This study introduces Halamphora witkowskii sp. nov., a newly discovered diatom species from Lake Van, the world's largest soda lake known for its high alkalinity. Detailed morphological and morphometric analyses using light and scanning electron microscopy effectively distinguished this new species from closely related taxa, including Halamphora minima, H. coffeaeformis, and H. tumida. Additionally, culturing studies from live cells led to the establishment of two separate monocultures, which were utilized for phylogenetic analysis using the rbcL molecular marker, further confirming the distinctiveness of Halamphora witkowskii from other known species.

Tryblionella punctata was described by W. Smith in 1853, transferred to Nitzschia by Grunow in 1880, and returned to Tryblionella in 1990 by Round et al., when the genus was restored and 41 species transferred into it from Nitzschia. Meanwhile, the species was claimed to be a synonym of Pyxidicula compressa by Boyer, who made the new combination Nitzschia compressa, though most authors continued to use N. punctata. The 1990 restoration of Tryblionella was not accepted by some, including Andrzej Witkowski et al. in their key work on coastal marine diatoms [2000, Diatom Flora of Marine Coasts], but others did accept it and M. Poulin accordingly made the new combination T. compressa. Although we demonstrate that T. compressa (as currently understood in the literature and exemplified by isolates from a coastal lagoon) has wide valves, an almost marginal raphe system, a strongly undulate valve face and a marginal ridge, like most species assigned to Tryblionella, molecular evidence indicates that it is not related to the group containing the type of Tryblionella, T. acuminata. Instead, it belongs to a clade (Bacillariaceae clade 8B) containing lanceolate Nitzschia species such as N. amphibia, N. inconspicua and N. reskoi, as well as Denticula kuetzingii. Some morphological characters support the same relationship, notably the structure of the girdle, which has alternating wide and narrow bands in the epitheca. Morphology suggests further that T. lanceola is a close relative. Both species need to be removed from Tryblionella but Bacillariaceae clade 8B does not correspond to any single accepted, named genus and there is no named monophyletic group to which they can yet be transferred. Until a consensus is reached about generic circumscriptions in the Bacillariaceae, we suggest that the classification of the two species should revert to the pre-1990 state (i.e. assignment to Nitzschia), to maximize continuity with older literature and maintain the current naming of isolate CCMP561, which is a strain widely used in genomic and biochemical studies. This also solves the problem that, on the basis of Bailey's description and illustrations, 'compressa' cannot be used for this species. In contrast, authentic Smith material and illustrations of T. punctata agree well morphologically, though not perfectly, with the three genetically characterized isolates, allowing them all to be assigned for the moment to N. punctata sensu lato. Witkowski et al.'s caution about the 1990 changes was abundantly justified.

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

Aiming to gain a general picture of rbcL diversity within freshwater diatom species, this study assembles and analyzes multiple metabarcoding datasets spanning various geographical regions. From these datasets, we inferred >10,000 amplicon sequence variants (ASVs) of 263-bp length. More than half of the 1000 most abundant ASVs were recorded in both Eurasia and N America and there was only limited evidence for continent-specific lineages. The geographical range was extended for some species, illustrating the potential of metabarcoding datasets for such checks. For detailed analysis of intraspecific diversity, 73 freshwater species were selected, corresponding to 360 ASVs assigned phylogenetically. We found notable variation, some species being represented by only one or a few ASVs, while others were represented by a higher number. Furthermore, within species, ASVs exhibited different dominance and distribution patterns, in some cases with a head-tail pattern, in others a more equal spread of abundance or unresolved reticulate relationships. Except for Ulnaria ulna, no geographical structure among species' ASVs was detectable in haplotype networks using the 263-bp rbcL marker. Observed heterogeneity within species was categorized by computing several metrics of genetic variation and classified into three groups, reflecting optimal sampling strategies based on the patterns of intraspecific variation in the 73 target species There was a significant relationship between intraspecific diversity and the traditional separation between 'centric' and 'pennate' diatoms, with centric species exhibiting significantly fewer variants than pennates, possibly because of different plastid inheritance patterns.

The development of the auxospore is reported in the surirelloid diatom Campylodiscus cf. neofastuosus. As in most pennate diatoms, growth of the auxospore is accompanied and constrained by the formation of a transverse perizonium composed of finely structured bands, which are added one after another as the auxospore expands. However, unlike in most pennate diatoms, in which the transverse perizonium develops bidirectionally from a hoop-like or shortly cylindrical primary band formed around the equator of the zygote, in C. cf. neofastuosus, development is unidirectional, outward from a heart-shaped cap at one end of the auxospore. Limited evidence from other surirelloid diatoms suggests that unidirectional perizonium development may be typical of the group. This developmental pattern correlates with the profound symmetry shifts that have occurred during the evolution of the circumferential raphe system of Surirellaceae and may have a common origin with these, despite the difference in life-cycle stage. The longitudinal perizonium of Campylodiscus also exhibits differences from the arrangement typical of most raphid diatoms, since it comprises three concentrically organized bands, including a unique, heavily silicified secondary band, which runs around the whole circumference of the wide primary band. In this and some other Surirellaceae, the longitudinal perizonium seems to play an important role in the morphogenesis of the initial cells by controlling the shape of the protoplast during a contraction before the formation of the initial epivalve. Small extra perizonial elements, unlike any reported previously in raphid diatoms, were detected beneath the suture formed by the ends of the transverse perizonial bands.

