Shotgun metagenomics and microscopy indicate diverse cyanophytes, other bacteria, and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria).

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free-living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.

Immunofluorescence localization of the tubulin cytoskeleton during cell division and cell growth in members of the Coleochaetales (Streptophyta).

Study of charophycean green algae, including the Coleochaetales, may shed light on the evolutionary history of characters they share with their land plant relatives. We examined the tubulin cytoskeleton during mitosis, cytokinesis, and growth in members of the Coleochaetales with diverse morphologies to determine if phragmoplasts occurred throughout this order and to identify microtubular patterns associated with cell growth. Species representing three subgroups of Coleochaete and its sister genus Chaetosphaeridium were studied. Cytokinesis involving a phragmoplast was found in the four taxa examined. Differential interference contrast microscopy of living cells confirmed that polar cytokinesis like that described in the model flowering plant Arabidopsis occurred in all species when the forming cell plate traversed a vacuole. Calcofluor labeling of cell walls demonstrated directed growth from particular cell regions of all taxa. Electron microscopy confirmed directed growth in the unusual growth pattern of Chaetosphaeridium. All four species exhibited unordered microtubule patterns associated with diffuse growth in early cell expansion. In subsequent elongating cells, Coleochaete irregularis Pringsheim and Chaetosphaeridium globosum (Nordstedt) Klebahn exhibited tubulin cytoskeleton arrays corresponding to growth patterns associated with tip growth in plants, fungi, and other charophycean algae. Hoop-shaped microtubules frequently associated with diffuse growth of elongating cells in plants were not observed in any of these species. Presence of phragmoplasts in the diverse species studied supports the hypothesis that cytokinesis involving a phragmoplast originated in a common ancestor of the Coleochaetales, and possibly in a common ancestor of Charales, Coleochaetales, Zygnematales, and plants.

Lacustrine Nostoc (Nostocales) and associated microbiome generate a new type of modern clotted microbialite.

Microbialites are mineral formations formed by microbial communities that are often dominated by cyanobacteria. Carbonate microbialites, known from Proterozoic times through the present, are recognized for sequestering globally significant amounts of inorganic carbon. Recent ecological work has focused on microbial communities dominated by cyanobacteria that produce microbial mats and laminate microbialites (stromatolites). However, the taxonomic composition and functions of microbial communities that generate distinctive clotted microbialites (thrombolites) are less well understood. Here, microscopy and deep shotgun sequencing were used to characterize the microbiome (microbial taxa and their genomes) associated with a single cyanobacterial host linked by 16S sequences to Nostoc commune Vaucher ex Bornet & Flahault, which dominates abundant littoral clotted microbialites in shallow, subpolar, freshwater Laguna Larga in southern Chile. Microscopy and energy-dispersive X-ray spectroscopy suggested the hypothesis that adherent hollow carbonate spheres typical of the clotted microbialite begin development on the rigid curved outer surfaces of the Nostoc balls. A surface biofilm included >50 nonoxygenic bacterial genera (taxa other than Nostoc) that indicate diverse ecological functions. The Laguna Larga Nostoc microbiome included the sulfate reducers Desulfomicrobium and Sulfospirillum and genes encoding all known proteins specific to sulfate reduction, a process known to facilitate carbonate deposition by increasing pH. Sequences indicating presence of nostocalean and other types of nifH, nostocalean sulfide:ferredoxin oxidoreductase (indicating anoxygenic photosynthesis), and biosynthetic pathways for the secondary products scytonemin, mycosporine, and microviridin toxin were identified. These results allow comparisons with microbiota and microbiomes of other algae and illuminate biogeochemical roles of ancient microbialites.

Aeroterrestrial Coleochaete (Streptophyta, Coleochaetales) models early plant adaptation to land.

