Search

Durinskia capensis is a kleptoplastic dinoflagellate species from high intertidal marine rock pools, which can use a variety of diatoms for photosynthesis. However, very few of the diatoms permit indefinite survival of the dinoflagellate and rbcL sequences show that D. capensis isolated from nature contains one of two closely related Nitzschia species as its kleptoplastids. In culture, without a supply of these ‘essential’ Nitzschia cells to replenish the intracellular store of diatom plastids and other organelles, D. capensis eventually loses all its kleptoplastids and dies. Inside Durinskia, diatoms do not possess frustules and so cannot be compared morphologically with free-living forms. Recently, one of the essential Nitzschia species was isolated from the type locality of D. capensis and grown in culture, allowing comparison with similar Nitzschia species, particularly N. agnita and N. kuetzingioides, examined from type material. We conclude that the ‘essential diatom’ of D. capensis differs morphologically from these and other Nitzschia species and it is therefore described as N. captiva sp. nov. Nitzschia agnita and N. kuetzingioides, on the other hand, are conspecific and N. agnita has priority. Nitzschia captiva and N. agnita are extremely similar in valve shape, dimensions, pattern and ultrastructure, but can be separated by their girdle structure. Nitzschia agnita appears to be a freshwater species, though somewhat salt-tolerant. In contrast, N. captiva, which is known principally from records of the kleptoplastids of D. capensis rather than from frustules, is so far marine. info:eu-repo/semantics/acceptedVersion

Two short diatom rbcL barcodes, 331-bp and 263-bp in length, have frequently been used in diatom metabarcoding studies. They overlap in a common 263-bp region but differ in the presence or absence of a 68-bp tail at the 5′ end. Though the effectiveness of both has been demonstrated in separate biomonitoring and diversity studies, the impact of the 68-bp non-shared region has not been evaluated. Here we compare the two barcodes in terms of the values of a biotic index (IPS) and the ecological status classes derived from their application to an extensive metabarcoding dataset from United Kingdom rivers; this comprised 1703 samples and was produced using the 331-bp primers. In addition, we assess the effectiveness of each barcode for discrimination of genetic variants around and below the species level. The strong correlation found in IPS values between barcodes (Pearson's R = 0.98) indicates that the choice of the barcode does not have major implications for current WFD ecological assessments, although a very few sites (55: 3.23% of those analysed) were downgraded from an acceptable WFD class (“good”) to an unacceptable one (“moderate”). Analyses of the taxonomic resolution of the two barcodes indicate that for many ASVs, the use of either marker – 263-bp and 331-bp – gives unambiguous assignations at species level though with differences in bootstrap confidence values. Such differences are caused by the stochasticity involved in the naïve Bayesian classifier used and by the fact that genetic distance, regarding closely related species, is increased when using the 331-bp barcode. However, in three cases, species differentiation fails with the shorter marker, leading to underestimates of species diversity. Finally, two ASVs from Nitzschia species evidenced that the use of the shorter marker can sometimes lead to false positives when the extent and nature of infraspecific variation are poorly known. info:eu-repo/semantics/acceptedVersion

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings the shape of the terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear-encoded SSU (18S) rDNA and chloroplast-encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under-scoring of this genus in marine plankton.

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (, The authors wish to thank the financial support of the European Union through the project LIFE18 ENV/ES/000199 and the Spanish Ministry of Science, Innovation and Universities through Project CTM2017-91355-EXP. Finally, European Commission (Erasmus program) and Government of Chile for supporting Lorenzo Rambaldo and Héctor Avila Cortés's visit at IDAEA-CSIC in Barcelona, Spain.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically based taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Subsequent gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies and a resulting putative recent hybrid. Our study shows that phylogenomic analyses of many unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

Our study evaluates differences in the distribution and ecology of genetic variants within several ecologically important diatom species that are also key for Water Framework Directive monitoring of European rivers: Fistulifera saprophila (FSAP), Achnanthidium minutissimum (ADMI), Nitzschia inconspicua (NINC) and Nitzschia soratensis (NSTS). We used DADA2 to infer amplicon sequence variants (ASVs) of a short rbcL barcode in 531 environmental samples from biomonitoring campaigns in Catalonia and France. ASVs within each species showed different distribution patterns. Threshold Indicator Taxa ANalysis revealed three ecological groupings of ASVs in both ADMI and FSAP. Two of these in each species were separated by opposite responses to calcium and conductivity. Boosted regression trees additionally showed that both variables greatly influenced the occurrence of these groupings. A third grouping in FSAP was characterized by a negative response to total organic carbon and hence was better represented in waters with higher ecological status than the other FSAP ASVs, contrasting with what is generally assumed for the species. In the two Nitzschia species, our analyses confirmed earlier studies: NINC preferred higher levels of calcium and conductivity. Our findings suggest that the broad ecological tolerance of some diatom species results from overlapping preferences among genetic variants, which individually show much more restricted preferences and distributions. This work shows the importance of studying the ecological preferences of genetic variants within species complexes, now possible with DNA metabarcoding. The results will help reveal and understand biogeographical distributions and facilitate the development of more accurate biological indexes for biomonitoring programmes. info:eu-repo/semantics/acceptedVersion

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. info:eu-repo/semantics/acceptedVersion

