Diversity of wood-inhabiting fungi in woodpecker nest cavities in southern Poland

Abstract

Globally, tree-holes are important ecological component of forest and woodlands. Numerous microorganisms rely on cavities, both natural and those excavated by primary cavity nesting birds, mainly by woodpeckers, for their survival and reproduction. However, the fungi occurring in cavities are not well characterized. Specifically, very little is known about the fungal communities inhabiting the woodpecker nest cavities. Therefore, in this study, we investigated the fungal diversity of cavities in southern Poland. The samples were collected from freshly excavated woodpecker nest cavities using a nondestructive method (ND). The spatial distribution of fungal communities within the cavities was evaluated by sampling different parts of a single cavity using a destructive method (D). We detected 598 fungal isolates that included 64 species in three phyla and 16 orders using the ND method. Most of the fungi isolated from the cavities represented the phylum Ascomycota (73.9% of the isolates) with 11 orders, and Microascales was the predominant order (30% of the isolates). The most common species detected was Petriella musispora , which was isolated from 65% of the cavities. A total of 150 isolates (25%) were members of Basidiomycota, with Hymenochaetales being the dominant order (16% of the isolates). The basidiomycetous fungi were isolated from 55% of the cavities. Several taxa closely related to the pathogenic fungi and associated with secondary animal infections were detected in the wood of cavities. We identified different fungal communities in the three cavity parts using the D method. The cavity entrance had more number of species than the middle and bottom parts. The results of this study advanced our current knowledge on the mycobiota in woodpecker nest cavities and provided preliminary evidence for tree cavities being the hotspot for fungal diversity.

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Acta Mycologica
ORIGINAL RESEARCH PAPER
Diversity of wood-inhabiting fungi in
woodpecker nest cavities in southern
Poland
Robert Jankowiak1*, Michał Ciach2, Piotr Bilański3, Riikka
Linnakoski4
1 Department of Forest Pathology, Mycology and Tree Physiology, Institute of Forest Ecosystem
Protection, University of Agriculture in Krakow, 29 Listopada 46, 31-425 Krakow, Poland
2 Department of Forest Biodiversity, Institute of Forest Ecology and Silviculture, University of
Agriculture in Krakow, 29 Listopada 46, 31-425 Krakow, Poland
3 Department of Forest Protection, Entomology and Forest Climatology, Institute of Forest
Ecosystem Protection, University of Agriculture in Krakow, 29 Listopada 46, 31-425 Krakow,
Poland
4 Natural Resources Institute Finland (Luke), Latokartanonkaari 9, 00790 Helsinki, Finland
* Corresponding author. Email: rljankow@cyf-kr.edu.pl
Abstract
Globally, tree-holes are important ecological component of forest and woodlands.
Numerous microorganisms rely on cavities, both natural and those excavated by
primary cavity nesting birds, mainly by woodpeckers, for their survival and reproduc-
tion. However, the fungi occurring in cavities are not well characterized. Specically,
very little is known about the fungal communities inhabiting the woodpecker nest
cavities. erefore, in this study, we investigated the fungal diversity of cavities in
southern Poland. e samples were collected from freshly excavated woodpecker
nest cavities using a nondestructive method (ND). The spatial distribution of
fungal communities within the cavities was evaluated by sampling dierent parts
of a single cavity using a destructive method (D). We detected 598 fungal isolates
that included 64 species in three phyla and 16 orders using the ND method. Most
of the fungi isolated from the cavities represented the phylum Ascomycota (73.9%
of the isolates) with 11 orders, and Microascales was the predominant order (30%
of the isolates). e most common species detected was Petriella musispora, which
was isolated from 65% of the cavities. A total of 150 isolates (25%) were members of
Basidiomycota, with Hymenochaetales being the dominant order (16% of the isolates).
e basidiomycetous fungi were isolated from 55% of the cavities. Several taxa closely
related to the pathogenic fungi and associated with secondary animal infections
were detected in the wood of cavities. We identied dierent fungal communities
in the three cavity parts using the D method. e cavity entrance had more number
of species than the middle and bottom parts. e results of this study advanced our
current knowledge on the mycobiota in woodpecker nest cavities and provided
preliminary evidence for tree cavities being the hotspot for fungal diversity.
