Toward the robust resolution of taxonomic ambiguity within Lepocinclis (Euglenida) based on DNA sequencing and morphology.

DNA sequences were analyzed for three groups of species from the Lepocinclis genus (L. acus-like, L. oxyuris-like, and L. tripteris-like) along with cellular morphology. Phylogenetic analyses were based on nuclear SSU rDNA, LSU rDNA, and plastid-encoded LSU rDNA. DNA sequences were obtained from species available in culture collections (L. acus SAG 1224-1a and UTEX 1316) and those isolated directly from the environment in Poland (48 isolates), resulting in 79 new sequences. The obtained phylogenetic tree of Lepocinclis included 27 taxa, five of which are presented for the first time (L. convoluta, L. gracillimoides, L. longissima, L. pseudospiroides, and L. torta) and nine taxonomically verified and described. Based on morphology, literature data, and phylogenetic analyses, the following species were distinguished: in the L. acus-like group, L. longissima and L. acus; in the L. tripteris-like group, L. pseudospiroides, L. torta, and L. tripteris; in the L. oxyuris-like group, L. gracillimoides, L. oxyuris var. oxyuris, and L. oxyuris var. helicoidea. For all verified species, diagnostic descriptions were emended, nomenclatural adjustments were made, and epitypes were designated.

Evaluation of V2 18S rDNA barcode marker and assessment of sample collection and DNA extraction methods for metabarcoding of autotrophic euglenids.

Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species-level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB-based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.

Description of Flexiglena gen. nov. and new members of Discoplastis and Euglenaformis (Euglenida).

Environmental sampling in Poland and the United States and phylogenetic analyses based on 567 sequences of four genes (155 sequences of nuclear SSU rDNA, 139 of nuclear LSU rDNA, 135 of plastid-encoded SSU rDNA, and 138 of plastid-encoded LSU rDNA) resulted in description of the new genus Flexiglena, which has been erected by accommodating Euglena variabilis, and enriching the Discoplastis and Euglenaformis genera with five new species. Four of them have joined the Discoplastis genus, currently consisting of six representatives: D. adunca, D. angusta (=Euglena angusta), D. constricta (=Lepocinclis constricta), D. excavata (=E. excavata), D. gasterosteus (=E. gasterosteus), and D. spathirhyncha. One of them has enriched the Euglenaformis genus, currently represented by two species: Euf. chlorophoenicea (= E. chlorophoenicea) and Euf. proxima. For most studied species, the diagnostic descriptions have been emended and epitypes were designated. Furthermore, the emending of Discoplastis and Euglenaformis diagnoses was performed.

Molecular and Morphological Delimitation of Species in the Group of Lepocinclis Ovum-like taxa (Euglenida).

Although Lepocinclis ovum is recognized as a cosmopolitan and common species, and Lepocinclis globulus is the type species of the genus Lepocinclis, their correct identification is nearly impossible. The reason is that over 30 morphologically similar taxa appear in the literature, but no good diagnostic features exist to distinguish amongst them. Using environmental sampling and nuclear SSU rDNA sequencing, we delimited species within the group of Lepocinclis ovum-like taxa. Morphological and molecular features were analyzed for taxa isolated from Poland and six cultured strains from algal collections. In the case of environmental sampling, DNA was obtained from a small number of cells (20-400) isolated with a micropipette without setting up laboratory cultures (52 isolates), and phylogenetic analyses were based on the variation in nSSU rDNA. Apart from L. ovum and L. globulus, 13 other species were distinguished and four taxa (Lepocinclis conica comb. nov., L. fominii comb. nov., L. gracilicauda comb. nov., and L. pseudofominii nom. nov.) had their taxonomic ranks changed. For all verified species, diagnostic descriptions were emended and epitypes designated. The only exception was L. ovum, for which the epitype was questioned and thus, a new candidate for the epitype was suggested for future adoption.

Taxon-rich phylogeny and taxonomy of the genus Phacus (Euglenida) based on morphological and molecular data.

