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\begin{document}

\title{Multiple cell wall chemical components drive the decomposition of
ectomycorrhizal necromass over time}



\author[1]{Riccardo Mancinelli}%
\author[1]{Peter van Bodegom}%
\author[1]{Jan Lankhorst}%
\author[2]{Nadia Soudzilovskaia}%
\affil[1]{Leiden University Faculty of Science}%
\affil[2]{Leiden University}%


\vspace{-1em}



  
  \date{May 29, 2024}


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\selectlanguage{english}
\begin{abstract}
The extramatrical mycelium of ectomycorrhizal fungi (EMF) is an
important source of soil carbon. While the importance of recalcitrant
compounds in the fungal cell wall has been reported earlier, the
contribution of abundant and more labile components, like glucans, and
the role of their temporal dynamics during decomposition remains
unknown. We examined how the decomposition of EMF mycelium is related to
the dynamics of 3 main fungal cell wall components; chitin, melanin, and
glucans. Across six EMF species, the initial concentrations of the three
components were not a good predictor of necromass loss after 6 weeks.
However, the dynamics of chitin concentration during the decomposition
process contributed to the weekly necromass degradation, with trends of
chitin loss dynamics being dissimilar across the fungal species. In
contrast, the dynamics of loss of melanin and glucans was not related to
the weekly necromass loss. Given similar total necromass loss across
species, we suggest that glucans and chitin are not discriminated by
decomposers. This mechanism compensates for the interspecific difference
in their initial concentration. Our results indicate that fungal
necromass decomposition does not behave similarly to plant litter, while
it is critical to soil carbon dynamics.%
\end{abstract}%



\sloppy


\textbf{Multiple cell wall chemical components drive the decomposition
of ectomycorrhizal necromass over time}

\textbf{Authors:} R. Mancinelli\textsuperscript{1}*, P.M. van
Bodegom\textsuperscript{1}, J. Lankhorst\textsuperscript{1}, N.A.
Soudsilovskaia\textsuperscript{1,2}

\textbf{Affiliations:}

\textsuperscript{1}Institute of Environmental Sciences, Leiden
University, 2300 RA Leiden, the Netherlands

\textsuperscript{2}Centre of Environmental Sciences, Hasselt University,
Martelarenlaan 42, 3500 Hasselt, Belgium

*To whom correspondence should be addressed:
r.mancinelli@cml.leidenuniv.nl.

\textbf{Keywords:} fungal litter, Ectomycorrhizal extramatrical
mycelium, fungal cell wall, chitin, glucans.

\section*{Abstract:}

{\label{abstract}}

The extramatrical mycelium of ectomycorrhizal fungi (EMF) is an
important source of soil carbon. While the importance of recalcitrant
compounds in the fungal cell wall has been reported earlier, the
contribution of abundant and more labile components, like glucans, and
the role of their temporal dynamics during decomposition remains
unknown. We examined how the decomposition of EMF mycelium is related to
the dynamics of 3 main fungal cell wall components; chitin, melanin, and
glucans. Across six EMF species, the initial concentrations of the three
components were not a good predictor of necromass loss after 6 weeks.
However, the dynamics of chitin concentration during the decomposition
process contributed to the weekly necromass degradation, with trends of
chitin loss dynamics being dissimilar across the fungal species. In
contrast, the dynamics of loss of melanin and glucans was not related to
the weekly necromass loss. Given similar total necromass loss across
species, we suggest that glucans and chitin are not discriminated by
decomposers. This mechanism compensates for the interspecific difference
in their initial concentration. Our results indicate that fungal
necromass decomposition does not behave similarly to plant litter, while
it is critical to soil carbon dynamics.

