Abstract Forest canopies harbour strong microclimatic gradients that shape plant performance, species interactions and ecosystem processes. Yet, despite renewed interest sparked by global change, forest canopies remain difficult-to-access experimental spaces. With the goal to expand access to tree canopies as experimental arenas, we designed, built, and tested TreeTOP, a standardized experimental platform that opens canopy space for manipulative ecological experiments, specifically with potted plants. TreeTOP features lightweight aluminum frames placed in mature tree canopies non-invasively, allowing potted plants to be placed in three different heights, ground level, shade canopy, and sun canopy. We implemented TreeTOP using two contrasting infrastructure concepts to demonstrate its applicability in both highly equipped canopy research facilities and forests without permanent canopy infrastructure. One installation relied on a canopy crane, grid power and fully automated irrigation, whereas the second was built by certified tree climbers and was equipped with an autonomous solar-powered, battery-operated irrigation system. At both sites, environmental sensor networks monitor the experiment. TreeTOP successfully reproduced characteristic canopy microclimatic gradients, including increasing light availability, daytime air temperatures and thermal extremes with canopy height. Despite differing infrastructures, both implementations generated comparable microclimatic patterns, demonstrating that standardized canopy experiments are feasible in forests with or without permanent canopy access. By opening canopy space for manipulative experiments, TreeTOP provides a transferable framework for investigating plant performance, phenology, species interactions and microbiome assembly under realistic forest conditions.

Transposable elements (TEs) play crucial roles in genome evolution and ecological adaptation in fungi, yet their dynamics in ectomycorrhizal species remain poorly understood. Cenococcum geophilum, the most widespread ectomycorrhizal fungus in boreal and temperate forests with its large, repeat-rich genome, represents an ideal system to investigate TE-mediated adaptation to the physical environment and symbiotic lifestyle. However, previous studies have been limited by fragmented genome assemblies that prevented the resolution of repeat-rich regions. We assembled a telomere-to-telomere reference genome of C. geophilum strain 1.58 using PacBio HiFi and Hi-C datasets, resulting in a 178.54 Mbp genome with seven contiguous chromosomes. We identified 14,145 genes and over 78% of the genome consists of transposable elements (TEs). Of these, 94% are affected by repeat-induced point mutations (RIP), a genome defense mechanism that acts during the sexual reproduction phase, indicating cryptic or ancient sexual reproduction in this putatively asexual fungus. Long terminal repeat retrotransposons, LINEs, and DNA transposons dominate, with three TE families (Ty3, Ty1, and Tad1) contributing over 60% of the genome size, indicating recent transposition bursts. Screening of 15 additional C. geophilum strains revealed recent and lineage-specific TE expansions, implying that several TEs escaped the RIP machinery and retained potential activity. Supporting TE activity in the context of symbiosis, we found 56 TEs differentially transcribed between ectomycorrhizal and free-living mycelium tissues. An even higher number (n = 66) of TEs were differentially expressed between stress resistance morphology (i.e. sclerotia) and free-living mycelium. This supports that TEs are differentially regulated as a response to symbiotic and stress-related conditions. Our results demonstrate that the C. geophilum genome expansion was driven by a few lineage-specific TE families in recent history, with high RIP activity attesting to sexual reproduction. We also provide insights how TEs could respond to lifestyle transitions and traits associated with desiccation resistance.

Ericoid mycorrhizal (ErM) fungi (ErMF) are crucial for the establishment of thousands of ericaceous species in heathlands and wetlands by increasing their tolerance to harsh conditions and improving nutrient uptake. However, ErM research has largely focused on a limited number of host species and four ErMF species (especially Hyaloscypha hepaticicola and Oidiodendron maius, to a lesser extent H. bicolor/H. finlandica and H. variabilis). Therefore, the degree to which other ericaceous plants and ErMF form functional associations, and corresponding benefits for plant growth, are not well understood. As such, we lack a clear understanding of how changes in fungal partners may influence plant fitness. To address this gap, we conducted a greenhouse experiment with nine ericaceous plant species and eight ErMF isolates to expand baseline knowledge regarding the effects of the ErM symbiosis on host plant growth. By analyzing ErM root colonization and host plant growth response, we observed that the mycorrhizal growth response (MGR) was variable and depended on plant and fungal identity. Moreover, overall inoculation effects on plant growth were independent from colonization levels. Finally, we found evidence that MGR was influenced by plant phylogeny. These results expand our basic understanding of the ErM symbiosis and provide valuable information for future restoration and conservation efforts.

