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Growth rate trades off with enzymatic investment in soil filamentous fungi

Saprobic soil fungi drive many important ecosystem processes, including decomposition, and many of their effects are related to growth rate and enzymatic ability. In mycology, there has long been the implicit assumption of a trade-off between growth and enzymatic investment, which we test here using a set of filamentous fungi from the same soil. For these fungi we measured growth rate (as colony radial extension) and enzymatic repertoire (activities of four enzymes: laccase, cellobiohydrolase, leucine aminopeptidase and acid phosphatase), and explored the interaction between the traits based on phylogenetically corrected methods. Our results support the existence of a trade-off, however only for the enzymes presumably representing a larger metabolic cost (laccase and cellobiohydrolase). Our study offers new insights into potential functional complementarity within the soil fungal community in ecosystem processes, and experimentally supports an enzymatic investment/growth rate trade-off underpinning phenomena including substrate succession.


Fungal material
The filamentous saprobic fungi used in our experiments were isolated from soil samples collected in Oderhänge Mallnow (Germany,52°27.778'N,14°29.349'E) -a 304 ha nature conservation area (grassland). The set comprised 31 fungal isolates from the phyla Ascomycota, Basidiomycota and Mucoromycota, which are all saprobic (Fig. S1). The technical details of isolation, identification and phylogenetic tree building have been published (Lehmann et al. (in press)). In general, the phylogenetic tree was reconstructed based on the complete intergenic transcribed spacer (ITS) and a part of the large rRNA subunit (LSU). Thereafter, the taxon identifications of strains were determined (Table   S1). Fungal strains have been deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ ; Table S2).

Fig. S1
. Phylogenetic (neighborjoining) tree of the 31 fungal strains belonging to the phyla Ascomycota (white), Basidiomycota (grey) and Mucoromycota (black). Colony pictures are from four-week old cultures grown on potato dextrose agar, and their order follows the order of the phylogenetic tree.

Cultivation periods
Hyphae can grow as fast as few micrometers to millimeters per hour and develop different growth strategies in different phases. In general, we harvest fungal tissue when the individual colony in the late linear growth phase (Table S2). Therefore, for each strain, the duration and onset of the linear growth phase were measured in a preliminary experiment, and the related data is given in Table S2.

Enzyme activity measurements
In order to profile fungal enzyme activities in the linear growth phase, we collected younger mycelia from the outer zone of the colony. For each enzyme, two small pieces of mycelium (3-5 mm 2 ) was cut, weighed freshly and stored at 4°C in a 1 ml Eppendorf tube. For each plate, eight pieces of mycelium were prepared for the enzyme measurements, which were conducted within 24 hours. The data present for each unit were the average of two subsamples.
The activities of enzymes were tested by a microplate photometric method.
The acid phosphatase activity was tested based on the hydrolysis of pNPP(paranitrophenylphosphate, a synthetic substrate) into pNP(para-nitrophenol) + P. The reaction mixture contained 100 µl of acetate buffer (50 mM, pH 5.5) with 100 µl substrate (5 mM). After incubation at 37 ˚C for 15 min, the absorbance at 410 nm (ε410= 18.3 mM −1 cm −1 ) was measured (microplate reader, Bio-RAD, USA). One unit (U) of acid phosphatase activity was defined as the amount of enzyme releasing 1µmol of pNP min-1. The assays for leucine aminopeptidase and cellobiohydrolase activity followed the same protocol with adjustments in incubation time, temperature, buffer pH and the substrate concentration (Table S2). For laccase activity, the protocol was as follows: the mycelial samples were placed in wells of a 96-well microplate with 100 µl of acetate buffer (pH 5) and 100 µl of 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) solution (2 mM). After incubating at 25 ˚C for 25 min, the mycelia were removed from the plate and the oxidization of ABTS was monitored by determining the absorbance at 405 (ε405 = 36.8 mM −1 cm −1 ) with a plate reader. One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS min-1. The final enzyme activity was standardized by the dry weight of mycelial sample, expressed as U mg -1 (dw).

The Comparison of the traits at phylum-level
To compare the difference of traits at phylum-level, the data were tested by analysis of variance and TukeyHSD test. The statistics are summarized in Table S5.

Regression between K r and the enzyme activities.
Linear regression and quantile regression are applied between Kr and each of the enzyme activities one by one. Table S6. gives the statistical summary of the tests.