Tryblionella (Bacillariaceae) was described by Smith in 1853, and for many years, the diagnosis of this genus was questionable. Recent molecular analysis based on the rbcL gene marker suggests that Tryblionella is a polyphyletic genus with T. apiculata, T. hungarica, and T. gaoana forming a distinct group from other Tryblionella representatives. Therefore, this study aimed to clarify the diagnosis of Tryblionella as a genus. The focus of this study was a selected group of species previously categorized within Grunow's section Apiculatae, which includes the type species T. acuminata. This classification serves as a foundation for conducting morphological and molecular comparisons with taxa of Tryblionella sensu lato, which are likely to represent distinct and highly diverse genera. Our review includes a detailed examination of frustule ultrastructure and ontogeny combined with a new molecular phylogenetic analysis derived from a three-gene concatenated dataset. The results of our research indicated that among Tryblionella, several monophyletic groups of taxa can be distinguished, including Tryblionella sensu stricto (s.s.), by three key characters: a porose valve cross-section; longitudinal valve undulation, where the peak of the undulation is located on the proximal side of the valve; and the presence of an axial sternum with thickened and relief virgae. Tryblionella s.s. taxa share a similar girdle structure: The girdle is graded, except that the first band bears a single row of poroids in the pars exterior and a crenulate margin on the side corresponding to the distal valve mantle.

In this study, we describe Halamphora vantushpaensissp. nov., a newly identified diatom species found in the highly alkaline Lake Van in Eastern Turkey (Türkiye). This new species is characterized morphologically by light and scanning electron microscopy, performed on both wild and cultivated samples. Two monoclonal cultures were submitted to a genome-skimming approach, giving access to the complete sequence of their nuclear rRNA cluster of genes, mitochondrial and plastid genomes. Both strains were highly similar from the genomic point of view, with few mutations noted, although in organellar genomes some of them concerned protein coding genes and were non-silent. Also, the group II intron in the mitochondrial cox1 gene was found to display a relatively high number of polymorphisms. The plastome also distinguishes itself from other Halamphora spp. by the extension of its inverted repeat at the expense of the two single copy regions of the genome. Maximum likelihood molecular phylogeny inferred from a concatenated three genes dataset (18S, psbC and rbcL) positions this species within the K clade, which is known to contain hypersaline to freshwater species.

In most diatoms, cell size decreases progressively during vegetative divisions and recovers after sexual reproduction, as a special zygote called the auxospore expands in volume and forms the maximum cell size for the species within it. Although auxospore structure shows significant diversity, including incunabular scales, bands, and strips, there is still limited information available on them compared to vegetative cells. This is mainly due to the short duration of the sexual phase in the diatoms' life cycle compared to the vegetative phase and the challenge of observing these stages in natural samples. In this study, we provide a comprehensive account of sexual reproduction in Amphora commutata, where two cells pair via the girdle, each forming two gametes that fuse isogamously in the space between the gametangia. The auxospores expand parallel to each other and perpendicular to the long axes of the gametangia. Nuclear behaviour during sexual reproduction was revealed using Feulgen staining, which is uncommon in diatom research, showing an intense DNA-specific stain against a colourless background. The fine structure of the auxospore wall is also documented: the perizonium is composed of transverse and longitudinal perizonial bands, with incunabular scales that vary in shape from circular to highly elongated. The elongated scales possess a unique structure in this species, having a straight rib along their long axis, clearly differing from the 'simple scales' reported in many diatoms, which are circular or elliptical in outline with a ring-like annulus as a pattern centre.

