Fungal Ecology 22 (2016) 106e114

Contents lists available at ScienceDirect

Fungal Ecology
journal homepage: www.elsevier.com/locate/funeco

Correlations between the composition of modular fungal communities

and litter decomposition-associated ecosystem functions
Witoon Purahong a, *, 1, Dirk Krger a, Franois Buscot a, b, Tesfaye Wubet a, b, *, 1
a
b

UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Theodor-Lieser-Str. 4, D-06120, Halle (Saale), Germany
German Centre for Integrative Biodiversity Research (iDiv), Halle-Jena-Leipzig, Deutscher Platz 5e, D-04103, Leipzig, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 10 July 2015
Received in revised form
14 March 2016
Accepted 21 April 2016
Available online 3 June 2016

Microbial co-occurrence network analyses provide important information on ecological interactions

between different microbial Operational Taxonomic Units (OTUs), and often indicate that total microbial
communities are sub-structured into modules. Here we investigate the fungal communities associated
with beech leaf litter decomposition over 473 days using pyrotag sequencing of the fungal ITS rRNA
genes. Our results demonstrate that the total fungal communities present during the two major
decomposition stages are sub-structured into four modules each, giving eight modular fungal communities in total. These modular communities displayed different relationships with leaf litter physicochemical properties and ecological functions. During the early decomposition stage, modules 2
(dominated by Gyoerffyella sp.1 and Mycosphaerella sp.) and 4 (Xylariales OTU3, Cylindrosympodium sp.1
and Leotiomycetes OTU12) correlated signicantly with both hydrolytic and oxidative enzyme activities
(P < 0.05). During the later decomposition stage, module 7 (Clitocybe phaeophthalma and Ceratobasidium
sp.) correlated signicantly with the activities of laccase and general peroxidase (P < 0.05). Our results
demonstrate that individual fungal subcommunities are largely responsible for providing specic
ecosystem functions in litter decomposition, which improves our understanding of the factors that
determine the distribution patterns of fungi and their potential roles in key ecosystem processes.
2016 Elsevier Ltd and British Mycological Society. All rights reserved.

Corresponding editor: Kevin D. Hyde

Keywords:
Litter decomposition
Fagus sylvatica
Pyrosequencing
Ecosystem functions
Fungal community structure

1. Introduction
Leaf litter decomposition is a complex ecological process that is
regulated by three main drivers: climate, litter quality, and the
decomposer communities (Coteaux et al., 1995). Fungi are regarded as the primary decomposers of leaf litter because they secrete
various digestive enzymes that breakdown polymeric organic plant
compounds such as cellulose, hemicellulose and lignin (Keiblinger
 and Baldrian, 2013;
et al., 2012; Schneider et al., 2012; Vo
r
skova
Purahong et al., 2014a). The distribution patterns of fungi provide
important information about their potential roles in ecosystem
 et al., 2012). Some studies refunctions and stability (Kubartova
ported that the overall composition of the fungal communities in
leaf litter correlates with the physicochemical properties of the

* Corresponding authors. UFZ-Helmholtz Centre for Environmental Research,

Department of Soil Ecology, Theodor-Lieser-Str. 4, D-06120, Halle (Saale), Germany.
E-mail addresses: witoon.purahong@ufz.de (W. Purahong), tesfaye.wubet@ufz.
de (T. Wubet).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.funeco.2016.04.009
1754-5048/ 2016 Elsevier Ltd and British Mycological Society. All rights reserved.

litter and with the microbially-mediated ecosystem functions

(Burke et al., 2011; Per
soh, 2015; Purahong et al., 2015). However, it
is not possible to generalize these observations as there is still no
consistent support for this aspect and most of the studies may
contain pseudocorrelations due to spatial distances, which is
probably the most important driver of fungal community assembly
(Talbot et al., 2014; Per
soh, 2015). Microbial communities are often
built around complex networks of interactions such that the overall
community is structured into smaller sub-communities or modules
of co-occurring taxa. Studies on the relationships between such
OTU co-occurrence modules and ecological variables can provide
more signicant insights into the ecosystem functions of fungi than
could be obtained by considering relationships at the level of the
whole fungal community (de Menezes et al., 2014). In addition,
analyses of such modular- or sub-communities may enable better
resolution of the factors that structure the microbial community in
a given ecosystem while also facilitating the identication of
different microbial functional groups and drivers of community
composition (de Menezes et al., 2014).
Leaf litter decomposition is a key process of nutrient recycling

