure, developed by Youhua Chen and Fangliang He, represents clustered species patterns more accurately than the older deviation based on spatial homogeneity. The newer measure made only modest adjustments to observed beta diversity and showed no vulnerability to sample size effects, whereas the older measure substantially altered observed values and increased with greater sampling effort.

The results were strikingly consistent. For all four taxa, beta diversity peaked at intermediate, regional spatial grains. Trees showed peak values at roughly 300 square kilometers, remarkably stable across four evaluated years spanning two decades. Winter birds peaked at approximately 500 square kilometers, while summer birds and ants both peaked near 2,000 square kilometers. Diatoms, the microscopic algae inhabiting lake bottoms, showed the largest peak at about 11,000 square kilometers. Hump-shaped quadratic curves fit the data far better than linear models, with Akaike Information Criterion weights exceeding 0.95 in all cases.

These rankings defied the team’s initial expectations. They had predicted that diatoms, confined to discrete lake habitats, would show the smallest community scales, followed by ants, trees, and birds. Instead, diatoms exhibited the largest peak, and trees the smallest. The researchers suggest that diatom community size roughly matched the average density of sampled lakes across the contiguous US, meaning the apparent regional scale may actually reflect the average dispersion of discrete diatom habitats rather than long-range dispersal. For terrestrial taxa inhabiting intergrading habitats, the regional peaks may reflect genuinely regional processes.

The repeatability of the patterns adds considerable weight to the findings. Bird beta diversity scaling was consistent across six consecutive years, though sensitive to seasonality, with winter communities concentrating at smaller spatial scales than summer ones. Tree patterns showed very little within- or among-annual variation at their peak grain. Given that birds and trees differ so enormously in life history and mobility, this commonality suggests that community assembly processes provide a strong and general basis for the observed regional scaling.

The results broadly support the regional community concept advanced by Robert Ricklefs, who argued in 2008 that regional scales should be preferred to local ones to capture the full suite of selective conditions driving diversity. The relatively low beta diversity at fine spatial grains in this study supports Ricklefs’ argument that local communities are one end of spatiotemporal continua rather than empirically distinct entities. The authors suggest that metacommunity ecology may be wise to treat local communities as sample points rather than bounded units, steering the field toward a species-centric approach.

Beyond its conceptual implications, the work bears fundamental consequences for conservation biogeography in the Anthropocene. If ecological communities are regional in scale, then local conservation efforts can best ensure success when coordinated regionally, through corridors and networks among conserved sites. The findings may also help researchers reconsider the appropriate spatial grains to use when studying diversity responses to land use and climate change, suggesting that rasterized study units matching peak beta diversity would obtain maximal resolution. The authors note that similar peaked spatial scaling may exist for other biogeographical patterns, potentially revealing maximal signal amidst noise and better representing natural patterns than maps designed for visual appeal.

Subject of Research: Peak beta diversity occurs at regional spatial grains

Article Title: Peak beta diversity occurs at regional spatial grains

Article References: Ohyama, L., Bogota-Gregory, J. D., & Jenkins, D. G. (2024). Peak beta diversity occurs at regional spatial grains. Frontiers of Biogeography, 17, Article e132675. https://doi.org/10.21425/fob.17.132675

Image Credits: AI Generated

DOI: 10.21425/fob.17.132675

Keywords: Peak, beta, diversity, occurs, regional, spatial, grains, scientific research

The stakes are higher than they might first appear. Across Europe, the ecological health of rivers and lakes is formally assessed under the Water Framework Directive using biological indicators, and in the United Kingdom one of the key tools is the Trophic Diatom Index, or TDI. Diatoms, the single-celled algae with ornate silica cell walls that dominate the phytobenthos of freshwaters, are scraped from biofilm-covered stones, identified either under the light microscope or, increasingly, through high-throughput DNA sequencing. The composition of the diatom assemblage reflects nutrient enrichment and other pressures, and the resulting Ecological Quality Ratio feeds directly into official classifications of whether a river is in good status or failing. But biofilms are far more than diatoms. They are slimy microbial cities, embedding bacteria, fungi, diverse algae, and protozoa within a protective extracellular matrix, and together these organisms drive primary productivity, decomposition, biogeochemical cycling, and pollutant degradation. Scientists have long argued that this broader microbial diversity could yield new diagnostic indicators of human impacts, and recent advances in machine learning and network analysis have made the search for such microbial metrics technically feasible.

The bottleneck has always been data. To identify reliable microbial features that can be upscaled into bioindicators of ecosystem function, researchers need thousands of samples spanning wide geographic areas and meaningful timescales. Generating such datasets from scratch is prohibitively expensive, requiring extensive field campaigns and laboratory analysis. This is precisely where the archive becomes revolutionary. Every year, the Environment Agency of England collects biofilm samples from rivers across the country as part of routine diatom monitoring, and each sample is preserved in an RNAlater-based nucleic acid preservative, a solution of 3.5 M ammonium sulphate, 17 mM sodium citrate, and 13 mM EDTA, before being frozen at -20 °C. A previous study by Baricevic and colleagues in 2022 had shown that such preserved samples remained stable for twelve months, with no significant impact on DNA quality, yield, or the composition of the diatom assemblages. But nobody knew whether the samples would survive intact over multiple years, and the answer to that question determined whether the archive could be treated as a genuine scientific resource or merely as regulatory paperwork.

