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Comparison of intergeneric ? 15 N and ? 13 C variations in Hygrophoraceae genera across all sites and average ? 15 N and ? 13 C for ectomycorrhizal and saprotrophic fungi from 16 published woodland studies. Genera of family Hygrophoraceae are shown with inverted black triangles; Mean for grassland saprotrophic fungi (GS) is indicated with green square. Mean values for ectomycorrhizal fungi (blue circles) and woodland saprotrophic fungi (red triangles) from published studies are also shown. Error bars in grey indicate standard error. For Hygrophoraceae genera are labelled as follows: Cuphophyllus (Cu), Gliophorus (Gl), Humidicutis (Hu), Hygrocybe (Hc), Hygrophorus (Hp), Neohygrocybe (Ne), Porpolomopsis (Po). Raw data and keys to published study labels (including numbers of replicates) are presented in Suppdata 10.

Comparison of intergeneric ? 15 N and ? 13 C variations in Hygrophoraceae genera across all sites and average ? 15 N and ? 13 C for ectomycorrhizal and saprotrophic fungi from 16 published woodland studies. Genera of family Hygrophoraceae are shown with inverted black triangles; Mean for grassland saprotrophic fungi (GS) is indicated with green square. Mean values for ectomycorrhizal fungi (blue circles) and woodland saprotrophic fungi (red triangles) from published studies are also shown. Error bars in grey indicate standard error. For Hygrophoraceae genera are labelled as follows: Cuphophyllus (Cu), Gliophorus (Gl), Humidicutis (Hu), Hygrocybe (Hc), Hygrophorus (Hp), Neohygrocybe (Ne), Porpolomopsis (Po). Raw data and keys to published study labels (including numbers of replicates) are presented in Suppdata 10.

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Article
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Several lines of evidence suggest that the agaricoid, non‐ectomycorrhizal members of the family Hygrophoraceae (waxcaps) are biotrophic with unusual nitrogen nutrition. However, methods for the axenic culture and lab‐based study of these organisms remain to be developed, so our current knowledge is limited to field‐based investigations. Addition of...

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... 277 (Table 1) A) δ 13 C natural abundance. Waxcap basidiocarps were lower in δ 13 C than basidiocarp cap tissues of 303 ectomycorrhizal and saprotrophic fungi reported here and elsewhere. However, across all samples, 304 δ 13 C values for waxcaps were distributed within a small range (-26.5 to -31.3‰ across all European 305 samples; 4.8‰ range) ( Fig. 5; Suppdata 3). The data presented here for more than 20 species in 6 306 genera and including data from several previous studies show that mean δ 13 (Table 2). However, for the three Hygrophoraceae, the midpoint 14 C age was 318 younger, in two cases a few months but for the third it was 1.8 years. 319 ...

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... However, their biology remains a mystery since isotopic signatures indicate that they are neither mycorrhizal nor saprotrophic (Seitzman et al. 2011, Halbwachs et al. 2013. Recent studies revealed that some of the waxcaps can be associated with plant roots and they probably have a biotrophic lifestyle with plants (Halbwachs et al. 2013(Halbwachs et al. , 2018. Tello et al. (2013) proved that at least one species, Hygrocybe virginea, is a maternally transmitted endophytic fungus associated with Plantago lanceolata. ...
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Neohygrocybe pseudoingrat a, a new waxcap species known from Slovakia and the Czech Republic, is characterised by its pale greyish coloured and often robust basidiomata (or sporocarps), nitrous smell, context without colour changes, hollow, contorted and compressed stipe and smooth or slightly fibrillose pileus surface. Based on morphology and DNA analysis of ITS and LSU sequences of the collected specimens, N. pseudoingrata belongs to Neohygrocybe sect. Neohygrocybe together with N. ovina , N. nitrata and N. ingrata . Collections of N. pseudoingrata form a well-supported clade in phylogenetic trees.
... The dominant family in NS was Hygrophoraceae, whose representatives are primarily found in undisturbed grassland habitats and are much rarer or absent in grasslands subject to agricultural intensification, which can characterize them as ecological indicators (Halbwachs et al., 2018). Thus, it is evident that the fungal community needs more time to recover and to approximate from the native area than the bacterial community, mainly because those are more sensitive to environmental changes (Genevieve et al., 2019). ...
