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Comparative Investigation of Marine Benthic Diatom Assemblages with Morphological and Metabarcoding Approaches Along the Coasts of Istanbul Metropolis
Abstract
The importance of sustainable bioassessment and management of coasts to maintain good trophic status is increasing due to the high anthropogenic impacts on marine ecosystems. Diatoms are one of the most important assemblages and useful tools for assessing the state of the environment due to their advantages over other organisms such as short life span and rapid reaction to environmental changes in aquatic systems. In this study, our objective was to investigate marine benthic diatoms and compare them using three different methods: light microscopy (LM), scanning electron microscopy (SEM), and molecular metabarcoding (MB). LM observations were supported by SEM; in addition, 18S rRNA metabarcoding was used to reveal the diatom flora. The results showed that the highest number of genera was observed with LM (49), followed by SEM (27) and MB (21). Eight genera were found to be common by all three methods. Although MB identified a small number of genera, it verified the presence of common taxa through a rigorous analysis. However, all the methods used gave consistent results in confirming that the diatom assemblages found along the coasts of Istanbul in the Sea of Marmara exhibited low diversity. Our results suggest that the morphological approach to the study of marine benthic diatoms can be complemented by the metabarcoding approach, which showed encouraging results, and that both approaches can be mutually supportive.
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Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.
Environmental DNA (eDNA) detection methods can complement traditional biomonitoring to yield new ecological insights in aquatic systems. However, the conceptual and methodological frameworks for aquatic eDNA detection and interpretation were developed primarily in freshwater environments and have not been well established for estuaries and marine environments that are by nature dynamic, turbid, and hydrologically complex. Environmental context and species life history are critical for successful application of eDNA methods, and the challenges associated with eDNA detection in estuaries were the subject of a symposium held at the University of California Davis on January 29, 2020 ( https://marinescience.ucdavis.edu/engagement/past-events/edna ). Here, we elaborate upon topics addressed in the symposium to evaluate eDNA methods in the context of monitoring and biodiversity studies in estuaries. We first provide a concise overview of eDNA science and methods, and then examine the San Francisco Estuary (SFE) as a case study to illustrate how eDNA detection can complement traditional monitoring programs and provide regional guidance on future potential eDNA applications. Additionally, we offer recommendations for enhancing communication between eDNA scientists and natural resource managers, which is essential for integrating eDNA methods into existing monitoring programs. Our intent is to create a resource that is accessible to those outside the field of eDNA, especially managers, without oversimplifying the challenges or advantages of these methods.
Benthic diatoms are the main primary producers and are sensitive to environmental changes in the estuarine ecosystem. Therefore, it is critical to evaluate the impact of environmental stress on the benthic diatom community in the estuarine ecosystem. In this study, the sediment samples from the five sampling sites were collected from the Yellow River Delta in the four seasons, and the abundance of benthic diatoms were determined using the high-throughput sequencing of 18S rRNA genes. The results showed that the motile guild taxa, such as Navicula, Nitzschia, and Amphora, was dominated in the benthic diatom the community throughout the sampling period. The structure of the benthic diatom community was significantly different among seasons (ANOSIM P < 0.01), especially between summer and winter. Redundancy analysis showed that water temperature and the concentrations of silicate, nitrate, ammonium, and pH value are the main driving factors shaping the seasonal assembly of the benthic diatom community. The results will improve knowledge about the benthic diatom community in the estuarine ecosystem and provide a theoretical foundation for estuary environmental management.
In this study, samples were collected from the Java Sea coasts, from the South China Sea in Hainan Island coasts and Quảng Yên region and Rú Chá mangrove near Hue in Central Vietnam. In studied samples a total of eight Luticola species have been observed. Three of the taxa studied are described herein as species new to science-Luticola orientalis sp. nov., L. cribriareolata sp. nov. and L. halongiana sp. nov. Under light microscopy (LM) L. orientalis sp. nov. and L. cribriareolata sp. nov. are similar with rhombic-lan-A peer-reviewed open-access journal Mateusz Rybak et al. / PhytoKeys 183: 115-142 (2021) 116 ceolate to rhombic/ elliptic-lanceolate to elliptic valve shapes and narrowly rounded apices. Both species can be easily distinguished by stria density (higher density in L. orientalis). Under SEM L. cribriareolata is characterized by cribrate areola occlusions, a character thus far observed only in three established species. The remaining species of the whole genus known thus far are characterized by hymenate areola. Similar morphology Luticola species have been observed from tropical mangrove forests from Madagascar but they all can be easily distinguished based on the lack of grooves in the central area. The third species-L. halongiana sp. nov. has rhombic-elliptic to rhombic-lanceolate valves with broadly rounded to slightly protracted apices in larger specimens. This species has a relatively broad central area. Also unique among brackish-water Luticola is the small, rounded stigma positioned almost midway between the valve center and valve margin. In the habitats from which the new species are described we also identified five established Luticola taxa including, L. belawanensis, L. celebesica, L. inserata, L. seposita and L. tropica. For those species we provide detailed SEM characteristics of valve ultrastructure, as well as the range of environmental conditions and geographic distribution within the study area.
Benthic diatoms have traditionally been used as bioindicators of aquatic ecosystems. Because diatom-based monitoring of water quality is required by European legislation, molecular-based methods had emerged as useful alternatives to classical methods based on morphological identification using light microscopy. The aim of this study was to test the reliability of DNA metabarcoding combined with High-Throughput Sequencing (HTS) techniques in the bioassessment of the trophic status of 22 Mediterranean shallow ponds in NW Spain. For each pond, the Trophic Diatom Index (TDI) was calculated from inventories obtained by identification using light microscopy (LM) followed by high-throughput sequencing (HTS) at the molecular level. Ponds were subsequently classified into five water quality classes. The results showed a good correspondence between both methods, especially after applying a correction factor that depended on the biovolume of the cells. This correspondence led to the assignment to the same quality class in 59% of the ponds. The determination and quantification of valves or DNA sequences was one of the main pitfalls, which mainly included those related to the variability in the relative abundances of some species. Accordingly, ponds with similar relative abundances for the dominant species were assigned to the same quality class. Moreover, other difficulties leading the discrepancies were the misidentification of some species due to the presence of semi-cryptic taxa, the incompleteness of the reference database and the bioinformatic protocol. Thus, the validation of DNA-based methods for the identification of freshwater diatoms represents an important goal, as an alternative to using traditional methods in Mediterranean shallow ponds.
