PHYLOGENY AND SYSTEMATICS OF EUGLENA (EUGLENACEAE) SPECIES WITH AXIAL,STELLATE CHLOROPLASTS BASED ON MORPHOLOGICAL AND MOLECULAR DATA—NEW TAXA,EMENDED DIAGNOSES,AND EPITYPIFICATIONS1

Morphological and molecular studies, as well as original literature reexamination, necessitate establishment of five Euglena species with a single axial, stellate chloroplast [Euglena viridis (O. F. Müller) Ehrenberg 1830 , Euglena pseudoviridis  Chadefaud 1937 , Euglena stellata  Mainx 1926 , Euglena pseudostellata sp. nov., and Euglena cantabrica  Pringsheim 1956 ], three species with two chloroplasts (Euglena geniculata Dujardin ex Schmitz 1884 , Euglena chadefaudii  Bourrelly 1951 , and Euglena pseudochadefaudii sp. nov.), and one species with three chloroplasts (Euglena tristella  Chu 1946 ). The primary morphological features, allowing distinction of the considered species are the presence and the shape of mucocysts, as well as the number of chloroplasts. Spherical mucocysts occur in E. cantabrica and E. geniculata, while spindle‐shaped mucocysts are present in E. stellata, E. pseudostellata, E. chadefaudii, E. pseudochadefaudii, and E. tristella. No mucocysts are observed in E. viridis and E. pseudoviridis. Two new species (E. pseudochadefaudii sp. nov. and E. pseudostellata sp. nov.) differ from the respective species, E. chadefaudii and E. stellata, only at the molecular level. Molecular signatures and characteristic sequences are designated for nine distinguished species. Emended diagnoses for all and delimitation of epitypes for seven species (except E. viridis and E. tristella) are proposed.     

151 Phylogenetic Analysis of The Subgenus Euglena with Particualr Reference to the Type Species Euglena Viridis (Euglenophyceae)

Euglena viridis was first described by Antony van Leeuwenhoek in 1674. This taxon later became the type for the genus Euglena erected by Ehrenberg in 1838. The primary characters that distinguish this taxon are the single stellate chloroplast and spherical mucocysts. A number of related Euglena species are similar in size, bear one or two stellate plastids and possess spherical or spindle-shaped mucocysts. We conducted morphological and molecular studies on taxa in the subgenus Euglena (all of which bear stellate chloroplasts) and compared this to genera in the subgenus Calliglena (non-stellate chloroplasts). Morphologically the strains in subgenus Euglena were very similar, except for chloroplast number and mucocyst shape. The E. stellata group has one chloroplast and a distinctive spindle-shaped mucocyst; the E. geniculata group has two chloroplasts and spherical mucocysts; the E. viridis group has one chloroplast and spherical mucocysts. Molecular analyses using SSU and LSU rDNA demonstrated that the subgenus Euglena is not monophyletic. The combined SSU/LSU trees provide strong support for a stellate clade (subgenus Euglena ), but one strain of E. viridis diverges at the base of the Euglena/Calliglena lineage. Multiple subclades are found within the main stellate clade. E. tristellata forms a separate divergence and four E. stellata strains form a single, well-supported subclade. Two E. viridis strains are among the E. geniculata group clade, while six others form two separate, but well-supported clades. This study demonstrates that the type species, E. viridis , is paraphyletic and will need to be redefined.     

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.     

PLASTID MORPHOLOGY,ULTRASTRUCTURE, AND DEVELOPMENT IN COLACIUM AND THE LORICATE EUGLENOPHYTES (EUGLENOPHYCEAE)1

Chloroplast morphology was investigated in five species of euglenophytes: Trachelomonas volvocinopsis Swirenko, Strombomonas verrucosa (Daday) Deflandre, Strombomonas costata Deflandre, Colacium mucronatum Bourrelly et Chafaud, and Colacium vesiculosum Ehrenberg. All five species share a common plastid morphotype: disk‐shaped plastids with a pyrenoid that protrudes asymmetrically toward the center of the cell and is capped by a single large grain of paramylon that conforms to the shape of the pyrenoid. Although plastids demonstrated some degree of diversity among the species studied, it was not consistent with current generic boundaries. The plastids of S. verrucosa show a developmental pattern similar to that of Euglena gracilis. The plastids divide during the early portion of the light phase after cell division, and pyrenoids are reduced or absent in dividing plastids. Developmental patterns of plastid replication also suggest that these five taxa share recent common ancestry with members of the genus Euglena subgenus Calliglena.     

