Abstract. Understanding features that fostered the persistence of Equisetum–Earth's oldest extant vascular plant genus–since Mesozoic times and through episodes of significant global environmental change, is of current interest in view of modern challenges to plant survival. In addition to known structural and physiological adaptations, we hypothesized that microscopy and shotgun metagenomic sequencing might reveal eukaryotic microorganisms such as fungi that may aid Equisetum survival. Here, we report evidence for several lineages of eukaryotic microbes associated with giant Equisetum xylochaetum, which dominates vegetation in saline streambeds of remote valleys in the hyper-arid Atacama Desert, Chile. Plant material was collected and field-preserved at two comparatively low-disturbance sites; DNA extracted in Chile using low-shear methods was later sequenced, 18S and 28S rDNA taxonomic marker sequences were selected for SILVAngs classification, allowing comparisons to eukaryotic microorganisms previously inferred for earlier-diverging plant lineages. SEM, fluorescence microscopy, and/or LM of toluidine blue-stained sections of roots indicated protists, epiphytic and endophytic fungi, and cortical nematodes. Eukaryotic genera inferred from 18S rDNA at >100X mean sequencing depth included the ciliate Engelmanniella, hyphal chytrid Monoblepharella, predatory ascomycete Cephaliophora, a salpingoecid choanoflagellate, and an annelid worm. 23S rDNA sequences indicated ascomycete Capnodiales fungi at one site and four types of Pezizomycotina fungi at the other. No evidence for vesicular-arbuscular mycorrhizal fungi was found, but we hypothesized that Equisetum may benefit from other types of fungal associations, some possibly inherited from ancestral plant lineages.

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.

Since rooted vascular plants rose to dominance, associated microbiota have powerfully influenced global biogeochemistry by mobilizing N and P and otherwise aiding plant health, thereby fostering sequestration of CO2 into coal and soil organics. Less well understood are evolutionary history and biogeochemical roles of microbiomes of ecologically significant nonvascular plants and related algae, whose lineages penetrate more deeply into time. Because analyses of diverse host-microbiome systems indicate that evolutionary history commonly influences microbiome composition and function, we mapped onto a geological time frame biogeochemical features inferred in our previous metagenomic studies of a phylogenetic spectrum of multicellular, freshwater-terrestrial, nonvascular autotroph-microbiome systems: Nostoc commune, representing microbialite-forming cyanobacteria; the Nostoc lichen Peltigera ponojensis; chlorophyte Cladophora glomerata; streptophyte alga Coleochaete pulvinata; moss Sphagnum fimbriatum; and liverwort Conocephalum conicum. Widespread and often abundant today, these species represent ancient lineages of minimal ages ranging from 1450 to 380 Ma, information we used to estimate present global annual magnitudes of organic/inorganic C sequestration, N fixation, and methane oxidation and to calculate biogeochemical impacts during periods from lineage origin to the rise of vascular plants and from lineage origins to present. Functional comparisons indicated that (1) functionally diverse bacterial associates have likely enhanced global C- and N-cycle roles of cyanobacteria for ∼1.5 billion years; (2) by 700–800 Ma, chlorophyte microbiomes retaining ancient functional features (e.g., vitamin B12 biosynthesis and N fixation) had added diverse methane-oxidizing bacteria and eukaryotes, including early metazoans; (3) by ∼500 Ma, streptophyte algal microbiomes possessing ancient functions had incorporated early-diverging fungi; (4) by 450 Ma, microbiomes of early plants included diverse prokaryotes, protists, fungi, and early terrestrial animals; and (5) by 385 Ma, plant microbiomes included P-mobilizing fungi. Autotroph-microbiome systems appear to have accreted functions of global significance over deep time, a new concept that illuminates the early evolution of terrestrial life and that aids modeling past, present, and future biogeochemical impacts.