Zooplankton community inventories are the basis of fisheries management for containing fish larvae and their preys; however, the visual identification of early-stage larvae (the “missing biomass”) is difficult and laborious. Here, eDNA metabarcoding was employed to detect zooplankton species of interest for fisheries from open and coastal waters. High-Throughput sequencing (HTS) from environmental samples using small water volumes has been proposed to detect species of interest whose DNA is the most abundant. We analyzed 6-L water samples taken from subtropical and tropical waters using Cytochrome oxidase I (COI) gene as metabarcode. In the open ocean, several commercial fish larvae and invertebrate species important in fish diet were found from metabarcodes and confirmed from individual barcoding. Comparing Atlantic, Mediterranean, Red Sea, and Pacific samples we found a lower taxonomic depth of OTU assignments in samples from tropical waters than in those from temperate ones, suggesting large gaps in reference databases for those areas; thus a higher effort of zooplankton barcoding in tropical oceans is highly recommended. This and similar simplified sampling protocols could be applied in early detection of species important for fisheries.
The phytoplankton community of Liaodong Bay was characterized in 2014. Considering that the continuous change in taxonomic composition and abundance was a significant characteristic of phytoplankton communities in the sea, phytoplankton community was continuously detected in 2015. Results showed that the average phytoplankton operational taxonomic units (OTUs) of each sample was 200-561. The same as in 2014, Dinophyceae abundance was still the highest in June and August of 2015 according to OTUs. Nineteen algal species could cause toxicity to disrupt food webs, and thirty-one species had been reported as causing algal blooms, four more than in 2014. Among algae documented as bloom-forming species, Akashiwo sanguinea, Amylax triacantha, Ostreococcus tauri, and Kryptoperidinium foliaceum were observed in August, while Pyrophacus steinii, Phaeocystis globosa, and Nannochloropsis granulata appeared only in June. Other algae with documented blooms were detected both in June and August of 2015. In the near-shore (NS) group, inner-shore (IS) group, and off-shore (OS) group, the number of algal species in August was higher than that in June in 2015. In NS and IS group, the dominant species were mainly Cryptophyceae and Haptophyta, respectively, while they belonged to Haptophyta and Bacillariophytina in OS.
Diatoms (Bacillariophyceae) are characterized by silicified cell walls that favor their long-term preservation in sediments, therefore widely used as bioindicators of present and past water conditions. Alongside with traditional morphological analyses, metabarcoding has become a valuable tool to study the community structures of various organisms, including diatoms. Here, we test whether the quantity of sediment sample used for DNA extraction affects the results obtained from high-throughput sequencing (metabarcoding) of the diatom rbcL region by isolating DNA from 10 g and 0.5 g (wet weight) of lake surface sediment samples. Because bioinformatics processing of metabarcoding data may affect the outcome, we also tested the consistency of the results from three different pipelines: 1) ESVs (exact sequence variants) pipeline; 2) clustering sequences at 95% sequence identity to form OTUs (operational taxonomic units; 95% OTUs); and 3) 97% OTUs pipeline. Additionally, the agreement between metabarcoding data and morphological inventories of corresponding samples were compared. Our results demonstrate highly uniform patterns between the diatom rbcL amplicons from 10 g and 0.5 g of sedimentary DNA (sedDNA) extracts (HTS 10 and HTS 0.5, respectively). Furthermore, after the careful curation of the sequencing data, metabarcoding results were highly consistent among the data sets produced by different bioinformatics pipelines. Comparing results from metabarcoding and microscopy, we identified some taxonomic mismatches: morphological analyses identified 59 diatom genera, whereas metabarcoding 49 to 54 genera. These mismatches are related to incompleteness of the sequence databases, but also to inconsistencies in diatom taxonomy in general and potential dissolution effects of diatom valves caused by high alkalinity of the investigated lake waters. Nevertheless, multivariate community analysis revealed consistent results between data sets identified by microscopy and metabarcoding – water depth and conductivity as the most significant variables in driving diatom communities in Lake Nam Co – further confirming that metabarcoding
is a viable method for identifying diatom-environment relationships.
The initial colonization of bacteria and diatoms on a immersed artificial substrate and the development of diatom assemblages in relation to physico-chemical parameters were investigated on a weekly basis at one station in the marine Lake Mrtvo More, South Croatia, from April to October 2016. According to TRIX trophic index, lake showed different trophic character: (i) oligotrophic (at the beginning and the end of the study), (ii) mesotrophic (the end of June to mid-July), (iii) eutrophic (the end of July to mid-September). Heterotrophic bacteria increased to peak abundances (69,268 cells cm–2) at the beginning of June when the diatoms abundances start to increase. The lake has high diatom species richness (285 diatom taxa within 72 genera), with the highest species diversity index in August. Among diatoms, adnate were the primary colonizers, particularly Cocconeis dirupta W.Gregory var. flexella (Janisch and Rabenhorst) Grunow and Cocconeis scutellum Ehrenberg var. scutellum, while motile taxa joined the fouling communities from July to September. This study showed close relationship between diatom species composition and changes of physico-chemical parameters, particularly the nutrient concentrations.