Morphological and molecular features were analyzed for a species of Phacus to better understand the phylogenetic relationships among them and establish the taxonomy. Phylogenetic analyses were based on nSSU rDNA and the research resulted in 55 new sequences. The study included species available in algal collections and those isolated directly from the environment in Poland and the Czech Republic. As a result, the obtained phylogenetic tree of Phacus includes 50 species, out of which 7 are represented on a tree for the first time (Phacus anacoelus, P. anomalus, P. curvicauda, P. elegans, P. lismorensis, P. minutus and P. stokesii) and many have been taxonomically verified. For all verified species, diagnostic descriptions were amended, the naming was reordered and epitypes were designated.

DNA barcoding in autotrophic euglenids: evaluation of COI and 18s rDNA.

Autotrophic euglenids (Euglenophyceae) are a common and abundant group of microbial eukaryotes in freshwater habitats. They have a limited number of features, which can be observed using light microscopy, thus species identification is often problematic. Establishing a barcode for this group is therefore an important step toward the molecular identification of autotrophic euglenids. Based on the literature, we selected verified species and used a plethora of available methods to validate two molecular markers: COI and 18S rDNA (the whole sequence and three fragments separately) as potential DNA barcodes. Analyses of the COI gene were performed based on the data set of 43 sequences (42 obtained in this study) representing 24 species and the COI gene was discarded as a DNA barcode mainly due to a lack of universal primer sites. For 18S rDNA analyses we used a data set containing 263 sequences belonging to 86 taxonomically verified species. We demonstrated that the whole 18S rDNA is too long to be a useful marker, but from the three shorter analyzed variable regions we recommend variable regions V2V3 and V4 of 18S rDNA as autotrophic euglenid barcodes due to their high efficiency (above 95% and 90%, respectively).

Delimiting species in the Phacus longicauda complex (Euglenida) through morphological and molecular analyses.

Although Phacus longicauda is the type species of the genus Phacus and one of the most common species among autotrophic euglenids, its correct identification is nearly impossible. Over 30 morphologically similar taxa appear in the literature, but there are no good diagnostic features to distinguish them. Using environmental sampling and whole genome amplification, we delimited species within the Phacus longicauda complex. Morphological and molecular characters were analyzed for 36 strains isolated from environmental samples (mainly from Poland). DNA was obtained from a small number of cells (20-30) isolated with a micropipette from every sample (i.e., without setting up laboratory cultures), and phylogenetic analyses were based on variation in nSSU rDNA. Apart from Phacus longicauda, three other species (Phacus circumflexus, Phacus helikoides, and Phacus tortus) were distinguished. Phacus cordata comb. nov. Zakrys et M. Lukomska and Phacus rotunda comb. nov. Zakrys et M. Lukomska had their taxonomic ranks changed and two species new to science, Phacus cristatus sp. nov. Zakrys et M. Lukomska and Phacus crassus sp. nov. Zakrys et M. Lukomska, were described. For all verified species, diagnostic descriptions were amended and epitypes designated.

Discovery of a new photosynthetic euglenoid in Poland: Euglena mazurica sp. nov. (Euglenales, Euglenaceae).

Herein we describe a new photosynthetic euglenoid species found in Poland - Euglena mazurica. A large population exists in a small, eutrophic body of water located in a pasture near Mikolajki town inside the Masurian Landscape Park (covering a part of the Masurian Lake District in Poland). The unique cell shape (corkscrew-like) discerns it well from other previously described euglenoid species with metabolic cells. The new species possesses two plate-like chloroplasts each with a pyrenoid accompanied by two paramylon caps placed on either side of it (diplopyrenoids). On the phylogenetic tree, the new species is situated within the Euglena clade. Though it is a sister branch to three clades - one representing the similar Euglena agilis, characterized by its fusiform cells and two chloroplasts with diplopyrenoids, the two species are clearly morphologically and molecularly distinct.

Biodiversity of autotrophic euglenids based on the group specific DNA metabarcoding approach.

This study reports a comprehensive analysis of photoautotrophic euglenids' distribution and biodiversity in 16 small water bodies of various types (including fish ponds, field ponds, rural ponds and park ponds) located in three regions of Poland: Masovia, Masuria and Pomerania during a period of three years. By employing a euglenid specific barcode marker and a curated database of V2 18S rDNA sequences it was possible to identify 97.7 % of euglenid reads at species level. A total of 152 species classified in 13 genera were identified. The number of euglenid species found in one pond varied from 40 to 102. The most common species were Euglena agilis and Euglenaria caudata, found in every analysed waterbody. The highest number of observed species belonged to Trachelomonas and Phacus. Certain species exhibited a tendency to coexist, suggesting the presence of distinct species assemblages. Among them, the most distinctive cluster was associated with water bodies located in the Masuria region, characterized also by the greatest species richness, including many very rare species: Euglenaformis chlorophoenicea, Lepocinclis autumnalis, L. marssonii, Trachelomonas eurystoma, T. manschurica, T. mucosa, T. zuberi, T. zuberi var. nepos.