\section*{Introduction}

{\label{introduction}}

Soil organic matter (SOM) is known to be the largest C pool in
terrestrial ecosystems, ranging from 1500 to 2000 Pg in the top meter.
This equals two to three times the amount of C stored in the vegetation,
which is approximately 500 Pg (Amundson, 2001; Butler \& Day, 1998;
Dixon et al., 1994). Among the various sources of SOM in forest
ecosystems, dead mycelia are probably among the largest ones. It has
been suggested that the majority of C stored in the deeper horizons of
boreal forest soils originates from the allocation of photosynthate to
ectomycorrhizal fungi (Clemmensen et al., 2013). The annual allocation
of carbon into mycorrhizal fungi can constitute up to 22\% of the annual
net primary productivity (Hobbie \& Hobbie, 2006). This major
contribution suggests the profound importance of these fungi in the
overall allocation of carbon in both litter and soil compartments.
Estimates of ectomycorrhizal (EM) biomass, which constitutes the major
fraction of soil fungal biomass, typically range from 100 to 600 kg
ha\textsuperscript{-1} (Hendricks, Mitchell, Kuehn, \& Pecot, 2016;
Wallander, G\selectlanguage{ngerman}öransson, \& Rosengren, 2004) and, together with roots,
produces more than 50\% of the dissolved organic carbon in forest's soil
(Högberg \& Högberg, 2002). Significant amounts of mycelia are produced
in soil layers below the depth of 10 cm (Boström, Comstedt, \& Ekblad,
2007) and in the litter, where the dry fungal biomass per g substrate
can be more than 10-fold that of soil (Baldrian et al., 2013). In forest
soil and litter, which are often N limited, fungal mycelia are more
attractive than plant residues as a target for decomposers, given their
high content of N, which can be 3 up to 30 times greater than in plant
litter (Koide \& Malcolm, 2009; Mouginot et al., 2014). Due to the large
fluxes of C and nutrients into the soil through fungal necromass, a
better understanding of the decomposition dynamics of fresh fungal
litter may improve our understanding of SOM dynamics, and how these two
are related.

A major driving factor of litter decomposition is the initial litter
``quality'', i.e. litter chemical composition (Berg, 1984). In general,
decomposition is favoured by relatively low litter C:N ratios (Cleveland
\& Liptzin, 2007), which might help to maintain the low C:N ratio of
microbial cells (Manzoni, Trofymow, Jackson, \& Porporato, 2010). Yet,
there is increasing evidence that other aspects of chemical litter
quality beyond basic stoichiometry also affect decomposition rates
(Wilkinson, Alexander, \& Johnson, 2011).

To date, the number of observations about the decomposition rates of EM
fungi is limited, as well as our knowledge on their main drivers. It has
been hypothesised that EM fungal necromass decomposition is driven
mainly by its necromass ``quality'', which, similarly to plant litter,
refers to fungal necromass chemistry and morphology. Previous research
found a significant positive relationship between the N concentration in
the fungal tissues and its decomposition (Christopher W Fernandez \&
Koide, 2014; Koide \& Malcolm, 2009).

The chemical composition of ectomycorrhizal fungal mycelium varies
widely among fungal species and possibly contributes to the variation in
decomposition rates of EM fungal necromass. While the cytoplasmic
fraction does not likely play a significant role in the decomposition of
EM necromass due to its high lability (Drigo, Anderson, Kannangara,
Cairney, \& Johnson, 2012), it seems that the composition of the cell
wall cell cel fraction drives the long-term decomposition of EM
extramatrical necromass. The chemical composition of the cell wall of EM
fungi varies with age, genotype, taxon, and environment (Bowman \& Free,
2006; Feofilova, 2010). EM fungal cell walls are composed mainly by
polysaccharides such as glucans and chitin (70-90\% of dry biomass),
proteins, which contains from 40 to 60\% of cell wall total N, and
melanins. The latter, despite being a minor component and highly
variable in concentration across different taxa (Butler \& Day, 1998a),
is linked to the tolerance of various environmental stressors
(Christopher W Fernandez \& Koide, 2013; Rosas \& Casadevall, 1997; Wang
\& Casadevall, 1994).

During the last decades, several studies suggested that differences in
initial concentrations of the main classes of chemical fungal cell-wall
compounds (proteins, carbohydrates, melanin) underpin necromass
degradation (Christopher W Fernandez \& Koide, 2012, 2014). Yet, there
are many uncertainties concerning the mechanisms behind this
relationship. These uncertainties range from the dynamics of secondary
compounds contents during the decomposition process to the actual
interaction between the components themselves. Moreover, we may not
assume that the initial concentration of cell-wall compounds (Berg,
2000; Melillo, Aber, \& Muratore, 1982), is a dominant predictor of the
decomposition rate and dynamics. The fungal wall structure is highly
variable. In contrast to plant litter, the fungal cell wall retains
considerable plasticity despite the presence of a relatively rigid
scaffold formed by chitin fibrils and \selectlanguage{greek}α-\selectlanguage{english}1,3-glucans. This plasticity
allows to easily reshape the molecular architecture of mycelia to
survive through different external stress. In addition, the fungal cell
walls contain an extensive covalent cross-linking of glucans and chitin
(Cairney, 2012; Kang et al., 2018). These dissimilarities suggest that
we might need to consider different drivers to predict its decomposition
compared to plants. So far, no previous research has explored the role
of the interaction between cell-wall components in the decomposition of
EM necromass. Nor did previous research consider the impacts of changes
in these cell wall components during decomposition. Since all these
components are highly interconnected and each one has a specific role in
maintaining the cell wall structure and function, an extensive temporal
analysis of dynamics of their composition through the fungal litter
degradation process will improve our understanding of the mechanisms
behind fungal necromass decomposition.