Background and aims – The diatom genus Simonsenia has been considered for some time a minor taxon, limited in its distribution to fresh and slightly brackish waters. Recently, knowledge of its diversity and geographic distribution has been enhanced with new species described from brackish-marine waters of the southern Iberian Peninsula and from inland freshwaters of South China, and here we report novel Simonsenia from fully marine waters. Methods – New isolates of Simonsenia species were obtained from marine waters, the littoral zone of the Korean Yellow Sea coast and the Gulf of Mexico in Corpus Christi (Texas), and documented in LM, SEM and with DNA sequence data (plastid-encoded rbcL and psbC). Phylogenetic trees of raphid diatoms were constructed to assess the relationships of the new species and of the genus as a whole. Key results and conclusions – Two novel species of Simonsenia (S. eileencoxiae and S. paucistriata) are described and a further putative taxon is characterized morphologically. The molecular phylogeny of the new Simonsenia species and previously sequenced species supports both the monophyly of the genus and its place within the Bacillariaceae. The Simonsenia clade clusters with clades composed of Cylindrotheca, Denticula and some Nitzschia spp. (including N. amphibia, N. frustulum, N. inconspicua). Hence Simonsenia is firmly positioned within the Bacillariaceae by molecular phylogenies, confirming its position within this group based on the possession of a canal raphe and its ultrastructure, and rejecting its classification within the Surirellaceae. Morphological data from the new Simonsenia species is typical for the genus, with a “simonsenioid” canal raphe type supported over the valve face with fenestral braces, alar canals connecting the canal raphe with the cell lumen, and the presence of fenestrae between the alar canals externally. Our results indicate unequivocally that the biogeography and the biodiversity of Simonsenia remain highly underestimated. info:eu-repo/semantics/publishedVersion

Diatoms are unicellular eukaryotic organisms, which have been exploited over the years for effective freshwater bioassessment. Therefore, they are excellent bioindicators, routinely used in national environmental monitoring programs all over Europe within the Water Framework Directive (WFD) 2000/60 /EC (Foster et al., 2000) and CEN standards (CEN, 2018). Over the years, new technologies have been introduced to this field to maximise and improve the time and cost required for freshwater bioassessment. The application of DNA metabarcoding for the characterisation of benthic diatom communities for WFD was recently introduced (Vasselon et al. 2017, Kelly et al. 2018). Through this technique, the identification of the species present in one environmental sample, is established using genetic variability and is characterised by a short DNA fragment called a barcode (Vasselon et al., 2019). The Wat-Dimon Eurostars project aim at creating a novel genomic test for the identification of European benthic diatoms. This new DNA-based test could be routinely implemented in national environmental monitoring programs all over Europe within the Water Framework Directive (WFD) 2000/60 /EC and CEN standards. In the near future, metabarcoding can complement and/or replace the traditional ecological assessments based on the morpho-taxonomy methodology approach needing taxonomic expertise and been subjected to scientific bias. Additionally, the project aims at developing a complementary bioinformatics tool for the biotechnological interpretation of the results. Such product will allow the prompt response to the environmental needs, the early assessment of environmental quality and early treatment response. The study will be developed and validated along a longitudinal gradient in the south part of Europe (Portugal, Spain, Cyprus), including four different biogeographical regions (Macaronesia, Atlantic, Alpine and Mediterranean). The method will cover all steps, from sampling and DNA extraction of diatom assemblages and amplification of DNA barcodes using universal primers for diatoms. The amplified products will be sequenced using Illumina MiSeq. Then, existing bioinformatic pipelines will be adjusted to quality-filter the high number of sequences from the samples and identify them by comparison with reference databases (Diat.Barcode, BOLD, GenBank). Enhancing these databases with diatom species prevalent in the different biogeographical regions assayed will be essential as existing databases are biased to more northerly regions and do not take into consideration harsh, extreme climatic conditions which are prominent in the Mediterranean and Macaronesia regions (Fig. 1). The project focuses on the rbcL gene and will used 18S gene only as an alternative or complementary tool if any problematic taxa appear. CEN, 2018. CEN/TR 17245: Water quality – Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN/TC 230/WG 23 – Aquat. Macrophytes Algae 1–8. https://doi.org/CEN/TR 17245:2018 Foster, D., Wood, A., Griffiths, M., 2000. THE WATER FRAMEWORK DIRECTIVE (2000/60/EC) – AN INTRODUCTION Dave Foster – Policy Advisor (Europe), Aram Wood EP Scientist (Water), Dr Martin Griffiths – Head of Water Quality, Environment Agency, Head Office, Rio House, Waterside Drive, Aztec West, Almon 7–9. Kelly et al. (2018). A DNA based diatom metabarcoding approach for Water Framework Directive classification of rivers. Environment Agency. Vasselon et al. (2017). Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France). Ecological Indicators. 82:1-12 Vasselon, V., Rimet, F., Domaizon, I., Monnier, O., Reyjol, Y., Bouchez, A., 2019. Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: Experience and developments from France Water Framework Directive networks. Metabarcoding and Metagenomics 3, 101–115. https://doi.org/10.3897/mbmg.3.39646