The structure of microbial communities, microalgae, heterotrophic protozoa and fungi contributes to characterize food webs and productivity and, from an anthropogenic point of view, the qualitative characteristics of water bodies. Traditionally, in freshwater environments many investigations have been directed to the study of pelagic microalgae (“phytoplankton”) and periphyton (i.e., photosynthetic and mixotrophic protists) through the use of light microscopy (LM). While the number of studies on bacterioplankton communities have shown a substantial increase after the advent of high-throughput sequencing (HTS) approaches, the study of the composition, structure, and spatio-temporal patterns of microbial eukaryotes in freshwater environments was much less widespread. Moreover, the understanding of the correspondence between the relative phytoplankton abundances estimated by HTS and LM is still incomplete. Taking into account these limitations, this study examined the biodiversity and seasonality of the community of eukaryotic microplankton in the epilimnetic layer of a large and deep perialpine lake (Lake Garda) using HTS. The analyses were carried out at monthly frequency during 2014 and 2015. The results highlighted the existence of a rich and well diversified community and the presence of numerous phytoplankton taxa that were never identified by LM in previous investigations. Furthermore, the relative abundances of phytoplankton estimated by HTS and LM showed a significant relationship at different taxonomic ranks. In the 2 years of investigation, the temporal development of the whole micro-eukaryotic community showed a clear non-random and comparable distribution pattern, with the main taxonomic groups coherently distributed in the individual seasons. In perspective, the results obtained in this study highlight the importance of HTS approaches in assessing biodiversity and the relative importance of the main protist groups along environmental gradients, including those caused by anthropogenic impacts (e.g., eutrophication and climate change).
In many marine littoral and sublittoral benthic habitats, we find small diatoms with few features resolvable with light microscopy (LM) other than internal costae across their valves. While classically those internal costae have defined their identification and classification, the use of electron microscopy and of molecular data have started to reveal the true diversity of unrelated forms and genera (e.g., Anaulus, Eunotogramma, Hustedtiella, or Plagiogramma) which possess these structures. Here we describe the new genus Ambo, in an attempt to clarify some of the polyphyly of taxa with internal costa by formally transferring Anaulus balticus, Anaulus simonsenii, and Plagiogramma tenuissimum as well as Ambo gallaeciae, described here. Related to this, we attempt to document and characterize the genus Anaulus itself, which was formally described by Ehrenberg with an illustration. A search by LM of mica designated by Ehrenberg as the holotype of Anaulus scalaris, the generitype of Anaulus, failed to recover a specimen which adequately describes the genus to the exclusion of other genera with internal costa. We also present morphological and molecular data for Anaulus creticus and suggest a new genus—Ceratanaulus—to reflect the distinct morphological and molecular characters we documented.
Recent years provided intense progression in the implementation of molecular techniques in a wide variety of research fields in ecology. Biomonitoring and bioassessment can greatly benefit from DNA metabarcoding and High-Throughput Sequencing (HTS) methods that potentially provide reliable, high quantity and quality standardized data in a cost- and time-efficient way. However, DNA metabarcoding has its drawbacks, introducing biases at all the steps of the process, particularly during bioinformatics treatments used to prepare HTS data for ecological analyses. The high diversity of bioinformatics methods (e.g., OTU clustering, chimera detection, taxonomic assignment) and parameters (e.g., percentage similarity threshold used to define OTUs) make inter-studies comparison difficult, limiting the development of standardized and easy-accessible bioassessment procedures for routine freshwater monitoring. In order to study and overcome these drawbacks, we constructed four de novo indices to assess river ecological status based on the same biological samples of diatoms analyzed with morphological and molecular methods. The biological inventories produced are (i) morphospecies identified by microscopy, (ii) OTUs provided via metabarcoding and hierarchical clustering of sequences using a 95% similarity threshold, (iii) individual sequence units (ISUs) via metabarcoding and only minimal bioinformatical quality filtering, and (iv) exact sequence variants (ESVs) using DADA2 denoising algorithm. The indices based on molecular data operated directly with ecological values estimated for OTUs/ ISUs/ ESVs. Our study used an approach of bypassing taxonomic assignment, so bias related to unclassified sequences missing from reference libraries could be handled and no information on ecology of sequences is lost. Additionally, we showed that the indices based on ISUs and ESVs were equivalent, outperforming the OTU-based one in terms of predictive power and accuracy by revealing the hidden ecological information of sequences that are otherwise clustered in the same OTU (intra-species/intra-population variability). Furthermore, ISUs, ESVs, and morphospecies indices provided similar estimation of site ecological status, validating that ISUs with limited bioinformatics treatments may be used for DNA freshwater monitoring. Our study is a proof of concept where taxonomy- and clustering-free approach is presented, that we believe is a step forward a standardized and comparable DNA bioassessment, complementary to morphological methods.
Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbcL, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbcL sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database.
Suspended marine particles constitute most of the particulate organic matter pool in the oceans thereby providing substantial substrates for heterotrophs, especially in the mesopelagic. Conversely, sinking particles are major contributors to carbon fluxes defining the strength of the biological carbon pump (BCP). This study is the first to investigate the differential influence of eukaryotic communities to suspended and sinking particles, using 18S rRNA gene sequencing on particles collected with a marine snow catcher in the mixed layer and upper-mesopelagic of the Scotia Sea, Southern Ocean. In the upper-mesopelagic, most eukaryotic phytoplankton sequences belonged to chain-forming diatoms in sinking particles and to prymnesiophytes in suspended particles. This suggests that diatom-enriched particles are more efficient in carbon transfer to the upper-mesopelagic than those enriched in prymnesiophytes in the Scotia Sea, the latter more easily disintegrating into suspended particles. In the upper-mesopelagic, copepods appeared most influential on sinking particles whereas soft-tissue metazoan sequences contributed more to suspended particles. Heterotrophic protists and fungi communities were distinct between mixed layer and upper mesopelagic, implying that few protists ride along on sinking particles. Furthermore, differences between predatory flagellates and radiolarians between suspended and sinking particles implied different ecological conditions between the two particles pools, and roles in the BCP. Molecular analyses of sinking and suspended particles constitute powerful diagnostic tools to study the eukaryotic influence on the BCP in a more holistic manner compared to classic carbon export studies focusing on sinking particles.
First described from the Mascarenes (Rodrigues Island, Indian Ocean), Olifantiella gorandiana Riaux-Gobin was later observed from several other oceanic basins. We here examine the morphology of this taxon from a scraping of a juvenile green turtle Chelonia mydas Linnaeus, named 'G5-Manahere', from Moorea Island (Society Archipelago, South Pacific). In this sample, a great range of morphologies, not previously described for the species, were encountered, demonstrating a high degree of polymorphism with regard to valve shape and also stria pattern. These morphological differences appear to be associated with size, as smaller cells become more rounded. The fine structure (e.g., stria density and pattern) may also be associated with changes in shape. These specimens may all belong to the same and unique taxon: Olifantiella gorandiana. This high degree of polymorphism is described, and put into the context of the constraining epizoic conditions. This study permits furthers the description of the fine structure of O. gorandiana, using focused ion beam (FIB) and scanning electron microscopy (SEM) techniques.