In this paper we examine the dynamics in the most abundant cell-wall
compounds (chitin, glucans and melanin) from EMF mycelia necromass
across 6 EM fungi species, seeking an answer to the question of whether
the dynamics in composition and concentration of these compounds is
linked to mycelium decomposition dynamics. Hereto we seek an answer to
three research questions: 1) Do the initial concentrations of the
cell-components alone predict fungal litter decomposition?; 2) Can the
concentration dynamics of chitin, glucans and melanin during the
decomposition process predict the dynamics of fungal litter mass loss?;
and 3) Do distinct EM fungal species exhibit a similar temporal dynamics
of losing cell wall components during the decomposition process?

\section*{Materials and Methods}

{\label{materials-and-methods}}

Fungal material and substrate

In this study, six ectomycorrhizal fungal species were selected based on
their representativeness and availability. The fungal species were
supplied by Westerdijk Fungal Biodiversity Institute (Utrecht, the
Netherlands) and by the Belgian Coordinated Collections of
Microorganisms/Mycoth\selectlanguage{ngerman}èque de L'Université catholique de Louvain
(BCCM/MUCL, Louvain-la-Nueve, Belgium). All the isolates were cultivated
in specific agar media (Table 2) for approximately 40 days until the
mycelium covered the entire surface of the petri dish. An average of 8
plates for each species was harvested. Upon harvesting, mycelium was
divided into three concentric layers, allowing comparing across
different ages of the same mycelium. Age was approximated as the average
of the growth between the inner edge and the outer edge. The layers were
removed from the plate, and the mycelium was then rinsed three times
with hot water (85 °C) to wash out the remaining agar without damaging
the mycelium. The mycelium was then dried overnight at 70\selectlanguage{english}. This
provided approximately 120 mg of dried mycelium for each layer. The
mycelium was put into a nylon mesh bag with 25 \selectlanguage{greek}µ\selectlanguage{english}m opening (Van Borselen
Filters B.V., Zoetermeer, The Netherlands) and heat-sealed before
incubation. Five replicates were prepared for each layer, resulting in
90 bags in total. One of these replicates was used to analyse the
concentrations of cell wall components of the initial necromass (Week
0). For \emph{Lactarius deliciosus} only 65 mg of dry mycelium per bag
could be prepared due to the scarcity of biomass. For the decomposition
assay, soil from a pine stand located in the Meijendel Dunes area
(Wassenaar, The Netherlands, 52\selectlanguage{ngerman}°08'57.0''N 4°22'21.6''E) was collected
and sieved with a 2 mm mesh sieve. Each bag was incubated in a pot with
approximately 400g of soil for a period of six weeks.

Mass loss evaluation

The mesh bags were harvested from the soil after one, two, four and six
weeks of decomposition. The bags were rinsed in deionised water to wash
off the remaining soil and dried overnight at 65\selectlanguage{english}. Mass loss was
calculated by weighing the dried bags and comparing their weight with
the dry weight before incubation. We further calculated the weekly mass
loss by dividing the mass difference between two consecutive mass
measurements by the number of weeks between the two measurements. The
dried mycelium was collected and ground with a pestle for
microcentrifuge tubes in a 1.5ml tube for chemical analysis.

