Piecing together the eophytes - a new group of ancient plants containing cryptospores.

The earliest evidence for land plants comes from dispersed cryptospores from the Ordovician, which dominated assemblages for 60 million years. Direct evidence of their parent plants comes from minute fossils in Welsh Borderland Upper Silurian to Lower Devonian rocks. We recognize a group that had forking, striated axes with rare stomata terminating in valvate sporangia containing permanent cryptospores, but their anatomy was unknown especially regarding conducting tissues. Charcoalified fossils extracted from the rock using HF were selected from macerates and observed using scanning electron microscopy. Promising examples were split for further examination and compared with electron micrographs of the anatomy of extant bryophytes. Fertile fossil axes possess central elongate cells with thick walls bearing globules, occasional strands and plasmodesmata-sized pores. The anatomy of these cells best matches desiccation-tolerant food-conducting cells (leptoids) of bryophytes. Together with thick-walled epidermal cells and extremely small size, these features suggest that these plants were poikilohydric. Our new data on conducting cells confirms a combination of characters that distinguish the permanent cryptospore-producers from bryophytes and tracheophytes. We therefore propose the erection of a new group, here named the Eophytidae (eophytes).

Earliest record of transfer cells in Lower Devonian plants.

Key sources of information on the nature of early terrestrial ecosystems are the fossilized remains of plants and associated organic encrustations, which are interpreted as either biofilms, biological soil crusts or lichens. The hypothesis that some of these encrustations might be the remains of the thalloid gametophytes of embryophytes provided the stimulus for this investigation. Fossils preserved in charcoal were extracted from Devonian Period (Lochkovian Stage, c. 410-419 Myr old) sediments at a geological site in Shropshire (UK). Scanning electron micrographs (SEMs) of the fossils were compared with new and published SEMs of extant bryophytes and tracheophytes, respectively. One specimen was further prepared and imaged by transmission electron microscopy. Fossils of thalloid morphology were composed almost entirely of cells with labyrinthine ingrowths; these also were present in fossils of axial morphology where they were associated with putative food-conducting cells. Comparison with modern embryophytes demonstrates that these distinctive cells are transfer cells (TCs). Our fossils provide by far the earliest geological evidence of TCs. They also show that some organic encrustations are the remains of thalloid land plants and that these are possibly part of the life cycle of a newly recognized group of plants called the eophytes.

Carbon for nutrient exchange between Lycopodiella inundata and Mucoromycotina fine root endophytes is unresponsive to high atmospheric CO2.

Non-vascular plants associating with arbuscular mycorrhizal (AMF) and Mucoromycotina 'fine root endophyte' (MFRE) fungi derive greater benefits from their fungal associates under higher atmospheric [CO2] (a[CO2]) than ambient; however, nothing is known about how changes in a[CO2] affect MFRE function in vascular plants. We measured movement of phosphorus (P), nitrogen (N) and carbon (C) between the lycophyte Lycopodiella inundata and Mucoromycotina fine root endophyte fungi using 33P-orthophosphate, 15 N-ammonium chloride and 14CO2 isotope tracers under ambient and elevated a[CO2] concentrations of 440 and 800 ppm, respectively. Transfers of 33P and 15 N from MFRE to plants were unaffected by changes in a[CO2]. There was a slight increase in C transfer from plants to MFRE under elevated a[CO2]. Our results demonstrate that the exchange of C-for-nutrients between a vascular plant and Mucoromycotina FRE is largely unaffected by changes in a[CO2]. Unravelling the role of MFRE in host plant nutrition and potential C-for-N trade changes between symbionts under different abiotic conditions is imperative to further our understanding of the past, present and future roles of plant-fungal symbioses in ecosystems.

Mucoromycotina Fine Root Endophyte Fungi Form Nutritional Mutualisms with Vascular Plants.

Fungi and plants have engaged in intimate symbioses that are globally widespread and have driven terrestrial biogeochemical processes since plant terrestrialization >500 million years ago. Recently, hitherto unknown nutritional mutualisms involving ancient lineages of fungi and nonvascular plants have been discovered, although their extent and functional significance in vascular plants remain uncertain. Here, we provide evidence of carbon-for-nitrogen exchange between an early-diverging vascular plant (Lycopodiella inundata) and Mucoromycotina (Endogonales) fine root endophyte fungi. Furthermore, we demonstrate that the same fungal symbionts colonize neighboring nonvascular and flowering plants. These findings fundamentally change our understanding of the physiology, interrelationships, and ecology of underground plant-fungal symbioses in modern terrestrial ecosystems by revealing the nutritional role of Mucoromycotina fungal symbionts in vascular plants.