W. Purahong et al. / Fungal Ecology 22 (2016) 106e114

that has two major phases (Berg, 2000). The rst phase (the early
decomposition stage) is regulated by the concentration of different
nutrients and readily available carbon while the second phase (the
later decomposition stage) is regulated by lignin decomposition.
While several studies have explored the roles of fungal communities in litter decomposition, most of them have focused on the
 and Baldrian,
total fungal diversity (Steffen et al., 2007; Vo
r
skova
2013; Purahong et al., 2015). Here we present the rst analysis of
the co-occurrence networks within the litter-decomposing fungal
community during the early and later stages of litter decomposition, and assess the composition of modular or sub-communities
that are more specically correlated with key enzymatic processes in litter decomposition.
A litterbag experiment was set up in a European beech (Fagus
sylvatica) dominated forest. The experiment was maintained for
473 d to ensure that the collected litter reached the later stage of
decomposition, which began after 284 d when signicant lignin
decomposition was observed (Purahong et al., 2014a). Indicators of
the microbial communities' ecological functions were investigated
by measuring eight enzyme activities at selected time points during
the experiment (Purahong et al., 2014b). The fungal communities
from the litter material were analyzed by pyrotag sequencing of the
fungal ITS rDNA. Our aim was to: (i) characterize the fungal cooccurrence networks and modular communities existing during
the early and later stages of decomposition; (ii) investigate the
correlation between each fungal modular community and the litter's physicochemical parameters; and (iii) assess the link between
the fungal modular communities and selected ecological functions
including the activities of ve hydrolytic enzymes important in the
acquisition of carbon (b-glucosidase (EC 3.2.1.21), cellobiohydrolase
(EC 3.2.1.91), and xylosidase (EC 3.2.1.37)), nitrogen (N-acetylglucosaminidase (EC 3.1.6.1)), and phosphorus (acid phosphatase (EC
3.1.3.2)), as well as three oxidative enzymes important for lignin
modication and degradation (laccase (EC 1.10.3.2), general
peroxidase (EC 1.11.1.7), and manganese peroxidase (EC 1.11.1.13),
MnP). We hypothesized that different fungal modular communities
would correlate with different physicochemical properties and
enzyme activities. We also expected that the correlations between
the modular communities and the activities of specic enzymes
would reect the taxonomic and functional signicance of the
communities' member OTUs.
2. Materials and methods
2.1. Study site
This study was carried out at the Hainich-Dn Biodiversity
Exploratory (about 1300 km2; 51
160 N 10
470 E) in Central Germany
(Fischer et al., 2010). The main soil type is Stagnosol on a limestone
bedrock (Fischer et al., 2010). The soil pH is weakly acidic (5.1 1.1;
mean SD) with a litter layer of 2 cme5 cm. The annual mean
temperature and precipitation ranges from 6.5
C e 8
C and
500 mme800 mm respectively. All information pertaining to the
experimental area has been described in detail in Fischer et al.
(2010). We assigned three replicate study plots (2 m  8 m)
located on at land within the experimental site (HEW12) characterized as an unmanaged deciduous forest reserve of 10,000 m2
dominated by European beech with an uneven-age distribution
(tree age up to >100 years) (Purahong et al., 2015).
2.2. Litterbag design and sampling
Freshly fallen leaves of European beech were collected from the
study site in October 2009. The leaves were air dried to constant
weight at room temperature. Ten grams of air-dried leaves were

