Silvia Pressel

First evidence of mutualism between ancient plant lineages (Haplomitriopsida liverworts) and Mucoromycotina fungi and its response to simulated Palaeozoic changes in atmospheric CO2

The discovery that Mucoromycotina, an ancient and partially saprotrophic fungal lineage, associates with the basal liverwort lineage Haplomitriopsida casts doubt on the widely held view that Glomeromycota formed the sole ancestral plant–fungus symbiosis. Whether this association is mutualistic, and how its functioning was affected by the fall in atmospheric CO2 concentration that followed plant terrestrialization in the Palaeozoic, remains unknown. We measured carbon-for-nutrient exchanges between Haplomitriopsida liverworts and Mucoromycotina fungi under simulated mid-Palaeozoic (1500 ppm) and near-contemporary (440 ppm) CO2 concentrations using isotope tracers, and analysed cytological differences in plant–fungal interactions. Concomitantly, we cultured both partners axenically, resynthesized the associations in vitro, and characterized their cytology. We demonstrate that liverwort–Mucoromycotina symbiosis is mutualistic and mycorrhiza-like, but differs from liverwort–Glomeromycota symbiosis in maintaining functional efficiency of carbon-for-nutrient exchange between partners across CO2 concentrations. Inoculation of axenic plants with Mucoromycotina caused major cytological changes affecting the anatomy of plant tissues, similar to that observed in wild-collected plants colonized by Mucoromycotina fungi. By demonstrating reciprocal exchange of carbon for nutrients between partners, our results provide support for Mucoromycotina establishing the earliest mutualistic symbiosis with land plants. As symbiotic functional efficiency was not compromised by reduced CO2, we suggest that other factors led to the modern predominance of the Glomeromycota symbiosis.

Symbiotic options for the conquest of land

The domination of the landmasses of Earth by plants starting during the Ordovician Period drastically altered the development of the biosphere and the composition of the atmosphere, with far-reaching consequences for all life ever since. It is widely thought that symbiotic soil fungi facilitated the colonization of the terrestrial environment by plants. However, recent discoveries in molecular ecology, physiology, cytology, and paleontology have brought into question the hitherto-assumed identity and biology of the fungi engaged in symbiosis with the earliest-diverging lineages of extant land plants. Here, we reconsider the existing paradigm and show that the symbiotic options available to the first plants emerging onto the land were more varied than previously thought.

Exploding a myth: the capsule dehiscence mechanism and the function of pseudostomata in Sphagnum

The nineteenth century air-gun explanation for explosive spore discharge in Sphagnum has never been tested experimentally. Similarly, the function of the numerous stomata ubiquitous in the capsule walls has never been investigated. Both intact and pricked Sphagnum capsules, that were allowed to dry out, all dehisced over an 8-12 h period during which time the stomatal guard cells gradually collapsed and their potassium content, measured by X-ray microanalysis in a cryoscanning electron microscope, gradually increased. By contrast, guard cell potassium fell in water-stressed Arabidopsis. The pricking experiments demonstrate that the air-gun notion for explosive spore discharge in Sphagnum is inaccurate; differential shrinkage of the capsule walls causes popping off the rigid operculum. The absence of evidence for a potassium-regulating mechanism in the stomatal guard cells and their gradual collapse before spore discharge indicates that their sole role is facilitation of sporophyte desiccation that ultimately leads to capsule dehiscence. Our novel functional data on Sphagnum, when considered in relation to bryophyte phylogeny, suggest the possibility that stomata first appeared in land plants as structures that facilitated sporophyte drying out before spore discharge and only subsequently acquired their role in the regulation of gaseous exchange.

Fungal symbioses in hornworts: a chequered history

Hornworts are considered the sister group to vascular plants, but their fungal associations remain largely unexplored. The ancestral symbiotic condition for all plants is, nonetheless, widely assumed to be arbuscular mycorrhizal with Glomeromycota fungi. Owing to a recent report of other fungi in some non-vascular plants, here we investigate the fungi associated with diverse hornworts worldwide, using electron microscopy and molecular phylogenetics. We found that both Glomeromycota and Mucoromycotina fungi can form symbioses with most hornworts, often simultaneously. This discovery indicates that ancient terrestrial plants relied on a wider and more versatile symbiotic repertoire than previously thought, and it highlights the so far unappreciated ecological and evolutionary role of Mucoromycotina fungi.