To find out, the research team selected 64 frozen biofilm samples that had originally been collected and analysed in 2019 and 2020 as part of routine monitoring, chosen to cover a broad geographic spread across England and to have passed rigorous quality control with more than 50,000 reads in the original sequencing run. In December 2022, after two or three years in the freezer, the samples were thawed and reanalysed, and crucially the reanalysis was performed twice on separate sub-samples from each archived specimen. This duplication was the study’s methodological masterstroke, because it allowed the researchers to disentangle any genuine changes caused by storage from the inevitable stochastic noise introduced by sub-sampling, PCR amplification, and sequencing. In total, 192 samples were sequenced across the three sub-sample groups, designated OG for the original analysis and RA and RB for the post-storage replicates, generating 12.9 million quality-controlled reads and revealing 5,138 unique amplicon sequence variants.

The laboratory workflow followed the standard protocol established for routine monitoring. Biofilm-covered stones had been scrubbed with a clean toothbrush in deionised or tap water, the suspension mixed with preservative and couriered overnight to the laboratory, and the samples stored as concentrated pellets at -20 °C. For the reanalysis, samples were thawed, centrifuged at 3000 × g to reform a concentrated pellet, and DNA was extracted using a Qiagen blood tissue kit with an extended overnight lysis step at 56 °C. The target region was the rbcL chloroplast gene, amplified with primers rbcL-646F and rbcL-998R, which capture diatoms but also amplify other non-diatom phytobenthos. The resulting amplicon libraries were purified with SPRI beads, tagged with Illumina Nextera XT unique dual-indexed adapters, normalised, pooled, and sequenced on an Illumina MiSeq. Bioinformatic processing in QIIME2 employed cutadapt for primer removal, DADA2 for denoising and chimera removal, and a multinomial naïve Bayes classifier trained on the diat.barcode reference database for taxonomic assignment, with no filtering so that both diatom and non-target phytobenthic sequences were retained throughout the analysis.

The statistical verdict was unambiguous. At the amplicon sequence variant level, differences in diatom assemblages were overwhelmingly explained by differences between sample sites, accounting for 38.3 per cent of the variation, while storage conditions between the original and post-storage sub-samples explained less than 1 per cent and were not statistically significant. When the researchers specifically compared two versus three years of storage, the result was equally insignificant. At the genus and species levels, no taxa were detected at significantly higher abundance after storage, and the vast majority of genera, 84.0 per cent, were found in all replicate groups. The few genera and species that appeared unevenly between replicates were detected at very low levels, typically fewer than 1,000 reads across only one to three samples, and almost all belonged to genera that were more widely detected across the dataset. Only two diatom species, Surirella solea and Placoneis constans, were uniquely detected in post-storage replicates without their genus being more widely found, and even these appeared at negligible abundance. Curiously, the non-target species Quercus robur, the English oak tree, was detected in three post-storage replicates only, almost certainly the result of trace contamination or incidental amplification rather than any meaningful biological signal.

Perhaps most importantly for regulators, the ecological status metrics derived from the DNA were remarkably consistent. The mean difference in TDI values before and after storage was just 3.18, and Pearson’s correlation coefficients between the three replicate groups ranged from 0.892 to 0.980, all highly significant. When ecological status classes were assigned, 75.8 per cent of samples received the same classification across all replicates, and 79.0 per cent were either identical or differed by only one class. In pairwise comparisons, the original and first post-storage replicate agreed on the same class for 90.4 per cent of samples. Only four samples stood out as clear outliers, with differences greater than one class depending on which replicates were compared. In one of these, the post-storage replicate contained a much larger proportion of the diatom Amphora pediculus, at 47.1 per cent, than the original and first replicate, at 22.2 and 17.5 per cent respectively. But because no species were differentially abundant between replicate groups across the dataset as a whole, the researchers concluded that these outlier differences were not caused by fundamental changes from storage, but rather by the same sub-sampling and PCR variation that plagues any routine monitoring data.

This interpretation places the study in a broader scientific context. The magnitude of the insignificant differences observed between original and post-storage analyses was similar to that reported in previous work when comparing differences between different analysts and different sequencing instruments, suggesting that the noise inherent in the analytical pipeline itself is comparable to or greater than any effect of multi-year storage. Comparable findings have emerged from studies of other microbial sample types. Researchers examining bacterial communities in faecal samples stored for five years in preservative at -80 °C found them comparable to reference samples, and multiple shorter-term studies have reported similar stability. However, the literature is not entirely uniform, as one study found that RNAlater significantly affected bacterial communities compared to liquid nitrogen freezing at one of two sites tested, while finding no significant differences for fungal communities. The authors of the current study caution that extrapolating their results to the wider microbial community requires care, because research on the stability of bacterial communities by 16S metabarcoding is contradictory and difficult to compare directly, and little research has been conducted on the impact of storage conditions on fungi or protists within biofilms.

The practical implications extend well beyond diatoms. The study further evidences RNAlater-based preservation of freshwater biofilms as a viable alternative to ethanol, one of the standard recommended methods under European standardisation guidelines. For logistical, health, and safety reasons, the Environment Agency has restrictions on the use of ethanol, and therefore sought alternatives for the sampling and storage of biofilm samples. The confirmation that these preserved samples remain stable for up to three years means that regulators can now maximise the value of their historic raw biofilm samples, reusing them for purposes far beyond their original intent, including monitoring other non-microbial taxonomic groups and exploring the responses of broader microbial communities to environmental change. What was once a freezer full of completed regulatory assessments has been recast as a living archive, ready to yield the large, cost-effective datasets that will underpin the next generation of microbial metrics for freshwater biomonitoring, and with them, a far richer understanding of how river ecosystems respond to the mounting pressures of a changing world.

