Cuticle ultrastructure of the Early Devonian trigonotarbid arachnid Palaeocharinus.

The cuticle is a key evolutionary innovation that played a crucial role in arthropod terrestrialization. Extensive research has elucidated the chemical and structural composition of the cuticle in extant arthropods, while fossil studies have further informed our understanding of cuticle evolution. This study examines the three-dimensionally preserved cuticular structure of the Early Devonian trigonotarbid arachnid genus Palaeocharinus, from the Rhynie chert of Scotland (∼408 Ma). Trigonotarbids, an extinct group of tetrapulmonate arachnids, are among the earliest known unequivocally terrestrial arthropods, and thus may shed light on the evolution of terrestriality. Using high-resolution Confocal Laser Scanning Microscopy (CLSM), we reveal detailed morphological features at the nanometre level. The external cuticle surface of Palaeocharinus is characterized by polygonal scales, sensilla, and small pores identified as the openings of dermal glands and wax canals. Internally, the cuticle exhibits polygonal clusters of pore canals, through which wax was transported from the epidermis to the cuticular surface. The pore canals twist along their vertical axes, reflecting the "twisted plywood" or Bouligand arrangement of chitin-protein microfibril planes characteristic of modern arthropod cuticles. Overall, the cuticle of Palaeocharinus is characteristically thick relative to those of other extinct and extant chelicerates, such thickening being a possible adaptation to terrestrial life.

A fungal plant pathogen discovered in the Devonian Rhynie Chert.

Fungi are integral to well-functioning ecosystems, and their broader impact on Earth systems is widely acknowledged. Fossil evidence from the Rhynie Chert (Scotland, UK) shows that Fungi were already diverse in terrestrial ecosystems over 407-million-years-ago, yet evidence for the occurrence of Dikarya (the subkingdom of Fungi that includes the phyla Ascomycota and Basidiomycota) in this site is scant. Here we describe a particularly well-preserved asexual fungus from the Rhynie Chert which we examined using brightfield and confocal microscopy. We document Potteromyces asteroxylicola gen. et sp. nov. that we attribute to Ascomycota incertae sedis (Dikarya). The fungus forms a stroma-like structure with conidiophores arising in tufts outside the cuticle on aerial axes and leaf-like appendages of the lycopsid plant Asteroxylon mackiei. It causes a reaction in the plant that gives rise to dome-shaped surface projections. This suite of features in the fungus together with the plant reaction tissues provides evidence of it being a plant pathogenic fungus. The fungus evidently belongs to an extinct lineage of ascomycetes that could serve as a minimum node age calibration point for the Ascomycota as a whole, or even the Dikarya crown group, along with some other Ascomycota previously documented in the Rhynie Chert.

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.

Hapalosiphonacean cyanobacteria (Nostocales) thrived amid emerging embryophytes in an early Devonian (407-million-year-old) landscape.

Cyanobacteria have a long evolutionary history, well documented in marine rocks. They are also abundant and diverse in terrestrial environments; however, although phylogenies suggest that the group colonized land early in its history, paleontological documentation of this remains limited. The Rhynie chert (407 Ma), our best preserved record of early terrestrial ecosystems, provides an opportunity to illuminate aspects of cyanobacterial diversity and ecology as plants began to radiate across the land surface. We used light microscopy and super-resolution confocal laser scanning microscopy to study a new population of Rhynie cyanobacteria; we also reinvestigated previously described specimens that resemble the new fossils. Our study demonstrates that all are part of a single fossil species belonging to the Hapalosiphonaceae (Nostocales). Along with other Rhynie microfossils, these remains show that the accommodation of morphologically complex cyanobacteria to terrestrial ecosystems transformed by embryophytes was well underway more than 400 million years ago.

Terrestrial surface stabilisation by modern analogues of the earliest land plants: A multi-dimensional imaging study.