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.
Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data.
Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding)requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD)and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD)and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants)are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%)in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.
Benthic macrophytes (macroalgae and angiosperms) are proposed as biological elements to assess the ecological status of coastal and transitional waters within the EU Water Framework Directive. In the present paper, a sampling of benthic macrophytes was made from 0-5 m depth at 25 different sites of the Sea of Marmara (Turkey) in 2015 and the Marine Floristic Ecological Index (MARFEI) was applied. Macrophytes were classified into five ecological status groups: ESGI (IA, IB, IC; late-successional taxa such as Cystoseira spp., angiosperms, calcareous algae) and ESGII (IIA, IIB; opportunistic taxa such as filamentous and sheet-like green algae). The study revealed good ecological status class (ESC) for 1 site, moderate for 11 sites, poor for 10 sites, and bad for 3 sites. MARFEIEQR showed a strong negative linear relationship with the pressure index MALUSI. Nevertheless, the results of this study have to be validated by similar studies in other georgaphical areas.
Diatoms constitute a diverse lineage of unicellular organisms abundant and ecologically important in aquatic ecosystems. Compared to other protists, their biology and taxonomy are well-studied, offering the opportunity to combine traditional approaches and new technologies. We examined a dataset of diatom 18S rRNA- and rDNA- (V4 region) reads from different plankton size-fractions and sediments from six European coastal marine sites, with the aim of identifying peculiarities and commonalities with respect to the whole protistan community. Almost all metabarcodes (99.6%) were assigned to known genera (121) and species (236), the most abundant of which were those already known from classic studies and coincided with those seen in light microscopy. rDNA and rRNA showed comparable patterns for the dominant taxa, but rRNA revealed a much higher diversity particularly in the sediment communities. Peculiar to diatoms is a tight bentho-pelagic coupling, with many benthic or planktonic species colonizing both water column and sediments and the dominance of planktonic species in both habitats. Overall metabarcoding results reflected the marked specificity of diatoms compared to other protistan groups in terms of morphological and ecological characteristics, at the same time confirming their great potential in the description of protist communities.
We obtained the complete mitogenome of Proschkinia sp. strain SZCZR1824, a strain belonging to a poorly known diatom genus with no previous molecular data. This genome is 48,863 bp long, with two group I introns in rnl and three group II introns in cox1. Using mitogenomic data, Proschkinia sp. was recovered with Fistulifera solaris, far distant from Navicula and Nitzschia, two genera with which Proschkinia has sometimes been associated based on morphology.
Benthic diatoms are ubiquitous in tidal flats and play major roles in maintaining coastal ecosystems. Spatio-temporal variations in diatom diversity have not been well-studied, mainly because of difficulties in morphological identification and the lack of appropriate genetic tools. To overcome these problems, we used the gene encoding the ribulose bisphosphate carboxylase large-subunit (rbcL) as a molecular marker, and sequenced these genes with the aid of the MiSeq platform. In this manner, we explored the genetic diversity of benthic diatoms in tidal flats of Guenso Bay on the west coast of Korea; differences in the spatial distributions of benthic diatoms were evident. The diatom communities were dominated by Nitzschia, Navicula, and Amphora; their relative distributions were affected by the sand proportion, grain size, and air exposure time. Our results suggest that meta-barcoding of the rbcL gene and next-generation sequencing can be used to explore the diversity of benthic diatoms.
In order to contribute understanding of the biodiversity of seaweed-associated diatoms at Moen Island in Chuuk lagoon, Micronesia in the tropical western Pacific, we collected four macroalgae (Halimeda sp., Padina sp., Sargassum sp., Dictyota sp.) that were distributed throughout at 5 subtidal sites of reef flat. These are the first records of diatoms from Chuuk, Federated States of Micronesia. A total 148 taxa belonging to 30 families and 49 genera were briefly documented with the identification references, dimension and illustrations, but many taxa could only be identified to genus. There is much similarity to the flora of Guam, but 16 records are new for the region. Four nomenclatural changes were necessary in consequence of the findings of Ruck et al. (2016) on Surirellales and Rhopalodiales.
A DNA based metabarcoding approach to assess diatom communities in rivers
Project summary SC140024
This project has established a novel, DNA based method to monitor and assess the make-up of diatom communities in rivers. We report the results of the first large scale development and testing of a metabarcoding method (Figure 1); combining DNA barcodes with high throughput next generation sequencing (NGS), for the ecological assessment of diatoms.
We collect data on animal and plant life in the river to help us to understand, assess and manage the health of the environment. Diatoms, with around 2800 UK freshwater species recorded, are a type of microscopic algae used alongside other organisms to assess the ecological status of a river.
Diatoms can be found attached to submerged surfaces such as stones and on plant stems in a river, and are commonly referred to as phytobenthos. They are sensitive to changes in environmental conditions and the diatom community (type of diatom species and their relative abundance) in a river is largely influenced by the surrounding water quality. This means that diatoms are valuable indicators of nutrient enrichment and other pressures. We have used the information from diatom communities to develop indices that allow us to detect the impact of nutrient enrichment, primarily phosphorus, on a river. To determine the make-up of these communities we have to date used a light microscope (LM) approach which is a time-consuming process, requiring highly skilled biologists to identify the diatoms.
This new metabarcoding approach will mean we can analyse a lot more samples much quicker using high throughput technology. For example using metabarcoding, diatom samples will be analysed in batches of around 200. The typical analysis time (Figure 1 Steps 2–5) for 200 samples will be approximately 15 working days. It currently takes 3–4 hours to analyse one diatom sample using LM which adds up to about 100 working days to analyse the same 200 samples.
About the new method
The new method for diatom analysis uses NGS to sequence small pieces of DNA (DNA barcodes) that are unique to different diatom species and which can be used to identify the different diatom species in a community. The new method developed was tested by comparing it to the LM method using approximately 620 diatom samples from UK rivers with different water quality.
