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•Diatom DNA metabarcoding was implemented in the Joint Danube Survey in 2019.•Communities analysed by different methods share common taxa but contain discrepancies.•IPS obtained by ...different methods correlate but affected by community discrepancies.•Metabarcoding should be used along morphology-based methods in future campaigns.
The Joint Danube Survey (JDS) is a regular transnational survey to monitor the quality of the Danube and its main tributaries, in accordance with the EU Water Framework Directive. During the JDS4 in 2019, conventional methods to monitor selected biological quality elements were complemented with DNA metabarcoding.
All together 72 phytobenthos samples were collected along the Danube and its major tributaries within the JDS4, using light microscopy and DNA metabarcoding amplifying a fragment of the rbcL marker gene. The (i) applicability of DNA metabarcoding to identify diatom communities compared to microscopy; (ii) diversity metrics between DNA metabarcoding and microscopy analysis and (iii) the usability of DNA metabarcoding for routine monitoring and assessment of the Danube under future JDS surveys were investigated.
Diatom communities resulting from light microscopy and DNA metabarcoding assessments share 26.5% of all taxa which corresponds to 64.3% when considering relative abundances. Discrepancies originate from biases both from metabarcoding, e.g. missing taxa from the reference library, and from microscopy, e.g. overlooking of hardly visible taxa. Microscopy detected more taxa in total but metabarcoding revealed a higher alpha diversity, detecting also very rare taxa in a given sample. Molecular and microscopy based Specific Pollution Sensitivity Index (IPS) values correlated significantly but differences were detected at several sites due to the differences in community composition and the overestimation of large taxa by metabarcoding. Although both methods showed a decreasing trend of IPS along the Danube, the metabarcoding based IPS covered a higher range of quality classes indicating lower values for downstream sites and the tributaries.
We suggest that metabarcoding provides a standardisable and efficient tool in biomonitoring, being more distinctive among quality classes than microscopy. Due to the high sequencing depth, it is able to detect a higher diversity on genetic level in a time- and cost-efficient manner that should be implemented in future quality assessment tools. We recommend its use in future biomonitoring surveys, for now, as a complementary method to conventional ones.
Received June 2, 2020, Accepted August 1, 2020 Cocconeis coreana and C. sijunghoensis are described as new based on micromorphological and molecular data. C. coreana is represented by five unialgal ...cultures from four different freshwater bodies, two from North Korea and three from South Korea. C. sijunghoensis is represented by two unialgal cultures from a brackish water body in North Korea. Except for one, all of the strains auxosporulated and showed an almost quadrupling of size in length and width. Morphologically, these species with their two different elliptical valves belong to the Cocconeis placentula group. The raphe valve has striae with uniseriate areolae continuing across a pronounced submarginal hyaline rim to the edge of the valve. The sternum valve has uniseriate dash-like areolae continuously from the valve face until the valve edge. Micromorpho-logically, these species possess two different open valvocopulae: only the raphe valvocopula has fimbriae; the sternum valvocopulahas none. Based on p-distances of currently available DNA sequence data, i.e., rbcL and 18SV4, both species are pronouncedly different from the epitype strain of C. placentula, with C. coreana closest to the published molecular data of the strain UTEX FD23 named C. placentula from Iowa, USA, while C. sijunghoensis is closest but not the same as die published molecular data of strain D36_012, die epitype strain of C. placentula from Berlin, Germany. Based on scanning electron microscope observations, differentiating features are discussed concerning valvocopula fimbriae, central area, areolation of die sternum valve and on die raphe valve especially between die submarginal hyaline rim and edge.
