Evolutionary analysis and quality assessment of ζ-carbonic anhydrase sequences from environmental microbiome.

Carbonic anhydrase (CA) is one of the most vital enzymes in living cells. This study has been performed due to the significance of this metalloenzyme for life and the novelty of some CA families like ζ-CA to evaluate evolutionary processes and quality check their sequences. In this study, bioinformatics methods revealed the presence of ζ-CA in some eukaryotic and prokaryotic microorganisms. Notably, it has not been previously reported in prokaryotes. The coexistence of ß- and ζ-CAs in some microorganisms is also a novel finding as well. Also, our analysis identified several CA proteins with 6-14 amino acid intervals between histidine and cysteine in the second highly conserved motif, which can be classified as the novel ζ-CA subfamily members that emerged under the Zn deficiency of aquatic ecosystems and selection pressure in these environments. There is also a possibility that the achieved results are rooted in the contamination of samples from the environmental microbiome genome with genomes of diatom species and the occurrence of errors was observed in the DNA sequencing outcomes. Combining of all results from evolutionary analysis to quality control of ζ-CA DNA sequences is the incentive motivation to explore more the hidden aspects of ζ-CAs.

Diatom endemism and taxonomic turnover: Assessment in high-altitude alpine lakes covering a large geographical range.

Diatoms are widely used as ecological indicators and show various degrees of endemism. Many studies that support the idea of endemic species integrate several climate zones, a variety of ecosystem types, and often focus on a global scale. Here, we investigated whether endemism could be detected when considering a homogeneous type of ecosystem in a single climate zone. We sampled stone biofilms at 40-50 cm depth in high-altitude lakes in the Alpine climate zone. A total of 149 samples were obtained from the French and Georgian mountains, two areas separated by ∼3000 km. Using Amplicon Sequence Variants derived from DNA metabarcoding, we assessed taxonomic turnover and Zeta-diversity (a measure of endemism). We ran haplotype networks and phylogenetic tests to measure geographical signal in the phylogenies of dominant taxa. The French and Georgian communities shared 51 % of species. Species that were not shared across both regions were mostly rare, and often not characteristic of lakes but of neighboring habitats instead. In contrast, at the sub-species level, 87 % of the genotypes showed restricted distributions. Whereas endemism was the rule at sub-species level, most species were shared across both French and Georgian lakes, suggesting that geographic barriers strongly limited dispersal at the sub-species level but not species level. Dominant species hosted higher levels of sub-specific diversity than rare species. In contrast to global-scale studies, we did not find any significant geographical structuring in the phylogeny of the investigated species. This could indicate ongoing dispersal at a frequency fast enough to prevent allopatric divergence, yet slow enough to prevent sharing most haplotypes between France and Georgia. These results have implications for biomonitoring: depending on the taxonomic level chosen, robust generic tools (species level) or tools dedicated to a region able to discriminate fine pressures differences (sub-species level) may be developed.

Evaluation of two short overlapping rbcL markers for diatom metabarcoding of environmental samples: Effects on biomonitoring assessment and species resolution.

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.

Biodiversity risk assessment of genetically modified Chaetoceros gracilis for outdoor cultivation.

A genetically modified (GM) strain of the diatom Chaetoceros gracilis expressing the phosphite dehydrogenase gene (ptxD), which is a useful gene both for the biological containment and the avoidance of microbial contamination, was characterized to estimate the risk against the biodiversity by laboratory experiments. GM strain could grow in the medium containing phosphite as a sole source of phosphorus, while its general characteristics such as growth, salt tolerance, heat and dehydration resistance in the normal phosphate-containing medium were equivalent to those of wild type (WT) strain. The increase in potential toxicity of GM strain against plant, crustacean, fish and mammal was also disproved. The dispersal ability of WT strain cultured in an outdoor raceway pond was investigated for 28 days by detecting the psb31 gene in vessels, settled at variable distances (between 5 and 60 m) from the pond. The diatom was detected only in one vessel placed 5 m apart. To estimate the influence on the environment, WT and GM strains were inoculated into freshwater, seawater and soil. The influence on the microbiome in those samples was assessed by 16S rRNA gene amplicon sequencing, in addition to the analysis of the survivability of those strains in the freshwater and the seawater. The results indicated that the effect to the microbiome and the survivability were comparable between WT and GM strains. All results showed that the introduction of the ptxD gene into the diatom had a low risk on biodiversity.

Assessment of marine benthic diatom communities: insights from a combined morphological-metabarcoding approach in Mediterranean shallow coastal waters.

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.

Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes.

ß-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of ß-chitin-derived high-value enzymes and products.

Cyprus' diatom diversity and the association of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.