Chemical analysis

Chitin was extracted according to the protocol of Fernandez \& Koide
(2012), with minor adjustments; the concentration in the samples was
determined with the spectrophotometer at 653 nm. Known amounts of pure
chitin (Merck KGaA, Darmstadt, Germany) were assayed to obtain a
standard curve which was used to obtain the concentrations in the
samples. Melanin concentrations were measured according to Fernandez et
al. (2014). The determination of melanin concentration was made by
placing 10 mg of sample in 2 mL of Azure A dye solution and incubating
for 24 hours. The solution was filtered through a 0.45 mm nitrocellulose
membrane, and the absorbance of the filtrate was measured using the
spectrophotometer at 610 nm. A standard curve was constructed using
known amounts of melanin previously isolated from the same material used
in the decomposition assay. The change in absorbance was used to
calculate the melanin content of the sample based on the standard curve.

Glucans were extracted with the Mushroom and yeast beta-glucans assay
kit (K-YBGL 08/18, Megazyme). The protocol was followed with a few
adjustments; a known amount of glucans was used to calculate the
standard curve, which was used to calculate the glucans concentration
after measuring the absorbance at 510nm.

\section*{Statistical analyses}

{\label{statistical-analyses}}

The mycelium age, i.e. layer, did not have any effect on the
decomposition (Table S1). Therefore they were treated as replicates,
allowing to use n=3 replicates for each week in subsequent statistical
analyses. A two-way ANOVA model with species, time and their
interactions was used to determine significant effects on the
decomposition of necromass (\% mass remaining). This was followed by a
post-hoc analysis to evaluate significant differences between species.

Differences in initial chitin, glucans and melanin contents among the
necromass of the six species were assessed with one-way ANOVAs.
Differences in the concentration of chitin, glucans and melanin between
species over time and their interactions were assessed through an
ANCOVA. The relationship between the weekly mass remaining and the
concentration of melanin, chitin and glucans were determined through
linear model analyses. All statistical analyses were conducted using R
studio (R Foundation for Statistical Computing, Vienna, Austria)



\section*{Results}

{\label{results}}

Within the six weeks of the decomposition experiment, the EM fungal
species lost on average 53.7\% of the necromass, ranging from a minimum
of 30.6\% \emph{(L. deliciosus} ) to a maximum of 68.1\% (\emph{S.
verrucosum} ). The differences in decomposition were highly significant
both between species and between weeks, but there was no significant
effect of their interaction (Species: F value= 6.55, p
value\textless{}0.001; Week: F value= 60.08, p value\textless{} 0.001;
Species*Week: F value= 1.29, p value= 0.22). This is visible from the
similar trends in mass loss, with an average loss of around 38\% of the
initial mass during the first week, followed by a slower degradation
during the last five weeks (Figure 1). \emph{L. deliciosus} was the only
species which decomposition was significantly lower than the others.

The six species showed different initial concentrations of glucans in
their necromass, where \emph{L. bicolor} , \emph{L. deliciosus}
and\emph{P. involutus} had significantly higher concentrations compared
to\emph{H. hiemale} , \emph{S. verrucosum} and \emph{I. rimosa} . On the
other hand, melanin and chitin initial concentrations did not show any
significant difference among species (Figure 2).

After six weeks, concentrations of glucans and melanin did not differ
between species, while chitin concentration of \emph{H. hiemale} was
higher than in the rest of the species, suggesting that the loss of
glucans and chitin differed among species (Figure 3). The analysis of
covariance (Table 1) confirmed this supposition and provided more
detailed insights. The dynamics of compounds loss differed among fungal
species: glucans concentration increased over time in \emph{I. rimosa}
and \emph{L. bicolor} , while it decreased for the other species (Figure
3. B); chitin concentration tended to increase over time in every
species besides \emph{P. involutus} (Figure 3. C).

The initial concentrations of the secondary compounds within fungal
species biomass was not a good predictor of decomposition (melanin:
R\textsuperscript{2}= 0.015, p= 0.281; chitin: R\textsuperscript{2}=
0.007, p=0.542; glucans: R\textsuperscript{2}=0.003, p= 0.32) and
therefore could not explain the difference in decomposition
between\emph{L. deliciosus} and the other species. However, the change
in concentrations of cell-wall compounds during the decomposition
process was related to biomass loss. Specifically, the chitin
concentration in the necromass was negatively related to the weekly mass
loss (F value=6.34, df=53, P value=0.0149, Figure 4), while for melanin
and glucans this was not the case (melanin: F value=0.398, df=52, P
value=0.531; glucans: F value=2.75, df=42, P value=0.104)

Interestingly, we found that the relationship between chitin
concentration in the samples and the weekly mass loss changed over time
(Figure 4): in the first week, the effect of chitin concentration on
weekly mass loss was positive, while for the rest of the decomposition
period it was negative.