Whilst the two approaches measure different things, we have demonstrated good agreement between the two methods (significant correlation r = 0.75 and 0.77 in high and low alkalinity rivers respectively). The metabarcoding method uses many of the high priority species that inform our assessments (around 10% of freshwater diatom species described in the UK (Figure 1, Step 5). We recognise that incremental improvements could be made as more diatom species are added to the barcode database. In addition, we found a lot of information in the metabarcoding data (e.g. other types of algae) that is currently not used as part of the assessment. With further development we could take account of this added information to help improve our understanding of the environmental quality of our rivers.
Further work planned
Although we have demonstrated good agreement, there are some differences in the outputs of some samples that require further investigation. Work is underway to explore these. This, alongside further data collection by other UK regulatory agencies in 2017, will refine the method further in 2018.
This project is part of a wider programme of research by the Environment Agency and other UK agencies to explore and develop DNA based methods for ecological assessment. This work will help us to approach the development of DNA based methods for other organism groups and water body types. Examples of other work include assessing the feasibility of using DNA based methods for fish in lakes, macroinvertebrates in rivers and lakes, and for the monitoring and surveillance of non-native invasive species.
We present here an ultrastructure-based revision of the diatom genus Anorthoneis using both light (LM) and electron microscopy (SEM). The original slides and type material of all known Anorthoneis species were analysed and new, previously unseen, morphological features of the frustules, such as rota-like vela and ligulate structures around the valve margin, were revealed. The phylogenetic relationships among Anorthoneis taxa were investigated based on morphological characters using a maximum parsimony method. This analysis indicated a large, well-supported clade that included taxa that exhibited similarities in external and internal details of the central areas in both raphe-sternum and sternum valves. Revealed synapomorphies within the genus include externally and internally transversely dilated central area on both sides of the raphe-sternum valve, externally transversely dilated central area on both sides of the sternum valve, sternum valve striae parallel at the center and radiate at the apices, and areolae with hymenes in both valves. During a survey a new species Anorthoneis arthus-bertrandii sp. nov., was found on seagrass leaves collected from Siladen Island (Indonesia). This taxon is sister to A. hyalina, but can be easily differentiated from the latter by smaller valve dimensions, central area size and shape, and rota-like vela occluding sternum valve areolae - a feature that has never been observed in other Anorthoneis taxa.
High-throughput sequencing of 16S ribosomal RNA gene amplicons has facilitated understanding of complex microbial communities, but the inherent noise in PCR and DNA sequencing limits differentiation of closely related bacteria. Although many scientific questions can be addressed with broad taxonomic profiles, clinical, food safety, and some ecological applications require higher specificity. Here we introduce a novel sub-operational-taxonomic-unit (sOTU) approach, Deblur, that uses error profiles to obtain putative error-free sequences from Illumina MiSeq and HiSeq sequencing platforms. Deblur substantially reduces computational demands relative to similar sOTU methods and does so with similar or better sensitivity and specificity. Using simulations, mock mixtures, and real data sets, we detected closely related bacterial sequences with single nucleotide differences while removing false positives and maintaining stability in detection, suggesting that Deblur is limited only by read length and diversity within the amplicon sequences. Because Deblur operates on a per-sample level, it scales to modern data sets and meta-analyses. To highlight Deblur’s ability to integrate data sets, we include an interactive exploration of its application to multiple distinct sequencing rounds of the American Gut Project. Deblur is open source under the Berkeley Software Distribution (BSD) license, easily installable, and downloadable from https://github.com/biocore/deblur .
IMPORTANCE Deblur provides a rapid and sensitive means to assess ecological patterns driven by differentiation of closely related taxa. This algorithm provides a solution to the problem of identifying real ecological differences between taxa whose amplicons differ by a single base pair, is applicable in an automated fashion to large-scale sequencing data sets, and can integrate sequencing runs collected over time.
Sequence-based approaches to study microbiomes, such as 16S rRNA gene sequencing and metagenomics, are uncovering associations between microbial taxa and a myriad of factors. A drawback of these approaches is that the necessary sequencing library preparation and bioinformatic analyses are complicated and continuously changing, which can be a barrier for researchers new to the field. We present three essential components to conducting a microbiome experiment from start to finish: first, a simplified and step-by-step custom gene sequencing protocol that requires limited lab equipment, is cost-effective, and has been thoroughly tested and utilized on various sample types; second, a series of scripts to integrate various commonly used bioinformatic tools that is available as a standalone installation or as a single downloadable virtual image; and third, a set of bioinformatic workflows and tutorials to provide step-by-step guidance and education for those new to the microbiome field. This resource will provide the foundations for those newly entering the microbiome field and will provide much-needed guidance and best practices to ensure that quality microbiome research is undertaken. All protocols, scripts, workflows, tutorials, and virtual images are freely available through the Microbiome Helper website (https://github.com/mlangill/microbiome-helper/wiki).
Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom.
Database URL: http://www.rsyst.inra.fr/
Significance
Diatoms, considered one of the most diverse and ecologically important phytoplanktonic groups, contribute around 20% of global primary productivity. They are particularly abundant in nutrient-rich coastal ecosystems and at high latitudes. Here, we have explored the dataset generated by Tara Oceans from a wide range of oceanic regions to characterize diatom diversity patterns on a global scale. We confirm the dominance of diatoms as a major photosynthetic group and identify the most widespread and diverse genera. We also provide a new estimate of marine planktonic diatom diversity and a global view of their distribution in the world’s ocean.
Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present into the environment. In this study, we tested if an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro, and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) versus 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems. This article is protected by copyright. All rights reserved.