Recent metabarcoding work in European rivers, including our own studies of Mediterranean rivers in Catalonia (Pérez-Burillo et al. 2020), has shown promising agreement between morphology- (LM) and DNA-based (HTS) assessments of diatom periphyton for the Water Framework Directive. However, in 10 out of the 164 Catalan sites we analysed, the ecological status class was downgraded from “Good”/ “High” with LM to “Moderate”/ “Poor”/ “Bad”by HTS. We call these the “critical” sites and they are especially important because the WFD requires remedial action to be taken by water managers for any river stretch with Moderate or lower status. In order to discover the reasons for downgrading we investigated the contribution of each species to the Indice de Polluosensibilité Spécifique [Specific Pollution-sensitivity Index, IPS] using a “leave-one-out” sensitivity analysis, paying special attention to the critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of its very weakly-silicified frustules during sample preparation for LM. Another important bias was brought about by differences between species in the rbcL copy number per cell, which for example affected the relative abundance obtained by HTS for Nitzschia inconspicua and Ulnaria ulna; these species were also identified by the sensitivity analysis as important for the WFD. However, blanket application of a correction factor based on cell volume did not lead to a clear improvement in the agreement between HTS and LM assessments. Only minor IPS discrepancies could be attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a valuable method for identifying priority species for isolation and barcoding that are currently not, or only poorly, represented in the DNA reference database.

We applied DNA metabarcoding to evaluate the ecology of genetic variants within several diatom species that are important for biomonitoring. Benthic diatoms are widely used as bioindicators for biomonitoring programmes, including those for European rivers demanded by Water Framework Directive (WFD). Morphological identification of diatoms at species level is required for assessing the ecological status in biomonitoring programmes. However, this is a time-consuming task and requires expert knowledge. In addition, closely related species, which sometimes are scarcely distinguishable on the basis of their morphology, can show different ecological preferences; these may even vary within a single diatom species. Not being able to identify the different ecological preferences shown by the genetic variants of a single species or closely related species, might have consequences for biomonitoring programmes, especially if such differences occur within common species. The key diatom species that we studied were: Fistulifera saprophila (FSAP), widely regarded as a marker for elevated nutrient levels, organic pollution and hence poor ecological status; Achnanthidium minutissimum (ADMI), which usually indicates good ecological status; and Nitzschia inconspicua (NINC) and N. soratensis (NSTS), two species that are widely separated phylogenetically but almost impossible to distinguish in the light microscope. Our dataset was based on high-throughput sequencing using a 312-bp rbcL marker. We used the denoising pipeline DADA2 to infer amplicon sequence variants (ASVs) from 554 environmental samples from river biomonitoring campaigns in Catalonia (NE Spain) and France. Ecological groupings of ASVs were distinguished according to their environmental responses given by Threshold Indicator Taxa ANalysis (TITAN); the environmental parameters that most influenced the occurrence of these groupings were tested using boosted regression trees. We could distinguish three different ecological groupings of ASVs within ADMI and three within FSAP. In each species two of the groupings were clearly separated by their opposite responses to calcium and conductivity and boosted regression trees showed that for three out of four of these groupings, these two variables were among the most important variables for explaining the ASV distributions. The third grouping in FSAP had a negative response to total organic carbon and a positive response to altitude and hence was better represented in less organically polluted waters and higher ecological status than is generally assumed for FSAP. Our analyses did not identify ecological groupings of ASVs within NINC and NSTS but confirmed earlier studies, based on more limited sampling, that indicated different preferences of these species. Conductivity and calcium were the variables that most influenced the occurrence of NINC and NSTS, NINC being better distributed in waters with higher levels of calcium and conductivity than NSTS. Our findings indicate the potential use of DNA metabarcoding for distinguishing the ecological preferences of genetic variants within a single species or closely related species. This information, coupled with the broad knowledge generated over many years using traditional microscope-based identifications, will facilitate the development of more accurate biological indexes for the biomonitoring programmes of the future.

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

Diatoms (Bacillariophyta) are ubiquitous microalgae, which present a huge taxonomic diversity, changing in correlation with differing environmental conditions. This makes them excellent ecological indicators for various ecosystems and ecological problematics (ecotoxicology, biomonitoring, paleo-environmental reconstruction …). Current standardized methodologies for diatoms are based on microscopic determinations, which is time consuming and prone to identification uncertainties. DNA metabarcoding has been proposed as a way to avoid these flaws, enabling the sequencing of a large quantity of barcodes from natural samples. A taxonomic identity is given to these barcodes by comparing their sequences to a barcoding reference library. However, to identify environmental sequences correctly, the reference database should contain a representative number of reference sequences to ensure a good coverage of diatom diversity. Moreover, the reference database needs to be carefully taxonomically curated by experts, as its content has an obvious impact on species detection. Diat.barcode is an open-access library for diatoms linking diatom taxonomic identities to rbcL barcode sequences (a chloroplast marker suitable for species-level identification of diatoms), which has been maintained since 2012. Data are accumulated from three sources: (1) the NCBI nucleotide database, (2) unpublished sequencing data of culture collections and more recently (3) environmental sequences. Since 2017, an international network of experts in diatom taxonomy curate this library. The last version of the database (version 9.2), includes 8066 entries that correspond to more than 280 different genera and 1490 different species. In addition to the taxonomic information, morphological features (e.g. biovolumes, chloroplasts, etc.), life-forms (mobility, colony-type) and ecological features (taxa preferences to pollution) are given. The database can be downloaded from the website (www6.inrae.fr/carrtel-collection/Barcoding-database/) or directly through the R package diatbarcode. Ready-to-use files for commonly used metabarcoding pipelines (Mothur and DADA2) are also available.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