107

placed into nylon litterbags (25 cm  25 cm, mesh size 2 mm). At

the end of the litter fall period (13 November 2009) 15 litterbags
were placed horizontally in the upper litter horizon of each replicate study plot. Nine additional litterbags were retained to determine the initial dry mass (oven-dried at 105
C  24 h until
constant weight), nutrient element concentrations, and lignin
content of the litter. The chemical composition of the litter material
is given in Supplementary Table S1. Three randomly chosen litterbags per plot (nine litterbags in total) were retrieved on ve sampling dates: in 2010 on February 10th (89 d), May 12th (180 d),
August 24th (284 d), November 10th (362 d), and in 2011 on March
1st (473 d). Each bag was placed into a separate clean plastic bag to
reduce the loss of small fragments, and transported in an ice-box
(0
C) to the laboratory within 4 h, and processed immediately.
First, the three replicate litterbags retrieved from the same plot
were pooled and their wet weight was determined. Thus, three
composite samples were obtained for each sampling time. Each
composite sample was then homogenized, subsampled into two
parts (original homogenized and freeze-dried samples), and stored
at 20
C for further analysis.
2.3. Physicochemical analysis of leaf litter
The water content and pH (in 0.01 M CaCl2) of leaf litter samples
were determined. Total C and N were measured by dry combustion
at 1000
C with an Elementar Vario EL III elemental analyzer
(Elementar Analysensysteme GmbH, Hanau, Germany) according
to DIN/ISO 10694. Nutrient ions (Mg, K, P, Ca, Fe, Cu, V, Mn, Co) were
determined using inductively coupled plasma (ICP) optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS) according to the manufacturers' specications. Total lignin was
calculated by summing Klason lignin (acid insoluble lignin) and
acid soluble lignin (Raiskila et al., 2007). Klason lignin content was
determined gravimetrically as the dry mass of solids after
sequential hydrolysis with sulfuric acid (72% w/w); in a second step,
acid soluble lignin was measured UV-photometrically in 4% H2SO4
(Efand, 1977; Liers et al., 2011). All physicochemical analyses were
conducted in triplicate on the same subsample.
2.4. Enzyme assays
A total of eight potential enzyme activities were measured from
original homogenized leaf litter samples (ve sampling dates:
89e473 d). These include ve hydrolytic enzymes important for the
acquisition of polymeric carbon (b-glucosidase, cellobiohydrolase,
and xylosidase), nitrogen (N-acetylglucosaminidase) and phosphorus (acid phosphatase), and three oxidative enzymes related to
the chemical modication of lignin (laccase, general peroxidase,
and MnP). Hydrolytic enzyme analyses were done using 4methylumbelliferone (MUB) substrate as described elsewhere
(Sinsabaugh et al., 2003). The protocol was slightly modied by pretesting (German et al., 2011). Oxidative enzyme analyses were done
using ABTS (2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid) substrate (Purahong et al., 2014a, 2016).
2.5. Microbial DNA extraction and sequence library preparation
DNA was extracted from 100 mg of each homogenized and
freeze-dried leaf litter sample using the ZR Soil Microbe DNA
MiniPrep kit (Zymo Research, Irvine, CA, USA) according to the
manufacturer's protocol. The presence and quantity of genomic
DNA was checked using a NanoDrop ND-1000 spectrophotometer
(ThermoFisher Scientic, Dreieich, Germany). DNA extracts were
then stored at 20
C for further analysis.
Fungal amplicon libraries were obtained for pyrosequencing

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W. Purahong et al. / Fungal Ecology 22 (2016) 106e114

using custom fusion primers. We used the primer pair ITS1F

(Gardes and Bruns, 1993) and ITS4 (White et al., 1990) to amplify
the fungal internal transcribed spacer (ITS) rRNA region. The
custom primers were constructed with the barcodes and
sequencing primers attached at the ITS4 primer for unidirectional
sequencing (Wubet et al., 2012; Lentendu et al., 2014). PCR conditions are shown in the Supplementary Information. Amplication
products were visualized with eGels (Life Technologies, Grand Island, New York). Products (three replicate amplicons per sample)
were pooled in equimolar amounts and each pool was cleaned with
Difnity RapidTip (Difnity Genomics, West Henrietta, New York),
and size selected using Agencourt AMPure XP (Beckman Coulter,
Indianapolis, Indiana, USA) following Roche 454 protocols (454 Life
Sciences, Branford, Connecticut, USA). Size-selected pools were
then quantied and 150 ng of DNA was hybridized to Dynabeads M270 (Life Technologies, Norway) to create single stranded DNA.
Single stranded DNA was diluted and used in emPCR reactions,
which were subsequently enriched for amplicon sequencing
following the established manufacturer's protocols (454 Life
Sciences).

We extracted the modular communities and analyzed each

fungal modular community composition using principal component analysis (PCA). The rst and second axis scores of PCA were
used to represent the fungal community composition as shown in
some previous studies (Ramette, 2007; Regan et al., 2014). The
amounts of variance explained by two components were shown.
We determined the gradient length of each fungal modular community using detrended correspondence analysis (DCA) and the
results show that all modular communities have short gradients
(less than 2.7 SD) (Ramette, 2007). Thus, linear methods such as
PCA are appropriate for analysis of the fungal modular communities in this study. Correlations between rst and second axis
scores of PCA of each fungal modular community and physicochemical parameters or enzyme activities were calculated using
Pearson product-moment correlation (P < 0.05). All datasets were
tested for normality using the Jarque-Bera JB test. PCA, DCA and
correlation analysis were done using software PAST 3.0 (Hammer
et al., 2001).