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The Brazilian Cerrado is a highland tropical savanna considered a biodiversity hotspot with many endemic species of plants and animals. Over the years, most of the native areas of this biome became arable areas, and with inadequate management, some are nowadays at varying levels of degradation stage. Crop-livestock integrated systems (CLIS) are one option for the recovery of areas in degradation, improving the physicochemical and biological characteristics of the soil while increasing income and mitigating risks due to product diversification. Little is known about the effect of CLIS on the soil microbial community. Therefore, we perform this pilot case study to support further research on recovering degraded areas. The bacterial and fungal soil communities in the area with CLIS were compared to an area under moderate recovery (low-input recovering - LI) and native savanna (NS) area. Bacterial and fungal communities were investigated by 16S and ITS rRNA gene sequencing (deep rRNA sequencing). Ktedonobacteraceae and AD3 families were found predominantly in LI, confirming the relationship of the members of the Chloroflexi phylum in challenging environmental conditions, which can be evidenced in LI. The CLIS soil presented 63 exclusive bacterial families that were not found in LI or NS and presented a higher bacterial richness, which can be related to good land management. The NS area shared 21 and 6 families with CLIS and LI, respectively, suggesting that the intervention method used in the analyzed period brings microbial diversity closer to the conditions of the native area, demonstrating a trend of approximation between NS and CLIS even in the short term. The most abundant fungal phylum in NS treatment was Basidiomycota and Mucoromycota, whereas Ascomycota predominated in CLIS and LI. The fungal community needs more time to recover and to approximate from the native area than the bacterial community. However, according to the analysis of bacteria, the CLIS area behaved differently from the LI area, showing that this treatment induces a faster response to the increase in species richness, tending to more accelerated recovery. Results obtained herein encourage CLIS as a sustainable alternative for recovery and production in degraded areas.
... A total of 832 fungal ASVs were identified by FUNGUILD as being putative plant pathogens (Table S4). Botrytis and Hygrocybe were not included as pathogens in the original FUNGUILD output, but we added them to the putative pathogen dataset based on established trophic modes of these genera from grasslands in previous studies (Dean et al., 2012;Halbwachs et al., 2018). We also added three other genera to the putative pathogen subset for exploratory graphing and statistics because they were found in the morphological study: Bjerkandera, Penicillium and Sistotrema. ...
Article
•Demographic studies measure drivers of plant fecundity including seed production and survival, but few address both abiotic and biotic drivers of germination such as variation in climate among sites, population density, maternal plants, seed type and fungal pathogen abundance. •We examined germination and microbial communities of seeds of Danthonia californica, which are either chasmogamous (external, wind‐pollinated) or cleistogamous (internal, self‐fertilized) and Festuca roemeri, which are solely chasmogamous. Seed populations were sourced across environmental gradients. We tested germination and used high‐throughput sequencing to characterize seed fungal community structure. •For F. roemeri, maternal plants significantly influenced germination as did climate and pathogens; germination increased in wetter, cooler sites. For D. californica, the main drivers of germination were maternal plant, seed type and pathogens; on average, more chasmogamous seeds germinated. Fungal communities depended largely on seed type, with fewer fungi associated with cleistogamous seeds, but the communities also depended on site factors such as vapor pressure deficit, plant density and whether the seeds had germinated. •Putative pathogens that were negatively correlated with germination were more abundant for both D. californica and F. roemeri chasmogamous seeds than D. californica cleistogamous seeds. In D. californica, cleistogamous and chasmogamous seeds contain vastly different fungal communities.
... The so-called CHEGD species represent fungi that are highly sensitive to such inputs and are hence appropriate for biomonitoring purposes; the acronym stands for Clavariaceae-Hygrocybe-Entoloma-Geoglossaceae-Dermoloma [2]. It is thought that these praticolous fungal genera are well adapted to nutrient-poor environments because of their biotrophic [3, 4,5] and/or endophytic (in higher plants) lifestyles [6]. Not least, this may explain why CHEGD fungi respond highly sensitive to external nutrient impact, which makes them suitable bioindicators for unimproved "natural" grasslands. ...
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The Gottesacker (“God’s acre”) in Herrnhut (Upper Lusatia, Saxony) has been a graveyard for almost 300 years. G. Zschieschang has mapped its fungal community since the 1960ies. Combining these findings and those of other mycologists, mycological data that cover about 55 years are reported here. In this context, we discuss the fungal biodiversity with special emphasis on CHEGD species (Clavariaceae-Hygrocybe-Entoloma-Geoglossaceae-Dermoloma). These species are used to classify and assess the conservation value of grasslands by monitoring their fungal communities. According to the determined CHEGD profile, the Gottesacker graveyard is ranked as a grassland of international importance. In addition, we present macroscopic and microscopic characteristics of two rare CHEGD species: Entoloma brunneosericeum and Clavaria messapica. These are the first records of both species for Germany and Saxony, respectively.