Subject of Research: Environmental DNA

Article Title: Influence of storage time on the stability of diatom assemblages using DNA from riverine biofilm samples

Article References: Warren, J., Butler, S., Evens, N., Hunt, L., Kelly, M., Newbold, L., Read, D. S., Taylor, J. D., & Walsh, K. (2024). Influence of storage time on the stability of diatom assemblages using DNA from riverine biofilm samples. Metabarcoding and Metagenomics, 8, Article e129227. https://doi.org/10.3897/mbmg.8.129227

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.129227

Keywords: environmental DNA, diatoms, biofilm, biomonitoring, metabarcoding, river health, Influence, storage, time, stability, diatom, assemblages

The Euphrates-Tigris catchment is one of the major drainage basins of the Middle East, but decades of overexploitation have pushed the water system to its ecological limits. Iraqi waterbodies suffer from salinization driven by water losses caused by upstream dam construction, water policies during the Iraq wars and the intensifying impact of climate change on a region that already experiences minimal rainfall. Against this backdrop, diatoms, a diverse group of microalgae with silicified cell walls, serve as the world’s most widely used bioindicators of water quality, reflecting ecosystem conditions with unique sensitivity. Epiphytic diatoms, which live attached to submerged macrophytes, are particularly interesting because they interact with their living hosts in ways that can shape their own community structure.

Accurate taxonomic identification of diatoms is essential for sound biodiversity evaluation, yet classical identification by light microscopy requires highly specialized taxonomic skills and can produce discrepancies between researchers. Cryptic diversity, species that look identical under the microscope but are genetically distinct, further hinders accurate identification. In regions where literature on the local diatom flora is scarce, researchers often rely on European floras, which may force identifications into existing taxonomic concepts and overlook local variation or unique species. DNA metabarcoding, which enables the concurrent identification of numerous species from environmental samples, has become an increasingly popular alternative, but until now there has been a notable absence of such studies in Iraq.

The research team collected 47 samples during the summer of 2019, the dry season, and the winter of 2020, the rainy season, at six sites across two water bodies: the River Tigris in the city of Al-Qurnah and the River Euphrates in the city of Al-Midaina. Because no stones were available as habitats at the sampling stations, samples were taken separately from 11 different taxa of macrophytes and macroalgae, including common reed, cattail, hornwort, watermilfoil, several pondweed species, green algae of the genus Cladophora, naiad and sea aster. At least five stems and branches of each plant were shaken vigorously with distilled water to dislodge the attached diatoms, and the resulting suspensions were preserved with 70 percent alcohol for transport to the Botanic Garden and Botanical Museum at Freie Universität Berlin, where each sample was split for morphological and molecular analysis.

For the morphological analysis, samples were treated with 35 percent hydrogen peroxide to oxidize organic materials, and the cleaned diatom frustules were mounted in the high refractive index medium Naphrax. Using a Zeiss Axioplan microscope with differential interference contrast and a 100x Plan Apochromat objective, the researchers counted a minimum of 400 valves per sample to document the presence and relative abundance of each taxon, and turned to a Hitachi FE 8010 scanning electron microscope when finer details were needed. This meticulous dual-microscope approach identified 284 infrageneric taxa belonging to 59 genera, a species richness notably higher than in previous Iraqi studies. Remarkably, 46 species were recorded for the first time in the region, and 62 others could not be identified to species level and are possibly new to science, including Prestauroneis furatensis, which was newly described from the epiphytic community of the River Euphrates.

The metabarcoding analysis targeted the V4 region of the 18S rRNA gene, amplified with the DIV4 primer pair and sequenced on an Illumina MiSeq platform with 300-bp paired-end reads. After bioinformatic processing with the DADA2 pipeline, which denoises reads and infers amplicon sequence variants, and quality control with the metabaR package to remove contaminants and tag-jumps, 5,461,214 reads remained from 43 successfully amplified samples. These reads belonged to 2,953 ASVs, of which 1,454, or 49.2 percent, were classified as Bacillariophyta, the diatoms. The molecular approach recovered 54 genera and 108 species, with many ASVs assigned to the same taxa, and in most cases metabarcoding detected more taxa per sample than microscopy.

The comparison between the two methods exposed striking technical discrepancies. At the genus level, 38 genera were retrieved in both datasets, while 21 genera were identified only by morphology, including Amphipleura, Aulacoseira, Caloneis, Prestauroneis and Synedropsis, and 16 only by metabarcoding, including Campylodiscus, Chaetoceros, Eunotia, Fistulifera and Skeletonema. The incomplete taxonomic reference database for the studied sites turned out to be the major limitation of the molecular approach, as many morphologically identified species in genera such as Mastogloia and Achnanthidium are absent from any reference library. Intraspecific genetic variation, multiple rRNA operons within diatom genomes evolving at different rates, and sequencing artifacts can also generate multiple ASVs from a single species, artificially inflating molecular richness. Conversely, some abundant species identified microscopically, such as Navicula recens, Navicula rostellata and Nitzschia clausii, had no corresponding match in the metabarcoding inventories, possibly because of insufficient DNA concentrations in those samples.

Community-level statistics revealed further contrasts. Shannon diversity indices and average taxa richness were mostly higher in the metabarcoding analyses than in the morphological ones, with the highest molecular species count of 345 recorded at one winter Euphrates station and the lowest of 5 at another. Both approaches agreed that taxon richness was lowest at station E3 on the Euphrates and highest at station E1 on the same river. The non-metric multidimensional scaling plots for morphology showed a clear seasonal effect in diatom community composition, separating the dry summer from the rainy winter, and PERMANOVA confirmed statistically significant differences between the two seasons and the two rivers. In contrast, the NMDS plots for the 18SV4 analyses did not show a seasonal effect for the same samples, a discrepancy the authors attribute to gaps in the DNA barcode reference databases, particularly at the species level, which hinder accurate taxonomic assignment.