Keywords
Basidiomycetes; cavity; Microascales; wood-inhabiting fungi; wood-decay fungi;
woodpeckers; tree-hollow
Introduction
Globally, tree-holes are important ecological component of forest and woodlands.
Tree-holes are formed by three major mechanisms: natural formation, where the fungi
decompose the wood over time; excavation by birds, mainly by woodpeckers, which
are oen considered as habitat engineers that play a key role in forest ecosystems [1];
DOI: 10.5586/am.1126
Publication history
Received: 2019-03-15
Accepted: 2019-04-23
Published: 2019-06-28
Handling editor
Wojciech Pusz, Faculty of Life
Sciences and Technology,
Wrocław University of
Environmental and Life
Sciences, Poland
Authors’ contributions
RJ and MC designed the study;
RJ performed the phenotypic
and molecular characterization,
wrote the original draft; MC
collected samples, wrote the
original draft; PB performed
the phenotypic and molecular
characterization; RL edited the
original draft
Funding
This study was funded by
the National Science Center,
Poland (contract No. UMO-
510 2014/15/NZ9/00560). This
research was also supported
by statutory research activity
founds by Minister of Science
and Higher Education assigned
to Faculty of Forestry, University
of Agriculture in Krakow.
Competing interests
No competing interests have
been declared.
Copyright notice
© The Author(s) 2019. This is an
Open Access article distributed
under the terms of the
Creative Commons Attribution
License, which permits
redistribution, commercial and
noncommercial, provided that
the article is properly cited.
Citation
Jankowiak R, Ciach M, Bilański
P, Linnakoski R. Diversity of
wood-inhabiting fungi in
woodpecker nest cavities in
southern Poland. Acta Mycol.
2019;54(1):1126. https://doi.
org/10.5586/am.1126

2 of 20© The Author(s) 2019 Published by Po lish Botanical Socie ty Acta Mycol 54 (1):1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
wood-boring insects that attack and damage the bark and wood of trees [2]. More than
thousand vertebrate species across the globe rely heavily on tree cavities for reproduction
and roosting [3]. e cavities created by primary excavators are later used by a large
group of secondary cavity nesters, which are species that are unable to independently
excavate tree-holes [4].
Fungi, especially the wood-decay species, can assist avian species to excavate cavi-
ties in the stems and branches of trees by soening the wood [5–8]. Most Holarctic
woodpecker species are reported to select trees that are soened by fungi, including the
sections of tree bole that contain heart rot for cavity construction [6,7,9,10]. e trees
selected for cavity excavation by woodpeckers may be inhabited either by specic fungal
species [11] or by distinct host fungal communities [8,12]. Red-cockaded woodpecker
(Picoides borealis) prefers trees infected with the heart rot fungus, Porodaedalea pini
for cavity excavation [6,8]. Recently, Jusino et al. [13] reported that Picoides borealis
shares a symbiotic relationship with the fungi, where the fungi may help excavators by
soening the wood and the excavators may facilitate the fungal dispersal.
Woodpeckers excavate nest cavities in live trees, snags, dead parts of living trees, or
within the decaying limbs of living trees [10]. Most woodpeckers usually excavate a new
nest cavity each year. Additionally, most woodpecker species excavate cavity only 2–6
weeks before nesting. Cavity excavation in sowood (Populus spp. and Salix spp.) takes
about 2 weeks, while that in hardwood (Fagus spp. and Quercus spp.) takes about 3–4
weeks. However, large woodpecker species such as black woodpecker (Dryocopus mar-
tius) may excavate a cavity within 5–6 years and reuse it for several years [14,15].
Tree cavities oer a specic and more stable microclimate when compared to the
ambient conditions. e internal temperature of cavities change at a lower rate compared
to the outside temperature. Consequently, daily temperature extremes are reduced and
typically lag several hours behind the ambient temperature [16–18]. e mean daily
relative humidity in tree cavities is high (typically exceed 90%) and stable throughout
the day, which is in contrast to a much lower and highly uctuating ambient humidity
[19,20]. Compared to the majority of secondary-cavity nesting birds, woodpeckers
do not use any external materials to ll the nest cup and lay their eggs directly on the
cavity bottom, which only contains small wood debris, such as wood scrapes or rotten
wood fragments [21].
Generally, woodpeckers remove fecal material from the cavity while nesting [22,23].