... Among fungi occurring in semi-natural grasslands of Europe, macrofungi collectively referred as CHEGD fungi (an acronym of Hygrophoraceae, Entolomataceae, Clavariacaeae, Geoglossaceae and Dermoloma) are well known to be typically associated with undisturbed, unfertilised grasslands (Griffith et al., 2013). Natural abundance of 15 N, 14 C and 13 C isotopes in basidiomata of Hygrophoraceae, as well as 13 C pulse label experiments, further suggest that these fungi are not saprotrophs but rather biotrophic endophytes and possibly mycorrhizal symbionts (Halbwachs et al., 2018). The metabarcoding analyses of fungal microbial diversity proved that CHEGD fungi are often the most abundant group in oligotrophic grasslands (Detheridge et al., 2018;Hay, Thorn & Jacobs, 2019). ...
... There is a negative correlation between nitrogen levels and diversity of some functional fungal groups, including CHEGD fungi (Detheridge et al., 2018;Halbwachs et al., 2018). The changes in the abundance and diversity of CHEGD fungi seen between traditional managements and mulching experiments in this study may be the result of increased N levels due to elevated N input from cut vegetation (Fang, Xie & Zhang, 2007). ...
Article
Mulching (cutting of vegetation without removal of clippings) is used as a low-cost method for maintaining remote or abandoned grasslands in Slovakia. The likely consequence of mulching is seasonal nutrient enrichment resulting from decomposition of plant litter by saprotrophic organisms. The potential changes in biodiversity of the ecosystem caused by long-term application of mulching are to date only very poorly understood. In order to examine the impact of mulching on soil mycobiota, we compared six different grassland management regimes applied over nine years on a sub-montane oligotrophic Nardus pasture in the Central Slovakia. The diversity of soil fungi was assessed using DNA metabarcoding of the ITS2 regions of the nrRNA locus performed by Illumina MiSeq. We focused on a particular group of macrofungi which is characteristic of traditionally managed and undisturbed European grasslands, and which are often the dominant soil fungi in these habitats. These are collectively known as CHEGD fungi (the acronym of the constituent taxa: Clavariaceae, Hygrophoraceae, Entolomataceae, Geoglossaceae and Dermoloma). We compared the relative abundance and diversity of CHEGD fungi with the total fungal and plant diversity. CHEGD fungi were dominant across all management treatments. Although there were no statistical effects of treatments on total fungal richness and diversity, CHEGD fungi and vascular plants diversity and richness were lower on plots where mulching or no management were imposed, suggesting that such management regimes would have a negative impact on grassland fungi. However, no single treatment covered the total CHEGD diversity of the study, indicating that the localized use of mulching in addition to traditional managements can enhance overall diversity of grasslands in the area. Our results also suggest that the impact of mulching depends on the season when the grassland is mulched and it might be reduced by combination with other management treatments. The high relative abundance and sensitivity of CHEDG fungi in oligotrophic grasslands to management treatments makes them excellent indicators of grassland natural quality and is consistent with the ecological importance of this fungal group.
... Their ecology may have a strong link to trophy that is so far unresolved. Analysis of C and N isotopes and 13 C pulse label experiments and high natural abundance stable isotopes of 15 N and low of 13 C in basidiocarps suggest that they form unspecified biotrophic associations with plants (Halbwachs et al. 2018). They have a particular response to available nutrition changes linked to management practices when compared to other grassland fungi (Caboň et al., unpublished data). ...
