Christine Strullu-Derrien

Summary: Mycorrhizal associations between fungi and plants are a fundamental aspect of terrestrial ecosystems. Mycorrhizas occur in c . 85% of extant plants, yet their geological record remains sparse. Rare fossil evidence from early terrestrial environments offers crucial insights into these ancient symbioses, but visualizing fossil fungi at the microscale within plant tissues is challenging. Here, we combine confocal laser scanning microscopy and fluorescence lifetime imaging microscopy (FLIM) to investigate a newly identified fungus and cellular structures of a 407‐Myr‐old plant from the Windyfield Chert, a stratigraphically distinct fossiliferous unit from Rhynie (Scotland). We also applied Raman spectroscopy to investigate the carbon framework of both fungal and plant tissues. This integrative approach revealed fungal structures in unprecedented detail. The fungus, Rugososporomyces lavoisierae gen. nov., sp. nov., exhibits features resembling extant Glomeromycotina arbuscular mycorrhizal fungi. This is the first record of mycorrhizas from the Windyfield Chert. FLIM further distinguished features at the subcellular level, while Raman spectroscopy showed that fungal arbuscules and vesicles of the plant water‐conducting cells underwent geological alterations, resulting in a similar chemical composition. These findings expand our understanding of ancient and extremely rare plant–fungal symbioses and highlight the potential of confocal‐FLIM for advancing palaeobotanical research.

Mycorrhizal associations between fungi and plants are a fundamental aspect of terrestrial ecosystems. The occurrence of mycorrhizae is now well established in c. 85% of extant plants, yet the geological record of these associations is sparse. Fossil evidence from early Paleozoic terrestrial environments provides rare insights into these ancient symbioses, but imaging micro-scale fossil fungi in surrounding plant tissues is challenging. We employed brightfield microscopy, confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM), and Raman spectroscopy to investigate a newly identified endomycorrhizal fungus within a 407-million-year-old plant from the Windyfield Chert. An exquisite preservation and our advanced imaging approach enabled the description of the fungal structures to unprecedented detail. The fungus, Rugosomyces lavoisieriae gen. nov., & sp. nov., exhibits structural features resembling modern Glomeromycotina arbuscular mycorrhizal fungi. Combining CLSM with FLIM provides extra information by separating features based upon fluorescence lifetime. Raman spectroscopy indicates that fungal arbuscules and plant water-conducting cells have undergone geological alterations, resulting in a similar chemical composition. These findings enhance our understanding of ancient plant-fungal symbioses and demonstrate the potential of advanced imaging techniques in paleobotanical research. Significance statement: Most living plants form mycorrhizal associations with fungi, but how this ancient evolutionary relationship developed is poorly understood. Exceptional fossil materials preserve direct evidence of endomycorrhizae, but imaging micro-scale fossil fungi inside plant cells is challenging. Here we use confocal microscopy combined with fluorescence lifetime imaging and Raman spectroscopy to study a new endomycorrhizal fungus inside a 400-million-year-old plant. We document structures resembling modern Glomeromycotina. We show that the residual organic matter of the fungus and the plant cell walls has a very short fluorescence lifetime, which provides a new means of imaging these fossils. Raman spectroscopy indicates that both arbuscules and water-conducting cells were geologically altered by their diagenetic history and that they now have a similar chemical composition.

Leaf-associated fungi, the fungi that depend on leaves to sporulate, have a rich Cenozoic record, however their earlier diversity is poorly characterized. Here we describe Harristroma eboracense gen. et sp. nov., a Middle Jurassic leaf-associated fungus colonizing the leaf cuticle of Nilssonia tenuicaulis (cycadophyte). To place our newly described species into a picture of the diversification of Mesozoic fungi, we reassess fossils with leaf-associated stromata in the context of fungal molecular phylogeny. Being melanized, with radiate stromata, and on leaves, H. eboracense and other fossils from the Jurassic and earlier periods are probably related to filamentous Ascomycota in the superclass Leotiomyceta. Characters needed for further resolution of leaf-associated fungal biology and classification, such as the presence of an ostiole for spore discharge and appressoria for entry into leaf tissue first appear in the Mesozoic fossil record. Among Early Cretaceous fossils, Spataporthe taylorii represents the oldest unambiguous evidence of perithecial Sordariomycetes while Protographum luttrellii and Bleximothyrium ostiolatum are the oldest Dothideomycetes thyriothecia. Environmental observations show that broad leaved gymnosperms (especially cycadophytes) growing in warm temperate wet forests might have been the first environment for the radiation of Leotiomyceta.