PHYLOGENY OF EUGLENOPHYCEAE BASED ON SMALL SUBUNIT rDNA SEQUENCES: TAXONOMIC IMPLICATIONS1

Small subunit rDNA sequences of 42 taxa belonging to 10 genera were used to infer phylogenetic relationships among euglenoids. Members of the phototrophic genera Euglena, Phacus, Lepocinclis, Colacium, Trachelomonas, and Strombomonas plus the osmotrophs Astasia longa, Khawkinea quartana, and Hyalophacus ocellatus were included. Six major clades were found in most trees using multiple methods. The utility of Bayesian analyses in resolving these clades is demonstrated. The genus Phacus was polyphyletic with taxa sorting into two main clades. The two clades correlated with overall morphology and corresponded in large part to the previously defined sections, Pleur‐ aspis Pochmann and Proterophacus Pochmann. Euglena was also polyphyletic and split into two clades. In Bayesian analyses species with less plastic pellicles and small disk‐like chloroplasts diverged at the base of the tree. They grouped into a single clade which included the two Lepocinclis spp., which also are rigid and bear similar chloroplasts. The metabolic Euglena species with larger plastids bearing pyrenoids and paramylon caps arose near the top of the tree. The loricates Strombomonas and Trachelomonas formed two well‐ supported, but paraphyletic, clades. The strong support for the individual clades confirmed the value of using lorica features as taxonomic criteria. The separation of the osmotrophic species A. longa, K. quartana, and H. ocellatus into different clades suggested that the loss of the photosynthetic ability has occurred multiple times.     

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.     

A new photosynthetic euglenoid isolated in Poland: Euglenaria clepsydroides sp. nov. (Euglenea)

In this paper, we describe a new photosynthetic euglenoid species, Euglenaria clepsydroides Zakry?, sp. nov., found in Poland. A large population of this species exists in a few, small, eutrophic bodies of water inside the Masurian Landscape Park (covering a part of the Masurian Lake District in Poland). The characteristic and atypical (hourglass-like) cell shape sets it well apart from the other species that have been described up to now. This atypical cell shape has so far been observed only in three species – Lepocinclis constricta, Euglena undulata and Euglena gymnodinioides – whose other morphological characteristics, such as the number and morphology of chloroplasts, the lack of mucocysts, and nuclear SSU rDNA sequence data, exclude the possibility that they could be close relatives of Euglenaria clepsydroides. On the phylogenetic tree, the new species is situated within the Euglenaria clade. While it is a sister group of the clade that includes representatives of Euglenaria anabaena, the two species are clearly morphologically distinct.     

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.     

TWO DIFFERENT SPECIES OF EUGLENA,E. GENICULATA AND E. MYXOCYLINDRACEA (EUGLENOPHYCEAE), ARE VIRTUALLY GENETICALLY AND MORPHOLOGICALLY IDENTICAL1

We investigated the similarity of a single Euglena myxocylindracea strain, isolated originally by Bold and MacEntee, to several Euglena geniculata strains on both morphological and DNA levels. We found the three DNA stretches, consisting of fragments coding for the parts of cytoplasmic and chloroplast small subunit rRNA, and the internal transcribed spacer (ITS2) of cytoplasmic rDNA, with the combined length of 4332 nucleotides, are identical in E. myxocylindracea and E. geniculata, strain SAG 1224‐4b. Morphological differences between E. myxocylindracea and any E. geniculata strain examined were well within the range of E. geniculata variability as well. The only difference behind the distinction of E. myxocylindracea from E. geniculata is the presence of the second chloroplast in the latter. However, we were able to induce the appearance of the second chloroplast in the cells of E. myxocylindracea and its disappearance in the cells of E. geniculata by changing the composition of the culture media. We therefore conclude that E. myxocylindracea Bold and MacEntee should be regarded as an environmental form of E. geniculata Dujardin. For the first time the morphology of E. geniculata chloroplasts was shown as revealed by confocal laser microscopy.     