2.6. Bioinformatics analysis

The raw demultiplexed reads were rst quality trimmed using
MOTHUR 1.33.3 (Schloss et al., 2009). Reads satisfying the following
criteria were considered for further analyses: holding one of the
expected barcodes with maximum 1 mismatch; the forward primer
with a maximum of 4 mismatches; a minimum length of 350 nt; a
minimum average quality of 25 Phred score; containing homopolymers with a maximum length of 8 nt; and with a maximum of
8 ambiguous nucleotides. The quality ltered reads were shortened
to their rst 350 bases and normalized to the smallest read per
sample. Potential chimeras were removed using UCHIME 4.2.40
(Edgar et al., 2011) as implemented in MOTHUR. Unique sequences
were sorted according to decreasing abundance and were clustered
into OTUs using CD-HIT-EST 4.5.4 (Fu et al., 2012) at a threshold of
97% pairwise similarity. Fungal ITS OTU representative sequences
were rst classied against the dynamic version of the UNITE
fungal ITS sequence database (version 6, released on 15.01.2014)
~ ljalg et al., 2013). The sequences with only fungi identication
(Ko
were further classied against the full version of the UNITE database in order to improve their taxonomic annotation. OTUs from
non-target taxa and rare OTUs (singletons to tripletons), which
could potentially have originated from sequencing errors (Kunin
et al., 2010), were removed from the dataset. Thus we used the
abundant OTUs (OTUs with >3 reads) for further statistical analyses. Raw pyrosequencing reads and metadata were submitted to
the European Nucleotide Archive under study accession number
PRJEB9175.
2.7. Statistical analyses
We used the relative abundance OTU matrices of the fungal
communities for further analyses. We used the extended local
similarity analysis (eLSA) to calculate the LS scores and Pearson
correlations between fungal OTUs using 1000 permutations (Xia
et al., 2011) to assess the co-occurrence network patterns of the
fungal communities at early and later stages of litter decomposition. The correlation matrix with P < 0.01 was visualized and the
early and later stage networks were extracted using Cytoscape 3.0.2
(Shannon et al., 2003). Fungal modules or sub-communities of the
two decomposition stages were calculated using the Louvain algorithm (Blondel et al., 2008) and network properties were calculated using the statistics tools implemented in Gephi (Bastian et al.,
2009).

3. Results
3.1. Fungal modular communities present during the early and later
stages of litter decomposition
A total of 85,979 quality-ltered sequences of fungal internal
transcribed spacers (ITS) were obtained after removal of non-target
and chimeric sequences. The sequences were subsampled and
rareed to the smallest read number per sample (2471). These were
then clustered into 771 fungal OTUs (including 407 singletons, 81
doubletons, and 58 tripletons). Singletons, doubletons and tripletons were removed and a total of 225 fungal OTUs belonging to
three phyla (Ascomycota 69.9%; Basidiomycota 30.06%;
Zygomycota 0.04%) were used for further analysis. Co-occurrence
network analysis of fungal communities at the early (0e180 d) and
later (284e473 d) litter decomposition stages resulted in the
detection of eight fungal modular communities, with modules 1e4
being associated with the early stage and modules 5e8 with the
later stage (Fig. 1).
The number of fungal OTUs in the modular communities varied
(Table 1) from 21 in module 2 to 55 in module 1. The dominant
fungal OTUs in each module and the ratio of Ascomycota to Basidiomycota in each case are presented in Table 2. Based on the
presence/absence of individual taxa, the Ascomycota were dominant in all of the modular communities. In contrast, the relative
abundance data indicated that the Basidiomycota were dominant in
fungal modules 1, 5 and 7. Ceratobasidium sp, Xylariales OTU3 and
Cylindrosympodium sp.1 were dominant in at least two fungal
modules in both early and later decomposition stages. Gyoerffyella
sp.1 and Mycosphaerella sp. were among the most frequently
detected taxa during the initial decomposition stage, and dominated fungal module 2. Clitocybe phaeophthalma and Mycena spp.
were frequently detected during the later decomposition stage and
dominated fungal modules 7 and 8, respectively.
The modular communities also differ in their network properties (Fig. 1, Table 1). Module 2 had the highest weighted degree
(25.762), weighted clustering coefcient (0.6) and number of triangles (204.286) whereas module 5 had the lowest weighted degree (1.846), weight clustering coefcient (0.160) and number of
triangles (1.192). Closeness and betweenness centrality were
generally much higher for fungal modules associated with the later
decomposition stage.

Fig. 1. Co-occurrence network plots of fungal communities in beech leaf litter during the (A) early and (B) later decomposition stages. The different colors represent the four
modular communities identied at each stage. Only nodes with more than two degrees are presented. Red modules 1 (A) and 5 (B); green modules 2 (A) and 6 (B); Navy
blue modules 3 (A) and 7 (B); purple modules 4 (A) and 8 (B). Ascomy Ascomycota; Basidi Basidiomycota; Zygomy Zygomycota.