The ecological picture emerging from the morphological data is consistent with the environmental stress documented in the region. Pennate diatoms dominated the epiphytic assemblages, constituting 93.7 percent of identified species, while centric diatoms accounted for only 6.3 percent. The distribution of diatom assemblages showed a prevalence of freshwater species at 65.5 percent, followed by brackish species at 13.5 percent and marine species at 10 percent, with the remaining 11 percent being euryvalent species tolerant of a broad range of conditions. Several taxa of marine origin, including Tabularia fasciculata, Seminavis strigosa and Surirella striatula, were recorded, likely because the Qurna marsh connects directly with the Shatt Al-Arab River and receives water from the Arabian Gulf during high tides, creating a conduit for marine diatom migration to a region situated around 200 kilometers from the Gulf. Brackish diatoms such as Bacillaria paxillifera and Nitzschia filiformis reflect salinity fluctuations that intensify as less freshwater flows from the rivers and more seawater ingress from the Gulf.

The study demonstrates that combining metabarcoding with classical microscopy increases the detection and identification of diatom species and lays the foundation for understanding diatom biodiversity in this region. Even amid major advances in molecular tools for biodiversity assessment, the role of morphology in species discovery remains central. The authors recommend repeating the analysis with the rbcL marker, which has shown promise in providing higher resolution for diatom identification, and producing unialgal diatom strains from the studied sites to close the gaps in reference libraries. Such investigations are essential for illustrating the importance of sustainable ecosystem management and the conservation of water as a vital resource, both regionally and globally, in a catchment that climate change, overuse of waters and subsequent salinization have brought to the brink of ecological devastation.

Subject of Research: Diatom biodiversity in the Euphrates-Tigris rivers revealed by combined microscopy and DNA metabarcoding

Article Title: Comparison of the biodiversity of epiphytic diatoms in the Euphrates-Tigris rivers using morphological and metabarcoding analyses

Article References: Mohamad, H., Schimani, K., Al-Shaheen, M., Abarca, N., Jahn, R., Al-Handal, A., Kusber, W.-H., & Zimmermann, J. (2024). Comparison of the biodiversity of epiphytic diatoms in the Euphrates-Tigris rivers using morphological and metabarcoding analyses. Metabarcoding and Metagenomics, 8, Article e135082. https://doi.org/10.3897/mbmg.8.135082

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.135082

Keywords: diatoms, DNA metabarcoding, Euphrates, Tigris, biodiversity, bioindicators, water quality, Iraq, 18S rRNA, epiphytic algae, Comparison, epiphytic

A new study published in the journal Metabarcoding and Metagenomics tackles this problem head-on with one of the most ambitious interlaboratory exercises yet attempted for diatom metabarcoding. Under the coordination of a reference laboratory, 17 participants from 14 countries carried out DNA extraction and PCR amplification in parallel on a shared set of standardised samples, following both a fixed common protocol and their own preferred in-house methods. The goal was to measure precisely where variability creeps into the workflow and whether the ecological conclusions drawn from the data survive the journey between laboratories.

The stakes are higher than they might appear. Environmental legislation across Europe, including the Water Framework Directive, requires member states to classify the ecological status of water bodies using consistent methods. If one country’s DNA-based diatom index systematically produces different scores than another’s, regulators cannot pool results, benchmark progress or trust cross-border comparisons. The research team, led by Valentin Vasselon and colleagues, therefore designed their proficiency test around the two steps of the metabarcoding pipeline that are most notorious for introducing laboratory-specific bias: extracting DNA from biofilm samples and amplifying target genetic markers with the polymerase chain reaction.

Participants received an identical package of test materials, including DNA extracts and biofilm samples collected from rivers and lakes, together with an artificial mock community of known diatom composition. The mock community served as a critical control: because its true composition was known in advance, any deviation in the sequencing results could be traced directly to errors introduced during laboratory processing rather than to genuine ecological differences between samples. Each laboratory performed the work under both conditions, first adhering strictly to a prescribed reference protocol and then applying whatever extraction kit and PCR setup they would normally use in routine monitoring.

The central finding is strikingly positive. When laboratories followed the shared protocol, their results proved highly reproducible, demonstrating that the method can be transferred successfully between facilities in different countries without specialised equipment or bespoke reagents. Even more reassuring for the future of routine monitoring, the ecological assessment outcomes remained highly similar and consistent regardless of which protocol each participant chose to apply. In practical terms, this means that the biological quality scores derived from diatom metabarcoding were robust to the reasonable diversity of laboratory practices currently in use across the scientific community.

That robustness matters because the alternative, forcing every laboratory onto a single rigid protocol, would be both politically and practically difficult. Different countries have invested in different extraction chemistries, primer sets and sequencing platforms over years of method development, and a blanket mandate would discard that accumulated expertise. The study’s authors instead propose a strategy they describe as all for one but prove them all: laboratories may continue using their preferred protocols, provided each one demonstrates its consistency by meeting minimum standard requirements verified through proficiency testing. This reframes standardisation not as uniformity of method but as uniformity of demonstrated performance.

The experimental design also illuminated the specific sources of variability that future standardisation efforts must control. By comparing results across the paired experiments, the team could separate systematic errors, which bias results in a consistent direction, from random errors, which add noise unpredictably. The inclusion of river, lake and mock community samples allowed them to test whether certain sample types or taxonomic compositions were more vulnerable to laboratory effects than others. Such distinctions are essential for deciding which steps of the workflow genuinely need harmonisation and which can safely be left to local preference.