Nitzschia soratensis and N. inconspicua are two small diatoms that are extremely similar in the light microscope though separable in subtle aspects of valve and girdle ultrastructure. They are not closely related in molecular phylogenies and differ in their ecological preferences, though they sometimes co-occur in the same communities. To test further their functional equivalence, we investigated the reproductive biology of N. soratensis, for comparison with a previously published account of N. inconspicua. Both species are automictic, lacking pairing between gametangia and gamete exchange. Nuclear staining shows the presence of two nuclei in some auxospores of N. soratensis, which are smaller than the nuclei of vegetative cells and appear to be haploid, indicating prior meiosis. These auxospores, surrounded by incunabula containing tangles of silica strips, give rise to uninucleate initial cells 18-21 mu m long. Frequently, however, small N. soratensis cells produce two spherical 'pseudogametes', some of which abort while others expand and form initial valves shorter than those produced by binucleate auxospores (maximum length 14.5 mu m). Similar uniparental auxosporulation, with silica strip formation, occurs in N. aff. hantzschiana, N. acidoclinata, N. fonticola and N. angustata, which are close relatives of N. soratensis according to morphological and/or molecular evidence. This group and the N. inconspicua complex may be the diatom equivalents of apomictic angiosperms, e.g. dandelions, and likely contain a multitude of microspecies.

The current article describes Navicula vanseeasp. nov., a new species of diatom from Lake Van, a highly alkaline lake in Eastern Anatolia (Türkiye). The description is based on light and scanning electron microscopy performed on two monoclonal cultures. The complete nuclear rRNA clusters and plastid genomes have been sequenced for these two strains and the complete mitogenome for one of them. The plastome of both strains shows the probable loss of a functional ycf35 gene. They also exhibit two IB4 group I introns in their rrl, each encoding for a putative LAGLIDADG homing endonuclease, with the first L1917 IB4 intron reported amongst diatoms. The Maximum Likelihood phylogeny inferred from a concatenated alignment of 18S, rbcL and psbC distinguishes N. vanseea sp. nov. from the morphologically similar species Navicula cincta and Navicula microdigitoradiata.

DNA metabarcoding has been performed on a large number of river phytobenthos samples collected from the UK, using rbcL primers optimised for diatoms. Within this dataset the composition of non-diatom sequence reads was studied and the effect of including these in models for evaluating the nutrient gradient was assessed. Whilst many non-diatom taxonomic groups were detected, few contained the full diversity expected in riverine environments. This may be due to the performance of the current primers in characterising the wider phytobenthic community and influenced by the sampling method employed, as both were developed specifically for diatoms. Nevertheless, the study identified considerable diversity in some groups, e.g. Eustigmatophyceae and a wider distribution than previously thought for freshwater Phaeophyceae. These results offer a strong case for the benefits of metabarcoding for expanding knowledge of aquatic biodiversity in the UK and elsewhere. Many of the ASVs associated with non-diatoms showed significant pressure responses; however, models that included non-diatoms had similar predictive strength to those based on diatoms alone. Whilst limitations of the primers for assessing non-diatoms may play a role in explaining these results, the diatoms provide a strong signal along the nutrient gradient and other algae, therefore, add little unique information. We recommend that future developments should use ASVs to calculate metrics, with links to reference databases made as a final step to generate lists of taxa to support interpretation. Any further exploration of the potential of non-diatoms would benefit from access to a well-curated reference database, similar to diat.barcode. Such a database does not yet exist, and we caution against the indiscriminate use of NCBI GenBank as a taxonomic resource as many rbcL sequences deposited have not been curated.

Dinoflagellates of the family Kryptoperidiniaceae, known as "dinotoms", possess diatom-derived endosymbionts and contain individuals at three successive evolutionary stages: a transiently maintained kleptoplastic stage; a stage containing multiple permanently maintained diatom endosymbionts; and a further permanent stage containing a single diatom endosymbiont. Kleptoplastic dinotoms were discovered only recently, in Durinskia capensis; until now it has not been investigated kleptoplastic behavior and the metabolic and genetic integration of host and prey. Here, we show D. capensis is able to use various diatom species as kleptoplastids and exhibits different photosynthetic capacities depending on the diatom species. This is in contrast with the prey diatoms in their free-living stage, as there are no differences in their photosynthetic capacities. Complete photosynthesis including both the light reactions and the Calvin cycle remain active only when D. capensis feeds on its habitual associate, the "essential" diatom Nitzschia captiva. The organelles of another edible diatom, N. inconspicua, are preserved intact after ingestion by D. capensis and expresses the psbC gene of the photosynthetic light reaction, while RuBisCO gene expression is lost. Our results indicate that edible but non-essential, "supplemental" diatoms are used by D. capensis for producing ATP and NADPH, but not for carbon fixation. D. capensis has established a species-specifically designed metabolic system allowing carbon fixation to be performed only by its essential diatoms. The ability of D. capensis to ingest supplemental diatoms as kleptoplastids may be a flexible ecological strategy, to use these diatoms as "emergency supplies" while no essential diatoms are available.

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.

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.

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 (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.

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 defined taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies based 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. 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, resulting in a putative recent hybrid. Our study shows that phylogenomic analyses of unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

Two short diatom rbcL barcodes, 331 bp and 263 bp in length, have frequently been used in diatom meta-barcoding 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 naive 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.