In this article we describe Nitzschia anatoliensis Górecka, Gastineau & Solak sp. nov., an example of a diatom species inhabiting extreme habitats. The new species has been isolated and successfully grown from the highly alkaline Van Lake in East Turkey. The description is based on morphology (light and scanning electron microscopy), the sequencing of its organellar genomes and several molecular phylogenies. This species could easily be overlooked because of its extreme similarity to Nitzschia aurariae but molecular phylogenies indicate that they are only distantly related. Furthermore, molecular data suggest that N. anatoliensis may occur in several alkaline lakes of Asia Minor and Siberia, but was previously misidentified as Nitzschia communis. It also revealed the very close genetic proximity between N. anatoliensis and the endosymbiont of the dinotom Kryptoperidinium foliaceum, providing additional clues on what might have been the original species of diatoms to enter symbiosis.

Nitzschia is common in the phytoplankton of several East African lakes. A new species, Nitzschia fenestralis, sp. nov. D. Grady, D.G. Mann et Trobajo was encountered at numerous depths in a 16 m sediment core from Lake Babogaya, Ethiopia and is described using light and scanning electron microscopy. It is compared with several other morphologically similar taxa described from East and Central Africa (especially N. aequalis, N. mediocris, N. obsoleta and N. fabiennejansseniana), and from Europe (N. fruticosa). An unusual feature of some of these species (N. fenestralis, N. obsoleta and N. fabiennejansseniana) is that in the raphe canal each stria is represented by two narrower areolae (alternatively interpreted as a single subdivided areola). It is this feature that suggested the name of the new species (through the resemblance to a series of sash windows). Another characteristic of N. fenestralis and N. obsoleta, apparently never reported previously in any diatom, is that the more advalvar bands end approximately halfway along the frustules, rather than at the poles. In most respects (shape and size, stria and fibula densities, valve and girdle structure), N. fenestralis and N. obsoleta are very similar, but confusion is unlikely because they differ in whether central raphe endings are present (N. fenestralis) or absent (N. obsoleta). In Nitzschia fenestralis, and perhaps to a lesser extent in N. obsoleta, the striae usually become strongly radiate towards the poles. A preliminary assessment, based on the literature, suggests that N. fabiennejansseniana may be synonymous with N. obsoleta, which was described earlier. info:eu-repo/semantics/publishedVersion

Our study of 164 diatom samples from Catalonia (NE Spain) is the first to evaluate the applicability of DNA metabarcoding, based on high throughput sequencing (HTS) using a 312-bp rbcL marker, for biomonitoring Mediterranean rivers. For this, we compared the values of a biotic index (IPS) and the ecological status classes derived from them, between light microscope-based (LM) and HTS methods. Very good correspondence between methods gives encouraging results concerning the applicability of DNA metabarcoding for Catalan rivers for the EU Water Framework Directive (WFD). However, in 10 sites, the ecological status class was downgraded from “Good”/“High” obtained by LM to “Moderate”/“Poor”/“Bad” by HTS; these “critical” sites are especially important, because the WFD requires remedial action by water managers for any river with Moderate or lower status. We investigated the contribution of each species to the IPS using a “leave-one-out” sensitivity analysis, paying special attention to critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of weakly-silicified frustules during sample preparation. Differences between species in the rbcL copy number per cell affected the relative abundance obtained by HTS for Achnanthidium minutissimum, Nitzschia inconspicua and Ulnaria ulna, which were also identified by the sensitivity analysis as important for the WFD. Only minor IPS discrepancies were attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a good method for identifying priority species for isolation and barcoding. info:eu-repo/semantics/acceptedVersion

A previously unknown member of the Bacillariaceae was discovered almost simultaneously in four different brackish coastal wetlands on the Atlantic and Mediterranean coasts of the Iberian Peninsula. It appears to tolerate a wide range of salinities but was never common in samples where it occurred. The frustules were consistently hantzschioid (i.e. with the raphe systems always on the same side of the frustule) and the valve outline was asymmetrical about the apical plane, two features that have until recently been considered characteristic of Hantzschia. Molecular phylogenies based on rbcL and LSU rDNA indicated, however, that the new species does not belong in Hantzschia but among the several disparate lineages that comprise the paraphyletic genus Nitzschia. This finding, coupled with the recent discovery of other diatoms with constant hantzschioid symmetry but with a morphology very similar to the type species of Nitzschia, is discussed in relation to the status and characterization of Hantzschi...