\section*{Discussion}

{\label{discussion}}

Our study for the first time assesses how the temporal dynamics of
secondary components control the decomposition of the extramatrical
mycelium of ectomycorrhizal fungi. We have monitored the concentrations
of the main cell wall secondary components over time to evaluate how
these changes affect the decomposition process. In this way, we
demonstrated how the different loss of these components between species
does not translate into a different overall mass loss from the
decomposing necromass, which was comparable across species after six
weeks. Considering that the structure of our species was very similar,
since they were all cultivated in vitro in absence of external
stressors, we can be sure that our results reflect the actual effects of
fungal chemical composition.

Overall, the magnitude and rate of decomposition of the EM fungal
species were consistent with previous findings, especially so concerning
the rapid mass decline in the first seven to ten days (Drigo et al.,
2012; Ryan, Schreiner, Swenson, Gagne, \& Kennedy, 2020). Yet in
contrast to these earlier studies, we found hardly any differences
between fungal species in the decomposition of the necromass. Previous
research suggested that the initial concentrations of secondary
components, and especially of melanin (C. W. Fernandez, Heckman, Kolka,
\& Kennedy, 2019; Christopher W Fernandez \& Koide, 2014), and chitin
(Fernandes and Koide, 2012) underpin fungal decomposition rate. While
higher concentrations of melanin in fungal litter decreased the
decomposition of EM fungal necromass, a higher concentration of chitin
was shown to be generally positively related to the decomposition rate .
The fungal species here examined did not show a significant difference
in the initial concentrations of these two components, which likely
explains the similar mass loss among species. Yet, initial
concentrations of glucans differed between species. However, these
differences did not result in differences in mass loss during the
decomposition. This happened even though the concentrations in glucans
may translate into differences in nitrogen concentrations given that
glucans do not contain nitrogen

Remarkably, \emph{L. deliciosus} , the slowest decomposing species in
our species set did not stand out in terms of initial concentration of
cell-wall components. This suggests, in contrast to previous studies
which did not evaluate the full dynamics in secondary compounds during
decomposition, that differences in initial concentrations do not drive
differences in decomposition rates. Instead, and in contrast to the
initial concentration, the dynamics of loss of chitin was related to
fungal species biomass loss. This is in line with the recent findings of
Ryan et al. (2020), who also found that fungal species decomposition was
strongly underpinned by the loss of carbohydrates from fungal biomass.
However, our results indicate that this finding could not be generalized
for all carbohydrates, as there was no significant relationship between
necromass loss and the concentration of glucans or melanin. Moreover,
our results show how the impacts of chitin on decomposition vary over
time. We observed a positive relation between chitin and necromass loss
during the first week of decomposition when its rate is the highest.
During the remainder of the weeks, the necromass loss had a weak
negative relationship to chitin concentration. This might be the result
of the increased nitrogen availability in the previous stage of
decomposition, which might be sufficient for decomposers to support the
production of extracellular enzymes devoted to break down the organic
matter (Schimel \& Weintraub, 2003) later on.

The role of melanin in the decomposition process of fungi has been
demonstrated previously (Koide et al 2019), and specifically to a
slow-degrading necromass pool (Ryan et al, 2020). In our study, the loss
of melanin was not related to the necromass loss at all. This might be a
consequence of the small quantity of melanin in our samples. These
consistently low concentrations of melanin among the different species
might be caused by the absence of external stressors during the growth
and growing conditions were optimal in terms of temperature, light and
nutrients.

The patterns of loss of all cell-wall compounds examined in this study
were highly species-specific (Figure 3). Yet, these differences between
species in the pathways of the loss of cell-wall compounds were not
reflected in the inter-specific differences in decomposition patterns.
We hypothesize that the differences across fungal species in the loss of
individual types of polysaccharides such as chitin and glucans, which
constitute 80-90\% of the fungal cell wall (Bartnicki-Garcia, 1968), may
compensate each other. This likely happens due to how decomposers forage
on the fungal necromass, attacking labile polysaccharides, without
discriminating between these different compounds. Indeed hydrolytic
enzymes (e.g. endo-chitinase, N-acetylgucosaminidase, b-glucosidase; the
same enzymes that degrade plant litter compounds such as cellulose) are
produced by the majority of bacteria and saprotrophic fungi (Conn \&
Dighton, 2000). On the other hand, the oxidative enzymes used mainly for
degrading lignin and melanin, are less abundant because they are almost
exclusively secreted by lignin decomposer fungi (Butler \& Day, 1998).
The combined enzyme actions on cell-wall compounds may also explain for
the inversion of the relationship between chitin concentration and the
weekly mass loss as it may impose a structural change in the cell wall
or in the N availability in necromass in general. However, while these
mechanisms may explain the interactions among cell-wall compounds in
affecting fungal litter decomposition and the consequent absence of
significant relationships for individual cell-wall compounds, they do
not fully explain the species-specific decomposition patterns.