Premise of research.Although molecular analyses indicate that fungi likely began to diversify in the Proterozoic, fossil remains from this period have not been confidently linked to modern fungal lineages. Less ambiguous Early Paleozoic remains have been attributed to diverse modern fungal lineages, though some classifications have been questioned, and linking fragmentary fossils to modern fungal groups has generally been challenging. Better knowledge of fungal structures most likely to be preserved in the fossil record would aid classifying enigmatic remains. To achieve this, we tested a spectrum of modern fungi that have been linked to Paleozoic fossils and/or display environmental persistence for retention of diagnostic structural features after acetolysis, a high-temperature acid hydrolysis process employed as a proxy for harsh diagenetic processes.Methodology.Standard acetolysis was applied to cultures of blastocladialean, mucoralean, or ascomycete fungi having melanized structures hypothesized to resist hydrolytic degradation; glomalean fungi associated with greenhouse-grown and field-collected thalloid liverworts; and basidiocarp samples from two species of the woody basidiomycete Ganoderma.Pivotal results.Distinctive acetolysis-resistant fungal structures included Allomyces javanicus-resistant sporangia; glomalean hyphae and spores; tubular sporangiophores, globular asexual sporangia, zygosporangia with suspensors, and hyphal tangles of Phycomyces blakesleeanus (Mucoromycotina); conidiophores, phialides, chains of conidia, and cleistothecial walls of Aspergillus chevalieri (Ascomycota); and dense-walled septate hyphae and perithecia of Sordaria fimicola and Chaetomium globosum (Ascomycota). Several taxa left few (e.g., nondistinctive spores) or no identifiable cellular remains.Conclusions.Consistent with previous reports, melanized structures tended to be hydrolysis resistant. Retention by modern liverwort-associated glomalean fungi of diagnostic hyphal and spore features after acetolysis indicates high potential for fossilization even under suboptimal conditions, supporting a previous classification of microfossils extracted from Ordovician carbonates. Acetolysis results were also consistent with interpretation of particular Silurian-Devonian macrofossils as plant-microbial consortia.

The freshwater and terrestrial green algal lineages discussed in this chapter include the scaly flagellate Mesostigma, the sarcinoid form Chlorokybus, the unbranched filamentous members of the Klebsormidiophyceae, and the branched filamentous members of the Coleochaetophyceae. The lineages discussed here, together with two other green algal lineages (Charophyceae and Zygnematophyceae) and the land plants (embryophytes), form a monophyletic group known as Streptophyta or Charophyta. The streptophyte algae share cytological and biochemical characteristics with plants and may shed light on the evolution of plant features. Of special interest is the evolution of mechanisms associated with the transition from freshwater to dry land, a topic currently being energized by whole-genome analyses. Metagenomic studies of these organisms have revealed surprising features that might also have characterized the microbiomes of early streptophytes.

Premise of research. The origin of land plants catalyzed key changes in Earth's atmosphere and biota. Microbial associations likely nurtured earliest plants and influenced their biogeochemical roles. Because angiosperm and animal microbiomes-bacteria, archaea, microbial eukaryotes, and genes that promote host survival-are known to display lineage effects, we hypothesized that microbiomes of early-diverging modern bryophytes and phylogenetically closely related green algae might likewise reveal commonalities reflecting ancestral traits.Methodology. New metagenomic sequence data were obtained for the late-diverging streptophyte algae Chaetosphaeridium globosum and Coleochaete pulvinata and the liverwort Conocephalum conicum, representing early-diverging land plants. New 16S rDNA amplicon sequences were acquired for the charalean Nitella tenuissima. Sequence data were used to infer bacterial genera and fungi for comparisons among streptophyte microbiota and with our published microbiome data for the outgroup chlorophyte Cladophora. To enhance evolutionary signal, taxa were sampled in the same time frame and from geographically close locales. Streptophyte metagenomic data were also probed for protein markers of significant physiological and biogeochemical functions: NifH indicating nitrogen fixation, particulate MMo indicating methane oxidation, and vitamin B-12 (cobalamin) indicating biosynthetic pathway enzymes.Pivotal results. Microbiota of studied streptophytes consistently included diverse N-fixing cyanobacteria and/or Rhizobiales, as well as methanotrophs and early-diverging fungi, and were more similar to each other than to Cladophora microbiota. Streptophyte metagenomic data indicated diverse nifH (nitrogen fixation) and pMMo (methane oxidation) marker sequences and vitamin B-12 pathway genes. Glomalean fungi occurred with Conocephalum, consistent with field studies of modern liverworts and microfossil evidence for co-occurrence of glomaleans and early land plants.Conclusions. A suite of N fixers, methanotrophs, cobalamin producers, and early-diverging fungi was consistently associated with modern streptophyte algae and bryophytes studied, suggesting features of early land plants that have played significant, previously unrecognized roles in global nitrogen and carbon cycling for hundreds of millions of years.