Article
We present the first phylogenetic evaluation of the genus Dermoloma, which is resolved as monophyletic and closely related to Pseudotricholoma, a poorly known Dermoloma-like lineage within the family Tricholomataceae. The position of Dermoloma is confirmed by the placement of the type species, Dermoloma cuneifolium, represented by multiple samples including the neotype. Based on our phylogenetic analyses, we recognised 25 European operational taxonomic units (OTUs) but could only assign species names to ten of them based on ex-type sequences. Furthermore, only five additional published Dermoloma names of uncertain status are available for the remaining 16 potential European species, thus demonstrating an unexpected amount of taxonomic diversity. Samples from Europe and North America seem to be endemic on a continental scale. North American samples formed six unique OTUs, but only one could be reliably named, Dermoloma hymenocephalum. Dermoloma is morphologically defined by basidiomata with brown, grey and white colours with a farinaceous odour and a pluristratous hymeniderm type of pileipellis. Our phylogenetic analyses support the subdivision of the genus into two subgenera and four sections, and species with inamyloid basidiospores are placed in subg. Dermoloma and those with amyloid basidiospores in subg. Amylospora. Both subgenera are further divided into two sections. The analysis of spore morphology shows that sect. Conica of subg. Dermoloma and sect. Nigrescentia of subg. Amylospora have a very distinctive spore shape. Sect. Atrobrunnea of subg. Amylospora showed relatively high variability of spores among species, but spores of sect. Dermoloma were similar and not useful for species discrimination.
... Additionally, we examined the effect of different storage conditions on an additional functional group not yet included in FUNguild, the CHEGD fungi ). This guild of grassland macrofungi fungi (comprising members of the families Clavariaceae, Hygrophoraceae, Entolomataceae, Geoglossaceae and Dermoloma spp.) are dominant components of undisturbed grassland habitats and suspected to be mycorrhizal or with intricate biotrophic association with higher plants (Detheridge et al. 2018;Halbwachs et al. 2018). ...
... The CHEGD fungi (barring Entolomataceae) also showed substantial decline in relative abundance in freeze-thaw treatments. Like AMF, these fungi are obligate root-associated biotrophs (Halbwachs et al. 2013;Halbwachs et al. 2018) and are negatively affected by the killing of host vegetation (Griffith et al. 2014). The fact that Entolomataceae were differently affected, compared to other CHEGD fungi, suggests that they are nutritionally more flexible, potentially with some saprotrophic ability. ...
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Storage of soil samples prior to metagenomic analysis presents a problem. If field sites are remote or if samples are collected by third parties, transport to analytical laboratories may take several days or even weeks. The bulk of such samples and requirement for later homogenisation precludes the convenient use of a stabilisation buffer, so samples are usually cooled or frozen during transit. There has been limited testing of the most appropriate storage methods for later study of soil organisms by eDNA approaches. Here we tested a range of storage methods on two contrasting soils, comparing these methods to the control of freezing at-80 °C, followed by freeze-drying. To our knowledge, this is the first study to examine the effect of storage conditions on eukaryote DNA in soil, including both viable organisms (fungi) and DNA contained within dying/dead tissues (plants). For fungi, the best storage regimes (closest to the control) were storage at 4 °C (for up to 14 d) or active air-drying at room temperature. The worst treatments involved initial freezing, followed by thawing which led to significant later spoilage. The key spoilage organisms were identified as Metarhizium carneum and Mortierella spp., with a general increase in saprotrophic fungi and reduced abundances of mycorrhizal/biotrophic fungi. Plant data showed a similar pattern, but with greater variability in community structure, especially in the freeze-thaw treatments, probably due to stochastic variation in substrates for fungal decomposition, algal proliferation and some seed germination. In the absence of freeze drying facilities, samples should be shipped refrigerated, but not frozen if there is any risk of thawing.
... Their ecology may have a strong link to trophy that is so far unresolved. Analysis of C and N isotopes and 13 C pulse label experiments and high natural abundance stable isotopes of 15 N and low of 13 C in basidiocarps suggest that they form unspeci ed biotrophic associations with plants (Halbwachs et al. 2018). They have a particular response to available nutrition changes linked to management practices when comparing to other grassland fungi (Caboň et al. in review). ...
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We present the first phylogenetic evaluation of the genus Dermoloma , which is resolved as monophyletic and closely related to Pseudotricholoma , a poorly-known Dermoloma -like lineage within the family Tricholomataceae. The position of Dermoloma is confirmed by the placement of the type species, D. cuneifolium , represented by multiple samples including the neotype. Based on our phylogenetic analyses, we recognised 25 European operational taxonomic units (OTUs), but could only assign species names to ten of them based on ex-type sequences. Furthermore, only five additional published Dermoloma names of uncertain status are available for the remaining 16 potential European species, thus demonstrating an unexpected amount of taxonomic diversity. Samples from Europe and North America seem to be endemic on a continental scale. North American samples formed six unique OTUs, but only one could be reliably named, D. hymenocephalum . Dermoloma is morphologically defined by basidiomata with brown, grey and white colours with a farinaceous odour and a pluristratous hymeniderm type of pileipellis. Our phylogenetic analyses support the subdivision of the genus into two subgenera and four sections, species with inamyloid basidiospores are placed in subg. Dermoloma and those with amyloid basidiospores in subg. Amylospora . Both subgenera are further divided in two sections. The analysis of spore morphology shows that sect. Conica of subg. Dermoloma and sect. Nigrescentia of subg. Amylospora have a very distinctive spore shape. Sect. Atrobrunnea of subg. Amylospora showed relatively high variability of spores among species, but spores of sect. Dermoloma were similar and not useful for species discrimination.