Consequently, the nests of primary-cavity nesting birds remain fairly clean and rarely
have nonwooden organic material. However, during the breeding event, which includes
incubation, hatching, ospring feeding, and molting of adults, some small portions of
organic debris may be deposited and accumulated at the cavity bottom. Small fraction
of feces and other organic materials such as parts of eggshells, single feathers, or even
dead ospring could remain in the cavity bottom. e other unique trait of a bird nest,
including those located in cavities, is its thermal properties induced by the presence of
warm-blooded parents or nestlings [24], which promote the growth of thermophilus
fungi [25]. erefore, the specic climatic and nutritional conditions in the tree-cavities
may inuence the composition of fungal communities and promote ultra-rich and
specic fungal diversity.
Compared to the fungi inhabiting bark wounds on trees [26,27], the mycobiome of
tree cavities has been poorly studied. Most studies have reported a positive association
between wood-decay fungi and cavity excavating birds [3,6,7,10,28,29]. ese studies
have relied only on visual observation of fungal fruiting bodies. is technique could
lead to a poor measure of association because many fungi inhabit a tree for decades
without fruiting [30,31]. Recently, Jusino et al. [8] reported that specic fungal com-
munities inhabit the living pine cavity excavated by red-cockaded woodpeckers using
molecular methods.
Although several studies have explored the cavity ecology, very little is known on the
fungal composition of cavities excavated by woodpeckers. erefore, in this study we
identied the diverse of wood-inhabiting fungi in the cavities excavated by European
woodpeckers using DNA-based techniques. We excluded old cavities as their age and
the presence of secondary-nesting species might inuence the fungal community
composition and dynamics. erefore, only the new cavities excavated in a given year
were used in this study.

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Material and methods
Study site
e study was conducted in southern Poland in 2014–2015 at two study regions (Kra-
kow region 50°05' N, 19°55' E and Western Carpathians 49°40' N, 19°30' E). Krakow
region represents a human-modied landscape type. e area is characterized by a
broad urbanization gradient from a densely built-up city center to the suburbs with
a moderate number of buildings and from the scattered buildings, typical of a rural
landscape to farmlands with minor fraction of tree vegetation (urban greenery, orchards,
and woodlots). e Western Carpathians represent forest-dominated landscape type.
Various plant communities dominate, including the fertile Carpathian beech forest
(Dentario glandulosae-Fagetum), r-spruce forest (Abieti-Piceetum), and the upper
subalpine acidophilous Carpathian spruce forest (Plagiothecio-Piceetum). e climate
is temperate, transitional from maritime to continental. e mean annual tempera-
ture is 7°C (maximum 17°C in July, minimum −3°C in January) and the total annual
precipitation is 800 mm.
Collection of samples and fungal isolations
e study regions were surveyed from early spring to identify the breeding territories of
woodpeckers. We search for the cavities at the stage of excavation from these breeding
territories. is approach enables us to study only the cavities that were excavated in a
given breeding season and exclude the older cavities. is standardization was necessary
as the composition of fungal community may potentially change with time. Moreover,
cavities were sampled only if they were completed, successful broods were recorded,
and the ospring had le the nest. We used these criteria as the interior of unnished
cavities may have a dierent microclimate than the completed cavities, which may in
turn inuence the fungal community. Moreover, the lack of breeding event may aect
thermal and trophic conditions within a cavity as incubating parents and sitting o-
spring may potentially increase the thermal properties of a cavity interior. Additionally,
continuous feeding may potentially provide external nutrients to a cavity interior.
Upon completion of breeding event, the wood samples from the cavity were col-
lected. To capture maximal fungal communities in the cavity, we analyzed 20 cavities
(C1 to C20), representing seven woodpecker species, sampled from eight tree species
(Tab. 1). We collected the samples from the interior of woodpecker cavities using a
nondestructive method (ND) as described previously by Jusino et al. [32]. e wood
samples were collected using a specially designed tool, which was a 40-cm-long steel
pipe with 2-cm diameter. e edge at one end was fabricated to form a chisel-like
blade. e sample collection did not cause extensive destruction of the cavity, as the
sample was a puck wood of ca. 2 × 2 cm size. From each cavity, three wood fragments
were collected from the central part of the hole for the isolation of fungi. e sampling
tool was ame-sterilized between each sampling location. As some of the cavities were
located very high in the trees, trained and certied arborists surveyed the trees using
ladders or by climbing.