110

W. Purahong et al. / Fungal Ecology 22 (2016) 106e114

Table 1
Network properties of early and later stage leaf litter decomposer fungal modules.
Network characteristics

Number of OTUs
Weighted Degree
Weighted Clustering Coefcient
Closeness Centrality
Betweenness Centrality
Number of triangles

Early decomposition stage

Later decomposition stage

Module1

Module 2

Module 3

Module 4

Module 5

Module 6

Module 7

Module 8

55
7.600
0.298
0.292
0.006
26.018

21
25.762
0.600
0.500
0.015
204.286

24
9.875
0.406
0.409
0.010
41.333

27
22.667
0.555
0.486
0.016
169.259

26
1.846
0.160
1.458
42.376
1.192

41
19.854
0.523
2.303
121.904
121.488

50
11.820
0.376
2.436
71.245
33.340

41
21.098
0.484
2.282
119.609
116.244

Table 2
Composition of different fungal modular community at phylum level (based on relative abundance and presence/absence data), and list of the dominant fungal taxa in the
respective fungal module.
Fungal modular
community

Decomposition
stage

Dominant fungal taxa

Abundance of Ascomycota (%)

(presence/absence)

Abundance of Basidiomycota (%)

(presence/absence)

Module
Module
Module
Module

1
2
3
4

Early
Early
Early
Early

34.9
92.3
84.3
99.6

65.1
7.7
15.7
0.4

Module 5
Module 6

Later
Later

Module 7
Module 8

Later
Later

Ceratobasidium sp., Flagellospora sp.

Gyoerffyella sp.1, Mycosphaerella sp.
Cryptosporiopsis sp.
Xylariales OTU3, Cylindrosympodium sp.1,
Leotiomycetes OTU12
Ceratobasidiaceae OTU79,
Xylariales OTU3, Cylindrosympodium sp.1,
Cylindrosympodium sp.2
Clitocybe phaeophthalma, Ceratobasidium sp.
Mycena terena, Mycena sp.3, Mycena pura, Apodus
sp.

3.2. Correlation between the fungal modular communities and litter

physicochemical parameters
The overall strength of the correlations between the fungal
modular community composition (PCA axis scores) and leaf litter
physicochemical properties was higher in the early stage of
decomposition than in the later stage. Most modules other than
module 5 showed contrasting relationships to leaf litter physicochemical properties (Table 3). Module 5 exhibited no correlations
with any leaf litter physicochemical properties. The litter quality
parameter (Lignin/N), macronutrient levels (N, K, Ca and P) and the
level of one micronutrient (Cu) correlated signicantly (P < 0.05)
with fungal modular communities 2 and 4 associated with the early
decomposition stage, but the directions of the correlations (positive
vs. negative) varied markedly between different modules. During
the later decomposition stage, only a few leaf litter physicochemical
properties correlated signicantly with fungal modular communities: module 6 with Mg, module 7 with Fe and V and module 8
with C, N and K (P < 0.05).

3.3. Correlations between fungal modular communities and

ecological functions
The correlations between the composition of the fungal
modular communities and enzyme activities were not always signicant (Table 4), but some highly signicant correlations were
identied. Only three of the fungal modular communities exhibited
signicant correlations with enzyme activity. During the early
decomposition stage, module 2 correlated signicantly with the
greatest number of enzyme activities, including those of b-glucosidase, cellobiohydrolase, xylosidase, N-acetylglucosaminidase and
MnP (P < 0.05). In addition, module 2 correlated marginally with
the activities of acid phosphatase and general peroxidase (P < 0.1).
Module 4 also correlated with the hydrolytic and oxidative enzyme
activities xylosidase and MnP; (P < 0.05). The only module exhibiting signicant correlations with enzyme activity during the later

(69.1)
(66.7)
(70.8)
(92.6)

(30.9)
(33.3)
(29.2)
(7.4)

30.5 (57.7)
99.5 (92.7)

69.5 (42.3)
0.5 (7.3)

21.9 (66.0)
66.8 (60.0)

78.1 (34.0)
33.1 (40.0)

stage was module 7, which correlated with laccase and general

peroxidase activity (P < 0.05).
4. Discussion
Our work combines a high-resolution molecular cultureindependent fungal meta-barcoding technique and co-occurrence
network analysis to characterize the fungal modular communities
present during the early and later stages of decomposition of European beech leaf litter. This combination represents an approach
for investigating the relationships between fungal community
composition, ecosystem functions, and related environmental
properties. To improve the resolution of the analysis, we focused on
the relationships between fungal sub-communities or modules
(rather than the fungal community as a whole) and environmental
parameters. Our results provide new insights into the factors that
affect the composition of the litter fungal community and its potential contributions to ecosystem functions. Nevertheless, when
considering them it is important to recall the potential biases