The distribution and evolution of fungal symbioses in ancient lineages of land plants.

An accurate understanding of the diversity and distribution of fungal symbioses in land plants is essential for mycorrhizal research. Here we update the seminal work of Wang and Qiu (Mycorrhiza 16:299-363, 2006) with a long-overdue focus on early-diverging land plant lineages, which were considerably under-represented in their survey, by examining the published literature to compile data on the status of fungal symbioses in liverworts, hornworts and lycophytes. Our survey combines data from 84 publications, including recent, post-2006, reports of Mucoromycotina associations in these lineages, to produce a list of at least 591 species with known fungal symbiosis status, 180 of which were included in Wang and Qiu (Mycorrhiza 16:299-363, 2006). Using this up-to-date compilation, we estimate that fewer than 30% of liverwort species engage in symbiosis with fungi belonging to all three mycorrhizal phyla, Mucoromycota, Basidiomycota and Ascomycota, with the last being the most widespread (17%). Fungal symbioses in hornworts (78%) and lycophytes (up to 100%) appear to be more common but involve only members of the two Mucoromycota subphyla Mucoromycotina and Glomeromycotina, with Glomeromycotina prevailing in both plant groups. Our fungal symbiosis occurrence estimates are considerably more conservative than those published previously, but they too may represent overestimates due to currently unavoidable assumptions.

Functional complementarity of ancient plant-fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts.

Liverworts, which are amongst the earliest divergent plant lineages and important ecosystem pioneers, often form nutritional mutualisms with arbuscular mycorrhiza-forming Glomeromycotina and fine-root endophytic Mucoromycotina fungi, both of which coevolved with early land plants. Some liverworts, in common with many later divergent plants, harbour both fungal groups, suggesting these fungi may complementarily improve plant access to different soil nutrients. We tested this hypothesis by growing liverworts in single and dual fungal partnerships under a modern atmosphere and under 1500 ppm [CO2 ], as experienced by early land plants. Access to soil nutrients via fungal partners was investigated with 15 N-labelled algal necromass and 33 P orthophosphate. Photosynthate allocation to fungi was traced using 14 CO2 . Only Mucoromycotina fungal partners provided liverworts with substantial access to algal 15 N, irrespective of atmospheric CO2 concentration. Both symbionts increased 33 P uptake, but Glomeromycotina were often more effective. Dual partnerships showed complementarity of nutrient pool use and greatest photosynthate allocation to symbiotic fungi. We show there are important functional differences between the plant-fungal symbioses tested, providing new insights into the functional biology of Glomeromycotina and Mucoromycotina fungal groups that form symbioses with plants. This may explain the persistence of the two fungal lineages in symbioses across the evolution of land plants.

Evolution and networks in ancient and widespread symbioses between Mucoromycotina and liverworts.

Like the majority of land plants, liverworts regularly form intimate symbioses with arbuscular mycorrhizal fungi (Glomeromycotina). Recent phylogenetic and physiological studies report that they also form intimate symbioses with Mucoromycotina fungi and that some of these, like those involving Glomeromycotina, represent nutritional mutualisms. To compare these symbioses, we carried out a global analysis of Mucoromycotina fungi in liverworts and other plants using species delimitation, ancestral reconstruction, and network analyses. We found that Mucoromycotina are more common and diverse symbionts of liverworts than previously thought, globally distributed, ancestral, and often co-occur with Glomeromycotina within plants. However, our results also suggest that the associations formed by Mucoromycotina fungi are fundamentally different because, unlike Glomeromycotina, they may have evolved multiple times and their symbiotic networks are un-nested (i.e., not forming nested subsets of species). We infer that the global Mucoromycotina symbiosis is evolutionarily and ecologically distinctive.

Ancient plants with ancient fungi: liverworts associate with early-diverging arbuscular mycorrhizal fungi.