We describe here a protocol for cleaning diatoms when time is short and the amount of sample is very limited. Essentially, the method consists of drying material onto coverslips and cleaning it directly in situ using nitric acid (or hydrogen peroxide), which is evaporated to dryness. After washing twice or a few times with deionized water, the coverslips are ready for mounting in resin for light microscopy as usual, or attachment to stubs for scanning electron microscopy. Besides speed, the method has the advantage that it often preserves some frustules intact or leaves their different elements (and stages of valve formation) closely associated with each other. Examples where the method is especially advantageous are to clean small aliquots of cultures for identification or to act as vouchers, or to explore diversity of the most abundant species in natural material (e.g. periphyton). It is less suitable for counts in ecological or palaeoecological studies. We tabulate the many other cleaning methods to provide context for the new method described here. info:eu-repo/semantics/acceptedVersion

This issue is dedicated to Dr. Eileen Joan Cox on the occasion of her 70th birthday. Eileen is a very well-known phycologist who has made a major contribution to diatom research, for instance in terms of scientific papers published and species described (all listed in pages 412–414 of this issue) but also in addressing different topics (e.g. taxonomy of particular groups, the ecology of streams and tube dwelling diatoms, life-history and morphogenesis, valve terminology, the chloroplasts of living diatoms, and many others). The photos on the issue’s cover were chosen to reflect some of these aspects of Eileen’s work: Navicula oblonga refers to her work and advocacy of research on living diatoms; Encyonema refers to her earliest work on diatoms that live inside polysaccharide tubes; and Navicula refers to Eileen’s principal taxonomic focus over many years – the diatoms that used to be classified in the catch-all genus Navicula, which have now been split off and placed into many different genera. However, perhaps the most important contribution of a scientist is one that cannot be so easily measured, and that is the positive impact and influence the scientist makes on her/his colleagues. In this respect, Eileen’s contribution has been huge: she has always facilitated collaborations, welcoming students and visitors and putting facilities at their disposal, always taking care of the person as well as the work. She has also volunteered her time and energy generously to scientific societies, editing journals and many other good causes. Not surprisingly we (editors) did not have to make any effort to convince researchers to contribute to this issue. In fact, we faced the opposite problem in having to restrict the number of papers quite severely to keep the issue manageable. We would like to take this opportunity to apologize to all those who would have liked to contribute but have been left out. info:eu-repo/semantics/publishedVersion

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.

The Bacillariaceae is a very species-rich family of raphid diatoms and includes the large and taxonomically difficult genus Nitzschia, whose species are often small-celled and finely structured and have few discrete morphological characters visible in the light microscope. The classification of Nitzschia is still mostly based on one developed in the second half of the 19th century by Grunow, who separated the genus into a series of sections largely on cell shape and symmetry, the position of the raphe, transverse extension of the fibulae, and folding of the valve. We assembled and analysed single-gene and concatenated alignments of nSSU, nLSU, rbcL, psbC and cox1 to test Grunow’s and subsequent classifications and to examine selected morphological characters for their potential to help define monophyletic groups. The maximum likelihood trees were equivocal as to monophyly of the family itself but showed good support for each of eight main clades of Bacillariaceae, three of which corresponded more or less to existing genera (Hantzschia, Cylindrotheca and Bacillaria). The other five main clades and some subclades comprised groups of Nitzschia species or assemblies of Nitzschia species with other genera (Pseudo-nitzschia, Fragilariopsis, Neodenticula, Tryblionella, Psammodictyon). Relationships between most of the eight main clades were not resolved robustly but all analyses recovered Nitzschia as non-monophyletic. The Grunowian classification of Nitzschia into sections was not supported, though in some respects (e.g. treatment of sigmoid species) it is better than subsequent reclassifications. Several of the main clades and subclades are cryptic (lacking morphological synapomorphies) and homoplasy is common in both light microscopical and ultrastructural characters (to the extent that organisms initially assigned to the same species sometimes prove to belong to a different main clade). Nevertheless, some characters, including the structure of the raphe canal and girdle, seem to be sufficiently conservative evolutionarily to give a provisional estimate of relationships if molecular data are unavailable. No new formal classifications are proposed but various options are explored and research needs identified. info:eu-repo/semantics/acceptedVersion

The ecology and modern distribution of benthic foraminiferal assemblages were analysed in the Ebro Delta (NW Mediterranean Sea). Foraminiferal distributions were from 191 sediment surface samples covering a wide range of deltaic habitats and adjacent open sea areas. According to similarity in species composition, cluster analysis identified four habitat types: (1) offshore habitat, (2) nearshore and outer bays, (3) salt and brackish marshes and (4) coastal lagoons and inner bays. Canonical Correspondence Analysis identified water depth, salinity and sand content as the main environmental factors structuring living foraminiferal assemblages. Partial Canonical Correspondence Analysis revealed water depth as the most statistically significant associated with the distribution of modern foraminifera in the Ebro Delta. Thus, a transfer function for water depth using Weighted Average Partial Least Squares regression was successfully developed. Although depth per se is unlikely to affect the foraminifera directly but will exert its effects via various environmental variables that co-vary with depth in the deltaic habitats (e.g. hydrodynamics, oxygen, food availability, etc), the resulting model ( r 2 = 0.89; RMSEP = 0.32 log 10 m) suggested a strong correlation between observed and foraminifera-predicted water depths, and therefore provided a potentially useful tool for water-depth reconstructions in the Ebro Delta. This work indicated the potential role of modern foraminifera as quantitative indicators of water depth and habitat types in the Ebro Delta. This complementary approach (transfer function and indicator species) will allow reconstruction of the palaeoenvironmental changes that have occurred in the Ebro Delta based on the benthic foraminiferal record.