During 2014–2015, fungi were isolated from 60 wood fragments collected from
the interior of cavities. e wood fragments were placed individually in sterile plastic
containers and stored at 4°C for 1–2 days until fungal isolation. Each wood fragment
was surface sterilized in 96% ethanol for 15 s. e fragment was dried using sterile lter
paper and the wood surface was removed using a sterile scalpel. Each wood fragment
was divided into 12 pieces (4 × 4 mm) and placed in 9-cm Petri dishes containing the
following culture medium: malt extract agar [MEA; 20 g malt extract (Biocorp Polska
Sp. z.o.o., Poland), 20 g agar (Biocorp Polska Sp. z.o.o., Poland), 1,000 mL sterile water,
and 50 mg/L tetracycline (Polfa S.A., Poland)] to isolate the general mycobiota; malt
extract agar with cycloheximide [CMEA, MEA with 200 mg/L cycloheximide (Sigma-
Aldrich, St. Louis, Co. LLC.)] to isolate Ophiostoma/Leptographium spp. [33]; or malt
extract agar with benomyl [BMEA, MEA with 8 mg/L benomyl (Sigma-Aldrich)] to
isolate basidiomycetes [34]. We used 720 wood pieces for fungal culturing.

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e fungal communities inhabiting the woodpecker nest cavity were detected using
a total destructive method (D). In fall 2015, a European beech (Fagus sylvatica) tree
with a cavity excavated by white-backed woodpecker (Dendrocopos leucotos) (C16)
was felled aer the breeding event. A section including the whole cavity (150 cm long)
was excised out from the tree and transported to the laboratory. e next day, the sec-
tion was cut along the center of the trunk axis to expose the interior of the cavity. e
wood fragments were disinfected for approximately 15 s using 96% ethyl alcohol and
dried on lter paper. e fungi were isolated from the wood surrounding the cavity
entrance, and from the wood layers underneath the entrance to a depth of 5, 15, and
25 cm till the cavity bottom. e wood fragments (about 4 × 4 mm), were cut using
a sterile chisel, and placed on the culture medium. We collected 360 wood fragments
for isolating the fungus.
Fungal identication
e cultures were incubated at room temperature (22–25°C) in the dark for 16 weeks.
e cultures were puried by transferring small pieces of mycelium or spore masses
from the individual colonies to fresh MEA. e puried cultures were grouped into
morphotypes based on the morphological characteristics of asexual and sexual struc-
tures, and anverse and reverse colony color reported in the literature [35–41] using a
Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan) tted with an Invenio
5S digital camera (DeltaPix, Maalov, Denmark) and linked to the COOLVIEW 1.6.0
soware (Precoptic, Warsaw, Poland). Depending on the size of the morphological
group, one to nine representative strains of each morphotype were further subjected
to molecular identication based on internal transcribed spacer (ITS) and 28S large
ribosomal subunit (LSU) sequence comparison. We selected 146 isolates for molecular
identication (Tab. S2). Additionally, the protein coding genes (β-tubulin or the elonga-
tion factor 1-α) were sequenced to identify the Ophiostomatales order, Fusarium spp.,
Neonectria spp., and Trichoderma spp.
e isolates were subjected to DNA extraction, PCR amplication, and sequencing
following the methods used by Jankowiak et al. [42]. See Tab. S1 for primers used to
sequence ITS region (ITSI-5.8S-ITSII), LSU, and elongation factor 1-α (TEF 1-α).
e sequences (Tab. S2) were deposited in the GenBank of the National Center for
Biotechnology Information (NCBI) database. e sequences were aligned with those
available in GenBank using the BLASTn algorithm. Only a 99–100% match with a
reliable source (ex-type sequences, published taxonomic studies) was accepted as proof
of identication. e sequences were considered to belong to the same species when
sequences exhibited ≥99.0% similarity with the ITS or LSU region (400–500 bp). Ad-
ditionally, the β-tubulin or the TEF 1-α sequences were compared with the sequences
available in GenBank to identify Fusarium spp. and Trichoderma spp. All the sequenced
isolates are deposited in the Culture Collection of Fungi of the Laboratory of Depart-
ment of Forest Pathology, Mycology and Tree Physiology, University of Agriculture in
Krakow, Poland (Tab. S2).
