Jonas Zimmermann

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

We present a new version of a barcoding reference library dedicated to diatoms, Diat.barcode v12, with newly published sequences, annotated with ecological, and biological traits (size class, life forms, ecological guilds, etc ) and curated by a college of experts. Diat.barcode incorporates rbcL data on all diatoms, though freshwater taxa are better represented. We used this library in two different areas, one where the taxonomic coverage of the library was good (mainland France) and another where it was poor (French Guyana) with about 320 diatom samples collected for river monitoring across both regions. We show that a direct bioinformatic assignment of environmental sequences to traits (ecological guilds, habitats, morphology) has great potential in French Guyana where species knowledge is poor and therefore the proportion of assigned environmental sequences is much lower (12.8%) than trait assignation (30%). We used co-correspondence analyses to show that, unlike mainland France where all species and trait assignation datasets were significantly correlated, in French Guyana only 7 out of 13 trait categories showed a significant correlation. This indicates a significant loss of ecological information through species assignment in French Guyana. Consequently, directly assigning environmental sequences to traits can be useful and provide more ecological information in regions with poor taxonomic knowledge because of the many rbcL sequences without taxonomic assignations.

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

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

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

Monoclonal diatom strains are the basis for integrative taxonomical studies combining molecular and morphological data for better taxonomic resolution and identification. The identification of diatoms, based on morphological features of the frustule, is difficult due to the variability of characters within and amongst species. The longer the cultivation time, the more cell size is reduced during asexual reproduction. Many divisions of the cell complicate identification as morphological characters of the frustule change at different rates in different taxa and teratologies can develop. Morphological variability raises the question whether the investigated genetic markers are stable over the cultivation time span. Sixteen monoclonal pennate diatom strains, identified as 10 different species in six genera, were cultivated for up to 18 or 24 months. It was shown that molecular data for the 18S-V4, rbcL, and psbA markers were stable for the entire cultivation time of all taxa. Images of the valves of the strains were taken every six months and examined morphometrically. Depending on the time since the start of the cultures, the morphological features showed high variability, particularly in length and shape, whereas width and number of striae, costae or fibulae were more stable, depending on the taxon and strain. A limited time span and good culture conditions seem to be prerequisite to avoid the production of teratological frustules. The results of parallel cultures in a unique experimental design are compared to observations from the literature dealing with single strains or special cognitive interests.

Background and aims – Within the project “German Barcode of Life – Diatoms” common diatoms of German waters were routinely isolated and cultivated. In order to understand the taxonomy and phylogeny of the genus Gomphonema, one of the most common taxa of Central Europe, known currently either under the name Gomphonema olivaceum in Europe or Gomphoneis olivacea in America, was studied. Methods – Twenty unialgal strains were established from five different water bodies in Germany and one from Lake Balaton, Hungary, which supplied molecular data (18S V4 and rbcL) besides morphometric and ultrastructural data. In addition, on eight populations from different water bodies including the type from Denmark, morphometric and micromorphological studies by light and scanning electron microscopy were performed. Key results – Molecular and micromorphological data show that the target taxon neither belongs to Gomphonema Ehrenb. nor to Gomphoneis Cleve. By reinstating the genus name Gomphonella Rabenh., the nomenclatural and taxonomic enigma of this taxon is solved, and with the presentation of the type by Hornemann the authorship of the epithet is clarified. Molecular data for the unialgal strains and several environmental clones show that there is more diversity in the Gomphonella olivacea clade than can be identified morphologically. In addition, the establishment of the new species Gomphonella coxiae and Gomphonella acsiae is supported. The molecular data classified Gomphonella species as belonging to the Cymbellales but not to the Gomphonemataceae. In addition, molecular data put Gomphoneis tegelensis R.Jahn & N.Abarca also into Gomphonella. In order to make the genera Gomphoneis and Gomphonema monophyletic, their astigmate members are transferred to Gomphonella. Conclusions – The results clarify that the gomphonemoid outline is not restricted to the family Gomphonemataceae but seem to be distributed across the entire order Cymbellales. This is shown in this paper for the revived genus Gomphonella, which contains the astigmate group of Gomphoneis and Gomphonema besides the longly disputed G. olivacea. Only a polyphasic approach, combining molecular and micromorphological data for taxonomy, nomenclatural evaluation, and observations from clonal cultures can reveal the full intricacies of evolutionary relations.