Major Mediterranean deltas began to develop during a period between 8000 and 6000 yr BP when the rate of fluvial sediment input overtook the declining rate of sea-level rise. However, different authors have argued that the Ebro Delta primarily formed during the late Middle Ages as a consequence of increased anthropogenic pressure on its river basin and these arguments are supported by the scarcity of previous geological studies and available radiocarbon dates. To reconstruct the environmental evolution of the Ebro Delta during the Holocene, we used micropalaeontological analysis of continuous boreholes drilled in two different locations (Carlet and Sant Jaume) on the central delta plain. Different lithofacies distributions and associated environments of deposition were defined based on diagnostic foraminiferal assemblages and the application of a palaeowater-depth transfer function. The more landward Carlet sequence shows an older and more proximal progradational delta with a sedimentary record composed of inner bay, lagoonal and beach materials deposited between 7600 and >2000 yr BP under rising sea-level and highstand conditions. This phase was followed by a series of delta plain environments reflected in part by the Carlet deposits that formed before 2000 yr BP. The Sant Jaume borehole is located closer to the present coastline and contains a much younger sequence that accumulated in the last 2.0 ka during the development of three different deltaic lobes under highstand sea-level conditions. The results of this study reinforce the idea that the Ebro Delta dates to the early Holocene, similar to other large Mediterranean deltas.

Summary Studies quantifying the content of fine particulate matter (organic and inorganic) retained in macrophyte stands are scarce. Thus, little is known about the relationship between retention of fine particulate matter and macrophyte architecture, especially in rivers. Here, we quantified the amount of organic and inorganic fine particulate matter retained by three different macrophyte species having two contrasting plant architectures (‘mesh-like’ and ‘strap-like’), to test the role of plant architecture on retention. Results showed that plant architecture and water velocity were related and their interaction was important for particle sedimentation/retention by macrophyte leaves and stems. The complexity of plant architecture appeared to be the main factor explaining variation in the amount of trapped matter. Increases in water velocity led to a decline of both the organic and the inorganic particulate matter retained by macrophytes, while a decrease in water velocity was related to an increase in macrophyte cover. The concentrations of trapped matter (both organic and inorganic) also differed significantly among seasons, indicating the existence of a seasonal cycle in the retention of fine particulate matter mainly associated with macrophyte cover (and biomass) and water velocity. Although there were seasonal and spatial variations in sediment retention in both ‘mesh-like’ and ‘strap-like’ architectures, macrophytes were effective sediment traps.

A new global database of 86 deltas and river basins was analyzed to investigate the relative importance of deforestation and land use changes versus natural forcings in determining long-term total delta size. Results show that mean river flow and shelf slope were the most important variables, whereas population density and sediment load had a much lower importance. Deforestation and other variables related to land-use generally had a very small effect, but were more influential in a subset comprising Mediterranean and Black Sea deltas. As most deltas have developed over thousands of years, the much shorter-lived anthropogenic signals from deforestation and other landscape perturbations have had only secondary impact on the total area of deltas. Also, delta progradation is strongly influenced on sand deposition, whereas anthropogenic impacts on sediment load have more often impacted mostly the finer sediment being deposited offshore (prodelta deposits) or in the deltaic plain. These data disproves the hypothesis that delta size and growth is strongly influenced by human forcings, particularly for larger deltas, since Holocene delta building is mainly determined by natural forces. However, humans are influencing the geomorphology of deltas, particularly over the last century when the Anthropocene nature of deltas has become manifest. A more precise terminology is proposed to clarify concepts such as “human-made”, “human-engineered” or “human-influenced” deltas. info:eu-repo/semantics/acceptedVersion

Diatoms are used routinely to assess pollution level in rivers and lakes. Current methods are based on identification by light microscopy, which is laborious. An alternative is to identify species based on short DNA fragments and High-Throughput Sequencing (HTS). However a potential limitation is the incomplete coverage of species in reference barcode libraries. Usually these libraries are compiled by isolating cells, before culturing and sequencing them, which is tedious and often unsuccessful. Here we propose the use of rbcL sequences from environmental samples analysed by HTS. We set several criteria to ensure good sequence quality and correspondence with the target species observed in microscopy: the sequence needed to be abundant in the sample, and with no insertions nor deletions or stop codon, phylogenetic neighbour taxa had to correspond to neighbour taxonomic taxa expected from morphological observations. Four species from tropical rivers are given as examples, including one that is new to science. info:eu-repo/semantics/publishedVersion

International audience; 1. In recent years, remarkable progress has been made in developing environmental DNA metabarcoding. However, its ability to quantify species relative abundance remains uncertain, limiting its application for biomonitoring. In diatoms, although the rbcL gene appears to be a suitable barcode for diatoms, providing relevant qualitative data to describe taxonomic composition, improvement of species quantifica-tion is still required. 2. Here, we hypothesized that rbcL copy number is correlated with diatom cell biovol-ume (as previously described for the 18S gene) and that a correction factor (CF) based on cell biovolume should be applied to improve taxa quantification. We carried out a laboratory experiment using pure cultures of eight diatom species with contrasted cell biovolumes in order to (1) verify the relationship between rbcL copy numbers (estimated by qPCR) and diatom cell biovolumes and (2) define a potential CF. In order to evaluate CF efficiency, five mock communities were created by mixing different amounts of DNA from the eight species, and were sequenced using HTS and targeting the same rbcL barcode. 3. As expected, the correction of DNA reads proportions by the CF improved the congruence between morphological and molecular inventories. Final validation of the CF was obtained on environmental samples (metabarcoding data from 80 ben-thic biofilms) for which the application of CF allowed differences between molecular and morphological water quality indices to be reduced by 47%. 4. Overall, our results highlight the usefulness of applying a CF factor, which is effective in reducing over-estimation of high biovolume species, correcting quantitative biases in diatom metabarcoding studies and improving final water quality assessment. K E Y W O R D S benthic diatom, biovolume correction factor, freshwater ecosystems, gene copy number variation, quantitative metabarcoding