Composition of picocyanobacteria community in the Great Mazurian Lakes: isolation of phycoerythrin-rich and phycocyanin-rich ecotypes from the system--comparison of two methods.

The study showed that the picocyanobacteria community of the Great Mazurian Lakes system (GML) was dominated by phycoerythrin-rich (PE) ecotypes and demonstrated a gradual decrease of the ratio between PE and phycocyanin-rich (PC) ecotypes. The Great Mazurian Lakes offer better conditions for the PE ecotype than for the PC one, despite the considerably high trophic status, probably thanks to low turbidity and attenuation of light in the water column. The successful isolation of PE and PC picocyanobacteria was achieved by two methods: the classic plate method and a modified flow-cytometry method. The modified flow-cytometry method proved to be superior: being more selective for PE picocyanobacteria as well as less time consuming and less laborious. The modifications introduced to the method, such us concentration of cyanobacterial cells by centrifugation to the density required by the flow cytometer, did not hinder the isolation while allowing to skip an intermediate phase of enrichment cultures that had been formerly proposed. The first phylogenetic analyses based on cpcBA operon and 16S rRNA gene demonstrated that picocyanobacteria isolates from GML could, with a high bootstrap support, be grouped into five and four clusters, respectively. Based on a cpcBA-IGS analysis and IGS length the study suggests that at least one of the clusters is new and has not been previously described.

PCR identification of toxic euglenid species Euglena sanguinea.

Euglena sanguinea Ehrenberg is the only known species of euglenids which forms toxic blooms causing tangible losses to fish farms. Euglena sanguinea produces euglenophycin, a toxin similar in structure to solenopsin, an alkaloid found in fire ant venom. It was proved that euglenophycin exhibits not only ichthyotoxic but also herbicidal and anticancer activity. Recently, a specific mass spectrometric method of identification and quantitation of euglenophycin was developed to facilitate monitoring of that toxin in freshwater ponds. Despite the recent taxonomic verifications, proper identification of E. sanguinea is still difficult, especially for less experienced researchers. Herein, we describe a simple method based on nested PCR amplification of the nSSU rDNA fragments to identify a single E. sanguinea cell and its detection in a sample of water. The method will further facilitate monitoring of water reservoirs, especially estimating the risk of toxic blooms.

Calcification and silicification: fossilization potential of cyanobacteria from stromatolites of Niuafo'ou's Caldera Lakes (Tonga) and implications for the early fossil record.

Calcification and silicification processes of cyanobacterial mats that form stromatolites in two caldera lakes of Niuafo'ou Island (Vai Lahi and Vai Si'i) were evaluated, and their importance as analogues for interpreting the early fossil record are discussed. It has been shown that the potential for morphological preservation of Niuafo'ou cyanobacteria is highly dependent on the timing and type of mineral phase involved in the fossilization process. Four main modes of mineralization of cyanobacteria organic parts have been recognized: (i) primary early postmortem calcification by aragonite nanograins that transform quickly into larger needle-like crystals and almost totally destroy the cellular structures, (ii) primary early postmortem silicification of almost intact cyanobacterial cells that leave a record of spectacularly well-preserved cellular structures, (iii) replacement by silica of primary aragonite that has already recrystallized and obliterated the cellular structures, (iv) occasional replacement of primary aragonite precipitated in the mucopolysaccharide sheaths and extracellular polymeric substances by Al-Mg-Fe silicates. These observations suggest that the extremely scarce earliest fossil record may, in part, be the result of (a) secondary replacement by silica of primary carbonate minerals (aragonite, calcite, siderite), which, due to recrystallization, had already annihilated the cellular morphology of the mineralized microbiota or (b) relatively late primary silicification of already highly degraded and no longer morphologically identifiable microbial remains.