Low-inflow estuaries are naturally more susceptible to anthropogenic stressors, compared to well flushed systems, with
excessive nutrient loading posing a particular threat. This study investigated the benthic diatom community structure of two
eutrophic, microtidal estuaries impacted by daily wastewater effluent discharges. It was hypothesised that the community
structure would be similar between the warm-temperate Hartenbos and subtropical uThongathi estuaries due to disproportionally high dissolved inorganic nitrogen (H: 38 kg DIN d−1; uT: 67.5 kg DIN d−1) and phosphorus (H: 22 kg DIP d−1; uT:
29.7 kg DIP d−1) inputs from wastewater treatment works (WWTWs). Taxa tolerant of high nutrient conditions proliferated
in both systems. However, the dominant taxa differed with the brackish Halamphora cofeaeformis species occurring in the
Hartenbos Estuary and the freshwater Navicula rostellata, Sellaphora pupula and Navicula gregaria species in the uThongathi Estuary. The overall benthic diatom diversity in both systems was low (H’>0 but≤1.5) and indicative of a degraded
health state. Temporal differentiation driven by salinity was evident in the Hartenbos Estuary, while changes in community
structure were limited to periods of increased river inflow in the uThongathi Estuary. Therefore, while the trophic status of
the dominant taxa was determined by the nutrient stress (primary stressor), changes in salinity and river inflow (secondary
stressors) shaped the distinct community assemblages observed in each estuary. This study provides insight into the impact
of similar anthropogenic-induced pressures in different biogeographical regions and the importance of managing towards a
natural dynamic state of microtidal, low-inflow estuaries.
The Sea of Marmara, located in northwest Turkey, comprises challenging environmental conditions and is an important bridge between the Black Sea and the Aegean Sea. In this study, we assessed the trophic situation of the coasts of the Sea of Marmara using the benthic diatom composition and their relation to the environmental variables in the spring and autumn of 2019 and 2020. A total of 120 samples were collected and 452 species were observed; Navicula Bory and Nitzschia Hassal were represented with the highest numbers of taxa. Analysis of Similarity (ANOSIM) revealed that diatom communities differed significantly between the spring-autumn periods and years. Canonical Correspondence Analysis (CCA) showed that diatoms grouped underwater temperature, salinity, dissolved oxygen, and TIN influence. TRIX observations revealed a highly mesotrophic-eutrophic status along the coasts. Our study contributes to the knowledge of diatom diversity, distribution and community changes in spring and autumn on the coasts Sea of Marmara. Our findings suggest that marine benthic diatoms could be used as eutrophication indicators in the coastal waters for long-term monitoring with the support of environmental parameters.
Karst ecosystems play a unique role as exceptional natural habitats in sustaining biodiversity. This study focuses on diatoms, a diverse group of microeukaryotes in the periphytic community of a karstic river. In a multi-microhabitat study along the Krka River (Croatia), our goal was to obtain a detailed overview of diatom diversity and community structure using morphological and molecular approaches, and to assess the applicability of eDNA metabarcoding as a reliable tool for biomonitoring assessment. The results revealed a relatively low agreement in the diatom community composition between the two approaches, but also provided complementary information, with no differences in beta diversity detected between microhabitats. The SIMPER analysis underlined the importance of the molecular approach in identifying diatom community composition, due to errors in distinguishing between deposited diatom cells that occurred in the morphological analysis. In contrast, the morphological approach indicated a clear diatom community separation along the river with a strong location effect. Despite certain differences, both approaches provided a feasible assessment of the ecological status according to the relationship to environmental pressures, classifying the Krka River as High (morphological approach) or Good (molecular approach) throughout the most of its course. Moreover, diatom diversity based on both approaches provides a reliable dataset applicable in routine monitoring assessment and offers a deeper understanding of the presented ecological status. The incompleteness of a reference database presents one major drawback of the molecular approach, which needs further updating in order to improve routine diatom metabarcoding.
We investigated the advantages and disadvantages of light microscope (LM)-based identifications and DNA metabarcoding, based on a 312-bp rbcL marker, for examining benthic diatom communities from Mediterranean shallow coastal environments. For this, we used biofilm samples collected from different substrata in the Ebro delta bays. We show that 1) Ebro delta bays harbour high-diversity diatom communities [LM identified 249 taxa] and 2) DNA metabarcoding effectively reflects this diversity at genus- but not species level, because of the incompleteness of the DNA reference library. Nevertheless, DNA metabarcoding offers new opportunities for detecting small, delicate and rare diatom species missed by LM and diatoms that lack silica frustules. The primers used, though designed for diatoms, successfully amplified rarely reported members of other stramenopile groups. Combining LM and DNA approaches offers stronger support for ecological studies of benthic microalgal communities in shallow coastal environments than using either approach on its own.
We present the results of the first ever research on biodiversity and biogeography of marine benthic diatoms from coral reefs of the tropical islands of Indonesia, including West Borneo, East Java, South Celebes, Komodo, Rinca and Flores. The sampled islands offer a broad range of coral reef microhabitats and host diatom assemblages from seaweeds, sea grass, dead corals, scrapes from other biological surfaces, sand, rocks and other solid surfaces. The results of this study are based on the light (LM) and scanning electron microscopy (SEM) identification of diatoms only. Our taxonomic analysis revealed the presence of 911 diatom taxa representing 188 genera. Biodiversity indices such as Shannon, Simpson and Evenness as well as species richness and diatom relative abundance were calculated for each sample examined. We find that these indices were higher for Indonesia than those published for tropical oceanic islands or regions from both hemispheres, e.g. Galapagos and Martinique, but comparable to Madagascar. The non-metric multidimensional analysis showed that the type of habitat from which a particular sample was taken was of greater significance in the taxonomic composition than the geographical aspect. In terms of biogeography, cosmopolitan taxa were dominant in terms of relative abundance, but species richness was higher within tropical zonal and local forms. Many taxa were identified only at the genus level, and may turn out to be new to science.
