Paramylon Grains Research Articles

Phylogenetic and Morphological Evolution of Green Euglenophytes Based on 18S rRNA.

Green euglenophytes are a group of eukaryotes with ancient origin. In order to understand the evolution of the group, it is interesting to know which characteristics are more primitive. Here, a phylogenetic tree of green euglenophytes based on the 18S rRNA gene was constructed, and ancestral states were reconstructed based on eight morphological characters. This research clarifies the phylogenetic relationships of green euglenophytes and provides a basis for the study of the origin of these plants. The phylogenetic tree, which was constructed by Bayesian inference, revealed that: Eutreptia and Eutreptiella were sister groups and that Lepocinclis, Phacus, and Discoplastis were close relatives; Euglena, Cryptoglena, Monomorphina, and Colacium were closely related in addition to Trachelomonas and Strombomonas; and Euglena was not monophyletic. An ancestral reconstruction based on morphological characters revealed seven primitive character states: ductile surface, spirally striated, slightly narrowing or sharp elongated cauda, absence of a lorica, chloroplast lamellar, shield or large discoid, pyrenoid with sheath, and with many small paramylon grains. However, the ancestral state of the length of the flagellum could not be inferred. Euglena and Euglenaria, which both possessed all of the ancestral character states, might represent the most ancient lineages of green euglenophytes.

Ultrastructural Alterations in Lepocinclis acus (Euglenophyta) Induced by Medium with High Organic Matter Content

Ultrastructural changes induced by exposure to excess of organic matter were studied in Lepocinclis acus (ex Euglena acus). The cells isolated from the Matanza River, Buenos Aires, Argentina, were grown in soil water medium. When transferred to medium enriched with Bacteriological Peptone OXOID®, marked body deformation and a significant shortening and widening of the cells was observed. These changes were unexpected in a species with quite rigid cells, a condition previously shown in studies of the pellicle fine structure. Transmission electron microscopy observations suggest that cellular deformation might be facilitated by an increase in strip number, whereas in the original strips normal ultrastructure was maintained. An increase in number and volume of paramylon grains and vacuoles, as well as the presence of membrane whorls in vacuoles was observed. The fine structure of organisms grown in medium with and without organic matter enrichment was compared, and the systematic and ecological importance of morphological changes triggered by cell deformation was discussed.

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.

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.

Descripción ultraestructural de <i>Euglena pailasensis</i> (Euglenozoa) del Volcán Rincón de la Vieja, Guanacaste, Costa Rica

The euglenoids are unicellular eukaryotic flagellates living in a diversity of soils and aquatic environments and ecosystems. This study describes the ultrastructure of an euglenoid isolated from the surface of a boiling mud pool with temperatures ranging from 38 to 98 degrees C and pH 2 - 4. The hot mud pool is located in Area de Pailas de Barro, Las Pailas, Rincón de la Vieja Volcano, Guanacaste, Costa Rica. The morphological characterization of the Euglena pailasensis was performed by SEM and TEM. It was determined that, although the euglenoid was obtained from an extreme volcanic environment, the general morphology corresponds to that of a typical member of Euglena of 30-45 microm long and 8-10 microm wide, with membrane, pellicle, chloroplasts, mitochondria, nucleus, pigments and other cytoplasmic organelles. E. pailasensis is delimited by a membrane and by 40 to 90 pellicle strips. It was observed up to 5 elongated chloroplasts per cell. The chloroplast contains several osmiophilic globules and a pyrenoid penetrated by few thylakoid pairs. The nutritious material is reserved in numerous small paramylon grains located at the center of the cell, mitocondria are characterized by the presence of crests in radial disposition toward the interior of the lumen. It was also observed around the external surface "pili" like filaments originating from the pellicle strips. There is no evidence for the presence of flagella in the ampulla (reservoir/canal area), a fact confirmed by negative staining, and a difference regarding other species of Euglena. The observed ultrastructural characteristics are not sufficient to explain the adaptation of this species to acid and hot environments.

