Circular extrachromosomal DNA in Euglena gracilis under normal and stress conditions.

Extrachromosomal circular DNA (eccDNA) enhances genomic plasticity, augmenting its coding and regulatory potential. Advances in high-throughput sequencing have enabled the investigation of these structural variants. Although eccDNAs have been investigated in numerous taxa, they remained understudied in euglenids. Therefore, we examined eccDNAs predicted from Illumina sequencing data of Euglena gracilis Z SAG 1224-5/25, grown under optimal photoperiod and exposed to UV irradiation. We identified approximately 1000 unique eccDNA candidates, about 20% of which were shared across conditions. We also observed a significant enrichment of mitochondrially encoded eccDNA in the UV-irradiated sample. Furthermore, we found that the heterogeneity of eccDNA was reduced in UV-exposed samples compared to cells that were grown in optimal conditions. Hence, eccDNA appears to play a role in the response to oxidative stress in Euglena, as it does in other studied organisms. In addition to contributing to the understanding of Euglena genomes, our results contribute to the validation of bioinformatics pipelines on a large, non-model genome.

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.

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.

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.

A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids.

Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.

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.

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.

Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis.

Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.

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.

Evolutionary Origin of Euglena.

Euglenids (Excavata, Discoba, Euglenozoa, Euglenida) is a group of free-living, single-celled flagellates living in the aquatic environments. The uniting and unique morphological feature of euglenids is the presence of a cell covering called the pellicle. The morphology and organization of the pellicle correlate well with the mode of nutrition and cell movement. Euglenids exhibit diverse modes of nutrition, including phagotrophy and photosynthesis. Photosynthetic species (Euglenophyceae) constitute a single subclade within euglenids. Their plastids embedded by three membranes arose as the result of a secondary endosymbiosis between phagotrophic eukaryovorous euglenid and the Pyramimonas-related green alga. Within photosynthetic euglenids three evolutionary lineages can be distinguished. The most basal lineage is formed by one mixotrophic species, Rapaza viridis. Other photosynthetic euglenids are split into two groups: predominantly marine Eutreptiales and freshwater Euglenales. Euglenales are divided into two families: Phacaceae, comprising three monophyletic genera (Discoplastis, Lepocinclis, Phacus) and Euglenaceae with seven monophyletic genera (Euglenaformis, Euglenaria, Colacium, Cryptoglena, Strombomonas, Trachelomonas, Monomorphina) and polyphyletic genus Euglena. For 150 years researchers have been studying Euglena based solely on morphological features what resulted in hundreds of descriptions of new taxa and many artificial intra-generic classification systems. In spite of the progress towards defining Euglena, it still remains polyphyletic and morphologically almost undistinguishable from members of the recently described genus Euglenaria; members of both genera have cells undergoing metaboly (dynamic changes in cell shape), large chloroplasts with pyrenoids and monomorphic paramylon grains. Model organisms Euglena gracilis Klebs, the species of choice for addressing fundamental questions in eukaryotic biochemistry, cell and molecular biology, is a representative of the genus Euglena.

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).

Intermediate introns in nuclear genes of euglenids - are they a distinct type?

BACKGROUND: Nuclear genes of euglenids contain two major types of introns: conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. RESULTS: Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: two in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. CONCLUSIONS: Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/ -8, -7 and -6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.

Phylogenetic Relationships and Morphological Character Evolution of Photosynthetic Euglenids (Excavata) Inferred from Taxon-rich Analyses of Five Genes.

Photosynthetic euglenids acquired chloroplasts by secondary endosymbiosis, which resulted in changes to their mode of nutrition and affected the evolution of their morphological characters. Mapping morphological characters onto a reliable molecular tree could elucidate major trends of those changes. We analyzed nucleotide sequence data from regions of three nuclear-encoded genes (nSSU, nLSU, hsp90), one chloroplast-encoded gene (cpSSU) and one nuclear-encoded chloroplast gene (psbO) to estimate phylogenetic relationships among 59 photosynthetic euglenid species. Our results were consistent with previous works; most genera were monophyletic, except for the polyphyletic genus Euglena, and the paraphyletic genus Phacus. We also analyzed character evolution in photosynthetic euglenids using our phylogenetic tree and eight morphological traits commonly used for generic and species diagnoses, including: characters corresponding to well-defined clades, apomorphies like presence of lorica and mucilaginous stalks, and homoplastic characters like rigid cells and presence of large paramylon grains. This research indicated that pyrenoids were lost twice during the evolution of phototrophic euglenids, and that mucocysts, which only occur in the genus Euglena, evolved independently at least twice. In contrast, the evolution of cell shape and chloroplast morphology was difficult to elucidate, and could not be unambiguously reconstructed in our analyses.