Ectomycorrhizal symbiosis supports tree growth and is crucial for nutrient cycling and temperate and boreal ecosystems functioning. The establishment of functional ectomycorrhiza (ECM) first requires the association of compatible partners. However, host and fungal genetic determinants governing mycorrhizal compatibility are unknown. To identify such factors in poplar and its fungal associates, we mined existing and de novo tree and fungal transcriptional datasets. We identified co-expressed genes enabling ECM symbiosis at early and mature stages of the interaction. These sets of genes can be divided into general fungal-sensing and ECM-specific components. We highlight the importance of fungal modulation of plant JA-related defenses and the regulation of secretory pathways for ECM compatibility, including upregulation of key fungal small secreted proteins, the downregulation of plant secreted peroxidases, and the downregulation of plant cell wall remodeling proteins concomitantly with the upregulation of fungal glycosyl hydrolases acting on pectin. Not only gene regulation, but also its temporal scale and dynamics seem to play a crucial role for mycorrhizal compatibility. The expression profile of the host Common Symbiosis Pathway and nutrient transporters was also studied, revealing constitutive levels of expression and moderate upregulation in compatible ECM interactions. Overall, these results underscore the importance of novel biological functions during the establishment of ECM symbiosis, help us gain insights into the molecular events determining mycorrhiza compatibility, and serve as a data-rich transcriptomic resource to open new research questions in the field.

Ectomycorrhizal symbiosis supports tree growth and is crucial for nutrient cycling and temperate and boreal ecosystems functioning. The establishment of functional ECM first requires the association of compatible partners. However, host and fungal genetic determinants governing mycorrhizal compatibility are unknown. To identify such factors in poplar and its fungal associates, we mined existing and de-novo tree and fungal transcriptional datasets. We identified core plant regulons enabling ECM symbiosis at early and mature stages of the interaction. These regulons can be divided into general fungal-sensing and ECM-specific components. We highlight the importance of fungal modulation of plant JA-related defences and the regulation of secretory pathways for ECM compatibility, including upregulation of key fungal small secreted proteins, the downregulation of plant secreted peroxidases and the downregulation of plant cell-wall remodelling proteins concomitantly with the upregulation of fungal glycosyl hydrolases acting on pectin. Not only gene-regulation, but also its temporal scale and dynamics seems to play a crucial role for mycorrhizal compatibility. The expression profile of the host Common Symbiosis Pathway was also studied, revealing constitutive levels of expression of a part of the pathway and moderate upregulation in compatible ECM interactions. Overall, these results underscore the importance of novel biological functions during the establishment of ECM symbiosis, help us gain insights into the molecular events determining mycorrhiza compatibility and serve as a data-rich transcriptomic resource to open new research questions in the field. Significance statement Ectomycorrhizal symbiosis is essential for forest ecosystems, but the genetic determinants of host and fungal compatibility remain underexplored. Employing comparative transcriptomics, we identified plant general and ECM-specific gene regulons, outlining the expression profile of Common Symbiosis Pathway genes in multiple ECM interactions and highlighting fungal and plant genes associated to interkingdom crosstalk and cell wall remodelling. These findings help us gain insights into the molecular events determining mycorrhiza compatibility. ### Competing Interest Statement The authors have declared no competing interest.

