Fungal growth and stoichiometric analyses
At the end of experiments, agar media were melted in a microwave and
mycelium collected on a 20 µm mesh. To remove superficial element
traces, mycelia were washed with 1 L of >90 °C
demineralized H2O, and cleaned with 0.1 M HCl for one
minute. This washing method was inevitable to ensure complete removal of
agar medium from the mycelium (Maynard et al. 2017), but we
showed in a separate experiment that it did not alter element
concentrations or treatment effects (Fig. S1). Fungal material was
freeze-dried and kept at -20 °C. Fungal dry biomass was determined, as
well as fungal density [mg cm-2 mycelium] based on
the actual size of the mycelium, since mycelial density as a trait
proved to be a good estimate of fungal fitness and indicator of nutrient
limitations (Camenzind et al. 2020). Fungal enzymatic activity
was assessed as an additional important response trait (for details see
Supporting Information S1). For element analyses, fungi were milled and
C and N contents determined with an Elemental Analyzer (EuroEA,
HekaTech, Germany). P content was analyzed after aqua regia digestion
(1:4 HCl:HNO3) by ICP-OES analyses (Optima 2100 DV,
Perkin Elmer, Germany). In cellulose media, P contents were only
analyzed for four isolates (RLCS16, RLCS17, RLCS27 and RLCS28) in media
with C:N of 5 and 200, respectively. Likewise, in SEA media P contents
were only analyzed for four isolates (RLCS12, RLCS22, RLCS27 and
RLCS28).