Arbuscular mycorrhizas are widespread in land plants including liverworts, some of the closest living relatives of the first plants to colonize land 500 million years ago (MYA). Previous investigations reported near-exclusive colonization of liverworts by the most recently evolved arbuscular mycorrhizal fungi, the Glomeraceae, indicating a recent acquisition from flowering plants at odds with the widely held notion that arbuscular mycorrhizal-like associations in liverworts represent the ancestral symbiotic condition in land plants. We performed an analysis of symbiotic fungi in 674 globally collected liverworts using molecular phylogenetics and electron microscopy. Here, we show every order of arbuscular mycorrhizal fungi colonizes early-diverging liverworts, with non-Glomeraceae being at least 10 times more common than in flowering plants. Arbuscular mycorrhizal fungi in liverworts and other ancient plant lineages (hornworts, lycopods, and ferns) were delimited into 58 taxa and 36 singletons, of which at least 43 are novel and specific to liverworts. The discovery that early plant lineages are colonized by early-diverging fungi supports the hypothesis that arbuscular mycorrhizas are an ancestral symbiosis for all land plants.

From rhizoids to roots? Experimental evidence of mutualism between liverworts and ascomycete fungi.

Background and Aims: The rhizoids of leafy liverworts (Jungermanniales, Marchantiophyta) are commonly colonized by the ascomycete fungus Pezoloma ericae. These associations are hypothesized to be functionally analogous to the ericoid mycorrhizas (ErMs) formed by P. ericae with the roots of Ericaceae plants in terms of bi-directional phosphorus for carbon exchange; however, this remains unproven. Here, we test whether associations between the leafy liverwort Cephalozia bicuspidata and P. ericae are mutualistic. Methods: We measured movement of phosphorus and carbon between C. bicuspidata and P. ericae using [33P]orthophosphate and 14CO2 isotope tracers in monoxenic cultures. We also measured leafy liverwort growth, with and without P. ericae. Key Results: We present the first demonstration of nutritionally mutualistic symbiosis between a non-vascular plant and an ErM-forming fungus, showing transfer of fungal-acquired P to the liverwort and of liverwort-fixed C to the fungus alongside increased growth in fungus-colonized liverworts. Conclusions: Thus, this ascomycete-liverwort symbiosis can now be described as mycorrhiza-like, providing further insights into ericoid mycorrhizal evolution and adding Ascomycota fungi to mycorrhizal fungal groups engaging in mutualisms with plants across the land plant phylogeny. As P. ericae also colonizes the rhizoids of Schistochilaceae liverworts, which originated in the Triassic and are sister to all other jungermannialean liverworts associated with fungi, our findings point toward an early origin of ascomycete-liverwort symbioses, possibly pre-dating their evolution in the Ericales by some 150 million years.

Hornwort stomata do not respond actively to exogenous and environmental cues.

Backgrounds and Aims: Because stomata in bryophytes occur on sporangia, they are subject to different developmental and evolutionary constraints from those on leaves of tracheophytes. No conclusive experimental evidence exists on the responses of hornwort stomata to exogenous stimulation. Methods: Responses of hornwort stomata to abscisic acid (ABA), desiccation, darkness and plasmolysis were compared with those in tracheophyte leaves. Potassium ion concentrations in the guard cells and adjacent cells were analysed by X-ray microanalysis, and the ontogeny of the sporophytic intercellular spaces was compared with those of tracheophytes by cryo-scanning electron microscopy. Key Results: The apertures in hornwort stomata open early in development and thereafter remain open. In hornworts, the experimental treatments, based on measurements of >9000 stomata, produced only a slight reduction in aperture dimensions after desiccation and plasmolysis, and no changes following ABA treatments and darkness. In tracheophytes, all these treatments resulted in complete stomatal closure. Potassium concentrations are similar in hornwort guard cells and epidermal cells under all treatments at all times. The small changes in hornwort stomatal dimensions in response to desiccation and plasmolysis are probably mechanical and/or stress responses of all the epidermal and spongy chlorophyllose cells, affecting the guard cells. In contrast to their nascent gas-filled counterparts across tracheophytes, sporophytic intercellular spaces in hornworts are initially liquid filled. Conclusions: Our experiments demonstrate a lack of physiological regulation of opening and closing of stomata in hornworts compared with tracheophytes, and support accumulating developmental and structural evidence that stomata in hornworts are primarily involved in sporophyte desiccation and spore discharge rather than the regulation of photosynthesis-related gaseous exchange. Our results run counter to the notion of the early acquisition of active control of stomatal movements in bryophytes as proposed from previous experiments on mosses.

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.

Stomatal density and aperture in non-vascular land plants are non-responsive to above-ambient atmospheric CO2 concentrations.