Pedunculate oak (Quercus robur L.), a long-lived forest tree species, forms symbiotic relationships with ectomycorrhizal (ECM) fungi, which can promote nutrient uptake, stress resilience, and growth. Like other tropical and temperate tree species, pedunculate oak exhibits endogenous rhythmic growth (ERG), a trait conferring the ability to repeatedly alternate root and shoot flushes as well as growth cessation as response to changing environmental conditions. However, the effects of different ECM fungal species on the ERG dynamics remain largely unknown. Here, we investigated the impact of two ECM fungi-Piloderma croceum, a basidiomycete previously shown to promote growth while not found in natural oak stands, and Cenococcum geophilum, an oak-native ascomycete with broad ecological range-on growth performance, biomass partitioning, and ERG patterns in a clonal oak system (clone DF159). By combining in vitro experiments with Bayesian modelling, we show that P. croceum promotes tree growth among treatments, without disrupting the endogenous growth rhythm. In contrast, C. geophilum, while showing high mycorrhization rates, led to reduced biomass accumulation and altered developmental progression through the ERG stages, especially by prolonging the steady state development stage-part of the root flush and characterized by peak net carbon assimilation. Co-inoculation revealed a competitive advantage of C. geophilum in root colonization, yet growth responses resembled those of the control. Our findings demonstrate that ECM species exert species-specific effects on biomass production and temporal development of plants, underscoring the functional importance of ECM fungi in shaping host development. Assessing these interactions provides new insights into the functional diversity of ectomycorrhizal symbiosis and can inform forest management strategies aimed at enhanced resilience in oak-dominated ecosystems under rapidly changing climatic conditions.

Severe drought impacts soil organic carbon (SOC) cycling. Yet, there is limited understanding of drought effects on rhizosphere C allocation and its fate in the soil, since belowground C contributes to new SOC formation while fueling soil microbial communities and SOC mineralization. Here, we quantified rhizosphere C inputs and losses in a 17-year long irrigation experiment in a dry Scots pine forest using 13C-enriched soil ingrowth bags with different mesh sizes. Fungal and bacterial communities inside the ingrowth bags and in adjacent soils were analyzed by Illumina MiSeq sequencing. After two years, net new SOC accumulation was 5 times greater in root-accessible vs root-exclusion bags (1000- vs 20-μm mesh). Irrigation stimulated new SOC formation in the first year as compared to natural drought both in root-accessible (+26%) and root-exclusion bags (+47%). Losses of “old” 13C-enriched SOC increased under irrigation in root-accessible (+88%) and root-exclusion bags (+32%), resulting in an overall balanced effect of irrigation on SOC. After two years, irrigation showed a limited effect on net new SOC formation both in root-accessible and root-exclusion bags despite a 70% greater root ingrowth under irrigation. We attribute the lacking irrigation effect on rhizosphere-derived SOC to higher respiratory losses of new soil C, which is in line with +55% old C losses by irrigation in root-accessible bags after two years. These findings indicate a faster C cycling in the rhizosphere under irrigation with enhanced C inputs, which were however rapidly mineralized, resulting in negligible net effects. The increased belowground C allocation and increased C turnover in the rhizosphere under irrigation were paralleled by shifts in fungal and bacterial communities in ingrowth bags as well as in adjacent soils. Fungal and bacterial community structures were also shaped by the presence of roots in the bags. Overall, our results in this long-term irrigation experiment imply that naturally dry conditions slow SOC cycling, suppressing both rhizosphere C inputs and losses. The reduced supply of belowground C leads to cascading effects on soil microbial community composition under drought.

Forest trees rely on ectomycorrhizal (ECM) fungi for acquiring scarce resources such as water and nutrients. However, the molecular mechanisms governing ECM traits remain inadequately understood, particularly the role of intraspecific fungal variation in root-tip colonisation and trophic interactions. This study examined six ECM traits using Pisolithus microcarpus, an ECM fungus capable of forming ECM rootlets in poplar. A collection of 40 sibling monokaryons and their parental dikaryon was analysed through genome and transcriptome sequencing to examine quantitative trait loci, gene expression and mating-type loci. These findings revealed a pronounced phenotypic continuum in poplar root colonisation by sibling monokaryons, ranging from incompatible to fully compatible strains. Genetic recombination among monokaryons was demonstrated, and genomic regions potentially involved in ECM fungal traits were identified. Transcriptomic analysis revealed greater differentiation in transcriptomic profiles between fungal strains than between fungal tissues, and uncovered tissue-specific functional responses for ECM and free-living mycelia. Poplar exhibited distinct transcriptomic responses when interacting with different sibling monokaryons and the parental dikaryon. Allele sorting at 11 mating-type loci confirmed the species' heterothallic tetrapolar system. This study advances understanding of the genetic and transcriptomic mechanisms underlying ECM symbioses, highlighting intraspecific fungal diversity's role in forest ecosystem functioning.