The Lerma-Chapala Basin, in Central Mexico, is geologically heterogeneous, climatically diverse and boasts high biodiversity, lying within two Biodiversity Hotspots, namely Mesoamerica and the ...Madrean Pine-Oak Woodlands. Epilithon and water samples were collected in the basin from 14 sampling sites three times each, two sampling campaigns during the rainy season and one in the dry season. A total of 274 infrageneric taxa in 48 genera were recorded. The taxonomic composition observed was dominated by taxa from the genera
,
,
,
,
and
. About a third of the taxa found could not be identified to the species level. From those unidentified morphodemes, two are described as new species, namely
and
. Furthermore,
is transferred to
. Canonical Correspondence Analysis (CCA) revealed that specific conductivity and pH were the main environmental factors driving the community composition observed. Three groups of samples were identified after the CCA: 1) characterized by acidic waters and low conductivity; 2) with circumneutral waters, low specific conductivity and high temperature and phosphorous concentrations; and 3) characterized by circumneutral waters, high conductivity and low nitrogen concentrations. The indicator value method (IndVal), based on the relative abundance and relative frequency of the most abundant taxa was calculated based on the groups observed in the CCA, identifying the characteristic taxa for each of the three groups.
The nucleotide sequences (18S and rbcL) of the freshwater diatom strain UTEX FD23 were revisited and its morphology studied for the first time. This strain is maintained at the UTEX Culture ...Collection of Algae, where it is referred to as Cocconeis placentula var. lineata. The sequences of this strain had been deposited in NCBI and used in more than 30 studies mostly under the name C. placentula, but also C. placentula var. lineata. We describe this species as new, based on sequence data and morphology (LM and SEM), which did not correspond to the epitypes of C. placentula var. placentula, C. lineata or any other described member of the C. placentula group. According to genetic distances for 18SV4, this new species is closest to C. placentula sensu stricto (5.8%), and has a distance of 7.9% to C. lineata. For rbcL, C. czarneckii is closest to the recently described C. coreana (2.6%-2.8%), it has a distance of 4.1% to C. lineata, and of 4.2% to C. placentula. Morphologically, C. czarneckii shares similarities with C. sijunghoensis, C. amerieuglypta, C. klamathensis and C. grovei, which differ by valve size and outline, numbers and pattern of submarginal areolae, and ultrastructural details of the valvocopula of the raphe valve. Our study, integrating available nucleotide sequences with a detailed morphological characterization of the strain UTEX FD23, added to our understanding of the heterogeneity of the C. placentula group, and will benefit the scientific community interested in this strain (e.g. for taxonomy, molecular phylogenetics and eDNA metabarcoding).
Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is ...also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.
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•Bioinformatics pipelines used for diatom metabarcoding in six European countries are compared•A common curated reference database does not guarantee detection of the same species assemblages•Variation in filtering, clustering and taxonomic assignment drive discrepancies in the outputs•Decisions made in bioinformatics analyses have an impact on environmental assessment•Low reproducibility of outputs from the pipelines highlights the need for standardization.
•Prymnesium parvum was the cause of the ecological disaster in the Oder river in 2022.•A single phylotype was detected by ITS and 18S rRNA sequencing.•Clonal cultures were established and are ...maintained in an algal culture collection.•Prymnesin B1 was identified by mass spectrometry in cultures and water samples.•Established droplet digital PCR assay useful for low-abundance P. parvum monitoring.
Blooms of Prymnesium parvum, a unicellular alga globally distributed in marine and brackish environments, frequently result in massive fish kills due to the production of toxins called prymnesins by this haptophyte. In August 2022, a harmful algal bloom (HAB) of this species occurred in the lower Oder River (Poland and Germany), which caused mass mortalities of fish and other organisms. This HAB was linked to low discharge of the Oder and mining activities that caused a significant increase in salinity. In this context, we report on the molecular detection and screening of this haptophyte and its toxins in environmental samples and clonal cultures derived thereof. Both conventional PCR and droplet digital PCR assays reliably detected P. parvum in environmental samples. eDNA metabarcoding using the V4 region of the 18S rRNA gene revealed a single Prymnesium sequence variant, but failed to identify it to species level. Four clonal cultures established from environmental samples were unambiguously identified as P. parvum by molecular phylogenetics (near full-length 18S rRNA gene) and light microscopy. Phylogenetic analysis (ITS1–5.8S-ITS2 marker region) placed the cultured phylotype within a clade containing other P. parvum strains known to produce B-type prymnesins. Toxin-screening of the cultures using liquid chromatography-electrospray ionization - time of flight mass spectrometry identified B-type prymnesins, which were also detected in extracts of filter residues from water samples of the Oder collected during the HAB. Overall, our investigation provides a detailed characterization of P. parvum, including their prymnesins, during this HAB in the Oder River, contributing valuable insights into this ecological disaster. In addition, the droplet digital PCR assay established here will be useful for future monitoring of low levels of P. parvum on the Oder River or any other salt-impacted and brackish water bodies.