BACKGROUND AND AIMS: Following the consensus view for unitary origin and conserved function of stomata across over 400 million years of land plant evolution, stomatal abundance has been widely used to reconstruct palaeo-atmospheric environments. However, the responsiveness of stomata in mosses and hornworts, the most basal stomate lineages of extant land plants, has received relatively little attention. This study aimed to redress this imbalance and provide the first direct evidence of bryophyte stomatal responsiveness to atmospheric CO2. METHODS: A selection of hornwort (Anthoceros punctatus, Phaeoceros laevis) and moss (Polytrichum juniperinum, Mnium hornum, Funaria hygrometrica) sporophytes with contrasting stomatal morphologies were grown under different atmospheric CO2 concentrations ([CO2]) representing both modern (440 p.p.m. CO2) and ancient (1500 p.p.m. CO2) atmospheres. Upon sporophyte maturation, stomata from each bryophyte species were imaged, measured and quantified. KEY RESULTS: Densities and dimensions were unaffected by changes in [CO2], other than a slight increase in stomatal density in Funaria and abnormalities in Polytrichum stomata under elevated [CO2]. CONCLUSIONS: The changes to stomata in Funaria and Polytrichum are attributed to differential growth of the sporophytes rather than stomata-specific responses. The absence of responses to changes in [CO2] in bryophytes is in line with findings previously reported in other early lineages of vascular plants. These findings strengthen the hypothesis of an incremental acquisition of stomatal regulatory processes through land plant evolution and urge considerable caution in using stomatal densities as proxies for paleo-atmospheric CO2 concentrations.

Fungal associations in Horneophyton ligneri from the Rhynie Chert (c. 407 million year old) closely resemble those in extant lower land plants: novel insights into ancestral plant-fungus symbioses.

Fungi (Eumycota) form close associations with plants, with which they have co-existed since the dawn of life on land, but their diversity in early terrestrial ecosystems is still poorly understood. We studied petrographic sections of exceptionally well-preserved petrified plants from the 407 million yr-old Rhynie Chert (Scotland, UK). For comparative purposes, we illustrate fungal associations in four extant lower land plants. We document two new endophytes in the plant Horneophyton lignieri: Palaeoglomus boullardii (sp. nov. Glomeromycota) colonizes parenchyma in a discontinuous zone of the outer cortex of the aerial axes, forming arbuscule-like structures, vesicles and spores; Palaeoendogone gwynne-vaughaniae (gen. nov., sp. nov. Mucoromycotina) colonizes parenchyma in the basal part of the plant, where it is present in intercellular spaces and as intracellular coils but absent from rhizoids. Critical comparisons between the newly discovered Horneophyton endophytes, fungi previously described from the Rhynie Chert and fungal colonization in extant lower land plants reveal several features characteristic of both Mucoromycotina and Glomeromycota. A reappraisal of fungal associations in early land plants indicates that they are more diverse than assumed hitherto, overturning the long-held paradigm that the early endophytes were exclusively Glomeromycota.

The biology of Marchantia polymorpha subsp. ruderalis Bischl. & Boissel. Dub in nature.

Introduction: Though used as the model liverwort in culture for several decades, the biology of Marchantia polymorpha subsp. ruderalis in nature has never been documented in detail in a single account. Methods: Here we synthesize routine field observations documented with hundreds of images of M. ruderalis colonies (or groups) showing sex differentiation over 3 years on two populations of M. ruderalis after major heathland fires in 2020. Results: Initial post-fire establishment is from airborne spores rather than a spore bank but thereafter spread is via gemmae which have less exacting germination requirements. Young sporelings are highly gemmiferous but gemmae production becomes less frequent after sex organ formation. Over the course of a year there are up to three waves of carpocephalum production with the overwhelming majority of antheridiophores appearing 2-3 months ahead of the archegoniophores though no differences in growth rates were apparent between male and female thalli. Spermatozoids are produced almost continuously throughout the year, whilst sporophyte maturation is restricted to the summer months. Discussion: Because of the asynchrony between antheridiophore and archegoniophore production a 1:1 sex ratio is only apparent over this period. The spring months see an excess of males with more females in the summer. An almost 100% fertilization rate, with fertilization distances of up to 19 m far exceeding those in all other bryophytes, is attributed to vast spermatozoid production for most of the year, dispersal on surface oil films between thalli and highly effective intra-thallus spermatozoid transport via the pegged-rhizoid water-conducting system. Archegoniophores do develop on female-only populations but have shorter stalks than those where fertilization has occurred. Eventual disappearance post fires is attributed to a fall in topsoil nutrient levels preventing new sporeling establishment and competition from Ceratodon purpureus and Polytrichum spp. A major drought in the summer of 2022 almost wiped out the heathland Marchantia populations but all the other bryophytes survived.