The evolution of the first plant-based terrestrial ecosystems in the early Palaeozoic had a profound effect on the development of soils, the architecture of sedimentary systems, and shifts in global biogeochemical cycles. In part, this was due to the evolution of complex below-ground (root-like) anchorage systems in plants, which expanded and promoted plant-mineral interactions, weathering, and resulting surface sediment stabilisation. However, little is understood about how these micro-scale processes occurred, because of a lack of in situ plant fossils in sedimentary rocks/palaeosols that exhibit these interactions. Some modern plants (e.g., liverworts, mosses, lycophytes) share key features with the earliest land plants; these include uni- or multicellular rhizoid-like anchorage systems or simple roots, and the ability to develop below-ground networks through prostrate axes, and intimate associations with fungi, making them suitable analogues. Here, we investigated cryptogamic ground covers in Iceland and New Zealand to better understand these interactions, and how they initiate the sediment stabilisation process. We employed multi-dimensional and multi-scale imaging, including scanning electron microscopy (SEM) and X-ray Computed Tomography (µCT) of non-vascular liverworts (Haplomitriopsida and complex thalloids) and mosses, with additional imaging of vascular lycopods. We find that plants interact with their substrate in multiple ways, including: (1) through the development of extensive surface coverings as mats; (2) entrapment of sediment grains within and between networks of rhizoids; (3) grain entwining and adherence by rhizoids, through mucilage secretions, biofilm-like envelopment of thalli on surface grains; and (4) through grain entrapment within upright 'leafy' structures. Significantly, µCT imaging allows us to ascertain that rhizoids are the main method for entrapment and stabilisation of soil grains in the thalloid liverworts. This information provides us with details of how the earliest land plants may have significantly influenced early Palaeozoic sedimentary system architectures, promoted in situ weathering and proto-soil development, and how these interactions diversified over time with the evolution of new plant organ systems. Further, this study highlights the importance of cryptogamic organisms in the early stages of sediment stabilisation and soil formation today.

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.

An expanded diversity of oomycetes in Carboniferous forests: Reinterpretation of Oochytrium lepidodendri (Renault 1894) from the Esnost chert, Massif Central, France.

335-330 million-year-old cherts from the Massif Central, France, contain exceptionally well-preserved remains of an early forest ecosystem, including plants, fungi and other microorganisms. Here we reinvestigate the original material prepared by Renault and Roche from collections of the Muséum National d'Histoire Naturelle, Paris, and present a re-evaluation of Oochytrium lepidodendri (Renault 1894), originally described as a zoosporic fungus. Confocal laser scanning microscopy (CLSM) was used to study the microfossils, enabling us in software to digitally reconstruct them in three-dimensional detail. We reinterpret O. lepidodendri as a pseudofungus and favour placement within the oomycetes, a diverse clade of saprotrophs and both animal and plant parasites. Phylogenetically, O. lepidodendri appears to belong to a group of oomycetes distinct from those previously described from Paleozoic rocks and most likely related to the Peronosporales s.l. This study adds to our knowledge of Paleozoic eukaryotic diversity and reinforces the view that oomycetes were early and diverse constituents of terrestrial biotas, playing similar ecological roles to those they perform in modern ecosystems.

Cryptogamic ground covers as analogues for early terrestrial biospheres: Initiation and evolution of biologically mediated proto-soils.

Modern cryptogamic ground covers (CGCs), comprising assemblages of bryophytes (hornworts, liverworts, mosses), fungi, bacteria, lichens and algae, are thought to resemble early divergent terrestrial communities. However, limited in situ plant and other fossils in the rock record, and a lack of CGC-like soils reported in the pre-Silurian sedimentological record, have hindered understanding of the structure, composition and interactions within the earliest CGCs. A key question is how the earliest CGC-like organisms drove weathering on primordial terrestrial surfaces (regolith), leading to the early stages of soil development as proto-soils, and subsequently contributing to large-scale biogeochemical shifts in the Earth System. Here, we employed a novel qualitative, quantitative and multi-dimensional imaging approach through X-ray micro-computed tomography, scanning electron, and optical microscopy to investigate whether different combinations of modern CGC organisms from primordial-like settings in Iceland develop organism-specific soil forming features at the macro- and micro-scales. Additionally, we analysed CGCs growing on hard rocky substrates to investigate the initiation of weathering processes non-destructively in 3D. We show that thalloid CGC organisms (liverworts, hornworts) develop thin organic layers at the surface (<1 cm) with limited subsurface structural development, whereas leafy mosses and communities of mixed organisms form profiles that are thicker (up to ~ 7 cm), structurally more complex, and more organic-rich. We term these thin layers and profiles proto-soils. Component analyses from X-ray micro-computed tomography data show that thickness and structure of these proto-soils are determined by the type of colonising organism(s), suggesting that the evolution of more complex soils through the Palaeozoic may have been driven by a shift in body plan of CGC-like organisms from flattened and appressed to upright and leafy. Our results provide a framework for identifying CGC-like proto-soils in the rock record and a new proxy for understanding organism-soil interactions in ancient terrestrial biospheres and their contribution to the early stages of soil formation.

