We investigated the differences of fatty acids composition of particular edible mushrooom species mushrooms between plots. Mushrooms were collected in 1 and 3 tree species plots in FORBIO-Zedelgem and BIOTREE-Kaltenborn and in 1,2,3,4- tree species mixtures in Bialowieza. Fatty acids (FA) composition is part of the information about nutritional value. We compared them to investigate the impact of tree species richness and other environmental factors on mushroom nutritional values.

Sampling took place in every 1- or 3-species plot (VIP plots) - in Bialowieza also in 2 and 4 species mixtures. Mushrooms were collected at the entire plots (30x30m), extended with a 2,5m-wide stripe of land around (35mx35m). Mushrooms from each plot were collected to separate baskets. Then, we: 1) determined mushrooms species to the lowest possible level 2) weighed fresh mass of each species from every plot separately with 0,1 g accuracy 3) dried the samples in 50℃ for 18h (longer if needed) 4) weighed samples with 0.1 g accuracy 5) took samples of edible mushrooms present in more than 7 plots to a laboratory.

Mushrooms were collected in 1 and 3 tree species plots in FORBIO-Zedelgem and BIOTREE-Kaltenborn and in 1,2,3,4- tree species mixtures in Bialowieza. In Bialowieza we conducted two season long monitoring (June-half of November 2020-2021), collecting mushrooms every ten days (all sporocarps of particular edible species from one plot were pulled together). In Zedelgem and Kaltenborn monitoring was organised twice in 2021 thanks to local support. All mushrooms were identifies, categorised into edible and non-edible species and dried in food dehydrator at 50°C for about 24 hours. Mushrooms from particular species, collected in more than 7 plots (in case of Kaltenborn in 5 plots) were taken to the Laboratory of Department of Bromatology of the Warsaw Medical University for estimation of the content and composition of fatty acids. Powdered dried mushrooms in the amount of 100 g were extracted for 30 min in an ultrasonic bath using 9 ml of chloroform and the addition of 25 µl of internal standard (C19:0 in toluene, 1 mg/mL). The resulting mixture was evaporated to dryness with nitrogen. Extracted samples were hydrolysed with 1 ml of 0.5M NaOH methanolic solution at 80°C for 15 min and then a complete derivation was carried with 1 ml of BF3 solution in methanol (14% w/v) at 80ºC for 15 min. Then 2 ml of NaCl was added to the sample. The methyl esters were extracted with 2 ml of hexane. The upper layer was taken and dried with an anhydrous Na2SO4. The pure liquid was transferred to inserts for GC-MS analysis. Analyses were performed on a gas chromatograph with time-of-flight mass spectrometry (GCTOFMS) (Pegasus® BT, LECO Corporation, St. Joseph, MI, USA). Chromatographic separations were conducted on the capillary column TR-FAME (120 m/0.25 mm ID/film thickness 0.25 µm; Thermo Fisher Scientific Inc., Göteborg, Sweden). Helium was used as the carrier gas (flow: linear velocity at 1 mL/min), the injection was 1 µL, in the splitless mode. The injector and transfer line were heated to 240ºC. The temperature program: initial temperature was set for 110ºC for 16 min then increased 6ºC/min to 190ºC; 1ºC/min to 210ºC and 0.4ºC/min to 220ºC and then held for 10 min at 220ºC. Ion source temperature was 250ºC and energy 70 eV. To determine the FA types contained in the samples, the FAME standards (Supelco 37Component FAME Mix, Sigma, St. Louis, MO, USA) were applied. Then ChromaTOF software was used for the identification and quantitative analysis of fatty acids. This file contain the results of these analyses - the FA composition of edible species as content of particular fatty acids (µg/100mg dry mass)

edible mushroom species - soil-dwelling and epixylic macrofungi, excluding bracket fungi and those with sporocarp size <0.5 cm

sunny or cloudy (mushroom collection were postpone during intensive rain); irrelevant in case of laboratory analyses

Fatty acid composition and environmental factors affected it - tree species richness, proportion of deciduous tree, nitrogen concentration, C/N ratio, canopy cover, understorey cover.