inherent in molecular techniques (Tedersoo et al., 2010; Vo
r
skova
and Baldrian, 2013).
4.1. Contrasting correlations between fungal modular communities
and leaf litter physicochemical properties
Our results demonstrate that different fungal modular communities have contrasting relationships with the physicochemical
properties of the leaf litter. This is consistent with our hypothesis
and the results of an earlier study on modular bacterial and fungal
communities in woodland and pastures (de Menezes et al., 2014).
Different associations between fungal modular communities and
leaf litter physicochemical properties may reect the niches occupied by individual sub-communities and their specic nutrient
requirements (Dickie et al., 2002; de Menezes et al., 2014; Purahong
et al., 2015). We found that different taxonomic groups of fungi
clustered into different modular communities and that the co-

W. Purahong et al. / Fungal Ecology 22 (2016) 106e114

111

Table 3
Heatmap showing the strength of the correlations between individual fungal modular communities and leaf litter physicochemical properties. (A) Data for modules 1e4,
associated with early decomposition; (B) data for modules 5e8, associated with later decomposition. Signicant variables (P < 0.05) are shown in bold and marginally signicant variables (P < 0.10) are shown in italics.

A
Physicochemical
properties
Total C
Total N
C/N
pH
Mg
K
Ca
P
Mn
Fe
Cu

Module 1
PC1
PC2
(78%)
(15%)
0.05
0.71
-0.66
0.72
-0.64
-0.32
-0.18
0.82
-0.72
-0.59

Co
V
Water content

0.78
-0.46
-0.61
-0.19

Lignin
Lignin/N
B

-0.57
0.23
-0.27
0.44
-0.28
0.03
0.10
0.15
-0.36
-0.06
0.11

Module 2
PC1
PC2
(97%)
(2%)
0.85
-0.90
0.91
-0.79
0.00
0.85
-0.72
-0.82
0.81
0.15

Module 3
PC1
PC2
(57%)
(19%)

Module 4
PC1
PC2
(92%)
(4%)

-0.08
-0.24

-0.57
0.58

-0.52
0.02

0.16
-0.22
0.64
0.02
0.54

-0.60
0.35
0.48
-0.73
0.81

-0.10
0.10
0.28
-0.08
0.48

-0.78
0.84
-0.86
0.63
0.29

-0.37
0.28
0.64
-0.19

0.53
-0.36
0.27
0.56

-0.11
0.00
0.08
-0.11

0.84
0.34
0.23

0.39
0.08
-0.16

0.24
-0.07
0.73

-0.93
0.86
0.76
-0.64
0.04
0.70
0.22
-0.13
0.14

-0.27
-0.33
0.26
-0.27
0.47
-0.02
0.39
-0.50
0.30
0.49
-0.56

-0.03
-0.16
0.86

-0.74
-0.05
0.31
-0.37

-0.48

0.04

-0.43

0.49

0.56

0.51

0.56

0.60

-0.77

-0.26

0.85

0.29

-0.49

0.01

-0.76

0.38

0.47
0.49
0.66

Module 5
PC1
PC2
(94%)
(4%)

Module 6
PC1
PC2
(90%)
(5%)

Module 7
PC1
PC2
(62%)
(25%)

Module 8
PC1
PC2
(79%)
(10%)