Aim: This study aims to (1) test whether mapped soil properties can improve the performance of species distribution models (SDMs) for 162 terricolous macrofungi at a regional level, (2) identify relevant soil predictors for macrofungal regional distribution and (3) quantify the relative importance of soil properties as compared to climate and topography in explaining macrofungal regional distribution. Location: The forested area (similar to 12,000 km(2)) in Switzerland. Taxon: Terricolous Macrofungi. Methods: We collected occurrences (presence-only) for 162 species of terricolous macrofungi, including 111 ectomycorrhizal and 51 saprotrophic species, from the SwissFungi database. We used soil property maps, generated through digital soil mapping at a 25 m resolution, to enhance macrofungal SDMs. For each species, we selected two climate, two topography and two soil predictors by an automated variable selection procedure. We built SDMs with randomised soil properties for performance comparison. We quantified the importance of soil properties based on permutation and variance partitioning. Finally, we projected the SDMs for three representative species at 25 m resolution with and without soil properties to assess the role of soil properties in shaping their biogeographical distributions. Results: Soil properties significantly improved the median performance of the SDMs across the 162 species. Ectomycorrhizal fungi showed a significantly greater improvement than saprotrophic fungi. On average, our models were able to explain two-thirds of the variance in macrofungal distribution, of which 11% could be independently explained by soil properties. Air temperature and topographic slope were identified as additional important factors controlling macrofungal distribution. Evident changes in geographical distribution were observed for the three representative species after adding soil properties. Main Conclusions: High-resolution digital soil maps significantly improve the predictive accuracy of macrofungal regional distribution. They should therefore be taken into account when modelling the geographical distribution of macrofungi.

Tracking signatures of selection in natural populations of ectomycorrhizal fungiprogress, challenges, and prospects Environmental changes have a manifold effect on biodiversity.Although soil harbours most terrestrial biodiversity (Anthony et al., 2023), including c. 90% of the fungal kingdom, little is known about the adaptation processes of natural populations of soil-dwelling organisms.Among these, ectomycorrhizal (ECM) fungi are involved in key mutualist interactions in forest ecosystems by providing water and nutrients to the host trees (Van Der Heijden et al., 2015).Recent studies have shown large intraspecific genomic variation in ECM fungi (e.g.Tremble et al., 2023a), raising questions about how natural selection affects ECM fungal populations.What are the signatures of adaptation in ECM fungal genomes?What are the strongest selective pressures occurring in local environments (Dauphin & Peter, 2023)?Given that environmental changes have become more pronounced in recent years (Dai, 2013), it is critical to improve our understanding of local adaptation in natural populations of these important organisms.Against this backdrop, a 2-d symposium on the population and landscape genomics of ECM fungi was held at the Swiss Federal Institute for Forest, Snow and Landscape Research WSL.The event was held on 5 and 6 September 2023, bringing together leading experts in the field of ecological genomics, to explore the latest advances and promising prospects.Drawing on the lively discussions at the event, this meeting report retraces the complementary approaches for tracking patterns of local adaptation, the methodological and analytical limitations currently encountered, and the emerging model organisms used to study the genomics of environmental adaptation in natural ECM fungal populations.The final section outlines some promising avenues for advancing the field and the overall biological knowledge of ECM fungi and their interacting host species.

The ectomycorrhizal fungi Tuber melanosporum Vittad. and Tuber aestivum Vittad. produce highly valuable truffles, but little is known about the soil fungal communities associated with these truffle species in places where they co-occur. Here, we compared soil fungal communities present in wild and planted truffle sites, in which T. melanosporum and T. aestivum coexist, in Mediterranean and temperate regions over three sampling seasons spanning from 2018 to 2019. We showed that soil fungal community composition and ectomycorrhizal species composition are driven by habitat type rather than climate regions. Also, we observed the influence of soil pH, organic matter content and C:N ratio structuring total and ectomycorrhizal fungal assemblages. Soil fungal communities in wild sites revealed more compositional variability than those of plantations. Greater soil fungal diversity was found in temperate compared to Mediterranean sites when considering all fungal guilds. Ectomycorrhizal diversity was significantly higher in wild sites compared to plantations. Greater mould abundance at wild sites than those on plantation was observed while tree species and seasonal effects were not significant predictors in fungal community structure. Our results suggested a strong influence of both ecosystem age and management on the fungal taxa composition in truffle habitats.