PHYLOGENY OF THE EUGLENALES BASED UPON COMBINED SSU AND LSU RDNA SEQUENCE COMPARISONS AND DESCRIPTION OF DISCOPLASTIS GEN. NOV. (EUGLENOPHYTA)1

A Bayesian analysis, utilizing a combined data set developed from the small subunit (SSU) and large subunit (LSU) rDNA gene sequences, was used to resolve relationships and clarify generic boundaries among 84 strains of plastid‐containing euglenophytes representing 11 genera. The analysis produced a tree with three major clades: a Phacus and Lepocinlis clade, a Discoplastis clade, and a Euglena, Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena clade. The majority of the species in the genus Euglena formed a well‐supported clade, but two species formed a separate clade near the base of the tree. A new genus, Discoplastis, was erected to accommodate these taxa, thus making the genus Euglena monophyletic. The analysis also supported the monophyly of Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena, which formed two subclades sister to the Euglena clade. Colacium, Trachelomonas, and Strombomonas, all of which produce copious amounts of mucilage to form loricas or mucilaginous stalks, formed a well‐supported lineage. Our analysis supported retaining Strombomonas and Trachelomonas as separate genera. Monomorphina and Cryptoglena formed two well‐supported clades that were sister to the Colacium, Trachelomonas, and Strombomonas clade. Phacus and Lepocinclis, both of which have numerous small discoid chloroplasts without pyrenoids and lack peristaltic euglenoid movement (metaboly), formed a well‐supported monophyletic lineage that was sister to the larger Euglena through Cryptoglena containing clade. This study demonstrated that increased taxon sampling, multiple genes, and combined data sets provided increased support for internal nodes on the euglenoid phylogenetic tree and resolved relationships among the major genera in the photosynthetic euglenoid lineage.     

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

A CHLOROPLAST-DNA PHYLOGENY OF THE WILD PERENNIAL RELATIVES OF SOYBEAN (GLYCINE SUBGENUS GLYCINE): CONGRUENCE WITH MORPHOLOGICAL AND CROSSING GROUPS

Hypotheses of evolutionary relationships among the Australian wild perennial relatives of soybean (Glycine subgenus Glycine) are based largely on patterns of meiotic pairing in intra- and interspecific experimental hybrids. This evidence has indicated a number of genome groupings within the subgenus but has not resolved most phylogenetic relationships. Restriction-endonuclease site variation of chloroplast DNA (cpDNA) within the perennial subgenus is reported here, representing a sampling of approximately 3% of the approximately 150-kilobase plastome. Seven hundred twenty-one unique restriction sites were compared within Glycine using 29 restriction endonucleases; 157 sites varied within the genus. Distance and parsimony methods using these data yielded congruent results, recognizing the existence of three major groups within subgenus Glycine: the species-rich and geographically diverse A clade consisting of G. canescens and related taxa; the B clade, which includes the stoloniferous species; and the C group, containing two species with distinctive curved pods. These results are in general agreement with hypotheses based on genome analysis; inconsistencies involve the inclusion of genetically divergent taxa such as G. falcata in well-supported plastome clades comprised of otherwise interfertile species. Such findings are not unexpected if crossing barriers are considered to be unique features of such anomalous species, paralleling their often numerous morphological and cpDNA autapomorphies. Consideration of cpDNA divergence within the three major clades of subgenus Glycine indicates that the rate of plastome evolution is uncoupled from rates of morphological or ecological diversification.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

Ultrastructure of Euglena anabaena var. minor (Euglenophyceae)

The freshwater green euglenoid Euglena anabaena var. minor has a pellicle with groove‐ridge articulation, a chloroplast with pyrenoids doubly sheathed by two paramylon caps, and a nucleus with permanently condensed chromosomes and nucleolus. The flagellar apparatus basically resembles that of Euglena. The dorsal root (DR) originates at the dorsal basal body of the emergent flagellum, while both the intermediate root (IR) and ventral root (VR) originate at the ventral basal body of the non‐emergent flagellum. The cytoplasmic pocket is associated with the ventral root/ reinforcing microtubular band. However, ultrastructural characterization of E. anabaena var. minor shows the pocket to consist of five to seven microtubules, and flagellar roots with microtubule configuration of 3–4–6 in the DR‐IR‐VR. The dorsal band microtubules pair at the reservoir‐canal transition level. The doublet microtubules are formed into triplets and doublets at the lower canal level and then make pellicular microtubules at the upper canal level.     

Stress resistance induced by paramylon treatment in Artemia sp.