Total C
Total N
C/N
pH

0.26
0.27
-0.01
0.05

0.12
0.01
0.38
0.09

0.43
0.45
-0.01
-0.50

-0.31
-0.17
-0.50
-0.15

0.28
0.25
0.14
-0.30

0.07
0.19
-0.41
-0.53

-0.25
-0.21
-0.16
0.52

-0.84
-0.90
0.08
0.48

Mg
K
Ca

0.02
0.22
-0.04

-0.52
0.35
-0.66

0.76
0.50
0.40

0.24
-0.36
0.53

0.27
0.48
0.15

-0.26
0.38
-0.24

-0.46
-0.58
-0.25

-0.64

Physicochemical
properties

-0.70
-0.42

0.36

-0.02

0.66

-0.61

-0.31

-0.02

-0.05

-0.39

Mn
Fe
Cu
Co
V
Water content

-0.07
0.24
0.02
0.10
0.57
-0.01

-0.21
0.34
-0.52
0.07
0.47
-0.40

0.54
-0.21
0.30
0.37
-0.39
-0.45

0.22
-0.09
0.36
-0.62
-0.23
0.42

0.59
0.20
-0.04
-0.05
0.08
-0.59

0.03

0.73
-0.17

-0.62
-0.28
-0.27
-0.15
-0.33
0.63

-0.64
-0.30
-0.29
-0.31
-0.08
0.38

Lignin

0.26

-0.33

0.59

-0.01

-0.28

-0.48

0.13

-0.36

Lignin/N

0.07

-0.32

0.26

0.10

-0.43

-0.56

0.26

0.23

occurrence network structures of different fungal modules differed

sharply. The composition of fungal communities varied substantially even at the phylum level - based on the collected abundance
data, the abundance of Ascomycota in individual modules ranged
from 21.9 to 99.6% while that of Basidiomycota ranged from 0.4 to
78.1% (Table 2). The correlations between the composition of the
fungal modular communities and the leaf litter physicochemical
properties were stronger during the early stage of decomposition
than during the later stage. This may be related to the nature of
fungal communities and the nutrient status of the decomposing
leaf litter. Ascomycota are better equipped for life in leaf litter
during the early decomposition stage whereas Basidiomycota are

0.79
-0.01
0.04

more likely to thrive during the later stage, when their strong

production of oxidative enzymes becomes important (Vo
r
skova
and Baldrian, 2013). Ascomycota have limited ability to produce
oxidative enzymes especially MnP which is important for lignin
decomposition in leaf litter (Purahong et al., 2014a). Ascomycota
are also less capable of forming extended mycelial networks to
translocate resources from other substrates (Boberg et al., 2010).
While this is generally not a problem during the early stage when
the leaf litter contains large quantities of easily decomposable
substrates, it means that the relative abundance of Ascomycota can
be expected to depend strongly on the availability of nutrients in
the leaf litter and its physicochemical properties (Cairney, 2005;

112

W. Purahong et al. / Fungal Ecology 22 (2016) 106e114

Table 4
Heatmap showing the strength of the correlations between fungal modular communities and enzyme activities. (A) Data for modules 1e4, associated with early decomposition; (B) data for modules 5e8, associated with later decomposition. Signicant variables (P < 0.05) are shown in bold and marginally signicant variables (P < 0.10) are
shown in italics.

A
Enzyme

Module 1
PC1
PC2
(72%)
(22%)

Module 2
PC1
PC2
(89%)
(9%)

Module 3
PC1
PC2
(52%)
(23%)

Module 4
PC1
PC2
(83%)
(8%)

-glucosidase
Cellobiohydrolase
Xylosidase
N-acetylglucosaminidase
Acid phosphatase

0.70
0.68
0.62
0.71
0.56

0.00
-0.01
0.28
0.02
-0.17

0.85
0.91
0.88
0.88
0.74

0.41
0.30
0.06
0.29
0.59

-0.20
-0.28
-0.54
-0.25
0.00

-0.49
-0.60
-0.46
-0.56
-0.43

-0.63
-0.68
-0.82
-0.66
-0.47

-0.42
-0.48
-0.35
-0.55
-0.38

Laccase
General peroxidase

-0.01
0.64

-0.16
0.06

-0.32
0.75

0.29
0.33

0.49
-0.22

0.25
-0.39

0.34
-0.59

-0.24
-0.26

Manganese peroxidase

0.63

0.49

0.93

0.00

-0.72

-0.35

-0.96

-0.14

B
Enzyme

Module 5
PC1
PC2
(94%)
(4%)

Module 6
PC1
PC2
(90%)
(5%)

Module 7
PC1
PC2
(62%)
(25%)

Module 8
PC1
PC2
(79%)
(10%)