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.

Terrestrialization of planet Earth likely began more than a billion years ago with the colonization of land by bacteria, followed by eukaryotic algae much like those occupying modern soils and shallow freshwaters and the earliest embryophytes, close relatives of modern bryophytes. Colonization of land by algae and the first plants was prerequisite to the development of organic-rich soils that later supported more complex plant communities dominated by vascular plants, and the rise of land animals. Consequently, understanding terrestrialization sheds light on Earth's early biological carbon cycling processes, which aids our understanding of global biogeochemistry in particular, and planetary science in general.Comprehending the process and pattern of ancient terrestrialization requires both neontological and paleontological approaches. Molecular phylogenetics provides the necessary scaffold upon which terrestrialization processes can be analyzed by comparing the structures, physiologies, microbiomes, and genomes of earliest-branching lineages of modern liverworts and mosses to those of plants' closest modern green algal relatives, the streptophyte algae (also known as charophyte algae or charophycean green algae). Such studies reveal that modern bryophytes inherited spore and body desiccation-resistance, degradation-resistant lignin-like phenolic cell wall polymers, and other physiological traits useful in terrestrial habitats from ancestral algae, indicating that such features were also traits of the earliest land plants.Because modern algae and bryophytes possess degradation-resistant cells or tissues, artificially degrading them for comparison with enigmatic microscopic fossils has been a fruitful way to identify remains of early terrestrial photosynthesizers and thus illuminate terrestrialization patterns. Microfossils cited as evidence for terrestrial cyanobacteria occur beginning more than 1,000 million years ago in the Precambrian, as do probable remains of freshwater and terrestrial eukaryotic algae. Some microfossils obtained from 499 to 511 million year old deposits closely resemble the modern complex streptophyte alga Coleochaete when it has been cultivated subaerially, suggesting that streptophytes were able to photosynthesize on land by the Middle Cambrian. Other microfossils observed in Cambrian and early Middle Ordovician deposits may also be remains of land plants. Remains of early liverwort-like land plants are confidently known from 470 million year old mid-Ordovician deposits, as are possible fossils of early-divergent mosses. Microfossils and macrofossils that have been compared to modern liverwort and moss taxa occur in Silurian to Devonian deposits laid down before and during the first major diversification of the vascular plants in the Late Silurian to Early Devonian, 407-418 million years ago. Such evidence, together with molecular phylogenies and clock analyses, demonstrates that bryophytes and streptophyte algal relatives were the dominant eukaryotic photosynthesizers on land from about 500-400 million years ago, prior to and during the earliest stages of vascular plant evolution.Because bryophytes and streptophyte algae produce degradation-resistant carbon that can be sequestered, thereby reducing atmospheric carbon dioxide levels, models suggest that they had significant impacts on Earth's carbon cycle for at least 40 million years and perhaps more than 100 million years. We can thus predict that other Earth-like, habitable-zone planets may likewise experience long periods during which organisms equivalent to earthly terrestrial streptophyte algae and bryophytes impact planetary biogeochemistry.

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.

Premise of research. The taxonomic affinities of nonmarine Proterozoic and Paleozoic microfossils are often difficult to determine. Given that the preservability (degradation resistance) of cell walls displaying distinctive features is widely regarded as a key feature allowing the recognition and classification of fossil protists, we examined the retention of diagnostic cell wall features after high-temperature chemical hydrolysis of several modern filamentous algal genera previously hypothesized to be related to particular Proterozoic or Paleozoic microfossils.Methodology. We collected and in some cases cultured filamentous algae from modern terrestrial sites or freshwaters of arid locales hypothesized to model ancient nonmarine habitats. We subjected these and other samples of Vaucheria (Stramenopila, Xanthophyceae), Cladophora (Chlorophyta, Ulvophyceae), Stigeoclonium (Chlorophyta, Chlorophyceae), and Oedogonium (Chlorophyta, Chlorophyceae) to acetolysis, an extremely degradative hydrolytic process widely used in palynology to select for resistant organic materials. We imaged the remains using bright-field, polarizing, and fluorescence LM and also SEM.Pivotal results. Filaments of all xanthophycean and chlorophytan green algal genera tested resisted acetolysis and retained distinctive structural traits previously used to classify Proterozoic and Paleozoic microfossils as algae. Features of cell wall remains revealed by polarizing microscopy and SEM suggested that degradation resistance results largely from the presence in cell walls of cellulose types that are more resistant to degradation than are celluloses of land plants and streptophyte algae. In the case of Cladophora, specific autofluorescence properties also suggest the presence of a previously undetected phenolic layer in the primarily cellulosic cell wall.Conclusions. Our results are more or less consistent with previous classifications of certain ancient microfossils with genera of modern filamentous algae and explain degradation resistance of their cell walls. The results justify the use of cell wall features to classify filamentous microfossils and suggest steps that might yield even more convincing identifications.

