Fungi are important decomposers of organic matter in terrestrial trophic networks (Lindahl et al., 2002; Bai et al., 2021). They inhabit environments where sufficient carbon (C) is usually available, but this is not the case for the main macronutrients, nitrogen (N) and phosphorus (P), which are often limiting. While in agricultural soils N and P deficiency is often overcome by fertilization, in most natural ecosystems available N or P sources are limited (Du et al., 2020). Both C and N are mainly present in soil organic matter, with N also coming from wet or dry atmospheric deposition (van Breemen and van Dijk, 1988), often enhanced by human activities such as combustion processes; P is also mobilized in soil by mineral weathering (Larsen, 1967). The access of fungi to N and P in the environment varies depending on their lifestyle (Root, 1967; Põlme et al., 2020), reflecting the C resources used by fungi. The most important ecological guilds of fungi (Põlme et al., 2020) in forest trophic networks are (i) wood-decomposing saprotrophs/parasites that accumulate C from decaying wood, (ii) soil and litter saprotrophs that acquire C through litter decomposition, and (iii) mycorrhizal fungi that acquire C from their plant hosts.

Of the substrates used by fungi, wood from living or dead trees contains the least N and P (Watkinson et al., 2006), whereas soil, and especially topsoil, is richer in these nutrients (Mooshammer et al., 2014). In a typical forest, N or P content may limit fungal activity in dead wood even when content of nutrients in soil is high (Piché-Choquette et al., 2023). Thus, wood saprotrophs (WS) must cope with nutrient limitation in their habitat. Soil saprotrophic (SS) and ectomycorrhizal (EcM) fungi utilize soil N and P, ectomycorrhizal fungi utilize C provided by their host trees in exchange for N (Rayner and Boddy, 1989; Lindahl et al., 2007; Smith and Read, 2008), and therefore their C acquisition costs are reduced. In principle, the benefits of EcM symbiosis appear to be highest in N-limited soils (Johnson, 2010), because the provision of N by fungi to plants leads to higher primary production, thereby increasing C availability to fungi. The ratio between N and P also appears to be very important for mycorrhizal fungi, as higher N supply promotes photosynthetic activity of host plants, which in turn have a higher capacity to exchange C for P (Johnson, 2010).

Increased N availability associated with human activities has been shown to increase the ratio of bacterial and fungal biomass in soil and inhibit microbial respiration and abundance (Ramirez et al., 2012; Zhou et al., 2017). Soil fungal communities are also affected by increased N availability due to anthropogenic deposition (Baldrian et al., 2023), including changes in abundance and balance between nutrient guilds and species within guilds (van der Linde et al., 2018; Baldrian et al., 2022).

Due to both natural variation and anthropogenic deposition, the total amounts of C, N, and P, as well as their ratios, vary substantially among soils (Cleveland and Liptzin, 2007). The amounts and ratios of these elements in fungal biomass also appear to vary among species, both under laboratory conditions and in nature (Brabcová et al., 2018; Camenzind et al., 2021). The analysis of nutrient content in fungal biomass under real conditions is methodically challenging, as it is virtually impossible to separate the hyphae of individual fungal species from their habitat matrix—plant litter, soil, or wood. However, assuming that nutrient content may be reflected in the biomass of fungi growing under laboratory conditions (Camenzind et al., 2021), it is also evident in the biomass that forms the fungal fruiting bodies (Vogt et al., 1981).

For ectomycorrhizal fungi, it has been suggested that the C:N ratio in their biomass remains relatively constant (Mooshammer et al., 2014), as a result of providing a “surplus” of N to their tree hosts (Kranabetter et al., 2019). In saprotrophic fungi, the storage of P and C is expected in their biomass (Quinché, 1997; Kalač, 2019), while any excess nitrogen, if present, is likely excreted (Mooshammer et al., 2014). Although nutrient content can vary even between different parts of the same individual (Trocha et al., 2016), it can be assumed that differences between ecological guilds should be much more pronounced.

Occasionally, higher concentrations of N and therefore lower C:N ratios in fruiting bodies have been observed in saprotrophic fungi compared to EcM fungi (Vogt et al., 1981; Zhang and Elser, 2017). This may indicate the lower costs of C in EcM fungi. However, it is well known that fungal species exhibit a preference for environments with specific (high or low) N availability (van der Linde et al., 2018). It should be noted that the nutrient content in fungal biomass is not only a response variable to species traits and environmental conditions; it also influences the fate of microbial biomass in the ecosystem. The rate of decomposition of fungal mycelia in forest soils has been reported to increase with the N content (Brabcová et al., 2018), and it is evident that N is a primary target for guilds of microorganisms responsible for decomposing fungal biomass (López-Mondéjar et al., 2020; Starke et al., 2020). While N is present in many biomolecules, including proteins and nucleic acids (Marzluf, 1996), it also constitutes a significant component of the fungal cell wall polysaccharide chitin (Lenardon et al., 2010), whose content in fungal cell walls varies (Blumenthal and Saul, 1957) and under starvation conditions this compound may undergo enzymatic self-degradation (White et al., 2002; Gruber and Seidl-Seiboth, 2012). However, the chitin content in biomass may to some extent determine the N requirement of each fungal species and potentially correlate with the N content in their biomass.