Cellular Differentiation in Moss Protonemata: A Morphological and Experimental Study

BACKGROUND AND AIMS Previous studies of protonemal morphogenesis in mosses have focused on the cytoskeletal basis of tip growth and the production of asexual propagules. This study provides the first comprehensive description of the differentiation of caulonemata and rhizoids, which share the same cytology, and the roles of the cytoskeleton in organelle shaping and spatial arrangement. METHODS Light and electron microscope observations were carried out on in vitro cultured and wild protonemata from over 200 moss species. Oryzalin and cytochalasin D were used to investigate the role of the cytoskeleton in the cytological organization of fully differentiated protonemal cells; time-lapse photography was employed to monitor organelle positions. KEY RESULTS The onset of differentiation in initially highly vacuolate subapical cells is marked by the appearance of tubular endoplasmic reticulum (ER) profiles with crystalline inclusions, closely followed by an increase in rough endoplasmic reticulum (RER). The tonoplast disintegrates and the original vacuole is replaced by a population of vesicles and small vacuoles originating de novo from RER. The cytoplasm then becomes distributed throughout the cell lumen, an event closely followed by the appearance of endoplasmic microtubules (MTs) in association with sheets of ER, stacks of vesicles that subsequently disperse, elongate mitochondria and chloroplasts and long tubular extensions at both poles of the nucleus. The production of large vesicles by previously inactive dictysomes coincides with the deposition of additional cell wall layers. At maturity, the numbers of endoplasmic microtubules decline, dictyosomes become inactive and the ER is predominantly smooth. Fully developed cells remain largely unaffected by cytochalasin; oryzalin elicits profound cytological changes. Both inhibitors elicit the formation of giant plastids. The plastids and other organelles in fully developed cells are largely stationary. CONCLUSIONS Differentiation of caulonemata and rhizoids involves a remarkable series of cytological changes, some of which closely recall major events in sieve element ontogeny in tracheophytes. The cytology of fully differentiated cells is remarkably similar to that of moss food-conducting cells and, in both, is dependent on an intact microtubule cytoskeleton. The disappearance of the major vacuolar apparatus is probably related to the function of caulonema and rhizoids in solute transport. Failure of fully differentiated caulonema and rhizoid cells to regenerate is attributed to a combination of endo-reduplication and irreversible tonoplast fragmentation. The formation of giant plastids, most likely by fusion, following both oryzalin and cytochalasin treatments, suggests key roles for both microtubules and microfilaments in the spatial arrangement and replication of plastids.

Cytological insights into the desiccation biology of a model system: moss protonemata

*Set out here is the first generic account of the cytological effects of dehydration and rehydration and exogenous abscisic acid on moss protonemata. *Protonemal cells were subjected to slow and fast drying regimes, with and without prior exposure to abscisic acid. The cytological changes associated with de- and rehydration were analysed by light, fluorescence and transmission electron microscopy, together with pharmacological studies. *Protonemata survive slow but not fast drying, unless pretreated with abscisic acid. Dehydration elicits profound cytological changes, namely vacuolar fragmentation, reorganization of the endomembrane domains, changes in the thickness of the cell wall and in the morphology of plastids and mitochondria, and the controlled dismantling of the cytoskeleton; these dynamic events are prevented by fast drying. In control cells, abscisic acid elicits changes that partially mimic those associated with slow drying, including controlled disassembly of cytoskeletal elements, thus enabling protonemal cells to survive normally lethal rates of water loss. *Our demonstration that moss protonemata are an ideal system for visualizing and manipulating the cytological events associated with vegetative desiccation tolerance in land plants now opens up the way for genomic dissection of the underlying mechanisms.

A novel ascomycetous endophytic association in the rhizoids of the leafy liverwort family, Schistochilaceae (Jungermanniidae, Hepaticopsida).

Liverworts form diverse associations with endophytic fungi similar to mycorrhizas in vascular plants. Whereas the widespread occurrence of glomeromycotes in the basal liverwort lineages is well documented, knowledge of the distribution of ascomycetes and basidiomycetes in derived thalloid and leafy clades is more fragmented. Our discovery that the ramified and septate rhizoids of the Schistochilaceae, the sister group to all other ascomycete-containing liverworts, are packed with fungal hyphae prompted this study on the effects of the fungi on rhizoid morphology, host specificity, the cytology of the association, and a molecular analysis of the endophytes. Two species of Pachyschistochila and their fungi were grown axenically. Axenic rhizoids were unbranched and nonseptate. Reinfected with their own fungus and that from the other species, both Pachyschistochila species produced branched and septate rhizoids identical to those in nature. Woronin bodies and simple septa identified the fungus as an ascomycete referable, according to phylogenetic analyses of ITS sequences, to the Rhizoscyphus (Hymenoscyphus) ericae aggregate, also found in other liverwort-ascomycete associations and in mycorrhizas in the Ericales. Healthy hyphae and host cytoplasm suggest that the Schistochila-fungus association reflects a balanced mutualistic relationship. The recent dating of the divergence of the Jungermanniales from the fungus-free Porellales in the Permian and the origins of the Schistochilaceae in the Triassic indicate that these associations in liverworts predate the appearance of the Ericales.

Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromycotina fungi under a simulated Palaeozoic CO2 decline

Most land plants form mutualistic associations with arbuscular mycorrhizal fungi of the Glomeromycota, but recent studies have found that ancient plant lineages form mutualisms with Mucoromycotina fungi. Simultaneous associations with both fungal lineages have now been found in some plants, necessitating studies to understand the functional and evolutionary significance of these tripartite associations for the first time. We investigate the physiology and cytology of dual fungal symbioses in the early-diverging liverworts Allisonia and Neohodgsonia at modern and Palaeozoic-like elevated atmospheric CO2 concentrations under which they are thought to have evolved. We found enhanced carbon cost to liverworts with simultaneous Mucoromycotina and Glomeromycota associations, greater nutrient gain compared with those symbiotic with only one fungal group in previous experiments and contrasting responses to atmospheric CO2 among liverwort–fungal symbioses. In liverwort–Mucoromycotina symbioses, there is increased P-for-C and N-for-C exchange efficiency at 440 p.p.m. compared with 1500 p.p.m. CO2. In liverwort–Glomeromycota symbioses, P-for-C exchange is lower at ambient CO2 compared with elevated CO2. No characteristic cytologies of dual symbiosis were identified. We provide evidence of a distinct physiological niche for plant symbioses with Mucoromycotina fungi, giving novel insight into why dual symbioses with Mucoromycotina and Glomeromycota fungi persist to the present day.

The timescale of early land plant evolution

Significance Establishing the timescale of early land plant evolution is essential to testing hypotheses on the coevolution of land plants and Earth’s System. Here, we establish a timescale for early land plant evolution that integrates over competing hypotheses on bryophyte−tracheophyte relationships. We estimate land plants to have emerged in a middle Cambrian–Early Ordovocian interval, and vascular plants to have emerged in the Late Ordovician−Silurian. This timescale implies an early establishment of terrestrial ecosystems by land plants that is in close accord with recent estimates for the origin of terrestrial animal lineages. Biogeochemical models that are constrained by the fossil record of early land plants, or attempt to explain their impact, must consider a much earlier, middle Cambrian–Early Ordovician, origin. Establishing the timescale of early land plant evolution is essential for testing hypotheses on the coevolution of land plants and Earth’s System. The sparseness of early land plant megafossils and stratigraphic controls on their distribution make the fossil record an unreliable guide, leaving only the molecular clock. However, the application of molecular clock methodology is challenged by the current impasse in attempts to resolve the evolutionary relationships among the living bryophytes and tracheophytes. Here, we establish a timescale for early land plant evolution that integrates over topological uncertainty by exploring the impact of competing hypotheses on bryophyte−tracheophyte relationships, among other variables, on divergence time estimation. We codify 37 fossil calibrations for Viridiplantae following best practice. We apply these calibrations in a Bayesian relaxed molecular clock analysis of a phylogenomic dataset encompassing the diversity of Embryophyta and their relatives within Viridiplantae. Topology and dataset sizes have little impact on age estimates, with greater differences among alternative clock models and calibration strategies. For all analyses, a Cambrian origin of Embryophyta is recovered with highest probability. The estimated ages for crown tracheophytes range from Late Ordovician to late Silurian. This timescale implies an early establishment of terrestrial ecosystems by land plants that is in close accord with recent estimates for the origin of terrestrial animal lineages. Biogeochemical models that are constrained by the fossil record of early land plants, or attempt to explain their impact, must consider the implications of a much earlier, middle Cambrian–Early Ordovician, origin.

Stomatal differentiation and abnormal stomata in hornworts

Abstract This light and electron microscope study reveals considerable uniformity in hornwort stomata morphology and density in contrast to common spatial and developmental abnormalities in tracheophytes and mosses. Stomata arise from a median longitudinal division of sporophyte epidermal cells morphologically indistinguishable from their neighbours apart from the retention of a single chloroplast whilst those in the other epidermal cells fragment. Chloroplast division and side-by-side repositioning of the two daughter chloroplasts determines the division plane in the stomatal mother cell. The nascent guard cells contain giant, starch-filled chloroplasts which subsequently divide and, post aperture opening, regain their spherical shape. Accumulation of wall material over the guard cells and of wax rodlets lining the pores follows opening. While the majority of stomata are bilaterally symmetrical those lining the dehiscence furrows display dextral and sinistral asymmetry due to differential expansion of the adjacent epidermal cells. The ubiquity of open stomata suggests that these never close with the maturational wall changes rendering movement extremely unlikely. These structural limitations, a liquid-filled stage in the ontogeny of the intercellular spaces, and spores already at the tetrad stage when stomata open, suggest that their primary role is facilitating sporophyte desiccation leading to dehiscence and spore dispersal rather than gaseous exchange. Stomata ontogeny and very low densities, like those in Devonian fossils, suggest either ancient origins at a time when atmospheric carbon dioxide levels were much greater than today or a function other than gaseous exchange regulation. We found no evidence for stomatal homology between hornworts, mosses and tracheophytes.