Ancient origin of fucosylated xyloglucan in charophycean green algae.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.

Simultaneous use of Interphako interference contrast and polarization microscopy in the study of microorganisms.

Simultaneous application of polarization microscopy and Interphako interference contrast has been used to study the internal structure of algal cells. The interference contrast technique showed fine cell structures (important is the selection of interference colors according to the Mach-Zehnder interferometer setting). In a polarization microscope, the crossed polarization filters together with the first-order quartz compensator mounted turntable showed the maximum birefringence of the individual structures. Material containing green algae was collected in the villages Sýkorice and Zbecno, Protected Landscape Area (PLA) Krivoklátsko. The objects were studied in a Carl Zeiss Jena NfpK laboratory microscope equipped with an In 160 base body with an Interphako In contrast interference module including a Mach-Zehnder interferometer with variable phase contrast, a special condenser with interchangeable aperture plates, a turntable, a Meopta Praha polarizer, a LOMO Sankt Petersburg analyzer, and a quartz compensator with first-order red and the digital camera DSLR Nikon D 70. Green algae of three orders were studied: Siphonocladales, Zygnematales, and Desmidiales. Anisotropic structures were found in all studied representatives of the green algae of the phylum Chlorophyta. Especially their cell walls showed strong birefringence (in all representatives of these orders). On the other hand, a representative of the order Siphonocladales (the genus Cladophora, Cladophoraceae, Ulvophyceae) was rarely found to display weak birefringent granules of storage substances due to the setting of the Mach-Zehnder interferometer and the use of the first-order compensator (interference colors are intensified). In addition, a very weak birefringence of periphyton cells (microbial biofilm) was found. In the study of the second algae of the genus Spirogyra (Zygnemataceae, Zygnematales, Conjugatophyceae), a strongly birefringent connecting wall between algal cells was found in contrast to the weaker birefringence of the peripheral wall. It was the use of Interphako interference contrast together with polarization filters and a first-order quartz compensator that particularly emphasized the central part of the connecting wall. In the study of the twinned Pleurotaenium algae (Desmidiaceae, Desmidiales, Conjugatophyceae), a strongly birefringent wall was found along the periphery of the cell with a nucleus in the middle part (isthmus). In this narrowing in the center of the cell, a sharply delimited birefringent edge of the cell wall is visible, especially when using Interphako interference contrast along with crossed polarization filters and a first-order quartz compensator. In conclusion, Interphako interference contrast provides a high degree of image contrast in a microscope and, if suitably simultaneously complemented by polarization microscopy (including a first-order quartz compensator), it will allow us to infer some of the composition of the investigated structures. However, working with Interphako interference contrast is considerably more difficult (setting Mach-Zehnder interferometer) than using other contrast techniques (positive and negative phase contrast, color contrast, relief contrast, and dark field).

A natural toroidal microswimmer with a rotary eukaryotic flagellum.

We describe Idionectes vortex gen. nov., sp. nov., a unicellular microeukaryote that swims by continuous inversion of its surface, similar to a vortex ring. This previously unreported mode of motility approximates a hypothetical concept called the 'toroidal swimmer', in which a doughnut-shaped object rotates around its circular axis and travels in the opposite direction to its outer surface motion. During swimming, the flagellum of Idionectes rotates relative to its cell body, which is normally a hallmark of prokaryotic rather than eukaryotic flagella.

Identification of 13 Spirogyra species (Zygnemataceae) by traits of sexual reproduction induced under laboratory culture conditions.

The genus Spirogyra is abundant in freshwater habitats worldwide, and comprises approximately 380 species. Species assignment is often difficult because identification is based on the characteristics of sexual reproduction in wild-collected samples and spores produced in the field or laboratory culture. We developed an identification procedure based on an improved methodology for inducing sexual conjugation in laboratory-cultivated filaments. We tested the modified procedure on 52 newly established and genetically different strains collected from diverse localities in Japan. We induced conjugation or aplanospore formation under controlled laboratory conditions in 15 of the 52 strains, which allowed us to identify 13 species. Two of the thirteen species were assignable to a related but taxonomically uncertain genus, Temnogyra, based on the unique characteristics of sexual reproduction. Our phylogenetic analysis demonstrated that the two Temnogyra species are included in a large clade comprising many species of Spirogyra. Thus, separation of Temnogyra from Spirogyra may be untenable, much as the separation of Sirogonium from Spirogyra is not supported by molecular analyses.

Red And far-red regulation of filament movement correlates with the expression of phytochrome and FHY1 genes in Spirogyra varians (Zygnematales, Streptophyta)1.