This new volume in the Bibliotheca Diatomologica series is said to deal in some detail with ‘120 representative diatom taxa’ (Abstract) selected from 23 samples collected in spring 2010 from coasta...

A microalgal growth model has been developed based upon experiments using three species – (Cylindrotheca closterium, Nannochloropsis gaditana and Phaeodactylum tricornutum) – at three different levels of carbon dioxide (provided by aeration with ambient air, ambient air enriched with 0.5% carbon dioxide, and ambient air enriched with 1% carbon dioxide). We used a two-step growth model for phytoplankton comprising first uptake of the nutrients (C, N and P) and then growth based on the intracellular concentrations of the three nutrients. In addition, the model considered the fraction of cellular carbon that is lipid. The model did not require calibration, as the parameters were based on literature values, allometric principles and direct measurements. The validation of the model gave acceptable results. Based upon both the model itself and experimental results for P. tricornutum, it can be concluded that higher content of organic carbon and lipid would not be obtained by further addition of carbon dioxide beyond 0.5–1%. The production of organic carbon by this species with addition of 1% carbon dioxide could be about 90 mg/l in 24 h, which is consistent with other production results from the literature.

The distributions of Nitzschia inconspicua Grunow and N. soratensis E. Morales & Viz in response to environmental variables were compared using a dataset composed of samples from 46 locations in UK streams and rivers; they were also investigated using laboratory experiments. There were only three samples in which both species occurred with a relative abundance>1%. Nitzschia soratensis preferred water of lower alkalinity, conductivity, reactive phosphorus and total oxidized nitrogen. The different preferences for conductivity of the two species were consistent with the results of the laboratory experiments, although no difference in response to phosphorus was observed in these trials. Interpretation of field results was confounded by correlations among environmental variables. However, taken together with the laboratory experiments, the data suggest that N. inconspicua and N. soratensis are separated primarily by local geology and habitat rather than by human pressures. Discrepancies between the results fr...

The large diatom genus Nitzschia and the morphologically similar Hantzschia are currently distinguished on the basis of cell symmetry and division. In Hantzschia, the eccentrically placed raphe systems of the two valves are always on the same side of the frustule (‘hantzschioid’ symmetry), never diagonally opposite (‘nitzschioid’ symmetry). Although some Nitzschia species produce hantzschioid cells, it has previously been thought that these cells never breed true but divide to give one nitzschioid daughter and one hantzschioid daughter. Sublittoral marine epipelon from Loch Goil, W Scotland, contained three undescribed species of Bacillariaceae whose cells were always hantzschioid, but which possessed a valve structure (silica flaps on either side of the raphe, no central raphe endings, a slit-like entrance to the raphe canal that narrows towards the poles, and discrete bar-like fibulae) linking them to N. spathulata and the type species of Nitzschia, N. sigmoidea. They also agreed with these species in c...

Paedogamy (fusion of gametes produced within the same gametangium following meiosis) has rarely been reported in diatoms, with fewer than 10 confirmed examples. One of these, reported by L. Geitler, was in a diatom from Illmitz, Lake Neusiedl (Austria), identified as ‘Nitzschia frustulum var. perpusilla'. We observed uniparental auxosporulation in two Nitzschia clones isolated from the lower Ebro River (Catalonia, Spain), morphologically similar to Geitler's material and belonging to the N. inconspicua species complex. We established that the auxospores were formed paedogamously by Feulgen staining of the nuclei and time-lapse microscopy of living cells. However, reinvestigation of Geitler's original cytological preparations revealed differences between the Illmitz and Ebro material with respect to the length of the initial cells, the structure of the perizonium, and the timing of degeneration of superfluous haploid nuclei during gametogenesis, indicating a genetic and possibly a taxonomic separation. Sca...

The taxonomy of several small-celled, ecologically significant Nitzschia species, which are frequently confused with each other or whose names are misapplied, is clarified. Following an examination of type material and modern samples by light and electron microscopy, it was concluded that N. frustulum (Kutzing) Grunow, N. inconspicua Grunow, N. soratensis E. Morales & Vis and N. invisitata Hustedt are independent species. No morphological basis was found for separating N. frustulum var. subsalina Hustedt or N. boliviana E. Morales & Vis from N. inconspicua and they are therefore placed in synonymy with N. inconspicua. Nitzschia soratensis, described recently from Bolivia, has previously been misidentified in Europe, either as N. inconspicua (from which it differs most obviously in having more bluntly rounded poles, striae within the raphe canal that are composed of triplets, and fibulae that can be seen in light microscopy to widen at their bases) or as N. abbreviata Hustedt ex Simonsen (from which it dif...