The occurrence of harmful algal blooms (HABs) is increasing in frequency and intensity in South African estuaries because of eutrophication. This study used DNA metabarcoding to identify the benthic diatom community structure in the eutrophic Sundays Estuary. The recurrent HAB phytoplankton species Heterosigma akashiwo was recorded at bloom concentrations (∼100 µg Chl a l⁻¹) in the brackish, vertically stratified middle reaches of the estuary. In contrast, microphytobenthos biomass increased towards the upper reaches in response to elevated availability of total oxidised nitrogen (NOx) and increased sediment stability. A total of 76 diatom species were identified using the molecular technique. Seven known nutrient-tolerant diatom species indicate a preference for either ammonium or NOx enrichment. Benthic diatom community diversity (H′ < 1) and evenness (J′ < 0.25) were low in the brackish middle reaches of the estuary, where the HABs decreased available light. Heterosigma akashiwo has been found in previous studies to suppress the growth of co-occurring taxa. Future research efforts should focus on verifying this relationship over seasonal timescales given the dominance of H. akashiwo in spring/summer. As the persistently nutrient-enriched state and stable flow conditions of the Sundays Estuary is the key driver of change, management efforts should be geared towards re-establishing the estuary’s natural flow variations and to measures that can mitigate nutrient pollution.
Hyalosira gene sequences are divided into two clades within different families. We examined authentic material of Hyalosira (isotype material of H. obtusangula, synonymous with H. delicatula) and voucher specimens of published sequences, and pooled our observations of Hyalosira-like taxa from benthic and epizoic habitats in several parts of the globe. The two molecular clades corresponded to two morphological groups, with Hyalosira obtusangula associated with Grammatophoraceae. We emend the description and provide lectotypification for Hyalosira and propose Placosira to encompass the taxa in the other clade, associated with Rhabdonemataceae. We propose that Hyalosira has uniseriate to triseriate striae, sometimes different on valve face and mantle. Copulae in most species had shallow septa, though in one they were moderately deep. All species had girdle bands bearing two rows of areolae separated by a midrib. We name five new species of Hyalosira. Morphologies of taxa in the Placosira clade were superficially similar to Hyalosira but differed in having areolae with ricae, a single row of areolae on the girdle bands, and tubular rimoportulae on the valve-face mantle junction. Hyalosira hustedtiana Patrick should revert to its original position in Striatella until the appropriate genus can be determined. We emend Rhabdonematales to encompass Rhabdonemataceae, Grammatophoraceae and Tabellariaceae.
Nutrient stoichiometry and input of trace metals may profoundly affect the growth and community structure of phytoplankton. A bioassay experiment was designed to explore the key components in atmospheric deposition that affect marine phytoplankton growth by adding aerosols and analogues nutrients and Cu to the surface water of the coastal East China Sea (ECS). Our results showed that atmospheric deposition along with the input of phosphate could largely enhance the chlorophyll a (Chl a) concentrations in this eutrophic water. Phosphorus addition lifted the proportions of T. oceanica in Diatoms and B. brevisulcata in Dinoflagellates. T. oceanica replaced S. costatum and became the dominant diatom species after the Chl a peak, probably associated with the N/P ratio approaching to 16. Atmospheric aerosols containing affluent N and little P showed limited promotion to Chl a, and the positive effect was very likely due to the soluble Cu and other trace metals supplied by the aerosol. Moreover, soluble aerosol Cu was found to be conducive to the relative abundance of most dominant class Coscinodiscophyceae, and both soluble aerosol Fe and Cu seemed to be very important for increasing the proportion of S. costatum. Soluble metals could be the key components in aerosols controlling the phytoplankton composition in the eutrophic sea and such impact might exceed affluent P provided by other exogenous sources.
Benthic microalgae play important roles in energy flow and biogeochemistry of coastal ecosystems; however, factors influencing community composition remain largely unknown. Our purpose was to identify and compare spatial and environmental influences on benthic diatom biogeography at regional scales. In summer 2018, we sampled sediment at various spatial scales (0.0001–180 km) from five saltmarshes in South Carolina, USA, and characterized diatom assemblages using DNA metabarcoding. Twenty-three environmental variables from marsh sediments, adjacent creeks, and atmosphere were recorded. Multivariate analyses revealed that the saltmarsh communities were clearly distinct. Community dissimilarity was compared to both geographic distance and environmental differences to determine influences on community structure. Mantel tests and redundancy analysis revealed that spatial influences, in addition to several environmental factors (phosphate, grain size, sediment sorting, sediment moisture, and creek salinity), were significant. Variation partitioning revealed that 26% of community variation was explained by geographic distance alone, whereas 60% could be explained by combined spatial/environmental factors. In contrast, the independent explanatory power of individual environmental factors was negligible. Our findings suggest that spatially structured environmental variation mainly conditioned the saltmarsh diatom biogeography in this region. Dispersal limitation, as evinced by the large pure spatial effect and distance-decay pattern, was also important. Spatial effects were stronger relative to that previously observed in other microbial groups (marine bacteria and phytoplankton) and detected at finer spatial scales. Results support prior studies that suggest body size and dispersal mode are important drivers of metacommunity structure and therefore must be considered when studying aquatic microbial biogeography.
Marine macrophytes (macroalgae and angiosperms) were defined as one of the biological quality elements to assess the ecological quality status of transitional and coastal waters by the European Water Framework Directive (WFD, 2000/60/EC). In the present study, the Ecological Evaluation Index (EEI-c) was tested to assess the anthropogenic impact by using marine benthic macrophytes (macroalgae and angiosperms) and pressures by using MA-LUSI in the Turkish marine waters. Macrophyte samples were collected from 56 stations on the coasts of Turkey (at 17 Black Sea, 15 Marmara Sea, 13 Aegean Sea and 11 Mediterranean coastal water bodies) in the summer period between 2014 and 2016. In total, 209 marine macrophytes (macroalgae and angiosperms) taxa were found in all stations. The study revealed high ecological status class for 15 sites, good for 21 sites, moderate for 6 sites, poor for 10 sites, and bad for 4 sites. The MA-LUSI index was tested from the Corine land cover map which affects 1.5 km buffer zone around the sampling sites. The relationship between the pressure index MA-LUSI and EEI-ceqr (eqr: ecological quality ratio) was also tested, and a negative linear relationship was found between MA-LUSI and EEI-ceqr in the Turkish sites. The present study supports the use of the EEI-c index for ecological quality state classification in a wide area of Turkish coastal waters.
