DOI: 10.5061/dryad.nk98sf7qj
title: Substrate quality drives fungal necromass decay and decomposer community structure under contrasting vegetation types

description: 1. Fungal mycelium is increasingly recognized as a central component of
 soil biogeochemical cycling, yet our current understanding of the
 ecological controls on fungal necromass decomposition is limited to single
 sites and vegetation types. 2. By deploying common fungal necromass
 substrates in a temperate oak savannah and hardwood forest in the
 midwestern USA, we assessed the generality of the rate at which high- and
 low-quality fungal necromass decomposes; further, we investigated how the
 decomposer ‘necrobiome’ varies both across and within sites under
 vegetation types dominated by either arbuscular (AM) or ectomycorrhizal
 (EM) plants. 3. The effects of necromass quality on decay rate were robust
 to site and vegetation type differences, with high-quality fungal
 necromass decomposing, on average, 2.5 times faster during the initial
 stages of decay. Across vegetation types, bacterial and fungal communities
 present on decaying necromass differed from bulk soil microbial
 communities and were influenced by necromass quality. Moulds, yeasts and
 copiotrophic bacteria consistently dominated the necrobiome of
 high-quality fungal substrates. 4. Synthesis: We show that regardless of
 differences in decay environments, high-quality fungal substrates
 decompose faster and support different types of decomposer microorganisms
 when compared with low-quality fungal tissues. These findings help to
 refine our theoretical understanding of the dominant factors affecting
 fast cycling components of soil organic matter (SOM) and the microbial
 communities associated with rapid decay.
description: Parallel necromass decomposition experiments were
 conducted at two sites and under two different vegetation types at each
 site: a temperate savannah containing EM-associated trees and
 AM-associated grasses and a temperate hardwood forest containing adjacent
 EM- and AM-dominated stands. The savannah site was located at Cedar Creek
 Ecosystem Science Reserve in central Minnesota, USA (N 45.42577 W
 093.20852). Cedar Creek is a 2266 ha reserve affiliated with the
 University of Minnesota, which contains of a mix of prairie and forest
 ecosystems. The mean annual temperature at Cedar Creek is 6.7°C and the
 mean annual precipitation is 801 mm. The forest site was located at Moores
 Creek in south-central Indiana, USA (N  39.08333 W  086.46666). Moores
 Creek, which is part of the Indiana University Research and Teaching
 Preserve system, is comprised of 105 ha of mixed deciduous hardwood forest
 (~80 years in age). The mean annual temperature at Moores Creek is 11.6°C
 and the mean annual precipitation is 1200 mm. Within both sites,
 vegetation communities differed in their AM- and EM-associated plant
 species and edaphic characteristics.  Plot locations at
 each site were chosen based on dominant vegetation type and mycorrhizal
 association. Three replicate plots were established at two locations in
 the savannah site; 10 m into EM-dominated <i>Quercus</i>
 forest and 20 m into the adjacent AM-dominated grassland. These distances
 were chosen based on previous work at the same site by Dickie and Reich
 (2005), which found little to no EM colonization of
 <i>Quercus</i> seedlings at 20 m away from the forest edge. At
 the forest site, 7 replicate plots were established based on known
 mycorrhizal associations of dominant tree species. In all plots, trees
 from the dominant mycorrhizal type (AM or EM) represented &gt;85% of
 the basal area of the plot and AM and EM plots were paired according to
 geographic proximity. Two fungal
 species,<i> Mortierella elongata </i>and <i>Meliniomyces
 bicolor</i>, were chosen to represent high- and low-quality
 necromass. <i>M. elongata</i> is<i> </i>a
 fast-growing saprotrophic fungus in the phylum Mucromycota, which is
 frequently found in both forest and agricultural soils (Li <i>et
 al.,</i> 2018). <i>M. bicolor</i> is an EM and ericoid
 mycorrhizal (ErM) Ascomycotan fungus frequently found in temperate and
 boreal forest soils. These two species have contrasting melanin and
 nitrogen levels, with <i>M. elongata</i> representing a
 high-quality substrate and <i>M. bicolor</i> representing a
 low-quality substrate. Complete details on the methods used for the
 chemical characterization of both species are provided in the online
 Supplementary Information. Fungal biomass for
 both species was produced in liquid cultures by individually inoculating
 50 mL flasks containing half-strength potato dextrose broth with 3 mm
 diameter mycelial plugs (one plug per flask). Following inoculation,
 cultures were transferred to an orbital shaker and left to shake at 80 rpm
 for at least 30 days or until growth stopped. To produce fungal necromass,
 cultures were rinsed with distilled water and dried at 26°C for 24 hours.