Genomic and fossil windows into the secret lives of the most ancient fungi.

Fungi have crucial roles in modern ecosystems as decomposers and pathogens, and they engage in various mutualistic associations with other organisms, especially plants. They have a lengthy geological history, and there is an emerging understanding of their impact on the evolution of Earth systems on a large scale. In this Review, we focus on the roles of fungi in the establishment and early evolution of land and freshwater ecosystems. Today, questions of evolution over deep time are informed by discoveries of new fossils and evolutionary analysis of new genomes. Inferences can be drawn from evolutionary analysis by comparing the genes and genomes of fungi with the biochemistry and development of their plant and algal hosts. We then contrast this emerging picture against evidence from the fossil record to develop a new, integrated perspective on the origin and early evolution of fungi.

The Rhynie chert.

In 1912, William Mackie, a medical practitioner surveying the regional geology west of Aberdeen, Scotland, happened on some unusual rocks (Figure 1) near the village of Rhynie. Dark gray to nearly black and shot through with cylindrical structures a few millimeters in diameter, these rocks differed markedly from the shales and volcanic rocks of local hills. Mackie had discovered the Rhynie chert - paleobotany's most iconic deposit - with its exceptionally preserved fossils that provide a uniquely clear view of early terrestrial ecosystems in statu nascendi. Early research by Robert Kidston and William Lang showed that the cylindrical structures in Rhynie rocks were the axes of early plants, preserved in remarkable cellular detail. A century of subsequent research confirmed that Rhynie provides not only an unparalleled record of early tracheophyte (vascular plant) evolution, but also offers additional paleontological treasures, including animals (mostly arthropods) and microorganisms ranging from fungi, algae, and oomycetes to testate amoebozoans, and even cyanobacteria. A captivating snapshot of life on land more than 400 million years ago, the Rhynie chert provides our earliest and best view of how terrestrial ecosystems came to be.

Reconstructing trait evolution in plant evo-devo studies.

Our planet is teeming with an astounding diversity of plants. In a mere single group of closely related species, tremendous diversity can be observed in their form and function - the colour of petals in flowering plants, the shape of the fronds in ferns, and the branching pattern of the gametophyte in mosses. Diversity can also be found in subtler traits, such as the resistance to pathogens or the ability to recruit symbiotic microbes from the environment. Plant traits can also be highly conserved - at the cellular and metabolic levels, entire biosynthetic pathways are present in all plant groups, and morphological characteristics such as vascular tissues have been conserved for hundreds of millions of years. The research community that seeks to understand these traits - both the diverse and the conserved - by taking an evolutionary point-of-view on plant biology is growing. Here, we summarize a subset of the different aspects of plant evolutionary biology, provide a guide for structuring comparative biology approaches and discuss the pitfalls that (plant) researchers should avoid when embarking on such studies.

Testate Amoebae in the 407-Million-Year-Old Rhynie Chert.

The Lower Devonian Rhynie chert is justly famous for the clear glimpse it offers of early terrestrial ecosystems [1]. Seven species of stem- and crown-group vascular plants have been described from Rhynie, many preserved in growth position [2], as well as 14 species of invertebrate animals, all arthropods [3] save for a single nematode population [4]. While these shed welcome light on early tracheophytes and land animals, modern terrestrial ecosystems additionally contain a diversity of microscopic organisms that are key to ecosystem function, including fungi, protists, and bacteria. Fungi ranging from mycorrhizae to saprophytes are well preserved in Rhynie rocks ([5] and references therein), and oomycetes are also present [5]. Both green algae (charophytes) and cyanobacteria have also been documented locally [6, 7, 8]. To date, however, phagotrophic protists have not been observed in Rhynie cherts, even though such organisms contribute importantly to carbon, nitrogen, and silica cycling in modern terrestrial communities [9]. Here, we report a population of organic tests described as Palaeoleptochlamys hassii gen. nov., sp. nov. from a pond along the Rhynie alluvial plain, which we interpret as arcellinid amoebozoans. These fossils expand the ecological dimensions of the Rhynie biota and support the hypothesis that arcellinids transitioned from marine through freshwater environments to colonize soil ecosystems in synchrony with early vascular plants.