Vegetation changes in a warming Arctic may affect plant-associated soil microbial communities with possible consequences for the biogeochemical cycling of carbon (C) and nitrogen (N). In a sub-arctic tundra heath, we factorially removed plant species with ecto- and ericoid mycorrhizal associations. After two years, we explored how mycorrhizal type-specific plant removal influences microbial communities, soil and microbial C and N pools, and extracellular enzymatic activities. Removal of ecto- and ericoid mycorrhizal plants did not change the soil fungal or bacterial community composition or their extracellular enzyme activities. However, ericoid plant removal decreased microbial C:N ratio, suggesting a stoichiometric effect decoupled from microbial community composition. In other words, microbial communities appear to show initial plasticity in response to major changes in tundra vegetation. This highlights the importance of longer-term perspectives when investigating the effects of vegetation changes on biogeochemical processes in Arctic ecosystems.

Qua o là sta amo ün tal eroe plain fideltà sün seis post, cun büst sfrattamà, e stenda il bratsch our da spelm e grava, sco üna sgür pronta pel cumbat, o ch'el sta ingramüschà sco ün vegliet cha la burasca da la vita ha fat sgobar sün sias crotschas.

Ectomycorrhizal (ECM) fungi serve key functions in forest ecosystems by supplying water and nutrients to tree hosts, yet mutualistic plant-fungi interactions are jeopardised by environmental alterations. Here, we discuss the great potential and current limitations of landscape genomics in investigating signatures of local adaptation in natural populations of ECM fungi.

Soil organic matter (SOM) originates from various sources such as foliar litter, roots and microbial (e.g. fungal) components. The relative sources contribution represents one of the key unknowns in SOM dynamics. Our study aimed to explore whether stable isotope ratios of non-exchangeable hydrogen (Hn) bound to organic matter can be used to differentiate SOM sources, since natural 2Hn abundance can strongly differ between root and foliar tissues. We also investigated if long-term irrigation with 2H-depleted water in a pine forest can be used to track Hn incorporation into organic matter inputs and eventually in the soil pools.In a 17-year-long irrigation experiment in a dry pine forest, we assessed variations in natural abundance of 2Hn, 13C, and 15N in SOM sources (foliar litter, fine roots, fungal mycelia), decomposing litter, soil (organic layers and uppermost 5 cm-mineral soil) and particle-size fractions. We then applied a Bayesian mixing model (including δ2Hn,δ13C, and δ15N) to estimate the relative sources contribution to SOM.Natural 2Hn abundance was significantly higher in roots vs. foliar litter (up to +39‰), and in fungal mycelia vs. roots (up to +41‰). Results from Bayesian mixing model suggest that foliar litter contributed to approximately 68 ± 10% of SOM in organic layers and in coarse particulate organic matter (POM). Foliar litter and roots contributed similarly to upper 2 cm of mineral soil (46 ± 11%), while 2-5 cm of mineral soil were largely derived from roots (61 ± 13%). Fungal mycelia contributed to 18 ± 8% of mineral-associated organic matter (MOM), while only to 1-2% of coarse and fine POM. Bayesian mixing models provided only a general indication of the sources contribution to SOM, also considering that isotopic signatures shifted during decomposition. Measurements of isotope signatures in microbial necromass might allow a more accurate assessment of the different SOM sources contribution.The δ2Hn depletion of soil water under irrigation was paralleled by a comparable decrease in δ2Hn of roots (~12‰). In comparison, the natural 2Hn abundance in fresh needles and foliar litter decreased less strongly (~ 7‰ and 4‰, respectively), likely due to photosynthetic adjustments that may have counterbalanced the irrigation water 2H-depletion. Similar to soil water 2H-depletion, δ2Hn values in coarse POM were 11‰ lower in irrigated vs. dry plots, suggesting that nearly all organic Hn turned over or exchanged with soil water in less than two decades. In contrast, δ2Hn values in fine POM and MOM decreased only by 3‰ under irrigation, which indicate that these fractions comprise slower cycling Hn pools.Our study showed that the natural 2Hn abundance represents a promising tool to differentiate among SOM sources. While 13C and 15N did not clearly separate between roots and foliar litter, Hn isotopic signatures allowed a good discrimination between SOM sources. In addition, long-term irrigation can provide a potential in situ 2H-labelling of SOM, which may help to examine organic Hn turnover rates across SOM pools.