The study arrives at a moment when DNA-based biomonitoring is transitioning from research demonstration to regulatory deployment. National programmes in several European countries have already shown that diatom metabarcoding can reproduce the ecological classifications traditionally obtained by microscopic identification, and in some cases the genetic approach detects additional species and provides finer resolution. What has been missing is the layer of quality assurance that mature analytical disciplines take for granted: ring trials, certified reference materials and documented evidence that a result from one accredited laboratory means the same thing as a result from another. This proficiency test provides a template for building exactly that layer.

The collaborative scale of the exercise is itself noteworthy. Seventeen laboratories spanning 14 countries, from established national reference facilities to smaller academic groups, executed identical experimental workflows in parallel, generating a dataset rich enough to disentangle protocol effects from laboratory effects and sample effects. The author list reflects a genuinely pan-European network of diatom ecologists, molecular biologists and water managers, suggesting that the community has reached the critical mass needed to sustain long-term intercalibration exercises of this kind.

For water managers and regulators, the practical message is that diatom DNA metabarcoding is now technically ready for routine application, provided that laboratories participate in ongoing proficiency testing and demonstrate that their chosen protocols meet agreed minimum standards. For the research community, the study offers a roadmap: standardised sample sets, mock communities as benchmarks, paired protocol comparisons and a reference laboratory to anchor the exercise. If future rounds of testing confirm these findings across more laboratories and more diverse freshwater types, the microscopic algae that have quietly recorded the health of Europe’s rivers for over a century may soon have their status read not through a microscope lens but through the language of DNA, with results that laboratories across the continent can finally compare on equal terms.

Subject of Research: Cross-laboratory proficiency testing to standardise diatom DNA metabarcoding for freshwater biomonitoring

Article Title: Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring

Article References: Vasselon, V., Rivera, S. F., Ács, É., Almeida, S. F. P., Andree, K. B., Apothéloz-Perret-Gentil, L., Bailet, B., Baričević, A., Beentjes, K. K., Bettig, J., Bouchez, A., Capelli, C., Chardon, C., Duleba, M., Elersek, T., Genthon, C., Jablonska, M., Jacas, L., Kahlert, M., … Paix, B. (2025). Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring. Metabarcoding and Metagenomics, 9, Article e133264. https://doi.org/10.3897/mbmg.9.133264

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.133264

Keywords: diatoms, DNA metabarcoding, biomonitoring, freshwater, proficiency testing, standardisation, PCR amplification, DNA extraction, water quality, ecological assessment, interlaboratory comparison, mock community

Bernier Cave is the type locality for A. stygobita, which was described in 2011 as the world’s first known cave brittle star. The species is endemic to Bernier Cave and the nearby Lighthouse Cave, though it is significantly more abundant in the former. Finding these animals is challenging because their small disks, measuring only 3 to 4 millimeters in diameter, are filled with brown detritus that camouflages them against the cave walls. Laboratory experiments have shown that A. stygobita does not accept standard brittle star food but readily consumes the diatom-rich detritus collected from the cave. This specialized diet appears to be the primary reason the population remains stable in this unique habitat.

The cave environment plays a crucial role in supporting this food web. Bernier Cave features a large ceiling entrance that allows both substantial amounts of light and organic detritus to reach the aquatic ecosystem. The water in the cave is brackish, with salinities ranging from 14 to 28 parts per thousand. Tidal fluctuations are relatively slight compared to the surrounding ocean, resulting in slow water movement that allows sizeable accumulations of detritus to settle. A distinctive brown tide line visible on the cave walls is composed largely of C. neofastuosus and other microbes, indicating the prolific growth of this diatom in the entrance room where light is most available.

Campylodiscus neofastuosus, formerly known as Surirella fastuosa, is a large diatom that thrives in brackish water. In the detritus of Bernier Cave, it forms the backbone of the microbial community. While smaller diatom species such as Amphora, Achnanthes, and Orthoseira are more numerous in terms of individual count, C. neofastuosus dominates the biomass due to its large size. The detritus also contains a diverse array of other organisms, including filamentous Chlorophyta, nematodes, ostracods, harpacticoid copepods, ciliates, dinoflagellates, foraminifera, and various bacteria. White bacterial mats, primarily consisting of filamentous sulfur bacteria like Beggiatoa, are also present and are closely associated with the diatom-rich detritus.

One of the most fascinating aspects of C. neofastuosus is its morphological adaptation to the low-light conditions of the cave. The diatom possesses a distinctive cell wall, or frustule, that is concave and slightly heteropolar, resembling a plano-concave lens. This shape is ideal for projecting and focusing light. The frustule acts as an optical device, concentrating the limited available light onto the lens-shaped central area of the cell. Additionally, the diatom has a canal raphe that enhances its motility, allowing it to position itself at the optimal angle for light capture. This structural adaptation is critical for maximizing photosynthetic efficiency in an environment where light is scarce.

The chloroplasts of C. neofastuosus also exhibit remarkable plasticity in response to light levels. In total darkness, the chloroplasts fill almost the entire cell, appearing dark brown due to high concentrations of fucoxanthin. Fucoxanthin is a pigment that efficiently harvests light and transfers excitation energy to chlorophyll a. Under low light conditions, the chloroplasts become lobed and follow the costae, or ribs, of the cell wall, concentrating pigment in the lens-shaped interior. In brighter light, the chloroplasts become more compact and rounded around the nucleus, likely to protect DNA from ultraviolet radiation. This dynamic shape-shifting allows the diatom to optimize its light capture strategy based on environmental conditions.