For the identication of Microascales, the most dominant order in this study, the
individual data sets for the ITS and LSU gene regions were used for phylogenetic analysis.
e data sets were compiled and edited in MEGA ver. 6.06 [43]. Sequence alignments
were performed using the online version of MAFFT ver. 7 [44]. e ITS and LSU data
sets were aligned using the E-INS-i strategy with a 200PAM/κ=2 scoring matrix, a gap
opening penalty of 1.53, and an oset value of 0.00. For maximum likelihood (ML)
and Bayesian (BI) analyses, the best-t substitution models for each data set were
estimated using the corrected Akaike information criterion (AICc) in jModelTest ver.
2.1.10 [45,46]. Phylogenetic analyses were performed for each of the data sets using
two dierent methods: ML and BI. ML searches were conducted in PhyML 3.0 [47]
using the Montpelier online server (http://www.atgc-montpellier.fr/phyml/) with 1,000
bootstrap replicates. BI analyses based on a Markov chain Monte Carlo (MCMC) were
performed using MrBayes ver. 3.1.2 [48]. e MCMC chains were run for 10 million
generations using the best-t model. e trees were sampled every 100 generations,
resulting in 100,000 trees from both the runs. e burn-in value for each dataset was
determined in TRACER ver. 1.4.1 [49].

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Tab. 1 Characterization of woodpecker nest cavities in Poland.
No. of
cavity Species of woodpecker Species of cavity-tree
Tre e
height
(m)
Tre e
DBH
(cm)
Height
of cavity
location (m)
Entrance
exposition Tree health
Cavity
location
Fruiting
body
present Habitat type
Date of
collection
Latitude
(N)
Longitude
(E)
C1 Picus viridis Salix fragilis 19 36 2.0 SE Live Stem -Riparian woodlot 2014-07-16 49°58'20°11'
C2 Picus viridis Salix fragilis 19 36 3.0 SE Live Stem -Riparian woodlot 2014-07-16 49°58'20°11'
C3 Dendrocopos medius Malus domestica 7 24 1.5 SE Live Dead branch - Orchard 2014-07-22 50°26'20°09'
C4 Picus viridis Salix fragilis 24 38 8.0 NW Live Stem +Riparian woodlot 2014-10-15 49°43'19°29'
C5 Picus viridis Salix fragilis 24 38 8.0 NW Live Stem +Riparian woodlot 2014-10-15 49°43'19°29'
C6 Dendrocopos major Alnus incana 22 32 4.0 SW Live Stem -Riparian forest 2014-10-18 49°24'20°45'
C7 Dendrocopos leucotos Salix fragilis 15 35 8.0 E Live Stem -Riparian woodlot 2014-10-18 49°24'20°45'
C8 Dendrocopos major Fagus sylvatica 4 45 3.0 SE Snag Stem +Riparian forest 2014-11-27 49°38'19°39'
C9 Picoides tridactylus Picea abies 25 40 4.0 S Snag Stem - Coniferous forest 2014-11-28 49°35'19°31'
C10 Dendrocopos major Fagus sylvatica 11 25 8.0 SE Snag Stem + Mixed forest 2014-11-28 49°36'19°31'
C11 Dryocopus martius Abies alba 45 87 18.0 SE Live Stem - Coniferous forest 2014-12-04 49°34'19°35'
C12 Dendrocopos leucotos Fagus sylvatica 17 38 13.0 S Live Stem + Deciduous forest 2014-12-04 49°38'19°39'
C13 Dendrocopos major Acer pseudoplatanus 12 50 6.0 E Live Stem + Mixed forest 2014-11-28 49°36'19°28'
C14 Picus viridis Salix fragilis 17 35 4.5 E Live Stem +Riparian woodlot 2015-11-17 49°42'19°27'
C15 Dendrocopos major Fagus sylvatica 32 48 6.0 NE Live Stem - Deciduous forest 2015-11-17 49°36'19°31'
C16 Dendrocopos leucotos Fagus sylvatica 11 34 8.0 E Snag Stem + Deciduous forest 2015-11-17 49°36'19°31'
C17 Picoides tridactylus Picea abies 35 72 9.0 SE Live Stem - Coniferous forest 2015-11-17 49°35'19°31'
C18 Dendrocopos minor Prunus domestica 4 26 3.0 NE Live Dead branch + Orchard 2015-11-22 49°43'19°05'
C19 Dendrocopos medius Salix fragilis 15 45 6.0 SW Live Live branch + City park 2015-12-19 49°59'19°57'
C20 Dendrocopos major Salix f ragilis 22 38 7.0 S Live Stem -Riparian woodlot 2015-12-19 50°01'19°58'

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Statistical analyses
e Shannon [50] and Simpson [51] diversity indices were estimated for each cavity
(DM) and each cavity site (D). e fungal dominance was determined by Camargo’s
index (1/S), where S represents species richness. A species was dened as dominant if
Pi > 1/S, where Pi is the relative abundance of species i, which is dened as the number
of competing species present in the community [52].