The origin and evolution of mycorrhizal symbioses: from palaeomycology to phylogenomics.

Contents Summary 1012 I. Introduction 1013 II. The mycorrhizal symbiosis at the dawn and rise of the land flora 1014 III. From early land plants to early trees: the origin of roots and true mycorrhizas 1016 IV. The diversification of the AM symbiosis 1019 V. The ECM symbiosis 1021 VI. The recently evolved ericoid and orchid mycorrhizas 1023 VII. Limits of paleontological vs genetic approaches and perspectives 1023 Acknowledgements 1025 References 1025 SUMMARY: The ability of fungi to form mycorrhizas with plants is one of the most remarkable and enduring adaptations to life on land. The occurrence of mycorrhizas is now well established in c. 85% of extant plants, yet the geological record of these associations is sparse. Fossils preserved under exceptional conditions provide tantalizing glimpses into the evolutionary history of mycorrhizas, showing the extent of their occurrence and aspects of their evolution in extinct plants. The fossil record has important roles to play in establishing a chronology of when key fungal associations evolved and in understanding their importance in ecosystems through time. Together with calibrated phylogenetic trees, these approaches extend our understanding of when and how groups evolved in the context of major environmental change on a global scale. Phylogenomics furthers this understanding into the evolution of different types of mycorrhizal associations, and genomic studies of both plants and fungi are shedding light on how the complex set of symbiotic traits evolved. Here we present a review of the main phases of the evolution of mycorrhizal interactions from palaeontological, phylogenetic and genomic perspectives, with the aim of highlighting the potential of fossil material and a geological perspective in a cross-disciplinary approach.

The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte.

The evolutionary emergence of land plant body plans transformed the planet. However, our understanding of this formative episode is mired in the uncertainty associated with the phylogenetic relationships among bryophytes (hornworts, liverworts, and mosses) and tracheophytes (vascular plants). Here we attempt to clarify this problem by analyzing a large transcriptomic dataset with models that allow for compositional heterogeneity between sites. Zygnematophyceae is resolved as sister to land plants, but we obtain several distinct relationships between bryophytes and tracheophytes. Concatenated sequence analyses that can explicitly accommodate site-specific compositional heterogeneity give more support for a mosses-liverworts clade, "Setaphyta," as the sister to all other land plants, and weak support for hornworts as the sister to all other land plants. Bryophyte monophyly is supported by gene concatenation analyses using models explicitly accommodating lineage-specific compositional heterogeneity and analyses of gene trees. Both maximum-likelihood analyses that compare the fit of each gene tree to proposed species trees and Bayesian supertree estimation based on gene trees support bryophyte monophyly. Of the 15 distinct rooted relationships for embryophytes, we reject all but three hypotheses, which differ only in the position of hornworts. Our results imply that the ancestral embryophyte was more complex than has been envisaged based on topologies recognizing liverworts as the sister lineage to all other embryophytes. This requires many phenotypic character losses and transformations in the liverwort lineage, diminishes inconsistency between phylogeny and the fossil record, and prompts re-evaluation of the phylogenetic affinity of early land plant fossils, the majority of which are considered stem tracheophytes.

The timescale of early land plant evolution.

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.

History and contemporary significance of the Rhynie cherts-our earliest preserved terrestrial ecosystem.

The Rhynie cherts Unit is a 407 million-year old geological site in Scotland that preserves the most ancient known land plant ecosystem, including associated animals, fungi, algae and bacteria. The quality of preservation is astonishing, and the initial description of several plants 100 years ago had a huge impact on botany. Subsequent discoveries provided unparalleled insights into early life on land. These include the earliest records of plant life cycles and fungal symbioses, the nature of soil microorganisms and the diversity of arthropods. Today the Rhynie chert (here including the Rhynie and Windyfield cherts) takes on new relevance, especially in relation to advances in the fields of developmental genetics and Earth systems science. New methods and analytical techniques also contribute to a better understanding of the environment and its organisms. Key discoveries are reviewed, focusing on the geology of the site, the organisms and the palaeoenvironments. The plants and their symbionts are of particular relevance to understanding the early evolution of the plant life cycle and the origins of fundamental organs and tissue systems. The Rhynie chert provides remarkable insights into the structure and interactions of early terrestrial communities, and it has a significant role to play in developing our understanding of their broader impact on Earth systems.This article is part of a discussion meeting issue 'The Rhynie cherts: our earliest terrestrial ecosystem revisited'.