Diatoms are among the most commonly used bioindicators. Correct taxonomic identifications are critical to their use as bioindicators because closely related diatom species can respond differently to ...water physicochemical characteristics and pollutants. However, diatom identification based on morphology can be time consuming, and requires highly specialized taxonomic skills. To optimize diatom identification, DNA metabarcoding is increasingly used because it is generally less time consuming and may be more accurate than morphological identification. To date, however, neither DNA metabarcoding nor DNA barcoding diatom studies have been conducted in Mexico. Thus, we studied epilithic diatoms from streams in Central Mexico with a combination of morphological and metabarcoding techniques, and compared the diatoms identified and quantified by each method. We also assembled a barcode reference library based on clonal culturing. This library is composed of 190 strains that belong to 72 species in 24 genera. The morphological analysis of environmental samples resulted in the identification of 204 infrageneric taxa in 42 genera, but clonal culturing from the same samples retrieved 12 additional infrageneric taxa and 1 additional genus, thereby revealing concealed diversity. The metabarcoding approach resulted in the identification of 266 infrageneric taxa that belonged to 35 genera. Together, these methods detected 49 genera. Of these genera, 14 were identified only by morphology, 29 were identified by both methods, and 6 were only identified by metabarcoding. Of the 266 taxa we retrieved with metabarcoding, we confidently assigned 94 infrageneric taxa because a direct morphological or barcode sequence correlation was possible. Thirty-four of these 94 taxa were only detected with the metabarcoding method. One-fourth (23) of the assignments were only possible because of the barcode reference library we developed during this study, because there were no existing barcode sequences that matched these barcodes in the International Nucleotide Sequence Database Collaboration databases. Large disparities existed between relative abundances based on valve counts and sequence reads of the most abundant taxa, even after we corrected for cell biovolume. Overall, we conclude that the combination of morphological and molecular methods increases the detection and identification of diatoms.
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.
In order to understand the taxonomy and phylogeny of the genus Gomphonema, the type of the name of the genus, G. acuminatum, and related species complexes are studied with an integrative taxonomic ...approach. Sixty-seven unialgal Gomphonema strains were established from 34 different water bodies in Croatia, Faroe Islands, Finland, Iceland, Germany, Mexico, Spain, Switzerland and Korea, which provided molecular (18S and rbcL), morphometric and ultrastructural data. Our study was complemented by molecular data from INSDC, summing up the studied data to 82 freshwater Gomphonema strains from the G. acuminatum and related species complexes which are here defined as the Gomphonema core group. Except for outline variations which occur even within one clone, their micro-morphologies are indistinguishable. The molecular data of the two markers clustered the taxa of the core group into one clade, well differentiated from other Gomphonema taxa such as G. augur and G. parvulum. For differentiation within the clade, the plastid marker rbcL provided better species resolution comparable to traditionally recognized taxa. The combined analysis of the molecular as well as morphological data resulted in the defining of only 7 subclades in contrast to 20 currently assigned names for the taxa of the Gomphonema core group in this study. The Gomphonema core group has been overdescribed due to the use of outline as the main criterion for species delimitation. 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.
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