Beyond its role in photosynthesis, C. neofastuosus produces extracellular polymeric substances, or EPS, which are essential for the formation of the detritus biofilm. These EPS are rich in polysaccharides, monosaccharides, and proteins, and they contain diatom oils including Omega-3 fatty acids. The EPS acts as a sticky glue, binding together various particles and microorganisms to form a fabric-like consistency. This structure is similar to marine snow found in deep-sea environments, which transports nutrients from surface blooms to the abyss. In the cave, this biofilm provides a manageable food source for the brittle star, allowing it to pull the detritus into its mouth with ease.

The nutritional value of the detritus is further enhanced by the symbiotic relationships within the biofilm. The EPS layer, known as the phycosphere, attracts heterotrophic bacteria that supply the diatoms with essential nutrients and cofactors, such as vitamin B12, which diatoms cannot produce themselves. This exchange creates a rich, energy-dense food web that supports not only the brittle star but also a diverse population of detritivores, including nematodes, ostracods, and copepods. The presence of C. neofastuosus makes the detritus in Bernier Cave distinct from that found in other caves, providing a high-quality food source that sustains the endemic population of A. stygobita.

This research underscores the importance of understanding the complex interactions between microorganisms and their environments in subterranean ecosystems. The ability of C. neofastuosus to adapt its morphology and chloroplast structure to low light conditions, combined with its production of nutrient-rich EPS, creates a unique niche that supports rare species. As cave ecosystems continue to face threats from climate change and human activity, preserving these delicate microbial communities is crucial for the survival of endemic species like the cave brittle star. The findings from Bernier Cave offer valuable insights into the resilience of life in extreme environments and the intricate mechanisms that allow organisms to thrive in the absence of abundant light.

Subject of Research: Diatom biofilm ecology and cave brittle star nutrition

Article Title: A biofilm micro-community dominated by the diatom Campylodiscus neofastuosus ( Surirellales ) binds detritus used as food source for rare brittle stars endemic to two Bahamian caves

Article References: Steinitz-Kannan, M., Carpenter, J. H., & Nienaber, M. A. (2025). A biofilm micro-community dominated by the diatom Campylodiscus neofastuosus (Surirellales) binds detritus used as food source for rare brittle stars endemic to two Bahamian caves. Subterranean Biology, 51, 197-212. https://doi.org/10.3897/subtbiol.51.141192

Image Credits: AI Generated

DOI: 10.3897/subtbiol.51.141192

Keywords: diatoms, cave ecology, brittle stars, biofilm, photosynthesis, Bahamas, microbiology, subterranean biology, Campylodiscus neofastuosus, Amphicutis stygobita, micro-community, dominated

Discostella asterocostata was originally described from China in 1985 under the name Cyclotella asterocostata, based on scanning electron microscopic examination of samples collected in the 1970s. The species was later transferred to the genus Discostella in 2004, after researchers recognised that the position of its marginal fultoportulae, the strutted processes sitting between the valve costae, distinguished it from related centric diatoms. Over the following decades, the species turned up in Japan, South Korea and the Russian Far East. Then, in a publication in 2021, it was reported from North America, where re-examination of archived plankton samples revealed it had already been present in Beaver Lake, Arkansas and the Wabash River, Indiana since 2012. Sediment core analyses from several American lakes pushed its known presence on that continent back to at least the mid-1980s.

Europe, by contrast, remained a conspicuous gap. Freshwater centric diatoms have been intensively documented on the continent for decades through national floras, monitoring programmes and decades of electron microscopic surveys of the Danube phytoplankton. The species’ very distinctive morphology, a ring-shaped pattern in the central area of the valve, makes it difficult to miss under the microscope. The researchers therefore argue that its detection in the Mura River in 2016 roughly coincides with its first appearance in Europe, rather than representing an earlier overlooked record.

The new study draws on routine biomonitoring samples collected between 2016 and 2022 at eight sites along the Mura, Drava and Danube Rivers in Croatia, and monthly phytoplankton samples from thirteen sites along the Hungarian Danube and two of its side arms between May 2021 and April 2022. In the Croatian samples, the species was identified through light and scanning electron microscopy, appearing in plankton from April to September at all eight sites. Its contribution to total phytoplankton biomass was always modest, ranging from 0.2 to 3.2 percent within the class Mediophyceae, but its consistent presence across years and sites suggested it had already become a permanent member of the community.

The Hungarian story is more revealing. In the first scanning electron microscopic survey of 2021 samples, only a single valve of Discostella asterocostata was found, in a sample from the Ráckevei-Soroksári Danube side arm in July. The species was absent from the Diat.barcode database version used in the initial metabarcoding analysis, so the researchers could not identify it molecularly at first. When updated barcode sequences from China became available, the team re-analysed their previously unclassified Discostella sequences and found a near-perfect match, with uncorrected p-distances below 0.01 and at most one or two base pair differences across the 263-base-pair rbcL region used for metabarcoding.

The re-analysis transformed the picture. Molecular detection revealed the species throughout the entire Hungarian section of the Danube by 2021 to 2022, with higher relative read numbers during the warm water period and greatest abundance in the Ráckevei-Soroksári Danube. Even more strikingly, re-analysis of unclassified sequences from earlier phytobenthos surveys of 328 Hungarian surface water samples collected in 2019 and 2020 identified the species in five oxbow lakes along the southern Hungarian Danube, with the earliest molecular detections in September 2019. Two unassigned amplicon sequence variants from the 4th Joint Danube Survey, also collected in 2019, proved identical to the researchers’ sequences, providing independent confirmation that the species was already present in the region six years before its first microscopic record in Hungarian plankton.