PREMISE OF THE STUDY: The streptophyte water-to-land transition was a pivotal, but poorly understood event in Earth history. While some early-diverging modern streptophyte algae are aeroterrestrial (living in subaerial habitats), aeroterrestrial survival had not been tested for Coleochaete, widely regarded as obligately aquatic and one of the extant green algal genera most closely related to embryophytes. This relationship motivated a comparison of aeroterrestrial Coleochaete to lower Paleozoic microfossils whose relationships have been uncertain. METHODS: We tested the ability of two species of the experimentally tractable, complex streptophyte algal genus Coleochaete Bréb. to (1) grow and reproduce when cultivated under conditions that mimic humid subaerial habitats, (2) survive desiccation for some period of time, and (3) produce degradation-resistant remains comparable to enigmatic Cambrian microfossils. KEY RESULTS: When grown on mineral agar media or on quartz sand, both species displayed bodies structurally distinct from those expressed in aquatic habitats. Aeroterrestrial Coleochaete occurred as hairless, multistratose, hemispherical bodies having unistratose lobes or irregular clusters of cells with thick, layered, and chemically resistant walls that resemble certain enigmatic lower Paleozoic microfossils. Whether grown under humid conditions or air-dried for a week, then exposed to liquid water, aeroterrestrial Coleochaete produced typical asexual zoospores and germlings. Cells that had been air-dried for periods up to several months maintained their integrity and green pigmentation. CONCLUSIONS: Features of modern aeroterrestrial Coleochaete suggest that ancient complex streptophyte algae could grow and reproduce in moist subaerial habitats, persist through periods of desiccation, and leave behind distinctive microfossil remains.

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy.

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.

Rolled liverwort mats explain major Prototaxites features: Response to commentaries.

In volume 97(2) of the American Journal of Botany (pp. 268-275), we published an article entitled "Structural, physiological, and stable carbon isotopic evidence that the enigmatic Paleozoic fossil Prototaxites formed from rolled liverwort mats". Here, we respond to a letter and a commentary on our article in the present issue, welcoming this opportunity to continue the scientific dialogue about an issue that has long been stimulating and controversial. For the reader's benefit, we first briefly describe the recent scholarly context of our article.

Structural, physiological, and stable carbon isotopic evidence that the enigmatic Paleozoic fossil Prototaxites formed from rolled liverwort mats.

New structural, nutritional, and stable carbon isotope data may resolve a long-standing mystery-the biological affinities of the fossil Prototaxites, the largest organism on land during the Late Silurian to Late Devonian (420-370 Ma). The tree trunk-shaped specimens, of varying dimensions but consistent tubular anatomy, first formed prior to vascular plant dominance. Hence, Prototaxites has been proposed to represent giant algae, fungi, or lichens, despite incompatible biochemical and anatomical observations. Our comparative analyses instead indicate that Prototaxites formed from partially degraded, wind-, gravity-, or water-rolled mats of mixotrophic liverworts having fungal and cyanobacterial associates, much like the modern liverwort genus Marchantia. We propose that the fossil body is largely derived from abundant, highly degradation-resistant, tubular rhizoids of marchantioid liverworts, intermixed with tubular microbial elements. Our concept explains previously puzzling fossil features and is consistent with evidence for liverworts and microbial associates in Ordovician-Devonian deposits, extensive ancient and modern marchantioid mats, and modern associations of liverworts with cyanobacteria and diverse types of fungi. Our interpretation indicates that liverworts were important components of Devonian ecosystems, that some macrofossils and microfossils previously attributed to "nematophytes" actually represent remains of ancient liverworts, and that mixotrophy and microbial associations were features of early land plants.

Cytokinesis in Coleochaete orbicularis (Charophyceae): an ancestral mechanism inherited by plants.