 Dried fungal necromass (~25 mg) were then placed into nylon mesh litter
 bags constructed from 53-micron mesh (Elko, Minneapolis, MN, USA) and
 heat-sealed. The 53-micron mesh size excluded both tree and grass root
 in-growth. Separate litter bags
 were constructed for replicates of each fungal species. During deployment,
 litter bags were buried at organic-mineral soil interface (0-5 cm depth).
 To determine if there was any mass loss due to transport and handling, an
 additional set of litter bags was carried into the field (n = 3).
 Necromass recovery was greater than 98% and did not differ between fungal
 species, so masses were not corrected for any loss during transport. At
 each harvest, litter bags were individually bagged, placed on ice, and
 taken directly to the laboratory for processing. For each sample,
 necromass was carefully removed from the litterbag and dried at 30°C to a
 constant mass to determine mass remaining. While the preparation
 and processing of fungal necromass was standardized across the two sites,
 the specific incubation times varied slightly between studies due to
 logistical constraints. At the savannah site, fungal necromass was
 incubated for 14, 28, 42, and 56 days beginning in July 2017 (n = 3 litter
 bags of each fungal species for each vegetation type for each sampling
 date). At the forest site, fungal necromass was incubated for 14, 31, and
 92 days beginning in July 2017 (n = 7 litter bags of each fungal species
 for each vegetation type for each sampling date). Soil moisture
 measurements at both sites were taken at the time litter bags were
 harvested. Gravimetric soil moisture data was collected from the composite
 of two 5 × 10 cm soil cores per plot at the savannah site and three 6.35 ×
 10 cm soil cores per plot at the forest site. To determine pH, a
 sub-sample of soil collected at the time of the first litter bag harvest
 was air-dried and analyzed in a 0.01 M CaCl<sub>2</sub>
 solution using a bench-top pH meter.  Statistical analyses
 and data visualization were conducted in R version 3.5.1 (R Core Team,
 2018). Analysis of variance (ANOVA) was used to test for differences in
 soil pH between sites and vegetation types within sites. To test for
 differences in soil moisture, ANOVAs were run with vegetation type (AM-
 vs. EM-associated vegetation) and sampling date as the predictor variables
 for each site. Prior to running the ANOVAs, soil moisture data was
 log-transformed to meet the assumptions of normality. Linear mixed-effect
 (LME) models were used to analyze the amount of fungal necromass remaining
 within each site. Fixed predictor factors included vegetation type (AM-
 vs. EM-associated vegetation), necromass type (high- vs. low-quality),
 incubation period, and soil moisture. Replicate sampling locations (either
 plots or plot pairs) were designated as a random factor. Because pH was
 only measured during one harvest at each site it was not included in this
 analysis. Mass remaining data were log logit-transformed to meet
 statistical assumptions.To evaluate the significance of linear
 mixed-effects models the Kenward-Roger approximation was used to estimate
 F statistics and denominator degrees of freedom (Halekoh &
 Højsgaard, 2014). Least square means were computed for each fixed effect
 and post-hoc comparisons were carried out on pairs of the least-squares
 means using the Tukey’s adjustment for multiple comparisons.
 Decay constants were
 calculated separately for each necromass type at each site. To calculate
 decay constants, we fit the proportion of remaining necromass against
 incubation time (days) using single- and double-exponential decay models.
 The best fitting model was selected using Akaike’s Information Criteria
 (AIC). According to AIC values, a double-exponential decay model
 (Equation1) produced the best fit. <b>Equation 1</b>:
 <i>[mass]</i><sub><i>t</i></sub><i>= a</i><i>e<sup>-</sup></i><sup><sup><i>k<sub>1</sub>t</i></sup></sup><i>+(1-a) </i><i>e<sup>-k<sub>2</sub>t</sup></i> The proportional mass remaining ([mass]<sub>t</sub>) was calculated by dividing the mass remaining at time (t) by the initial mass for each litterbag. In equation 1, a refers to is the initial proportion of fast decomposing or labile material, 1-a is the initial proportion of slow decomposing or recalcitrant material. k<sub>1 </sub>and k<sub>2</sub> are the degradation rate constants of the labile (fast-decomposing) and recalcitrant (slow-decomposing pool), respectively. The nonlinear least-squares Levenberg-Marquardt algorithm used to estimate model parameters, a, k<sub>1</sub> and k<sub>2</sub>, using the ‘minpak.lm’ package. 
description: The microbial community data that accompanies the fungal
 necromass decomposition data is available under the following NCBI
 BioProject Accessions: Moores Creek Bacteria (PRJNA607032), Moores Creek Fungi (PRJNA607029), Cedar Creek Bacteria (PRJNA607034), Cedar Creek Fungi (PRJNA607030).