Ultrastructural alterations in Phacus brachykentron (Euglenophyta) due to excess of organic matter in the culture medium

Morphological and ultrastructural changes induced by exposure to excess of organic matter were analyzed in Phacus brachykentron (Pochm.). The cells were isolated from sites in Matanza River, Buenos Aires, Argentina, which have a high degree of organic matter contamination coming from waste waters discharges of the meat industry. Master strains were cultured on soil water medium and a toxicity bioassay was performed. As a result of the enriched medium, several morphological and ultrastructural cellular alterations were observed by optical, scanning, and transmission electron microscopy. Among these, we can point out changes in cell dimensions, remarkable widening of some pellicle bands, increased number and volume of paramylon grains, displacement of the nucleus from the central to the lateral position, some chloroplasts with their thylakoids disordered, and cell lysis. The response to organic enrichment was very fast, i.e. during the 48h of the bioassay. Therefore, any significant increase of organic matter would rapidly affect wild euglenoids. Our results suggest that the alterations observed, such as the presence of large intracellular paramylon bodies or the deformation of euglenoid cells in natural samples, have the potential to be used as environmental bioindicators.

An in situ test to explore the responses of Scenedesmus acutus and Lepocinclis acus as indicators of the changes in water quality in lowland streams

This bioassay was designed with the aim of exploring the responses of two wild planktonic microalgae exposed in situ (72 h) as indicators of the changes in water quality. Monocultures of both strains within dialysis membrane bags were placed at two sites in a small lowland stream. Site 1 is located at a suburban area with low horticultural activity and Site 2 is impacted by toxic industrial discharges and urban land use. There was a decrease in population growth of both species at Site 2 compared with Site 1. The comparison of the algae exposed in situ with the normal specimens cultured at the laboratory indicated a significant increase in the cellular volume for both species at both sites. Abnormal shape was recorded at both sites, the percentage being significantly greater for Scenedesmus acutus at Site 2. Significant changes in pyrenoids size were observed in S. acutus and in the percentage of fragmented nuclei in Lepocinclis acus. Also in the latter abnormal paramylon grains were observed. These responses were accentuated at Site 2. This bioassay was sensitive, short term, low cost, and therefore is a suitable tool to contribute with the monitoring and ecological risk assessment of lowland streams.

Characterization of paramylon morphological diversity in photosynthetic euglenoids (Euglenales, Euglenophyta)

Monfils A.K., Triemer R.E. and Bellairs E.F. 2011. Characterization of paramylon morphological diversity in photosynthetic euglenoids (Euglenales, Euglenophyta). Phycologia 50: 156–169. DOI: 10.2216/09-112.1A characteristic feature of euglenoid cells is the presence of a β1–3 glucan storage product called paramylon. The euglenoid lineage is tremendously diverse, with a great deal of variation in paramylon grain morphology. Number, shape, location and external morphology of paramylon have been used as diagnostic features for several euglenoid species. The goals of this study were to examine and characterize the paramylon grains from a variety of different species in vivo and in vitro, provide a consistent, descriptive terminology that can be used to describe paramylon grain types, and discuss the potential phylogenetic utility of paramylon grain morphology. Over 1000 light microscopy images were examined to survey paramylon diversity across the Euglenales, and scanning electron microscopy was used to examine paramylon in 11 representative species (Colacium vesiculosum, Cryptoglena skujae, Discoplastis spathirhyncha, Euglena gracilis, Lepocinclis acus, L. ovum, Monomorphina pyrum, Phacus orbicularis, P. pleuronectes, Strombomonas borystheniensis, and Trachelomonas ellipsoidalis). The various types of paramylon grains were separated into six distinct morphological categories: disk, ellipse, pyrenoid cap, plate, bobbin or rod, with further distinction for varieties found within each category. These categories were then applied to the diversity found in the genera and major lineages. This study was able to determine a high level of distinction among differing paramylon grain morphologies both within a species and among species, and variation was found in both large and small grains. Paramylon can be used at the generic level to support major clades and generic relationships and may provide insight into the taxonomic placement of euglenoids currently unavailable for sequencing.