Diatom indices are used to evaluate the ecological status of rivers but they have been rarely applied in estuaries. This study aimed to identify the diatom species indicating the main environmental gradients and pressures in a highly stratified estuary; and to evaluate the applicability of existing freshwater diatom indices. Marine influence due to salt-wedge intrusion and sea water mixing appeared as the main factor affecting diatom community. Three diatom assemblages were identified: indicators of riverine conditions (without marine influence), indicators of estuarine conditions (heterogeneous conditions with higher conductivities due to marine influence) and those specifically indicating well-established salt-wedge situations. Nowadays, the main human pressure affecting diatom community in the Ebro Estuary is the hydrological alteration resulting from flow regulation and abstraction. Several limitations were encountered in the application of diatom indices (e.g. inverse response with nutrients; ecologically important species not considered). Therefore, their use in estuaries should be done cautiously.

The lower Ebro River (Catalonia, Spain) has recently undergone a regime shift from a phytoplankton-dominated to a macrophyte-dominated system. This shift is well known in shallow lakes but apparently it has never been documented in rivers. Two initial hypotheses to explain the collapse of the phytoplankton were considered: a) the diminution of nutrients (bottom-up); b) the filtering effect due to the colonization of the zebra mussel (top-down). Data on water quality, hydrology and biological communities (phytoplankton, macrophytes and zebra mussel) was obtained both from existing data sets and new surveys. Results clearly indicate that the decrease in phosphorus is the main cause of a dramatic decrease in chlorophyll and large increase in water transparency, triggering the subsequent colonization of macrophytes in the river bed. A Generalized Linear Model analysis showed that the decrease in dissolved phosphorus had a relative importance 14 times higher than the increase in zebra mussel density to explain the variation of total chlorophyll. We suggest that the described changes in the lower Ebro River can be considered a novel ecosystem shift. This shift is triggering remarkable changes in the biological communities beyond the decrease of phytoplankton and the proliferation of macrophytes, such as massive colonization of Simulidae (black fly) and other changes in the benthic invertebrate communities that are currently investigated.

The purpose of this study was to explore efficient methods of harvesting the microalga Phaeodactylum tricornutum. Natural sedimentation experiments, performed at different light and temperature conditions, did not yield significant improvements in efficiency even after 1 week. When alkalinity-induced flocculation was performed, both the flocculation efficiency and the concentration factor dramatically improved at pH = 9.75 (0.5–0.7 units over the original pH of the culture) after 10 min settling time. Sedimentation rates are documented at pH ranging between pH 9.75 and 11.0. The results of the application of two conventional flocculants used in wastewater treatment, polyaluminium chloride and aluminium sulphate, are also presented. Chitosan was also used as a natural flocculating agent to improve possible contamination problems in the downstream process. pH was adjusted in order to determine optimum flocculation efficiency of chitosan in combination with a high concentration factor. Satisfactory results were found with chitosan at an adjusted pH of 9.9 using concentrations as low as 20 mg L−1, after testing a flocculant range of 5–200 mg L−1.

This study describes the diatom species Planothidium iberense sp. nov. from a salinity gradient of the Ebro River Estuary in northeastern Spain. A detailed description is given based on light and scanning electron microscopy observations, and the diatom species is compared with morphologically similar Planothidium species. Planothidium iberense is distinguished from P. linkei mainly by its morphometric characteristics, but also by the presence of multiseriate striae, in contrast to biseriate striae in P. linkei. Planothidium iberense is also distinguished from P. delicatulum and P. septentrionale by the presence of an one-side expanded central area on the sternum valve, which is absent in the latter two Planothidium species.

SUMMARY The effects of salinity on the growth and valve morphology of five benthic estuarine diatoms (Nitzschia pusilla, N. frustulum, N. palea, N. filiformis var. conferta and Eolimna subminuscula), isolated from both freshwater and brackish/marine habitats, were investigated. The four Nitzschia strains grew well over a broad salinity range, though some (N. pusilla, N. frustulum) showed a broader salinity range tolerance (from fully saline down to at least 9.5 ppt) than others (N. palea, N. filiformis var. conferta had reduced growth at salinities of 16 ppt and above). Salinity significantly affected the valve morphology of the five strains studied. However, there was no consistent pattern in either the morphological characters affected or the direction of the effects. Although significant, the effects of salinity on valve morphology were very small and therefore it seems that the taxonomic usefulness of some of the classical taxonomical characters is not undermined.

Cox1 has been suggested as a barcode marker for diatoms but it has not been tested intensively in any group of closely related species outside Sellaphora. We evaluated the use of cox1, LSU and rbcL for phylogeny and identification in the taxonomically problematic but ecologically important freshwater diatom Nitzschia palea, for which LSU rDNA sequences, mating and morphological data had already been published, and for which DNA and/or clonal isolates were still available; some new isolates were added. The previous and new information concur in suggesting that N. palea is a complex of several or many species. Where cox1 sequences could be obtained, they were more variable than LSU and rbcL and discriminated between lineages that differed in their morphologies, mating compatibilities, LSU sequences or rbcL sequences. Repeated failures for some strains to recover cox1 sequences from DNA that yielded LSU and rbcL suggest that cox1 will be impractical as a universal barcode marker in diatoms until better prime...