e chi-square test was performed to evaluate the dierence among the proportions,
followed by the Marascuilo procedure for pairwise comparison of the proportions, using
StatTools.net soware (http://www.statstodo.com/). ese procedures were performed
to determine the dierence in the frequency of a fungus among the cavity sites.
A principal component analysis (PCA) was used to understand the correlation
between the abundance of fungal species in the woodpecker species and the dierent
tree species. e data were log transformed prior to the analysis. is statistical analysis
was conducted using PAST 3.18 [53].
Results
Collection of isolates and fungal identication
We obtained 742 fungal isolates from 1,080 wood fragments of woodpecker nest cavities
using two sampling techniques. e isolates included 182 Basidiomycota, 554 Ascomy-
cotina, and six Mucoromycotina (Tab. 2 and Ta b. 3 ). e isolates were separated into
52 morphotypes based on the preliminary morphological investigation. As the initial
morphological survey of the isolated cultures and ITS sequence data revealed that our
morphotypic criteria were not stringent, the morphotypes were grouped into 69 species
based on the ITS and other gene sequence analysis (Tab. S2).
e ITS and LSU sequence analyses within the order Microascales revealed that 43
isolates resided in two major phylogenetic clades: Graphiaceae and Microascaceae (Fig. 1
and Fig. 2). Within Graphiaceae, two isolates named as Graphium sp. 1 were unknown
species that are closely related to Graphium penicillioides, while four other isolates named
as Graphium sp. 2 were phylogenetically related to Graphium madagascariense (Fig. 1
and Fig. 2). Within Microascaceae, the ITS and LSU trees identied Parascedosporium
putredinis (three isolates), Petriella musispora (nine isolates), Petriella guttulata (ve
isolates), and Petriella sordida (one isolate). Additionally, four unidentied isolates that
are closely related to Lophotrichus meti (Lophotrichus sp.) were also identied (Fig. 1
and Fig. 2). e ITS and LSU sequence analysis revealed that some isolates resided
in the Scopulariopsis and Acaulium genera. Among them, six isolates were identied
as Scopulariopsis candida, two isolates were closely related to Scopulariopsis soppii
(Scopulariopsis cf. soppii), three isolates were identied as Acaulium albonigrescens, and
two isolates, named as Acaulium sp. represented species that were closely related to
Acaulium acremonium. is family was also represented by Cephalotrichum stemonitis
and Wardomyces inatus (Fig. 1 and Fig. 2).
Diversity of fungal species isolated from dierent cavities by ND method
Among the 720 wood pieces (collected from woodpecker nest cavities) used for fungal
culturing, we obtained fungal growth from 418 (58.1%) wood pieces. Among these 418
wood pieces, we obtained between one and three dierent fungal cultures from each
wood piece. In total, we obtained 598 cultures. We did not observe any fungal growth
from the samples obtained from the cavity excavated by the great-spotted woodpecker
(Dendrocopos major) on sycamore (Acer pseudoplatanus) (C13). e 598 fungal isolates
included 64 fungal species that were assigned to three phyla, and 16 orders. Within
the phylum Basidiomycota, we isolated members belonging to the orders Agaricales,
Hymenochaetales, and Polyporales. Within the phylum Mucoromycotina, we isolated
the members belonging to the orders Mortierellales and Mucorales. Most of the fungi
isolated from the cavities represented the phylum Ascomycota (73.9% of total). ey

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Tab. 2 Wood-colonizing fungi and their diversity in dierent woodpecker nest cavities.