Human activities are the leading cause of environmental impairments. Appropriate biomonitoring of ecosystems is needed to assess these activities effectively. In freshwater ecosystems, periphytic and epilithic biofilms have diatom assemblages. These assemblages respond rapidly to environmental changes, making diatoms valuable bioindicators. For this reason, freshwater biomonitoring programs are currently using diatoms (e.g., Water Framework Directive). In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring. In this study, this approach is used for the first time in Cyprus by considering the association of environmental and anthropogenic pressures to diatom assemblages. Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physicochemical, human land-use pressures) variables' role in the observed diatom diversity. Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium caputium in perennial ones. Additionally, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), stations' local characteristics (e.g., elevation), and land use activities on the observed differences in diatom diversity. Finally, we conclude that multi-stressors and anthropogenic pressures together as multiple stressors have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status.

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization.

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.

Multifunctionalizing the marine diatom Phaeodactylum tricornutum for sustainable co-production of omega-3 long chain polyunsaturated fatty acids and recombinant phytase.

There is an urgent requirement for sustainable sources of food and feed due to world population growth. Aquaculture relies heavily on the fish meal and fish oils derived from capture fisheries, challenging sustainability of the production system. Furthermore, substitution of fish oil with vegetable oil and fish meal with plant seed meals in aquaculture feeds reduces the levels of valuable omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, and lowers the nutritional value due to the presence of phytate. Addition of exogenous phytase to fish feed is beneficial for enhancing animal health and reducing phosphorus pollution. We have engineered the marine diatom Phaeodactylum tricornutum, accumulating high levels of EPA and DHA together with recombinant proteins: the fungal Aspergillus niger PhyA or the bacterial Escherichia coli AppA phytases. The removal of the N-terminal signal peptide further increased phytase activity. Strains engineered with fcpA and CIP1 promoters showed the highest level of phytase activity. The best engineered strain achieved up to 40,000 phytase activity units (FTU) per gram of soluble protein, thus demonstrating the feasibility of development of multifunctionalized microalgae to simultaneously produce industrially useful proteins and fatty acids to meet the demand of intensive fish farming activity.

Phylogeny, ecology, morphological evolution, and reclassification of the diatom orders Surirellales and Rhopalodiales.

The Surirellales and Rhopalodiales are large, widespread, and morphologically diverse groups of raphid pennate diatoms (Bacillariphyta) whose raphe, a structure that facilitates active motility, opens internally into a siliceous canal. We collected 202 representatives of the lineage and sequenced genes from the nuclear, plastid, and mitochondrial genomes to infer phylogenetic relationships as a basis for comparative study of ecology and morphological evolution as well as reclassification. The lineage was ancestrally marine, and we report the first evidence for a 'stepping stone' model of marine-freshwater transitions in which freshwater invasions were preceded by adaptation to intermediate brackish habitats. Phylogenetic comparative analyses also showed that the shift from an apical (e.g., Entomoneis) to transapical major axis of development (e.g., Surirella) did not have to proceed through subcircular intermediate forms (i.e., Campylodiscus). Rather, subcircular forms evolved both within lineages with longer apical axis or longer transapical axis. We also used the inferred phylogeny as a basis for genus-level reclassification of the lineage. Campylodiscus now includes the fastuosoid members of Surirella and Campylodiscus, but excludes other marine Campylodiscus which are now classified as Coronia. Surirella includes the Surirella striatula clade, Surirella Pinnatae group, and species formerly classified as Cymatopleura. We resurrected the genus Iconella to accommodate Stenopterobia and the robustoid members of Surirella and Campylodiscus. We broadened Epithemia to include members of the paraphyletic genus Rhopalodia. Finally, we discuss the challenges of constructing a classification that best leverages available phylogenetic data, while minimizing disruption to the research community and recognizing practical considerations stemming from the slow rate of progress on systematic studies of understudied organisms.

16S rRNA assessment of the influence of shading on early-successional biofilms in experimental streams.

Elevated nutrient levels can lead to excessive biofilm growth, but reducing nutrient pollution is often challenging. There is therefore interest in developing control measures for biofilm growth in nutrient-rich rivers that could act as complement to direct reductions in nutrient load. Shading of rivers is one option that can mitigate blooms, but few studies have experimentally examined the differences in biofilm communities grown under shaded and unshaded conditions. We investigated the assembly and diversity of biofilm communities using in situ mesocosms within the River Thames (UK). Biofilm composition was surveyed by 454 sequencing of 16S amplicons (∼400 bp length covering regions V6/V7). The results confirm the importance of sunlight for biofilm community assembly; a resource that was utilized by a relatively small number of dominant taxa, leading to significantly less diversity than in shaded communities. These differences between unshaded and shaded treatments were either because of differences in resource utilization or loss of diatom-structures as habitats for bacteria. We observed more co-occurrence patterns and network interactions in the shaded communities. This lends further support to the proposal that increased river shading can help mitigate the effects from macronutrient pollution in rivers.

Assessment of species diversity and distribution of an ancient diatom lineage using a DNA metabarcoding approach.

