1. Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoringValentin Vasselon, Sinziana F. Rivera, Éva Ács, Tina Eleršek, Maša Jablonska, 2025, original scientific article Abstract: 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.
Keywords: cross-laboratory experiment, DNA-based approach, ecological status assessment, intercalibration, standardisation, environmental science, biomonitoring, DNA metabarcoding, benthic diatoms
Published in DiRROS: 17.01.2025; Views: 947; Downloads: 814
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2. Discovery of novel and known viruses associated with toxigenic and non-toxigenic bloom forming diatoms from the Northern Adriatic SeaTimotej Turk Dermastia, Denis Kutnjak, Ion Gutiérrez-Aguirre, Corina P. D. Brussaard, Katarina Bačnik, 2024, original scientific article Abstract: Algal blooms impact trophic interactions, community structure and element fluxes. Despite playing an important role in the demise of phytoplankton blooms, only few viruses infecting diatoms have been cultured. Pseudo-nitzschia is a widespread diatom genus that commonly blooms in coastal waters and contains toxin-producing species. This study describes the characterization of a novel virus infecting the toxigenic species Pseudo-nitzschia galaxiae isolated from the northern Adriatic Sea. The ssRNA virus PnGalRNAV has 29.5 nm ± 1.2 nm icosahedral virions and a genome size of 8.8 kb. It belongs to the picorna-like Marnaviridae family and shows high specificity for P. galaxiae infecting two genetically and morphologically distinct strains. We found two genetically distinct types of this virus and screening of the global virome database revealed matching sequences from the Mediterranean region and China, suggesting its global distribution. Another virus of similar shape and size infecting Pseudo-nitzschia calliantha was found, but its genome could not be determined. In addition, we have obtained and characterized a new virus that infects Chaetoceros tenuissimus. The replicase protein of this virus is very similar to the previously described ChTenDNAV type-II virus, but it has a unique genome and infection pattern. Our study is an important contribution to the collective diatom virus culture collection and will allow further investigation into how these viruses control diatom bloom termination, carbon export and toxin release in the case of Pseudo-nitzschia.
Keywords: algal blooms, diatoms, virus, PnGalRNAV, genome, Adriatic Sea, marine virology, phytoplankton ecology, marine biology
Published in DiRROS: 23.10.2024; Views: 1529; Downloads: 1546
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3. Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected generaTimotej Turk Dermastia, Ivano Vascotto, Janja Francé, David Stanković, Patricija Mozetič, 2023, original scientific article Abstract: Diatoms are one of the most important phytoplankton groups in the world’s oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks.
Keywords: rbcL, metabarcoding, monitoring, diatoms, population genetics, Pseudo-nitzschia, Adriatic
Published in DiRROS: 12.07.2024; Views: 1522; Downloads: 883
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