To further understand this pattern, not only chitin lability deserves
more attention, but especially glucans, as the most abundant
carbohydrate fraction of the cell wall, should be further investigated.
For instance, distinguishing between alpha- and beta-glucans could be an
interesting line of research, as these compounds serve different
functions in the cell wall compartment. Alpha-glucans contribute to the
cell wall matrix (Sietsma \& Wessels, 1994), while the beta-glucans play
a structural role, crosslinking the chitin fibers (Wessels, Mol,
Sietsma, \& Vermeulen, 1990). Once we can understand the magnitude and
the rate at which these components are lost from the necromass and later
stored in the soil organic matter, we can more precisely predict what
would be the C:N ratio of the soil organic matter, which in many cases
is essential to assess its chemical `lability' (Cui et al., 2020).
Further insights on this C input from ectomycorrhizal fungi might
provide a more comprehensive view of the contributions of these groups
of microorganisms to soil organic matter. While most of the research to
date has focused on their capacity to mobilize organic C by decomposing
organic matter (Leake et al., 2014; D. Read \& Perez\selectlanguage{english}-Moreno, 2003; D. J.
Read, Leake, \& Perez-Moreno, 2005), we emphasize the importance of the
decomposition of their necromass as a contribution to soil organic
matter dynamics.

Considering that glucans and chitin are both attacked by hydrolytic
enzymes, it is also worth further investigate the temporal activity of
these enzymes under the influence of external factors. Such analysis of
hydrolytic enzymes could provide more insights into the mechanisms that
drive the ectomycorrhizal decomposition, especially concerning
environmental conditions. The activity of many enzymes depend on soil
abiotic parameters (pH, temperature, moisture), which drastically change
between soil layers and seasonality (Wittmann, Kahkonen, Ilvesniemi,
Kurola, \& Salkinoja-Salonen, 2004). This can have a major effect on the
ectomycorrhizal necromass decomposition, which can be modelled more
easily once we can fine-tune them based on environmental conditions.

With the findings presented in this study, we encourage to follow two
main paths to further understand the contribution of ectomycorrhizal
necromass to SOM. First, there is a need to dig deeper into the nature
of interactions between the two most abundant polymers in the cell wall
(glucans and chitin), to see if those interactions may have a
significant effect on the decomposition of both single components and
the overall necromass. In particular, whether these interactions are
prone to change under the influence of environmental conditions or that
they are highly species-specific. Understanding these interactions may
take a long time, in analogy to the complications that arose to
understand the interaction between lignin and cellulose on plant litter
decomposition (Evans, Dutton, Guillen, \& Veness, 1994; Talbot \&
Treseder, 2012). Those interactions are particularly difficult to study
in natural conditions due to the external biotic factors that are
involved (Cuchietti, Marcotti, Gurvich, Cingolani, \& Harguindeguy,
2014). Secondly, when it comes to developing models which aim to predict
SOM dynamic, without knowing the nature of the interactions within
mycorrhizal necromass -- as highlighted above, we can still use the
decomposition estimates from our study and others to develop descriptive
models. This would demand considering mycorrhizal necromass as a
separate pool next to plant litter, instead of assuming that both pools
behave similarly. Either way, our results suggest that necromass
decomposition does not behave similarly to plant litter, while it is a
critical component to consider in estimating SOM dynamics.