Fungiare integral to well-functioning ecosystems, and their broader impact on Earth systems is widely acknowledged. Fossil evidence from the Rhynie Chert (Scotland, UK) shows thatFungiwere already diverse in terrestrial ecosystems over 407-million-years-ago, yet evidence for the occurrence ofDikarya (the subkingdom ofFungithat includes the phylaAscomycotaandBasidiomycota) 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 documentPotteromyces asteroxylicolagen. et sp. nov. that we attribute toAscomycota 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 plantAsteroxylon 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 theAscomycotaas a whole, or even theDikaryacrown group, along with some otherAscomycotapreviously documented in the Rhynie Chert.

Microorganisms are key to our understanding of early life on land, especially in terms of below-ground processes. In the fossil record, exceptionally preserved silicified systems are the best sources to document the diversity of Fungi and microorganisms and the roles that they played, including their interactions with plants. Continued advances in technology allow us to document these in unprecedent detail at sites like the Rhynie cherts (Scotland, UK), dating to 407 Ma, and the Grand Croix chert (Massif Central, France), dating to ca 307 ̶ 303 Ma. Techniques we used include Confocal Laser Scanning Microscopy (CLSM) and Laser-Induced Breakdown Spectroscopy (LIBS)-based imaging, among others.In the Rhynie cherts, plant-fungal associations and glomeromycotan spores have been observed as well as other diverse microorganisms colonizing the substrate. In modern vascular plants, endomycorrhizas are typically associated with roots, but most of the vascular plants at Rhynie were rootless, and endomycorrhizas developed in aerial axes. This is probably the plesiomorphic state for land plants. Data from Grand Croix demonstrate that by the end of the Carboniferous, endomycorrhizas had become associated with the root systems of trees. The evolution of the endomycorrhizal symbioses during the Paleozoic, from early plants to trees, is associated with important changes in the nature of the symbiosis, the structure of the soil, and changes in level of carbon dioxide gas in the atmosphere. Using a combination of techniques to decipher the nature of the organisms and their interactions is as an area of developing interest, particularly in the context of recent work on modern relatives.

We document the habit and affinity of the most complete Mesozoic Era tree to be excavated in the UK. The fossil was found in situ in a palaeosol of the Upper Jurassic Purbeck Group of southern England (Tithonian: ca. 150–145 million years). It comprises over 100 permineralized (silicified) pieces that represent a rooted stump and fallen trunk, together weighing more than two tonnes. This exceptional specimen was excavated in a manner that retained the original associations among its parts, providing a unique insight into the overall habit and mode of growth. A laser scanning approach was used to facilitate the investigation, producing the largest 3D reconstruction of a plant fossil. Anatomical details reveal that the wood belongs to the fossil-genus Agathoxylon. Despite an estimated growth age of more than 200 years, the tree was of modest size, not greatly exceeding 12 m in height. The main trunk bifurcated, developing into a decurrent, spreading crown. Its habit differed significantly from most modern arborescent conifers, which have pole-like central trunks and narrow, conical crowns, and from known growth forms in the Cheirolepidiaceae, an important extinct group of Mesozoic conifers. These findings extend our knowledge of the growth architecture of Jurassic conifers, which were prominent and diverse elements of seasonally arid, low to mid-latitude coastal communities during the Late Mesozoic. GRAPHICAL ABSTRACT