Morphometric comparison added another layer to the evidence. The researchers measured eight valve features, including five diameter measurements, the count of marginal strutted processes and the number of outer and central striae, on 35 Croatian and 10 Hungarian valves, then compared the results with published morphometric data from Chinese and American populations. Principal component analysis showed that the first principal component, accounting for 91.8 percent of the variance in the dataset, was strongly correlated with cell diameter. More importantly, the populations from Croatia, Hungary, the United States and Asia completely overlapped on the second and third principal component axes, indicating that the four populations are morphologically indistinguishable. Phylogenetic analysis of the rbcL sequences placed all Discostella asterocostata variants in a monophyletic group well separated from other Discostella species, reinforcing the conclusion that the European populations have not diverged from their Asian ancestors.

The ecological preferences of the species offer a clue to its future in Europe. Spearman correlation analysis of Croatian samples showed a significant positive correlation between the species’ biomass and water temperature, and significant negative correlations with total organic carbon, nitrogen forms and chloride. The Hungarian molecular data confirmed these patterns, with relative read numbers positively correlated with temperature and negatively correlated with total organic carbon, total nitrogen and nitrate. The species also showed a negative correlation with discharge, suggesting a preference for slow-flowing or stagnant waters. In short, Discostella asterocostata favours warmer waters with lower nutrient loads, a profile that differs from the dominant centric diatoms of very large European rivers and that aligns uncomfortably well with the trajectory of climate warming.

How the species reached Europe remains uncertain, but the researchers consider several plausible vectors. In North America, invasive Asian carp, particularly silver carp and bighead carp, have been suggested as carriers, since their filtering apparatus can collect algae larger than 10 micrometres from the water and some algae may survive digestion and be excreted alive in faeces. Asian carps were introduced to Hungary from East Asia as early as 1963, but the researchers consider it unlikely that they explain the species’ appearance there, given the timing. The pattern of spread, with the species appearing in floodplain and protected oxbow lakes and in the Ráckevei-Soroksári Danube before it was detected in the main channel of the Hungarian Danube, suggests that waterbirds may have played a role as dispersal vectors, carrying viable cells between waterbodies. Ship ballast water and fish transport water remain additional possibilities that cannot be excluded.

For now, the species has not shown invasive behaviour in European rivers. It has never been the dominant centric diatom in any phytoplankton sample, and its rapid spread, though indisputable, has not been accompanied by the high abundances that would trigger ecological alarm. But its clear temperature-related seasonal dynamics, its preference for warm, nutrient-poor and slow-flowing waters, and its expansion pattern across the Danube catchment all support the hypothesis that ongoing warming of freshwater systems could promote its further dispersal and increased abundance. The study’s authors emphasise that integrating traditional microscopy with high-throughput molecular techniques such as metabarcoding is essential for the early detection of non-indigenous diatom species, particularly when they occur at low densities that routine counts of 400 to 500 valves can easily miss. As rising water temperatures and enhanced dispersal pathways continue to reshape aquatic ecosystems worldwide, the quiet colonisation of the Danube by a tiny Asian diatom may prove an early warning of changes still to come.

Subject of Research: First European record of the non-indigenous diatom Discostella asterocostata in the Danube River

Article Title: From Asia through America to Europe: Molecular and morphological evidence for a non-indigenous diatom species, Discostella asterocostata (B.Q.Lin, S.Q.Xie & S.X.Cai) Houk & Klee in Hungary and Croatia

Article References: Stanković, I., Shemesh, S., Hanžek, N., Šušnjara, M., Gligora Udovič, M., Kiss, K. T., Trábert, Z., Udvari, Z., Bíró, T., Vadkerti, E., & Ács, É. (2025). From Asia through America to Europe: Molecular and morphological evidence for a non-indigenous diatom species, Discostella asterocostata (B.Q.Lin, S.Q.Xie & S.X.Cai) Houk & Klee in Hungary and Croatia. Metabarcoding and Metagenomics, 9, Article e154667. https://doi.org/10.3897/mbmg.9.154667

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.154667

Keywords: diatoms, invasive species, Danube River, metabarcoding, climate change, freshwater ecology, phytoplankton, non-indigenous species, Asia, America, Europe, Molecular

The study focused on Lake Aydat, a shallow, nutrient-rich lake that stratifies and mixes twice a year, making it a classic model system for freshwater ecology. From November 2018 to January 2020, the team collected water from the sunlit euphotic zone at the deepest point of the lake, sampling monthly and intensifying to twice weekly during late spring. Fourteen litres of water per sample were filtered to concentrate microbial biomass, which was preserved at minus 80 degrees Celsius until DNA could be extracted. This careful, time-resolved sampling design was essential: the goal was not merely to list the species present, but to track how entire communities of algae, ciliates, fungi and parasites rose and fell across a full annual cycle.

The short-read side of the comparison used Illumina sequencing of the V4 hypervariable region of the 18S ribosomal RNA gene, a fragment of roughly 380 base pairs that has become the standard marker for surveying eukaryotic diversity. The long-read side used Pacific Biosciences Sequel II technology with circular consensus sequencing, a method that reads the same DNA molecule repeatedly to correct errors, achieving accuracy comparable to Illumina. Instead of a single hypervariable region, the long-read approach amplified nearly the entire ribosomal RNA operon — approximately 4,500 base pairs spanning the 18S gene, the internal transcribed spacer regions, the 5.8S gene and part of the 28S gene. In total, the team recovered more than 153,000 filtered long reads, from which they extracted over 20,000 full-length 18S sequences averaging about 1,232 base pairs each.