... High-throughput sequencing has enabled assessments of fungal diversity in a wide range of ecosystems and substrates, ranging from local (Pickles et al., 2012;Anderson et al., 2014) to global scales (Tedersoo et al., 2010(Tedersoo et al., , 2014, and including substrates such as individual plant roots (Bahram et al., 2011;Botnen et al., 2014;Lorberau et al., 2017), soils (Kyaschenko et al., 2017;Sterkenburg et al., 2018) and dead wood (Baldrian & Val a skov a, 2008). As information on habitat occupation by species accumulates, it is becoming clear that some taxa with supposedly defined niche distributions might occupy more than one niche, and that current ideas and beliefs concerning the ecology and function of some fungal taxa will require revision (Selosse et al., , 2010Halbwachs et al., 2018;Schneider-Maunoury et al., 2018;Lofgren et al., 2018; see also Selosse et al., 2018). ...
Article
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The root‐associated habit has evolved on numerous occasions in different fungal lineages, suggesting a strong evolutionary pressure for saprotrophic fungi to switch to symbiotic associations with plants. Species within the ubiquitous, saprotrophic genus Mycena are frequently major components in molecular studies of root‐associated fungal communities, suggesting that an evaluation of their trophic status is warranted. Here, we report on interactions between a range of Mycena species and the plant Betula pendula. Seventeen Mycena species were inoculated onto B. pendula seedlings. Physical interactions between hyphae and fine roots were examined using differential staining and fluorescence microscopy. Physiological interactions were investigated using 14C and 32P to show potential transfer between symbionts. All Mycena species associated closely with fine roots, showing hyphal penetration into the roots, which in some cases were intracellular. Seven species formed mantle‐like structures around root tips, but none formed a Hartig net. Mycena pura and M. galopus both enhanced seedling growth, with M. pura showing significant transfer of 32P to the seedlings. Our results support the view that several Mycena species can associate closely with plant roots and some may potentially occupy a transitional state between saprotrophy and biotrophy.
... Several lines of evidence suggest that waxcaps (and likely other CHEGD) are not saprotrophs, as previously suspected, but rather mycorrhizal with grasses and/or herbs (Griffith et al., 2014). Halbwachs et al. (2018) presented isotopic evidence consistent with a mycorrhizal habit, and the hyphae of waxcap have been detected within the roots and shoots of putative plant hosts (Halbwachs et al., 2013;Tello et al., 2014). However, association with ericaceous hosts has not previously been observed. ...
Article
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Results of surveys of species of fungi in selected areas of Lundy which used fruitbodies to assess distribution and abundance are presented in comparison to data derived from identification of taxa from DNA sequences in extracts of soils (eDNA) from six sites on the island. Two sites were within acid grassland, four from heathland areas of Calluna vulgaris (Heather). The apparent restriction of Cuphophyllus (=Hygrocybe) lacmus (Grey Waxcap) to the Maritime heath on the North End as determined by past annual fruitbody surveys was confirmed by the abundance of its DNA in the two soil samples from the North End. C. lacmus DNA was absent in the soil samples from the two acid grassland sites and in one of the 'southern' Calluna sites, though it was present in the soil sample from the other 'southern' Calluna site, perhaps indicating a wider distribution on Lundy than that found by fruitbody surveys. The DNA sequence data for the soil sample from acid grassland on the Airfield are also compared with the fruitbody counts made over the past eleven years for this site and shows that for some taxa fruitbody abundance is reflected in the proportion of their DNA in the samples, for others it is not. In addition, some taxa found in the DNA profiles have yet to be found in any survey and may represent new records for Lundy. CHEGD scores derived from the DNA profiles and fruitbody surveys are compared with those of high diversity grassland sites on the mainland and confirm the high conservation status of Lundy grasslands.