A study was undertaken to investigate the effectiveness of paramylon extracted from the non-photosynthetic WZSL mutant of Euglena gracilis in potentiating the resistance of the brine shrimp Artemia sp. to stress conditions resulting from poor growth medium quality and daily handling. The experimental design incorporated four different treatments: I) paramylon addition/no growth medium replacement; II) no paramylon addition/no growth medium replacement; III) paramylon addition/growth medium replacement; IV) noparamylon addition/growth medium replacement. As shown by the survival curves of Artemia sp., the addition of paramylon significantly reduced susceptibility of animals to the stressors. Paramylon effectively increased also the ability of offspring to withstand poor water quality and handling damages. Without paramylon almost all offspring died before adulthood, whereas addition of paramylon allowed the animals to moult to the adult stage. Moreover, reproductive success (higher number of living offspring) was enhanced considerably in animals treated with paramylon treated under both stress conditions. These results show that paramylon extracted from Euglena represents a promising biologically active compound for aquaculture purposes. It could be used as a purified product or as component of whole cells, since the Euglena mutant, because of the high intracellular amount of paramylon it can accumulate, could be added to the feed or to water in tanks and ponds without prior processing. This revised version was published online in August 2006 with corrections to the Cover Date.     

MULTIGENE ANALYSES OF PHOTOSYNTHETIC EUGLENOIDS AND NEW FAMILY,PHACACEAE (EUGLENALES)1

Bayesian and maximum‐likelihood (ML) analyses of the combined multigene data (nuclear SSU rDNA, and plastid SSU and LSU rDNA) were conducted to evaluate the phylogeny of photosynthetic euglenoids. The combined data set consisted of 108 strains of photosynthetic euglenoids including a colorless sister taxon. Bayesian and ML analyses recovered trees of almost identical topology. The results indicated that photosynthetic euglenoids were divided into two major clades, the Euglenaceae clade (Euglena, Euglenaria, Trachelomonas, Strombomonas, Monomorphina, Cryptoglena, Colacium) and the Phacaceae clade (Phacus, Lepocinclis, Discoplastis). The Euglenaceae clade was monophyletic with high support and subdivided into four main clades: the Colacium, the Strombomonas and Trachelomonas, the Cryptoglena and Monomorphina, and the Euglena and Euglenaria clades. The genus Colacium was positioned at the base of the Euglenaceae and was well supported as a monophyletic lineage. The loricate genera (Strombomonas and Trachelomonas) were located at the middle of the Euglenaceae clade and formed a robust monophyletic lineage. The genera Cryptoglena and Monomorphina also formed a well‐supported monophyletic clade. Euglena and the recently erected genus Euglenaria emerged as sister groups. However, Euglena proxima branched off at the base of the Euglenaceae. The Phacaceae clade was also a monophyletic group with high support values and subdivided into three clades, the Discoplastis, Phacus, and Lepocinclis clades. The genus Discoplastis branched first, and then Phacus and Lepocinclis emerged as sister groups. These genera shared a common characteristic, numerous small discoid chloroplasts without pyrenoids. These results clearly separated the Phacaceae clade from the Euglenaceae clade. Therefore, we propose to limit the family Euglenaceae to the members of the Euglena clade and erect a new family, the Phacaceae, to house the genera Phacus, Lepocinclis, and Discoplastis.     

Chloroplast Genome Evolution in the Euglenaceae

Over the last few years multiple studies have been published outlining chloroplast genomes that represent many of the photosynthetic euglenid genera. However, these genomes were scattered throughout the euglenophyceaean phylogenetic tree, and focused on comparisons with Euglena gracilis. Here, we present a study exclusively on taxa within the Euglenaceae. Six new chloroplast genomes were characterized, those of Cryptoglena skujai, E. gracilis var. bacillaris, Euglena viridis, Euglenaria anabaena, Monomorphina parapyrum, and Trachelomonas volvocina, and added to six previously published chloroplast genomes to determine if trends existed within the family. With this study: at least one genome has now been characterized for each genus, the genomes of different strains from two taxa were characterized to explore intraspecific variability, and a second taxon has been characterized for the genus Monomorphina to examine intrageneric variability. Overall results showed a large amount of variability among the genomes, though a few trends could be identified both within Euglenaceae and within Euglenophyta. In addition, the intraspecific analysis indicated that the similarity of a genome sequence between strains was taxon dependent, and the intrageneric analysis indicated that the majority of the evolutionary changes within the Euglenaceae occurred intergenerically.