Substantial differences in the accessibility of C, N, and P by various ecological guilds of fungi raise the question of whether their amounts and ratios are conserved in fungal groups that obtain these nutrients from soil, plant litter, deadwood, or the roots of their host plants. In these environments, the relative content of C, N, and P can vary over more than an order of magnitude (Piché-Choquette et al., 2023). The composition of fungal biomass may also be conserved phylogenetically, at least over short phylogenetic distances (Mouginot et al., 2014), reflecting traits such as the proportion of protein or chitin content in their cell walls. Alternatively, the N and P content in fungal biomass may vary within each species, simply reflecting the accessibility of N and P in each individual site where the fungus is found.

We anticipate that the ecological traits of fungi, particularly those related to the mode and source of C and nutrient acquisition, are the primary drivers of the nutrient content in their biomass. This is because the differences in nutrient limitation between habitats are too significant to be overcome. Some level of phylogenetic conservation is also likely due to the evolutionary preservation of biochemical traits and the similarity in the ecology of closely related taxa. We analyzed the fruiting bodies of fungi from temperate forests to determine their C, N, and P contents, aiming to assess the contributions of ecology and phylogeny to biomass composition. We assume that N and P contents are higher in soil saprotrophic fungi than in wood-decomposing and ectomycorrhizal fungi. To je způsobeno nízkou koncentrací živin ve dřevě v případě dřevních saprotrofů a výměnou živin za uhlík s hostitelskou rostlinou v případě mykorhizních hub. Additionally, we expect a positive correlation between chitin content and N content in fungal biomass. Furthermore, our study was intended to provide data on the stoichiometric ratios of main macronutrients in the biomass of macrofungi, offering reliable information on this biogeochemically important feature.

The fungal fruiting bodies samples belonged to 13 orders, predominantly covering Basidiomycota, with some species of Ascomycota. Among the 282 samples, there were 212 species from 82 fungal genera and 53 families. These were classified according to Põlme et al. (2020) into EcM (154 samples), SS (73 samples), and WS (55 samples; Supplementary Table 1 and Figure 1).

Our results support the hypothesis that N and P content in fungal biomass differs among ecological guilds (Root, 1967; Põlme et al., 2020)—mycorrhizal, soil, and wood saprotrophic fungi. Thus, the nutrient content is, to some extent, determined by the environments to which these fungal groups have been adapted and the source of C that they use. In comparison to both saprotrophic groups, mycorrhizal fungi have an advantage of a stable supply of C as a result of their mutualistic relations with host plants (Bödeker et al., 2016). As a consequence of the trade with the host plants for C, N, and P, these fungi should pass the excess of N and P acquired from the soil to their host, retaining limited amounts of these nutrients in their mycelia (Trocha et al., 2016; Zhang and Elser, 2017; Kranabetter et al., 2019). This is reflected by lower contents of N and P in this guild, as well as lower variation in the content of these two nutrients. Conversely, the high average values discovered among soil saprotrophic species, where such nutrient trade does not take place, could be explained as an ability of soil saprotrophic fungi to store N and P in case of their excess (Quinché, 1997; Mooshammer et al., 2014; Trocha et al., 2016; Kalač, 2019; Kranabetter et al., 2019). The low content of N and P in wood saprotrophs compared to soil saprotrophs reflects their adaptation to the low nutrient contents in deadwood (Watkinson et al., 2006; Piché-Choquette et al., 2023).

Our results show—in addition to the effect of fungal lifestyle—the phylogenetic conservation of nutrient content. Lilleskov et al. (2011) showed that the response of fungi to elevated N content in soil is consistent only at the level of genera, and other ecological traits of fungi are also only rarely conserved at higher taxonomic levels (Zanne et al., 2020). This was also confirmed by the results of our study, where the conservation of nutrient content in biomass was highest at low taxonomic levels, while its variability increased with taxonomic rank. This may have been due to the fact that fungi have repeatedly evolved different lifestyles during evolution (Martin et al., 2016; Zanne et al., 2020), and therefore phylogenetic relationships at higher taxonomic levels may not correspond to the ecological features of fungal species. Although the differences in nutrient content between taxonomic groups are not as pronounced as differences between ecological guilds, phylogenetic relations seem to play some role in the determination of nutrient content in biomass.