Tax o n
Number of isolates obtained*
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20
Total
isolated
Basidiomycota
Fomes fomentarius 6 10 A16 A
Fomitiopora punctata 18 A18 A
Inonotus obliquus 2 19 A21 A
Ischnoderma benzoinum 14 A14 A
Phellinus alni 18 A18 A
Phellinus igniarius 18 A21 A39 A
Phellinus cf. igniarius 1 1
Trametes suaveolens 1 1
Trametes versicolor 22 A22 A
Ascomycota: Microascales
Acaulium sp. 1 1
Cephalotrichum stemonitis 2 1 3
Graphium sp. 1 2 1 3
Graphium sp. 2 2 2 4
Lophotrichus sp. 6 A6 A12 A24 A
Parascedosporium putredinis 2 2 4
Petriella guttulata 1 2 5 17 A25 A
Petriella musispora 2 7 A12 A18 A18 A3 1 5 16 A12 A1 3 1 99 A
Petriella sordida 1 1 2
Scopulariopsis candida 3 10 A13 A
Scopulariopsis cf. soppii 1 1 2
Wardomyces inatus 1 1 2
Ascomycota: other
Alternaria arborescens 1 1
Alternaria sp. 1 1
Arthrobotrys oligospora 4 4
Arthrobotrys sp. 6 1 7
Cadophora malorum 1 1
Chaetomium angustispirale 1 1

8 of 20© The Author(s) 2019 Published by Po lish Botanical Socie ty Acta Mycol 54 (1):1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
Tab . 2 Continued
Tax o n
Number of isolates obtained*
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20
Total
isolated
Chaetomium sp. 1 1 1 1 3
Chaetomium sp. 2 4 1 10 A1 12 A6 A1 35 AA
Chaetomium sp. 3 1 15 A7 1 2 26 A
Clonostachys rosea 2 1 3
Clonostachys sp. 1 9 A10 A
Cosmospora viridescens 1 1 2
Cosmospora sp. 1 1 2
Duddingtonia agrans 1 1
Epicoccum nigrum 1 1
Fusarium solani s. stricto 1 1
Fusarium sp. 1 (FSSC complex) 1 1 1 3
Fusarium sp. 2 (FSSC complex) 1 1
Fusarium sp. 3 (FSSC complex) 6 6
Galactomyces geotrichum 1 1
Geotrichum sp. 5 A5
Humicola fuscoatra 1 1
Neobulgaria sp. 1 1 1 5 A8
Neonectria sp. 1 1
Ophiostoma exuosum 14 A14 A
Paracremonium s p. 10 A12 A6 A7 A35 A
Penicillium brevicompactum 1 6 4 11 A
Phialemonium s p. 1 1
Phialophora sp. 7 1 8
Phoma sp. 1 1
Podospora sp. 1 1
Pseudocosmospora vilior 1 1
Pseudogymnoascus pannorum 3 2 5 A10 A
Sporothrix sp. 3 5 A8
Trichocladium cf. asperum 1 1
Trichoderma harzianum 1 12 A1 1 10 A25 A
Trichoderma longibrachiatum 1 1

9 of 20© The Author(s) 2019 Published by Po lish Botanical Socie ty Acta Mycol 54 (1):1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
were distributed in 11 orders, with Microascales
being the most abundant order (30% of the iso-
lates) (Tab. 2). e genus Petriella was the most
abundant (21%), which was isolated from 80%
of the woodpecker nest cavities. Additionally,
the predominant species was from the Hypoc-
reales order (15 species). We oen isolated the
members of Microascales on benomyl MEA
medium, which promotes the growth of basid-
iomycetes. A total of 150 isolates (25% of total)
were classied to the Basidiomycota division,
mainly to the Hymenochaetales order (16% of
the isolates). e basidiomycetous fungi were
isolated from 55% of the cavities. e members of
the Mucoromycotina were sparsely represented
(Tab. 2).
e predominant species was Petriella mu-
sispora (16.5% of the total number of fungal
isolates), which was isolated from 65% of the
cavities (Tab. 2 ). e other species were rarely
isolated from the cavities, although Chaetomium
sp. 2, Paracremonium sp., and Phellinus igniarius
comprised 5.8–6.5% of the total isolates and
detected in 10%, 30%, and 20% of the cavities,
respectively (Ta b. 2).
