FungalTraits vs. FUNGuild: Comparison of Ecological Functional Assignments of Leaf- and Needle-Associated Fungi Across 12 Temperate Tree Species

Recently, a new annotation tool “FungalTraits” was created based on the previous FUNGuild and FunFun databases, which has attracted high attention in the scientific community. These databases were widely used to gain more information from fungal sequencing datasets by assigning fungal functional traits. More than 1500 publications so far employed FUNGuild and the aim of this study is to compare this successful database with the recent FungalTraits database. Quality and quantity of the assignment by FUNGuild and FungalTraits to a fungal internal transcribed spacer (ITS)–based amplicon sequencing dataset on amplicon sequence variants (ASVs) were addressed. Sequencing dataset was derived from leaves and needles of 12 temperate broadleaved and coniferous tree species. We found that FungalTraits assigned more functional traits than FUNGuild, and especially the coverage of saprotrophs, plant pathogens, and endophytes was higher while lichenized fungi revealed similar findings. Moreover, ASVs derived from leaves and needles of each tree species were better assigned to all available fungal traits as well as to saprotrophs by FungalTraits compared to FUNGuild in particular for broadleaved tree species. Assigned ASV richness as well as fungal functional community composition was higher and more diverse after analyses with FungalTraits compared to FUNGuild. Moreover, datasets of both databases showed similar effect of environmental factors for saprotrophs but for endophytes, unidentical patterns of significant corresponding factors were obtained. As a conclusion, FungalTraits is superior to FUNGuild in assigning a higher quantity and quality of ASVs as well as a higher frequency of significant correlations with environmental factors. Supplementary Information The online version contains supplementary material available at 10.1007/s00248-022-01973-2.


Physiochemical analyses
Wet leaf and needle samples were shaken for 1 h in falcon tubes with 30 mL milliQ water to leach water-soluble components from their surfaces. The leachates were centrifuged for 5 min at 3500 rpm, decanted and filtered through pre-flushed 0.45 µm regenerated cellulose syringe filters. The remaining leaf/needle material was dried for two weeks at 40 °C for dry weight determination. All quantification results are given in reference to the dry weight. The pH of the leachates was determined using pH paper with a scale precision of 0.2 pH units.
Simultaneous analysis of total bound nitrogen (TN b ) and inorganic nitrogen (N min ) was performed to quantify organic nitrogen (N org ). N org was calculated as the difference: N org = TN b -N min . TN b was analyzed using a sum parameter analyzer with high temperature combustion and chemiluminescence detection (Mitsubishi TN-100; a1 envirosciences, Düsseldorf, Germany). An optimal volume of 4 ml per sample was required for all rinsing and analysis procedures.
All samples were measured as triplicates. A sample volume of 30 µL each was automatically injected into the combustion tube filled with a platinum catalyst. The samples were combusted at 800°C in pure oxygen. During this process, all organic and inorganic nitrogen compounds in the sample are oxidized to NO x and subsequently converted to NO. The NO reacts with ozone in the chemiluminescence detector according to the following oxidation reaction: NO + O 3  NO 2 + O 2 + hv. The energetically activated NO 2 emits light spontaneously, and the intensity of the emitted light is proportional to the NO concentration. Detection is performed by a photomultiplier. For N Min quantification, a flow injection analyzer (Quikchem QC85S5; Lachat Instruments, Hach Company, Loveland CO, USA) with corresponding manifolds for the measurement of ammonium nitrogen N NH 4 + , nitrite nitrogen N NO 2 − , and nitrate-plus nitrite nitrogen N NO 3 − +NO 2 − was used. In this instrument, samples and the required chemical reagents are pumped through a system of calibrated tubing and mixed together in a matrix-related carrier stream. After reaction with the reagents, the analytes are photometrically quantified by measuring the absorbance at a special wave length in a flowthrough cuvette. An optimal volume of 10 ml per sample was required for all rinsing, dilution and analysis procedures.
For this reason, only one single measurement could be performed per sample. N NH 4 + was determined by the gas diffusion method. For this purpose, samples were automatically mixed with an alkaline buffer at the manifold. The resulting NH 3 was passed through a gas diffusion membrane and absorbed into an acidic indicator stream. The resulting pH change causes a color change in the indicator solution, which was measured photometrically at λ = 590 nm. The intensity of the coloration is proportional to the N NH 4 + concentration in the sample. N NO 3 − was reduced to nitrite using a cadmium column in the manifold prior to the chemical reaction to form an azo dye. The nitrate reduced by cadmium and the nitrite originally present in the sample were analyzed using the Griess reaction by diazotization with sulfanilamide and coupling with N-(1-naphthyl) ethylenediamine dihydrochloride. The deep pink color of the resulting dye was measured at λ = 520 nm. N NO 2 − alone was determined after the same reaction, without using a cadmium column.
DOC was quantified as non-purgeable organic carbon (NPOC) with a sum parameter analyzer using high-temperature combustion and infrared detection (vario TOC cube, Elementar Analysensysteme GmbH, Langenselbold, Germany).  assigned by FUNGuild and FungalTraits: a) EcM ASV richness ± SE, b) EcM genera assigned by FUNGuild and FungalTraits, and cluster analysis of EcM community composition from c) FUNGuild and d) FungalTraits. This dataset is previously published elsewhere [1]. The statistical differences were accessed using t-test: ns = not significant, * = P < 0.05.

Table S1
Relative abundances (%) of fungal ASVs associated with leaves and needles of 12 temperate forest assigned functions by FUNGuild and FungalTraits (please see in another excel file). Table S2. Factors explain variations in saprotrophic and endophytic fungal community composition associated with leaves and needles of temperate tree species based on presence/absence data and Jaccard distance measure. The factors used in the variation analysis are the factors that significantly corresponded with the saprotrophic and endophytic fungal community (please see Table 2). ND = Not detected.