Spirogyra filaments show unique photomovement that differs in response to blue, red, and far-red light. Phototropins involved in the blue-light movement have been characterized together with downstream signaling components, but the photoreceptors and mechanical effectors of red- and far-red light movement are not yet characterized. The filaments of Spirogyra varians slowly bent and aggregated to form a tangled mass in red light. In far-red light, the filaments unbent, stretched rapidly, and separated from each other. Mannitol and/or sorbitol treatment significantly inhibited this far-red light movement suggesting that turgor pressure is the driving force of this movement. The bending and aggregating movements of filaments in red light were not affected by osmotic change. Three phytochrome homologues isolated from S. varians showed unique phylogenetic characteristics. Two canonical phytochromes, named SvPHY1 and SvPHY2, and a noncanonical phytochrome named SvPHYX2. SvPHY1 is the first PHY1 family phytochrome reported in zygnematalean algae. The gene involved in the transport of phytochromes into the nucleus was characterized, and its expression in response to red and far-red light was measured using quantitative PCR. Our results suggest that the phytochromes and the genes involved in the transport system into the nucleus are well conserved in S. varians.

The diversity of ice algal communities on the Greenland Ice Sheet as revealed by oligotyping.

The Arctic is being disproportionally affected by climate change compared with other geographic locations, and is currently experiencing unprecedented melt rates. The Greenland Ice Sheet (GrIS) can be regarded as the largest supraglacial ecosystem on Earth, and ice algae are the dominant primary producers on bare ice surfaces throughout the course of a melt season. Ice-algal-derived pigments cause a darkening of the ice surface, which in turn decreases albedo and increases melt rates. The important role of ice algae in changing melt rates has only recently been recognized, and we currently know little about their community compositions and functions. Here, we present the first analysis of ice algal communities across a 100 km transect on the GrIS by high-throughput sequencing and subsequent oligotyping of the most abundant taxa. Our data reveal an extremely low algal diversity with Ancylonema nordenskiöldii and a Mesotaenium species being by far the dominant taxa at all sites. We employed an oligotyping approach and revealed a hidden diversity not detectable by conventional clustering of operational taxonomic units and taxonomic classification. Oligotypes of the dominant taxa exhibit a site-specific distribution, which may be linked to differences in temperatures and subsequently the extent of the melting. Our results help to better understand the distribution patterns of ice algal communities that play a crucial role in the GrIS ecosystem.

Concomitant use of polarization and negative phase contrast microscopy for the study of microorganisms.

A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkorice and Zbecno (Protected Landscape Area Krivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus Euglena (Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus Pediastrum (Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence-an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus Spirogyra (Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus Pinnularia (Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator-an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.

Gadolinium-uptake by aquatic and terrestrial organisms-distribution determined by laser ablation inductively coupled plasma mass spectrometry.

Gadolinium (Gd) based contrast agents (CA) are used to enhance magnetic resonance imaging. As a consequence of excretion by patients and insufficient elimination in wastewater treatment plants they are detected in high concentrations in surface water. At present, little is known about the uptake of these species by living organisms in aquatic systems. Therefore the uptake of gadolinium containing chelates by plants and animals grown in exposed water or on soil irrigated with exposed water was investigated. For this purpose two types of plants were treated with two different contrast agents. The uptake of the Gd contrast agents was studied by monitoring the elemental distribution with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This technique allows the multi-elemental analysis of solid samples with high resolution and little sample preparation. The analysis of L. minor showed that the uptake of Gd correlated with the concentration of gadodiamide in the water. The higher the concentration in the exposed water, the larger the Gd signal in the LA-ICP-MS acquired image. Exposure time experiments showed saturation within one day. The L. minor had contact with the CAs through roots and fronds, whereas the L. sativum only showed uptake through the roots. These results show that an external absorption of the CA through the leaves of L. sativum was impossible. All the analyzed parts of the plant showed Gd signal from the CA; the highest being at the main vein of the leaf. It is shown that the CAs can be taken up from plants. Furthermore, the uptake and distribution of Gd in Daphnia magna were shown. The exposure via cultivation medium is followed by Gd signals on the skin and in the area of the intestine, while the uptake via exposed nutrition algae causes the significantly highest Gd intensities in the area of the intestine. Because there are hints of negative effects for human organism these findings are important as they show that Gd based CAs may reach the human food chain via plants and animals growing in contaminated water or plants growing in fields which are irrigated with surface water.

Removal and bioaccumulation of heavy metals from aqueous solutions using freshwater algae.

Four freshwater algae, including Cladophora glomerata, Oedogonium westii, Vaucheria debaryana and Zygnema insigne, were tested for their bioaccumulation capacity for cadmium (Cd), chromium (Cr) and lead (Pb) in a controlled environment with an average temperature of 18 °C, and light/dark duration of 12:12 h. Experiments were performed in aqueous solutions containing selected heavy metals (HM) (ranging from 0.05 to 1.5 mg L(-1)) with 0.5 g of living algae at 18 °C and pH 6.8. The results indicated that C. glomerata was observed to be the most competent species for the removal of Cr, Cd and Pb from aqueous solutions. HM removal trends were in the order of Cd>Cr>Pb while the removal efficiency of selected algae species was in the order of C. glomerata, O. westii, V. debaryana and Z. insigne. The bioaccumulation capacity of C. glomerata, V. debaryana and Z. insigne was observed for different HM. Removal of HM was higher with low levels of HM in aqueous solutions. The results indicated that C. glomerata, O. westii, V. debaryana and Z. insigne had significant (P≤0.01) diverse bioaccumulation capacity for Cr, Cd and Pb.