BACKGROUND: Continuous efforts to estimate actual diversity and to trace the species distribution and ranges in the natural environments have gone in equal pace with advancements of the technologies in the study of microbial species diversity from microscopic observations to DNA-based barcoding. DNA metabarcoding based on Next Generation Sequencing (NGS) constitutes the latest advancement in these efforts. Here we use NGS data from different sites to investigate the geographic range of six species of the diatom family Leptocylindraceae and to identify possible new taxa within the family. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the V4 and V9 regions of the nuclear-encoded SSU rDNA gene region in the NGS database of the European ERA-Biodiversa project BioMarKs, collected in plankton and sediments at six coastal sites in European coastal waters, as well as environmental sequences from the NCBI database. All species known in the family Leptocylindraceae were detected in both datasets, but the much larger Illumina V9 dataset showed a higher species coverage at the various sites than the 454 V4 dataset. Sequences identical or similar to the references of Leptocylindrus aporus, L. convexus, L. danicus/hargravesii and Tenuicylindrus belgicus were found in the Mediterranean Sea, North Atlantic Ocean and Black Sea as well as at locations outside Europe. Instead, sequences identical or close to that of L. minimus were found in the North Atlantic Ocean and the Black Sea but not in the Mediterranean Sea, while sequences belonging to a yet undescribed taxon were encountered only in Oslo Fjord and Baffin Bay. CONCLUSIONS/SIGNIFICANCE: Identification of Leptocylindraceae species in NGS datasets has expanded our knowledge of the species biogeographic distribution and of the overall diversity of this diatom family. Individual species appear to be widespread, but not all of them are found everywhere. Despite the sequencing depth allowed by NGS and the wide geographic area covered by this study, the diversity of this ancient diatom family appears to be low, at least at the level of the marker used in this study.

Phylogenetic analyses of a combined data set suggest that the Attheya lineage is the closest living relative of the pennate diatoms (Bacillariophyceae).

A Bayesian analysis of a seven gene data set was conducted to reconstruct phylogenetic relationships among a sample of centric and pennate diatoms and to test alternative hypotheses about the closest living relative of Bacillariophyceae. A lineage, composed of two Attheya species, was inferred to share the most recent common ancestor with Bacillariophyceae--a relationship that was also corroborated by the combined parsimony analysis. All competing hypotheses about the closest living relative of Bacillariophyceae were rejected because 100% of the trees in the post-burn-in sample in the Bayesian analysis supported the Attheya-Bacillariophyceae clade. According to a partitioned Bremer support analysis, the majority of the genes in the combined data matrix supported the Attheya--Bacillariophyceae relationship. The global topology of the phylogenetic tree indicated that a monophyletic group consisting of Thalassiosirales and Toxarium undulatum formed the deepest branch followed by a node uniting a clade composed of Bacillariophyceae/Attheya species and a lineage made up of Eucampia zoodiacus, Chaetocerotales, Lithodesmiales, Triceratiales, Biddulphiales and Cymatosirales. Except for the phylogenetic positions of Lithodesmiales, Thalassiosira sp and Skeletonema costatum, the optimal tree obtained from the combined parsimony analysis showed the same branching order of taxa as those seen in the consensus tree inferred from three independent Markov chain Monte Carlo analyses. Noteworthy findings are that Toxarium undulatum shares a strongly supported node with Thalassiosirales and that the genus Attheya is not a member of the Chaetocerotales lineage.

An assessment of potential diatom "barcode" genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta).

Due to limited morphological differentiation, diatoms can be very difficult to identify and cryptic speciation is widespread. There is a need for a narrower species concept if contentious issues such as diatom biodiversities and biogeographies are to be resolved. We assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model 'morphospecies', Sellaphora pupula agg. This is the first time that the suitability of cox1 as an identification tool for diatoms has been assessed. A range of cox1 primers was tested on Sellaphora and various outgroup taxa. Sequences were obtained for 34 isolates belonging to 22 Sellaphora taxa and three others (Pinnularia, Eunotia and Tabularia). Intraspecific divergences ranged from 0 to 5bp (=0.8%) and interspecific levels were at least 18bp (=c. 3%). Cox1 divergence was usually much greater than rbcL divergence and always much more variable than 18S rDNA. ITS rDNA sequences were more variable than cox1, but well-known problems concerning intragenomic variability caution against its use in identification. More information and less sequencing effort mean that cox1 can be a very useful aid in diatom identification. The usefulness of cox1 for determining phylogenetic relationships among Sellaphora species was also assessed and compared to rbcL. Tree topologies were very similar, although support values were generally lower for cox1.

Toward assigning helical regions in alignments of ribosomal RNA and testing the appropriateness of evolutionary models.

We suggest a nucleotide substitution model that takes correlation between base-paired nucleotides into account. The model includes the estimation of the transition-transversion ratio and allows inference of the shape parameter of a discrete gamma distribution to include rate heterogeneity. A Cox-test statistic, applied to a diatom ribosomal RNA alignment, shows that the suggested correlation model explains evolution of the stem region better than usual independence models. Moreover, the Cox-test procedure is extended to shed some light upon the problem of assigning helical regions in a secondary structure based alignment. This approach provides an estimate of the percentage of stem positions that do not appear to be correlated.