-glucosidase

0.28

0.45

-0.19

-0.16

0.30

0.18

0.20

-0.49

Cellobiohydrolase
Xylosidase
N-acetylglucosaminidase
Acid phosphatase
Laccase

-0.03
-0.03
-0.14
0.12
-0.14

0.49
0.00
0.48
-0.14
0.23

0.23
0.17
-0.40
-0.23
0.09

-0.15
0.11
0.10
0.30
-0.16

0.65
-0.09
0.19
0.00

-0.57
-0.24
-0.08
0.50
-0.30

-0.47
-0.07
0.16
-0.24
-0.41

General peroxidase

0.11

0.29

0.16

-0.20

0.77
0.70

0.34
0.04
0.35
0.07
-0.27
-0.12

-0.58

-0.39

Manganese peroxidase

0.31

0.42

-0.34

-0.21

-0.24

0.47

0.17

-0.10

Boberg et al., 2010, 2014). Some of the Basidiomycota that became

highly detected during the later stage of decomposition, such as
Clitocybe spp., Mycena spp., are known to form large mycelia during
the later decomposition stage and to efciently produce a wide
range of enzymes including oxidative enzymes (Dowson et al.,
1989; Boberg et al., 2010). Large mycelia, especially those formed
by Basidiomycota, are reported to have a high capacity for translocating resources (i.e. carbohydrates, nutrients and water) from
different substrates to locations that are far away from their original
source (Boberg et al., 2010, 2014). This may help these species to
solve the problem of nutrient limitation that arises during the later
stage, which would in turn explain why the correlations between
the composition of the fungal modular communities and the
physicochemical properties of the leaf litter became weaker in this
stage.
4.2. Links between fungal modular communities and ecological
functions
High levels of hydrolytic enzymes and one oxidative enzyme
(laccase) were detected across different sampling dates in the litterbag experiment (Purahong et al., 2014a, 2014b). MnP activity in the
leaf litter during the early stage of decomposition was low, whereas
that of general peroxidase varied between sampling times and
peaked during the later decomposition stage, at 362 d. We found
signicant correlations between modular community composition
and ecological functions, suggesting that the ecological functions
examined herein are provided by specic subgroups of the total
fungal community. In the early decomposition stage, fungal module
2 correlated with four hydrolytic enzymes important for C and N
acquisition. This module primarily contains members of the Ascomycota (92.3%) and is dominated by Gyoerffyella sp.1 and Mycosphaerella sp. While data on the enzymes produced by the fungal

genus Gyoerffyella are still limited, many studies have shown that
such hyphomycetes can produce a wide range of enzymes that
decompose cell wall polysaccharides (Abdel-Raheem and Ali, 2004;
Krauss et al., 2011). Mycosphaerella spp. have been described as
being able to adapt to various life strategies including endophytic,
saprotrophic and plant pathogenic (Crous, 2009; Unterseher et al.,
 and Baldrian, 2013). Our results suggest that
2013; Vo
r
skova
Mycosphaerella may play an important role as an early decomposer.
The composition of fungal module 4 also correlated with the activity of a hydrolytic enzyme important for C acquisition. This
module was dominated by Xylariales OTU3, Cylindrosympodium
sp.1 and Leotiomycetes OTU12, some of which are known to be
specialized biopolymer decomposers (Martnez et al., 2005; Nghi
et al., 2012; Schneider et al., 2012). Fungal modules 2 and 4 also
correlated signicantly with MnP activity, which was low during
the early decomposition stage. This nding reects the limited
ability of these fungi to produce MnP. In addition, the members of
Xylariales were reported to defend deadwood against secondary
basidiomycete saprophytes (Fukasawa et al., 2009). This may
explain the low levels of MnP activity observed during the early
decomposition of beech litter in this work.
During the later stage, only the composition of fungal module 7
correlated with studied enzyme activities (laccase and peroxidase
activities). This module consists primarily of Basidiomycota (78.1%)
which are represented by Clitocybe spp. and Ceratobasidium sp.
Clitocybe spp. are known to be efcient producers of peroxidases
and laccase (Steffen et al., 2000; Kellner et al., 2014). Interestingly,
the Fe concentration was the litter physicochemical parameter that
correlated most strongly with this module. Fe is a key element for
fungi because it is involved in many important metabolic processes
and the fungal ligninolytic peroxidases are heme-enzymes that
depend on Fe for their catalytic activity (Prescott et al., 1999; Ullrich
and Hofrichter, 2007).

W. Purahong et al. / Fungal Ecology 22 (2016) 106e114

We conclude that the total fungal communities associated with

European beech leaf litter decomposition are sub-structured into
different fungal modular communities. These modular communities differ in size, network structure, and taxonomical composition. Moreover, different modular communities showed contrasting
relationships with specic leaf litter physicochemical properties,
which may be explained by their different niche spaces and
nutrient requirements. Signicant correlations between the fungal
modular communities and the activities of individual enzymes
suggest that key ecological functions are provided by sub groups
(modular communities) of the total fungal community. Furthermore, these correlations reect the taxonomic and functional signicance of the different modular communities.
Acknowledgments
Our work was funded, in part, by contributing projects to the
DFG Priority Program 1374 on Infrastructure-Biodiversity Exploratories (BU 941/17-1, KR 3587/1-1, KR 3587/3-2, HO 1961/4-1, and
HO 1961/5-2). We thank the managers of the three exploratories,
Swen Renner, Sonja Gockel, Kerstin Wiesner, and Martin Gorke, for
their work in maintaining the plot and project infrastructure;
Simone Pfeiffer and Christiane Fischer for providing support
through the central ofce; Michael Owonibi for managing the
central database; and Markus Fischer, Eduard Linsenmair, Dominik
ller, Jens Nieschulze, Daniel Prati, Ingo Scho
ning, ErnstHessenmo
Detlef Schulze, Wolfgang W. Weisser, and the late Elisabeth Kalko
for their role in setting up the Biodiversity Exploratories project.
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.funeco.2016.04.009.
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