Osmotic stress and recovery in field populations of Zygnema sp. (Zygnematophyceae, Streptophyta) on Svalbard (High Arctic) subjected to natural desiccation.

Zygnema is a genus of filamentous green algae belonging to the class of Zygnematophyceae (Streptophyta). In the Arctic, it typically forms extensive mats in habitats that regularly dry out during summer, and therefore, mechanisms of stress resistance are expected. We investigated its natural populations with respect to production of specialized desiccation-resistant cells and osmotic acclimation. Six populations in various stages of natural desiccation were selected, from wet biomass floating in water to dried paper-like crusts. After rewetting, plasmolysis and osmotic stress effects were studied using hypertonic sorbitol solutions, and the physiological state was estimated using chlorophyll a fluorescence parameters. All populations of Zygnema sp. formed stationary-phase cells filled with storage products. In green algal research, such cells are traditionally called akinetes. However, the populations differed in their reaction to osmotic stress. Whereas the wet-collected samples were strongly impaired, the osmotic stress resistance of the naturally dried samples was comparable to that of true aeroterrestrial algae. We showed that arctic populations of Zygnema acclimate well to natural desiccation via hardening that is mediated by slow desiccation. As no other types of specialized cells were observed, we assume that the naturally hardened akinetes also play a key role in winter survival.

Osmotic stress in Arctic and Antarctic strains of the green alga Zygnema (Zygnematales, Streptophyta): effects on photosynthesis and ultrastructure.

The osmotic potential and effects of plasmolysis on photosynthetic oxygen evolution and chlorophyll fluorescence were studied in two Arctic Zygnema sp. (strain B, strain G) and two Antarctic Zygnema sp. (strain E, strain D). Antarctic strain D was newly characterized by rbcL sequence analysis in the present study. The two Antarctic strains, D and E, are most closely related and may represent different isolates of the same species, in contrast, strain B and G are separate lineages. Incipient plasmolysis in the cells was determined by light microscopy after incubating cells in sorbitol solutions ranging between 200 mM and 1000 mM sorbitol for 3, 6 and 24h. In Zygnema strain B and G incipient plasmolysis occurred at ~600 mM sorbitol solution (720 mOsmol kg(-1), ψ=-1.67 MPa) and in strains D and E at ~300 mM (318 mOsmol kg(-1), ψ=-0.8 MPa) sorbitol solution. Hechtian strands were visualized in all plasmolysed cells, which is particularly interesting, as these cells lack pores or plasmodesmata. Ultrastructural changes upon osmotic stress were a retraction of the condensed cytoplasm from the cell walls, damages to chloroplast and mitochondrial membranes, increasing numbers of plastoglobules in the chloroplasts and membrane enclosed particles in the extraplasmatic space. Maximum photosynthetic rates (P(max)) in light saturated range were between 145.5 µmol O(2) h(-1)mg(-1)Chl a in Zygnema G and 752.9 µmol O(2) h(-1)mg(-1)Chl a in Zygnema E. After incubation in 800 mM sorbitol for 3h P(max) decreased to the following percentage of the initial values: B: 16.3%, D: 16.8%, E: 26.1% and G: 35.0%. Osmotic stress (800 mM sorbitol) decreased maximum photochemical quantum yield of photosystem II (F(v)/F(m)) when compared to controls. Maximum values of relative electron transport rates of photosystem II (rETR(max)) decreased after incubation in 400 mM sorbitol in Zygnema D and E, while they decreased in Zygnema B and G only after incubation in 800 mM sorbitol. The kinetics of the rETR curves were similar for the Arctic strains Zygnema B and G, but distinct from the Antarctic strains Zygnema D and E, which were similar when compared with each other. This suggests that the investigated Arctic Zygnema sp. strains might be better adapted to tolerate osmotic water stress than the investigated strains from the Antarctic.

Broad phylogenomic sampling and the sister lineage of land plants.

The tremendous diversity of land plants all descended from a single charophyte green alga that colonized the land somewhere between 430 and 470 million years ago. Six orders of charophyte green algae, in addition to embryophytes, comprise the Streptophyta s.l. Previous studies have focused on reconstructing the phylogeny of organisms tied to this key colonization event, but wildly conflicting results have sparked a contentious debate over which lineage gave rise to land plants. The dominant view has been that 'stoneworts,' or Charales, are the sister lineage, but an alternative hypothesis supports the Zygnematales (often referred to as "pond scum") as the sister lineage. In this paper, we provide a well-supported, 160-nuclear-gene phylogenomic analysis supporting the Zygnematales as the closest living relative to land plants. Our study makes two key contributions to the field: 1) the use of an unbiased method to collect a large set of orthologs from deeply diverging species and 2) the use of these data in determining the sister lineage to land plants. We anticipate this updated phylogeny not only will hugely impact lesson plans in introductory biology courses, but also will provide a solid phylogenetic tree for future green-lineage research, whether it be related to plants or green algae.