Steadfast Swiss stone pines in a dynamic habitat: patchy, mixed-age stand above the Aletsch Glacier (

Global warming is pushing populations outside their range of physiological tolerance. According to the environmental envelope framework, the most vulnerable populations occur near the climatic edge of their species' distributions. In contrast, populations from the climatic center of the species range should be relatively buffered against climate warming. We tested this latter prediction using a combination of linear mixed effects and machine learning algorithms on an extensive, citizen-scientist generated dataset on the fruitbody productivity of the Burgundy (aka summer) truffle (Tuber aestivum Vittad.), a keystone, ectomycorrhizal tree-symbiont occurring on a wide range of temperate climates. T. aestivum's fruitbody productivity was monitored at 3-week resolution over up to 8 continuous years at 20 sites distributed in the climatic center of its European distribution in southwest Germany and Switzerland. We found that T. aestivum fruitbody production is more sensitive to summer drought than would be expected from the breadth of its species' climatic niche. The monitored populations occurring nearly 5°C colder than the edge of their species' climatic distribution. However, interannual fruitbody productivity (truffle mass year-1 ) fell by a median loss of 22% for every 1°C increase in summer temperature over a site's 30-year mean. Among the most productive monitored populations, the temperature sensitivity was even higher, with single summer temperature anomalies of 3°C sufficient to stop fruitbody production altogether. Interannual truffle productivity was also related to the phenology of host trees, with ~22 g less truffle mass for each 1-day reduction in the length of the tree growing season. Increasing summer drought extremes are therefore likely to reduce fruiting among summer truffle populations throughout Central Europe. Our results suggest that European T. aestivum may be a mosaic of vulnerable populations, sensitive to climate-driven declines at lower thresholds than implied by its species distribution model.

Climate change exposes ecosystems to strong and rapid changes in their environmental boundary conditions mainly due to the altered temperature and precipitation patterns. It is still poorly understood how fast interlinked ecosystem processes respond to altered environmental conditions, if these responses occur gradually or suddenly when thresholds are exceeded, and if the patterns of the responses will reach a stable state. We conducted an irrigation experiment in the Pfynwald, Switzerland from 2003-2018. A naturally dry Scots pine (Pinus sylvestris L.) forest was irrigated with amounts that doubled natural precipitation, thus releasing the forest stand from water limitation. The aim of this study was to provide a quantitative understanding on how different traits and functions of individual trees and the whole ecosystem responded to increased water availability, and how the patterns and magnitudes of these responses developed over time. We found that the response magnitude, the temporal trajectory of responses, and the length of initial lag period prior to significant response largely varied across traits. We detected rapid and stronger responses from above-ground tree traits (e.g., tree-ring width, needle length, and crown transparency) compared to below-ground tree traits (e.g., fine root biomass). The altered above-ground traits during the initial years of irrigation increased the water demand and trees adjusted by increasing root biomass during the later years of irrigation, resulting in an increased survival rate of Scots pine trees in irrigated plots. The irrigation also stimulated ecosystem-level foliar decomposition rate, fungal fruit body biomass, and regeneration abundances of broadleaved tree species. However, irrigation did not promote the regeneration of Scots pine trees which are reported to be vulnerable to extreme droughts. Our results provide extensive evidence that treeand ecosystem-level responses were pervasive across a number of traits on long-term temporal scales. However, after reaching a peak, the magnitude of these responses either decreased or reached a new stable state, providing important insights into how resource alterations could change the system functioning and its boundary conditions.

Fig. 1. Solides aroles dans l’environnement dynamique d’un peuplement équienne et clairiéré en amont du glacier d’Aletsch (Valais). «Ici et là se tient encore une sentinelle debout, tronc déchiqueté, fidèle à son poste, qui semble brandir un bras comme une hache de guerre surgie des éboulis de rochers ou se recroqueviller, tel un vieillard courbé sur ses béquilles par les tempêtes de la vie.» (Schnidrig 1935) Institut fédéral de recherches WSL CH-8903 Birmensdorf ISSN 2296-4436