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.

RESTING CYST FORMATION OF EUTREPTIELLA GYMNASTICA (EUGLENOPHYCEAE) IN THE NORTHERN COASTAL BALTIC SEA1

ABSTRACTResting cyst formation of Eutreptiella gymnastica Throndsen was observed during a mesocosm experiment, where nutrient enrichment had induced almost a unialgal bloom. Cells and resting cysts of E. gymnastica were examined in scanning (SEM) and transmission electron microscopy (TEM) and light microscopy. Mature cysts were spherical, with a smooth thick mucilaginous cover that appeared layered when observed with the TEM. Intermediate forms were spherical and lacking flagella and a mucilaginous cover; the euglenoid pellicular striation and canal opening were easily visible. The volume of these intermediate spherical cells and mature cysts was estimated to have increased threefold compared to flagellated cells and contained many paramylon grains. When the cells were grazed by zooplankton, the paramylon grains passed the gut intact and were packed into fecal pellets. Intact undigested paramylon grains were observed in SEM after the breaking up of the pellets.

Paramylon synthesis by Euglena gracilis photoheterotrophically grown under low O2 pressure

Special culture conditions for Euglena gracilis Z and ZR are described. They induce interactions between the chloroplast and mitochondrial metabolisms leading to paramylon synthesis. When grown in continuous light under pure nitrogen and in the presence of lactate as the sole carbon source, sugar synthesis occurs during the first 24 h of culture with the participation of both mitochondria (using lactate) and of chloroplasts (fixing CO2 from lactate decarboxylation). The activities of ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase are very high and mitochondria and chloroplasts develop then a common network of vesicles in which paramylon grains can be seen. Electron micrographs demonstrate membrane continuity between the two types of organelles. Occasionally the mitochondrial matrix and the chloroplast stroma are separated by only a unit membrane.

PARAMYLON SYNTHESIS IN HETEROTROPHIC AND PHOTOHETEROTROPHIC EUGLENA (EUGLENOPHYCEAE)1

ABSTRACTGrowth of Euglena gracilis Klebs in medium containing DL‐lactate as sole carbon source results in the synthesis of large quantities of paramylon at the beginning of exponential growth. The accumulation of paramylon grains in the cytoplasm is observable in both dark‐grown and light‐grown cells. Furthermore, the cells fix 14CO2 in darkness. Incorporated CO2 is found in paramylon which is function of the physiological state of the cells, being most intense in exponential phase heterotrophic Euglena. The mitochondria undergo structural modifications in older cultures, i.e., when the exogenous carbon source becomes exhausted. At the beginning of exponential growth, they are swollen and vesiculated with an electrontranslucent matrix containing an extensive internal membrane system. This vesiculated mitochondrial system is correlated with paramylon synthesis, since forming paramylon grains are seen within the vesiculated complex. Stationary phase cells present a compact chondriome with a dense matrix devoid of vesiculation. Membrane‐bound paramylon grains are found in the cytoplasm of these cells.

CRYPTOGLENA PIGRA: A EUGLENOID WITH ONE CHLOROPLAST12

ABSTRACT The taxonomic status of Cryptoglena pigra Ehrb., interpreted from observations based on bright‐field microscopy, has been uncertain. Examination with the electron microscope of a clone of C pigra isolated by E. G. Pringsheim reveals certain features which, collectively, are distinctly euglenoid: periplast associated with muciferous bodies and subpellicular microtubules; canal and reservoir with microtubules; one flagellum with a swelling and emergent through a canal, and a second flagellum without a swelling and nonremergent; stigma (eyetpot) closely apprrssed to but not part of the chloroplast; nucleus with permanently condensed chromosomes attached to the inner nuclrar membrane; mitochondria with disc‐shaped cristae constricted at the base; chloroplast with thylakoids often in triplets; and paramylon grains in the cytoplasm. Unlike most euglenoids, C. pigra possesses a single chloroplast that in transverse thin sections is U‐shaped.