Recently, highly vacuolate cells of Arabidopsis were shown to exhibit "polarized" cytokinesis, in which the phragmoplast and cell plate contact the mother cell wall and then progress from one side of the cell to the other, rather than forming uniformly outward from the cell center (Cutler and Ehrhardt, 2002, Proceedings of the National Academy of Sciences, USA 99: 2812-2817). It was not known if such a mechanism was unique to flowering plants or whether it occurred more broadly in the plant clade. To determine if a polar mechanism of cell division might have been characteristic of the first plants, differential interference contrast optics were used to examine living cells of the charophycean green alga Coleochaete orbicularis, a close relative of plants, with cytokinesis involving a phragmoplast. By recording images in different focal planes over time, such "polarized" cytokinesis was found in cells dividing either parallel or perpendicular to the edge of this radially symmetrical organism. Previously reported differences between these two types of division in Coleochaete were clarified. Polarized cytokinesis appears to be an ancestral mechanism of plant cell division inherited from the highly vacuolate cells of the charophycean algal ancestors of plants.

Ultrastructure of potato tubers formed in microgravity under controlled environmental conditions.

Previous spaceflight reports attribute changes in plant ultrastructure to microgravity, but it was thought that the changes might result from growth in uncontrolled environments during spaceflight. To test this possibility, potato explants were examined (a leaf, axillary bud, and small stem segment) grown in the ASTROCULTURETM plant growth unit, which provided a controlled environment. During the 16 d flight of space shuttle Columbia (STS-73), the axillary bud of each explant developed into a mature tuber. Upon return to Earth, tuber slices were examined by transmission electron microscopy. Results showed that the cell ultrastructure of flight-grown tubers could not be distinguished from that of tuber cells grown in the same growth unit on the ground. No differences were observed in cellular features such as protein crystals, plastids with starch grains, mitochondria, rough ER, or plasmodesmata. Cell wall structure, including underlying microtubules, was typical of ground-grown plants. Because cell walls of tubers formed in space were not required to provide support against the force due to gravity, it was hypothesized that these walls might exhibit differences in wall components as compared with walls formed in Earth-grown tubers. Wall components were immunolocalized at the TEM level using monoclonal antibodies JIM 5 and JIM 7, which recognize epitopes of pectins, molecules thought to contribute to wall rigidity and cell adhesion. No difference in presence, abundance or distribution of these pectin epitopes was seen between space- and Earth-grown tubers. This evidence indicates that for the parameters studied, microgravity does not affect the cellular structure of plants grown under controlled environmental conditions.

Resistant tissues of modern marchantioid liverworts resemble enigmatic Early Paleozoic microfossils.

Absence of a substantial pretracheophyte fossil record for bryophytes (otherwise predicted by molecular systematics) poses a major problem in our understanding of earliest land-plant structure. In contrast, there exist enigmatic Cambrian-Devonian microfossils (aggregations of tubes or sheets of cells or possibly a combination of both) controversially interpreted as an extinct group of early land plants known as nematophytes. We used an innovative approach to explore these issues: comparison of tube and cell-sheet microfossils with experimentally degraded modern liverworts as analogues of ancient early land plants. Lower epidermal surface tissues, including rhizoids, of Marchantia polymorpha and Conocephalum conicum were resistant to breakdown after rotting for extended periods or high-temperature acid treatment (acetolysis), suggesting fossilization potential. Cell-sheet and rhizoid remains occurred separately or together depending on the degree of body degradation. Rhizoid break-off at the lower epidermal surface left rimmed pores at the centers of cell rosettes; these were similar in structure, diameter, and distribution to pores characterizing nematophyte cell-sheet microfossils known as Cosmochlaina. The range of Marchantia rhizoid diameters overlapped that of Cosmochlaina pores. Approximately 14% of dry biomass of Marchantia vegetative thalli and 40% of gametangiophores was resistant to acetolysis. Pre- and posttreatment cell-wall autofluorescence suggested the presence of phenolic compounds that likely protect lower epidermal tissues from soil microbe attack and provide dimensional stability to gametangiophores. Our results suggest that at least some microfossils identified as nematophytes may be the remains of early marchantioid liverworts similar in some ways to modern Marchantia and Conocephalum.