Conclusions Our results show that the decomposition process of necromass
of ectomycorrhizal fungal mycelium is not species-specific overall, as
the rate and magnitude of decomposition do not show significant
differences as for the pathway of chemical component loss from their
cell walls. As evidence of this, the concentration changes of the cell
wall compounds over time do not correspond to the final biomass loss.
For this reason, looking at the concentration of single compounds cannot
ensure an accurate prediction of the necromass loss in ectomycorrhizal
fungi. Together, this brings evidence that distinct EM fungal species
affect SOM through distinct pathways of cell-wall compound loss, even if
these fungi show a similar overall mass loss. These pathways differ
remarkably from those of plant litter and need to be further understood
to be able to better predict the contribution of fungal necromass on
soil organic matter dynamics.



\section*{Acknowledgements}

{\label{acknowledgements}}

This research was supported by N. Soudzilovskaia and her VIDI grant
016.161.318 issued to NAS by The Netherlands Organization for Scientific
research.

\section*{Author contribution}

{\label{author-contribution}}

JL carried out the experiment and made preliminary statistical analysis.
RM planned and designed the research and wrote the first draft of the
manuscript, and performed the data analysis. PB and NS helped with the
experimental design, data analysis and reviewing/editing the manuscript.

\section*{Data availability}

{\label{data-availability}}

The data that support the findings of this study are openly available in
the public repository DRYAD at https://datadryad.org/

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\section*{Tables}

{\label{tables}}

Table 1. Outcomes of the ANCOVA on secondary component loss during 6
weeks decomposition of ectomycorrhizal fungal species (SSq= sum of
squares, df= degrees of freedom, F= F-statistic, p= p-value).\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
RESPONSE & \textbf{SSq} & \textbf{df} & \textbf{F} &
\textbf{p}\tabularnewline
\midrule
\endhead
CHITIN & & & &\tabularnewline
Week & 12720 & 1 & 24.16 & 5.38e-06 ***\tabularnewline
Species & 63093 & 5 & 23.97 & 4.11e-14 ***\tabularnewline
Week:Species & 8689 & 5 & 3.30 & 0.00969 **\tabularnewline
MELANIN & & & &\tabularnewline
Week & 8962 & 1 & 8.24 & 0.00530 **\tabularnewline
Species & 10542 & 5 & 1.93 & 0.0976\tabularnewline
Week:Species & 2896 & 5 & 0.533 & 0.751\tabularnewline
GLUCANS & & & &\tabularnewline
Week & 16 & 1 & 0.014 & 0.905\tabularnewline
Species & 201584 & 5 & 35.51 & 6.52e-16 ***\tabularnewline
Week:Species & 61296 & 5 & 2.66 & 0.0319 *\tabularnewline
\bottomrule
\end{longtable}

Table 2. Isolates of EMF with corresponding species names, collection
codes, culture medium and isolate supplier. \textsuperscript{a} BCCM
MUCL, Belgian Coordinated Collections of Microorganisms/Mycotheque de
L'Universite~\selectlanguage{english}catholique de Louvain, Louvain-la-Neuve,
Belgium.\textsuperscript{b} CBS Filamentous fungi and Yeast Collection,
Westerdijk Fungal Biodiversity Institute -- KNAW, Utrecht, the
Netherands.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Name & CBS/MUL NO. & Medium & Supplier\tabularnewline
\midrule
\endhead
\emph{Scleroderma verrucosum} & MUCL 34674 & MA2 &
BCCM/MUCL\textsuperscript{a}\tabularnewline
\emph{Paxillus involutus} & CBS 100140 & MOD &
CBS\textsuperscript{b}\tabularnewline
\emph{Laccaria bicolor} & CBS 445.79 & CHA &
CBS\textsuperscript{b}\tabularnewline
\emph{Inocybe rimosa} & CBS 210.55 & X agar &
CBS\textsuperscript{b}\tabularnewline
\emph{Hebeloma hiemale} & CBS 376.89 & MEA &
CBS\textsuperscript{b}\tabularnewline
\emph{Lactarius deliciosus} & CBS 582.63 & BAF &
CBS\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}

\section*{Figure legends}

{\label{figure-legends}}

Figure 1 Mean per cent mass remaining of necromass from the six
ectomycorrhizal fungal species. Individual points show the average of
EMF mass remaining, error bars show the standard error.

Figure 2. Initial concentrations {[}mg/g{]} of the secondary compounds
within mycorrhizal fungal necromass

Figure 3. Concentration {[}mg component / g total mass{]} of the
secondary compounds from the necromass of ECM fungal species during the
decomposition process.

Figure 4. The relationship between chitin concentration and average
weekly decomposition (\% mass loss) of EM fungal necromass.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}

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