Gareth Evans

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.