The use of diatoms as bioindicators to detect human-induced change is a globally accepted monitoring tool. DNA metabarcoding is an effective complementary tool to standard microscopic methods for species identification, providing reliable and timeous diatom assessments in a range of aquatic ecosystems. This study evaluated the suitability of the DNA metabarcoding approach for diatom monitoring in the St Lucia Estuary, South Africa. Results obtained from the standard microscopic (morphological) and molecular methods were compared and indicated a low similarity in the vegetated (11%), unvegetated (12%) and epiphytic (12%) habitats at species level for the epipelic and epiphytic diatom communities. The difference between the methods was expected given the challenges pertaining to the incomplete molecular reference database, intragenomic variability, physiological differences (number of chloroplasts) and highly variable microphytobenthos biomass. While the molecular method was unable to identify the presence of all the dominant diatom species (routinely used as ecological indicators), the available molecular operational taxonomic units (MOTUs) provided an effective complementary tool to determine the relevant community diversity estimates required for the application of the South African estuarine health index (EHI).
Dozens of monoclonal cultures of small-celled araphid diatoms from brackish or marine habitats worldwide were analyzed using morphological observations (light and electron microscopy) and molecular data (nuclear-encoded small subunit ribosomal RNA and chloroplast encoded rbcL and psbC). As a result, we established one new genus Gedaniella, distinguished by a shared morphological character (occlusions of branched volae, projecting from the apical sides of the areolae) and some molecular data, including five new species: G. alfred-wegeneri, G. arenaria, G. boltonii, G. panicellus, G. paucistriata and three new combinations: G. flavovirens, G. guenter-grassii and G. mutabilis. Additionally we describe eleven novel species within the existing genus Serratifera: S. andersonii, S. brevis, S. clavata, S. corallina, S. namibica, S. nosybeana, S. parkii, S. punctata, S. rhombica, S. sourniae, S. takanoi and one new combination S. opephoroides. Furthermore, five new taxa or combinations were described within other genera: such as Cratericulifera crinigera, Nanofrustulum wachnickianum, Plagiostriata baltica, Pseudostaurosira madagascariensis and Stauroforma rinceana. Detailed descriptions for established species are also provided: Nanofrustulum shiloi, Opephora pacifica and Pseudostaurosira elliptica. This study strongly suggests that the complete biodiversity of small-celled araphids is still far from known, and many species currently placed in Opephora and Pseudostaurosira need to be further re-investigated.
Although hypotheses have been proposed and developed to interpret the origins and functions of introns, substantial controversies remain about the mechanism of intron evolution. The availability of introns in the intermediate state is quite helpful for resolving this debate. In this study, a new strain of diatom (denominated as DB21-1) was isolated and identified as Luticola sp., in which multiple types of 18S rDNAs (obtained from genomic DNA; lengths ranged from 2,056 bp to 2,988 bp) were obtained. Based on the alignment between 18S rDNAs and 18S rRNA (obtained from cDNA; 1,783 bp), 7 intron insertion sites (IISs) located in the 18S rDNA were identified, each of which displaying the polymorphism of intron presence/absence. Specific primers around each IIS were designed to amplify the introns and the results indicated that introns in the same IIS varied in lengths but terminal sequences were conserved. Our study showed that the process of intron loss happens via a series of successive steps, and each step could derive corresponding introns under intermediate states. Moreover, these results indicate that the mechanism of genomic deletion that occurs at DNA level can also lead to exact intron loss.
Epiphytic diatoms are an important component of death and fossil assemblages in macrophyte-dominated shallow lakes. In the Argentinean Pampas, the lack of studies on the composition, diversity and distribution of epiphytic diatoms limits the extent of the paleoenvironmental inferences that can be made from their fossil record. We studied the composition of epiphytic diatom biofilms living on five macrophytes in a Pampean shallow lake (Nahuel Rucá, Buenos Aires Province). Samples of emergent, free-floating, rooted-floating and submersed macrophytes were collected in triplicate over one year and analyzed for diatoms. Epiphytic assemblages on the free-floating species Azolla filiculoides and Ricciocarpus natans were dominated by Lemnicola hungarica, whereas the remaining macrophytes (the emergent Schoenoplectus californicus, the submersed Ceratophyllum demersum and the rooted-floating Hydrocotyle ranunculoides) were dominated by Cocconeis placentula. Although all assemblages were dominated by epiphytic taxa, low proportions of planktonic and benthic taxa were also recorded. Composition, diversity, richness and evenness of diatom assemblages found on free-floating macrophytes differed significantly from the rest of the samples analyzed. The strong association of the epiphytic diatom, L. hungarica, with free-floating macrophytes, together with the high dominance of C. placentula on emergent and submersed plants provides a potential method for inferring past fluctuations in aquatic vegetation coverage in the palaeolimnological record of Pampean shallow lakes.
Diatoms are excellent ecological indicators of water quality because they are broadly distributed, they show high species diversity and they respond rapidly to human pressures. In Europe, the Water Framework Directive (WFD) gives the legal basis for the use of this indicator for water quality assessment and its management. Several quality indices, like the Specific Polluosensitivity Index (SPI), were developed to assess the ecological quality status of rivers based on diatom communities. It is based on morphological identifications and count of diatom species present in natural biofilms using a microscope. This methodology requires high taxonomic skills and several hours of analysis per sample as 400 individuals must be identified to species level. Since several years, a molecular approach based on DNA metabarcoding combined to High-Throughput Sequencing (HTS) is developed to characterize species assemblages in environmental samples which is potentially faster and cheaper. The ability of this approach to provide reliable diatom inventories has been demonstrated and its application to water quality assessment is currently being improved. Despite optimization of the DNA metabarcoding process with diatoms, few studies had yet extended it at the scale of a freshwater monitoring network and evaluated the reliability of its quality assessment compared to the classical morphological approach.
A new diatom genus, Labellicula, is described from soil samples taken from the subantarctic island Ile de la Possession (Crozet Archipelago). While this naviculoid genus shows some affinities with Brachysira, Diadesmis, Fistulifera, Neidium and Luticola, it presents a combination of characters that is unique: i.e. the presence of a stigma, large, elongated open alveoli and a relatively simple raphe structure. Thus far only one species, Labellicula subantarctica sp. nov., is placed in the genus. The ecology of this species is described and its valve structure compared to that of similar genera.