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.

PREMISE OF THE STUDYThe 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.METHODSWe 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 RESULTSWhen 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.CONCLUSIONSFeatures 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.

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.

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.

Plants—defined here as the monophyletic Phylum Embryophyta composed of bryophytes and vascular plants—achieved multicellularity independently of animals and fungi. Plant bodies typically exhibit both apical-basal and radial symmetry, and are able to detect and respond to gravity vectors. Insight into the origin and early evolution of plant body symmetry and gravity responses can be obtained by comparing early-divergent embryophytes—bryophytes—with the modern green algae closest to plant ancestry—charophyceans. The comparative approach suggests that basal-apical and radial symmetry and gravity response capabilities were established prior to colonization of land and may have contributed to the success of early plants on land. Mapping traits related to body symmetry and gravity responses onto increasingly more robust phylogenies helps to elucidate the order and mechanism of trait appearances, suggesting model taxa whose further study is likely to prove most illuminating. For example, among charophycean algae Zygnematales exhibit form and behavior that may reflect mechanisms underlying symmetry and growth polarities typical of Coleochaetales and Charales (which are more closely related to plant ancestry) and embryophytes. Molecular, biochemical, and cell-level approaches applied within a phylogenetic context provide powerful tools for understanding early events in the evolutionary origin of plant body symmetry and gravity responses. For example, molecular analyses have revealed that early divergent plants possess homologs of KNOX-like plant transcriptional regulators. The study of possible charophycean homologs is expected to yield additional information about the evolution of plant-specific developmental regulation. Body symmetry is among the intriguing ways that plants differ from other organisms. Plants typically exhibit both apical-basal symmetry (also known as longitudinal or translational symmetry) and radial symmetry (also known as rotational or transectional symmetry) (Hudson, 2000). For example, leaves and other lateral organs typically originate from the tips of plant shoots, and they do so in spiral, circular, or other radial patterns that are apparent to even the most casual observer. Both plants and animals (including early divergent metazoans) possess apical-basal organization,but the typical radial symmetry of plant bodies departs dramatically from the bilateral symmetry characteristic of most animals. What is the evolutionary basis for this difference?

This chapter maps the stress-related physiological traits onto a robust phylogeny for modern charophycean algae and bryophytes. Trait mapping suggests that early phenolics could have been preadaptive to the development of stable plant–microbe relationships. As in modern plants, phenolic compounds may have controlled microbial behavior, allowing microbes to live in close proximity to algae and early land plants without becoming pathogenic. The chapter also compares the aspects of phenolic chemistry among charophyceans, bryophytes, and pteridophytes and estimates the extent to which nonvascular plants could have contributed to carbon sequestration prior to the origin of vascular plants. Thioacidolysis was used as an assay for lignin-specific β-O-4 phenolic linkages in representative green algae and early-divergent land plants. Selected green algae and bryophytes were surveyed for the presence of resistant biomass and the percentages of resistant cell wall biomass were quantitatively determined. The amount of resistant organic carbon that might have been generated by early non-vascular land plants was also estimated. Adaptive utility for high levels of wall phenolics might include (1) resistance to attack by pathogenic bacteria, protists and fungi, (2) increased stability of cell walls, contributing to the ability to achieve increased height, (3) UV-damage resistance, and (4) desiccation resistance.

Friction on ice in driving, running, and walking situations is important for winter activities in Alaska. In order to simulate the friction, we designed a traction-measuring device fitting to low-temperature applications. By using a hydraulic system we apply a load ranging from less than one pound to as high as five hundred pounds. The static friction can be measured by moving the ice block against any materials desired. Results showed that the ice surface changes according to applied load, sliding speed, and temperature. This device is capable of providing insight of tribological behavior of ice and snow against materials such as automobile tires and shoe materials, etc.