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.

New methods reveal oldest known fossil epiphyllous moss: Bryiidites utahensis gen. et sp. nov. (Bryidae).

PREMISE OF THE STUDY: Epiphyllous bryophytes are a highly characteristic feature of many humid tropical forest ecosystems. In contrast to the extensive fossil record for the leaves of their host plants, the record is virtually nonexistent for the epiphylls themselves, despite a fossil record for mosses that begins in the Middle Carboniferous Period, 330 million years ago. METHODS: Epifluorescence optical microscopy, scanning electron microscopy, and atomic force microscopy were employed to investigate an intimate association between a newly discovered epiphyllous moss and a Lauraceae plant host from the middle Cretaceous. KEY RESULTS: We describe the oldest fossil specimen of an epiphyllous moss, Bryiidites utahensis gen. et sp. nov., identified from an individual specimen only 450 µm long, situated on an approximately one millimeter square fossil leaf fragment. The moss epiphyll is exquisitely preserved as germinating spores and short-celled protonemata with transverse and oblique cross-walls closely matching those of extant epiphyllous mosses on the surface of the plant-leaf hosts. CONCLUSIONS: The extension of the epiphyll record back to the middle Cretaceous provides fossil evidence for the appearance of epiphyllous mosses during the diversification of flowering plants, at least 95 million years ago. It also provides substantive evidence for a tropical maritime climate in central North America during the middle Cretaceous.

Evolution of phenotypic disparity in the plant kingdom.

The plant kingdom exhibits diverse bodyplans, from single-celled algae to complex multicellular land plants, but it is unclear how this phenotypic disparity was achieved. Here we show that the living divisions comprise discrete clusters within morphospace, separated largely by reproductive innovations, the extinction of evolutionary intermediates and lineage-specific evolution. Phenotypic complexity correlates not with disparity but with ploidy history, reflecting the role of genome duplication in plant macroevolution. Overall, the plant kingdom exhibits a pattern of episodically increasing disparity throughout its evolutionary history that mirrors the evolutionary floras and reflects ecological expansion facilitated by reproductive innovations. This pattern also parallels that seen in the animal and fungal kingdoms, suggesting a general pattern for the evolution of multicellular bodyplans.

The origin of the sporophyte shoot in land plants: a bryological perspective.

BACKGROUND: Land plants (embryophytes) are monophyletic and encompass four major clades: liverworts, mosses, hornworts and polysporangiophytes. The liverworts are resolved as the earliest divergent lineage and the mosses as sister to a crown clade formed by the hornworts and polysporangiophytes (lycophytes, monilophytes and seed plants). Alternative topologies resolving the hornworts as sister to mosses plus polysporangiophytes are less well supported. Sporophyte development in liverworts depends only on embryonic formative cell divisions. A transient basal meristem contributes part of the sporophyte in mosses. The sporophyte body in hornworts and polysporangiophytes develops predominantly by post-embryonic meristematic activity. SCOPE: This paper explores the origin of the sporophyte shoot in terms of changes in embryo organization. Pressure towards amplification of the sporangium-associated photosynthetic apparatus was a major driver of sporophyte evolution. Starting from a putative ancestral condition in which a transient basal meristem produced a sporangium-supporting seta, we postulate that in the hornwort-polysporangiophyte lineage the basal meristem acquired indeterminate meristematic activity and ectopically expressed the sporangium morphogenetic programme. The resulting sporophyte body plan remained substantially unaltered in hornworts, whereas in polysporangiophytes the persistent meristem shifted from a mid-embryo to a superficial position and was converted into an ancestral shoot apical meristem with the evolution of sequential vegetative and reproductive growth. CONCLUSIONS: The sporophyte shoot is interpreted as a sterilized sporangial axis interpolated between the embryo and the fertile sporangium. With reference to the putatively ancestral condition found in mosses, the sporophyte body plans in hornworts and polysporangiophytes are viewed as the product of opposite heterochronic events, i.e. an anticipation and a delay, respectively, in the development of the sporangium. In either case the result was a pedomorphic sporophyte permanently retaining juvenile characters.