Neither the phylogenetic relationship nor the membership in ecological guilds strongly determines the nutrient content in biomass, as indicated by the share of unexplained variation. This unexplained variation is likely associated with local, highly variable environmental conditions, where nutrient levels in fungi somewhat mirror those in the surroundings. This variability may also suggest an adaptive response to fluctuating environmental conditions (Vogt et al., 1981; Camenzind et al., 2020).

Due to dissimilar sources of N and P, their soil content could be highly variable. Such variability is associated with the type of geological bedrock, soil type, altitude, soil depth, and other factors; there can be many-fold differences in N content between various forest soils (Huntington et al., 1988; Batjes, 1996). Even higher is the variability of soil P content, exceeding differences of two orders of magnitude between various sites (Deng et al., 2017). Substantially low is nutrient content in wood, but the differences among tree species are considerable (Cowling and Merrill, 1966). Despite these dissimilarities and high, mutually independent variabilities of N and P causing considerable variability of their ratios within fungal habitats, the content of N and P in fungal biomass shows a significant positive correlation (r = 0.478, p < 0.01, Figure 2). No significant differences were found between all three ecological guilds in the N:P ratio, which could indicate that fungi in general maintain an approximately similar ratio of these nutrients, although the variability of this ratio is high, especially within both groups of saprotrophs. The N:P ratio is important particularly for mycorrhizal fungi, because it is tightly associated with their successful C, N, and P trade-off with the host plant (Johnson, 2010). This could explain the lower variability of the N:P ratio in the EcM group compared to the other two fungal groups (Table 1). Conversely, saprotrophic fungi may store both N and P independently of each other, according to their obtainable quantity, which could explain their higher variability in this ratio.

The content of chitin in fungal biomass was significantly lower in wood saprotrophic fungi than in the other two groups, although the variability in all three groups was high. As we hypothesized, the chitin content showed a positive correlation with the total N in mycelium (r = 0.298, p = 0.0239; Figure 2), indicating that fungi with sufficient N supply may synthesize more chitin. Chitin, as a chemical compound, contains approximately 6.9% of N (No and Meyers, 1995; Tracey, 1955), and N in chitin constitutes only 4.6–7.4% of the total N in the biomass (Supplementary Table 2). Therefore, the contribution of N bound in chitin to the differences in N content among fungal ecological guilds is limited. These differences are more likely caused by N in proteins and intracellular content rather than by N localized in chitin. This is further supported by the observation that soil saprotrophic fungi, which have the highest levels of total nitrogen, do not necessarily contain the highest levels of chitin.

Fungal mycelium is an important component of food chains, serving as a source of nutrients after its decomposition. Differences in N content in biomass between fungi of all three ecological guilds are significant, indicating variable attractiveness of dead mycelium for subsequent decomposition. Not surprisingly, N content in fungal biomass was the best predictor of its decomposition rate (Fernandez et al., 2016; Brabcová et al., 2018). In general, the biomass of soil saprotrophs is anticipated to decompose more rapidly than that of the ECM and wood saprotrophic fungi owing to the higher nitrogen content; however, the rate of decomposition is contingent upon the particular combination of environmental conditions and the unique properties of each fungus. The increasing deposition of N, which was frequently documented (Morrison et al., 2016; Hůnová et al., 2017; Baldrian et al., 2023), raises the question of its influence on fungi of various ecological guilds. While the wood, as the habitat of wood saprotrophic fungi, should be relatively little affected by N deposition, the impact on soil saprotrophic and mycorrhizal fungi could be more pronounced. Since the provision of N to plants is one of the essential functions of mycorrhizal symbioses (Kranabetter et al., 2019), increased N availability as a result of N deposition might affect the importance of mycorrhiza for plants. In such a case, at least the shift of the limitation to P could be expected. For the saprotrophic fungi, the direct impact of higher N availability should not be of cardinal significance since these fungi seem to be able to cope with various levels of nutrients and their ratios. But some indirect influence, such as increasing competition of other groups of microorganisms, could be assumed instead.

Our results indicate that both ecology and evolution determine nutrient contents in fungal biomass. In the light of ongoing global climatic change and continuing increased N deposition, it is especially important to assess how the differences in nutrient content among fungal guilds and the share of C that they receive within the soil affect the further fate of fungal biomass and whether it can impact organic matter accumulation or C storage in soils.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in this article/Supplementary material.

MP: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft. TV: Formal analysis, Resources, Writing – review & editing. TM: Formal analysis, Resources, Writing – review & editing. JB: Formal analysis, Resources, Writing – review & editing. LT: Resources, Writing – review & editing. BA: Formal analysis, Writing – review & editing. PB: Conceptualization, Methodology, Supervision, Writing – review & editing. RL-M: Conceptualization, Funding acquisition, Methodology, Writing – review & editing.