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.

Distribution of conventional and nonconventional introns in tubulin (α and ß) genes of euglenids.

The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns--positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism--intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns--because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns.

A redescription of morphologically similar species from the genus Euglena: E. laciniata, E. sanguinea, E. sociabilis, and E. splendens(1).

Euglena sanguinea (Ehrenberg 1831) was one of the first green euglenoid species described in the literature. At first, the species aroused the interest of researchers mainly due to the blood-red color of its cells, which, as it later turned out, is not a constant feature. Complicated chloroplast morphology, labeled by Pringsheim as the "peculiar chromatophore system", made the correct identification of the species difficult, which is the reason why, throughout the 20th century, new species resembling E. sanguinea were continually being named due to a lack of suitable diagnostic features to distinguish E. sanguinea. Interest in E. sanguinea has returned in recent years, following findings that the species can produce ichthyotoxins. This was followed by the need to classify E. sanguinea correctly, which was achieved through the verification of morphological and molecular data for all species similar to E. sanguinea. As the result of the analysis, the number of species sharing some morphological similarities with E. sanguinea could be reduced from 12, as described in the literature, to four, with established epitypes and updated diagnostic descriptions. The most important diagnostic features included: the presence of mucocysts (i.e., whether they were visible before and/or after staining), the number of chloroplasts, the size of the double-sheathed pyrenoids, and the presence of the large paramylon grain in the vicinity of the stigma. Moreover, sequence analysis revealed the presence of unusually long SSU rDNA sequences in E. sanguinea. Previously, SSU rDNA sequences of such length were known to be present in primary osmotrophic euglenoids.

TAXONOMIC REVISIONS OF MORPHOLOGICALLY SIMILAR SPECIES FROM TWO EUGLENOID GENERA: EUGLENA (E. GRANULATA AND E. VELATA) AND EUGLENARIA (EU. ANABAENA, EU. CAUDATA, AND EU. CLAVATA)(1).

The establishment of epitypes (together with the emended diagnoses) for three species of Euglenaria Karnkowska, E. W. Linton et Kwiatowski [Eu. anabaena (Mainx) Karnkowska et E. W. Linton; Eu. caudata (Hübner) Karnkowska et E. W. Linton; and Eu. clavata (Skuja) Karnkowska et E. W. Linton] and two species of Euglena Ehrenberg [E. granulata (Klebs) Schmitz and E. velata Klebs] was achieved due to literature studies, verification of morphological diagnostic features (cell size, cell shape, number of chloroplasts, the presence of mucocysts), as well as molecular characters (SSU rDNA). Now all these species are easy to identify and distinguish, despite their high morphological similarity, that is, spindle-shaped (or cylindrically spindle-shaped) cells and parietal, lobed chloroplasts with a single pyrenoid, accompanied by bilateral paramylon caps located on both sides of the chloroplast. E. granulata is the only species in this group that has spherical mucocysts. E. velata is distinguished by the largest cells (90-150 µm) and has the highest number of chloroplasts (>30). Eu. anabaena has the fewest chloroplasts (usually 3-6), and its cells are always (whether the organism is swimming or not) spindle-shaped or cylindrically spindle-shaped, in contrast to the cells of Eu. clavata, which are club-shaped (clavate) while swimming and only after stopping change to resemble the shape of a spindle or a cylindrical spindle; Eu. clavata has numerous chloroplasts (15-20). Eu. caudata is characterized by asymmetrical spindle-shaped (fusiform) cells, that is, with an elongated rear section and a shorter front section; the number of chloroplasts normally ranges from 7 to 15.

THE SPECIES EUGLENA DESES (EUGLENACEAE) REVISITED: NEW MORPHOLOGICAL AND MOLECULAR DATA1.

For this study, we have examined the literature and the morphological diversity, as well as analyzed the nuclear SSU rDNA sequences of two very common and cosmopolitan species formerly known as Euglena deses Ehrenb. and Euglena intermedia (G. A. Klebs) F. Schmitz. Our studies have shown that there is evidence for distinguishing only one species (E. deses). Here, we define new diagnostic features for E. deses, namely, periplast ornamentation (the presence of small papillae-discovered for the first time in this species) and the lateral location of the anterior canal opening, from which the flagellum emerges. We also designate the epitype and emend the diagnosis for E. deses.