Both datasets were processed with comparable pipelines, clustering sequences into operational taxonomic units at 97 percent identity and assigning taxonomy through phylogenetic placement against curated reference databases. After filtering, the short-read dataset contained 747 OTUs and the long-read dataset 546. To make a rigorous, like-for-like comparison, the researchers performed an elegant analytical trick: they extracted the V4 region computationally from the long-read sequences, creating a hybrid dataset that could be clustered together with the genuine short-read OTUs. This allowed them to ask precisely whether differences in taxonomic assignment arose from the length of the sequence itself or from the different organisms each method happened to capture.

The answer was unambiguous. At broad taxonomic levels — supergroups and divisions — the two methods agreed well, painting similar pictures of the lake’s major eukaryotic lineages. But at finer scales the approaches diverged sharply. Among clusters containing sequences from both datasets, more than a quarter showed deeper taxonomic assignment with the long-read data, in some cases resolving up to seven additional hierarchical ranks. Overall, nearly 30 percent of long-read OTUs could be classified to genus level, compared with only 11 percent of short-read OTUs. The long-read dataset also recovered 35 genera that the short-read approach missed entirely, against just 13 genera unique to the short reads — and those short-read-only sequences tended to be rare, low-abundance taxa, consistent with Illumina’s greater sequencing depth.

Certain groups benefited disproportionately from the longer sequences. Ciliates, single-celled predators that are central to aquatic food webs, were frequently assigned only to phylum by short reads, while long reads resolved 34 percent of ciliate OTUs to genus. Chytrid fungi, parasites of phytoplankton with major ecological influence, saw genus-level identification jump from 8 percent with short reads to 36 percent with long reads. Diatoms, the silica-shelled algae that underpin much of freshwater primary production, were classified to genus in 92 percent of long-read OTUs versus 56 percent of short-read OTUs. Where the short-read data suggested a lake dominated by the diatom genus Fragilaria, the long-read data revealed substantial populations of Cyclotella and Asterionella — a finding that matched earlier microscopic observations of the lake, underscoring how short-read surveys can misrepresent community composition.

The seasonal dynamics the long-read data revealed are where the study becomes genuinely vivid. Because taxa could be identified precisely, the researchers could watch individual genera succeed one another through the year. The ciliate Condylostoma peaked in June 2019, while Carchesium, a related filter-feeder with similar dietary preferences, showed a different temporal pattern — a succession that may reflect differing life-history strategies or tolerances to temperature, oxygen and nutrient fluctuations. Among the diatoms, Cyclotella flourished in spring, Fragilariforma dominated in July, and Asterionella reached its greatest abundance in the cold months of January and April. Such fine-grained temporal partitioning among close relatives is exactly the kind of ecological process that short-read data, with its blurred taxonomy, tends to obscure.

Perhaps the most ecologically compelling result concerns parasitism. Chytrid fungi infect and lyse phytoplankton, sometimes terminating algal blooms and reshaping entire food webs, yet host–parasite dynamics are notoriously difficult to observe in nature. The long-read data allowed the team to track the chytrid genus Zygorhizidium alongside its diatom hosts month by month. Zygorhizidium abundance rose sharply between March and May, coinciding with the spring diatom bloom — a pattern consistent with laboratory work showing that Zygorhizidium affluens infects the diatom Asterionella formosa, but now observable directly in a natural lake. A December peak in chytrids followed a November surge of the golden alga Mallomonas, echoing previous reports of chytrid-driven algal declines. The data also showed simultaneous peaks of Perkinsea parasites and colonial green algae, supporting emerging evidence that these parasites, better known from marine hosts, play a similar role in freshwaters.

The authors are careful to note that short-read sequencing retains real advantages: it is cheaper, generates far greater depth, benefits from mature analytical pipelines, and excels at detecting rare taxa. Long reads also demand higher-quality DNA, since degraded environmental samples can fail to amplify across 4,500 base pairs, and reference databases remain thinner for full-length sequences, which occasionally forced more conservative assignments. Indeed, in a small minority of cases the short reads achieved finer classification, likely because the V4 region is better represented in reference databases. These caveats matter for anyone designing a monitoring programme, and the study does not suggest that short reads should be abandoned.

But the trajectory is clear. As sequencing costs fall and reference databases grow, the study argues that long-read metabarcoding should move from specialist tool to routine instrument of environmental surveillance. The ability to resolve genera and species in time series opens the door to tracking bioindicator ciliates for water quality, detecting parasitic outbreaks before blooms collapse, and mapping the cryptic diversity that short fragments cannot distinguish. Combined with the 28S sequences the team plans to analyse next, and with emerging single-cell and genome-based reference collections, long-read surveys promise to close taxonomic gaps that have limited freshwater ecology for two decades. For the invisible majority of lake life, the picture is finally coming into focus — one long read at a time.

Subject of Research: Comparing short-read and long-read metabarcoding of eukaryotic diversity in a lake over one year

Article Title: Profiling the eukaryotic diversity over a year in a lake ecosystem through short- and long-read metabarcoding

Article References: Chauvet, M., Terzian, P., Moné, A., Debroas, D., Bronner, G., & Lepère, C. (2026). Profiling the eukaryotic diversity over a year in a lake ecosystem through short- and long-read metabarcoding. Metabarcoding and Metagenomics, 10, Article e184566. https://doi.org/10.3897/mbmg.10.184566

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.184566

Keywords: metabarcoding, long-read sequencing, PacBio, eukaryotic diversity, Lake Aydat, 18S rRNA, chytrid fungi, diatoms, ciliates, freshwater ecology, seasonal dynamics, host-parasite interactions