e number of isolates obtained from cavi-
ties excavated by woodpeckers ranged from 0
(C13) to 55 (C2). e species richness (S) for
all cavities was 64 and ranged from 0 (C13)
to 15 (C2). e fungal diversity values varied
widely among the cavities (Tab. 2). e fungal
community associated with the cavities C20 (D
= 0.83) and C14 (D = 0.82) exhibited the highest
diversity, while that associated with the cavities
C13 (D = 0.00) and C17 (D = 0.37) exhibited the
lowest diversity (Tab. 2).
e PCA analysis separated the samples along
the rst axis (22.3%) mainly based on the preva-
lence of Petriella musispora, Paracremonium sp.,
Lophotrichus sp., and Phellinus igniarius in the
cavities of Salix fragilis excavated by Picus viridis
woodpecker. e fungal communities associated
with the cavity excavated by Dendrocopos medius,
Dryocopus martius, and Piocoides tridactylus
were the most distant from those associated
with the cavity excavated by Picus viridis. e
second PCA axis revealed a variability of 19.6%
and separated the fungal communities associated
with the cavity excavated by woodpeckers that
generally use Salix fragilis from those associ-
ated with the cavity excavated by woodpeck-
ers that use other tree species. We observed a
strong correlation between Chaetomium sp. 2
and samples of Dendrocopos medius nest cavity,
while Chaetomium sp. 3 was strongly correlated
with the samples of Picoides tridactylus nest
cavity (Fig. 3).
Tab . 2 Continued
Tax o n
Number of isolates obtained*
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20
Total
isolated
Trichoderma paraviridescens 6 A6
Trichoderma trixiae 1 11 A12 A
Mucoromycotina
Mortierella cf. hyalina 1 1
Mortierella zychae 1 2 3
Mucor hiemalis 1 1
Mucor piriformis 1 1
No. of total fungal isolates 43 55 46 35 34 53 33 3 2 32 28 38 0 53 31 28 13 30 8 33 598
Species richness (S) 10 15 6 10 6 8 4 3 2 4 4 7 0 11 13 5 3 4 3 11 64
Camargo’s index (1/S) 0.100 0.067 0.167 0.100 0.167 0.125 0.250 0.333 0.500 0.250 0.250 0.143 0 0.091 0.077 0.200 0.333 0.250 0.333 0.091 0.016
Simpson’s index (D) 0.252 0.217 0.325 0.202 0.356 0.259 0.418 0.333 0.500 0.315 0.344 0.274 0 0.173 0.320 0.510 0.621 0.389 0.594 0.170 0.055
Simpson’s index of diversity [SID;
SID = (1 − D)]
0.749 0.784 0.675 0.798 0.644 0.741 0.582 0.667 0.500 0.686 0.656 0.726 0 0.827 0.681 0.490 0.379 0.611 0.406 0.830 0.945
Shannon index of diversity (H) 1.726 1.979 1.334 1.859 1.279 1.575 1.021 1.099 0.693 1.273 1.210 1.529 0 1.959 1.791 0.950 0.687 1.066 0.736 2.025 3.392
* Results obtained overall from selective and nonselective media. A – dominant species.

10 of 20© The Author(s) 2019 Published by Polis h Botanical Societ y Acta Mycol 54 (1):1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
0.2
P. guttulata MF782711
C. brevis tipita tum CBS 157.57 LN850984
M. campaniformis CBS 138126 LM652391
Lophotric hus sp . MF782700
Microascus cirros us CBS 217.31 LM652400
P. putredinis MF782706
Pseudoscopulariopsis schumacheri CBS 435.86 LM652455
S. candida MF782727
P. in term ediu s CBS 217.32 LM652450
A. acremonium MUCL 8274 LM652457
P. musispora MF782713
L. pro lifica ns CBS 467.74 AY882370
Lophotric hus sp . MF782702
A. albonigrescens MF782688
A. albonigrescens MF782690
Lophotric hus sp . MF782701
W. infla tus CBS 367.62 LN850994
Lomentospora pro lifica ns CBS 452.89 HQ185322
G. pseudormiticum CMW5611 AY148185
Graphiums p. 2 MF782699
W. inflatus MF782730
S. dehoogiiCBS 117393 KT008553
G. pen icillioid es CBS 102631 AB038431
ScedosporiumaurantiacumCBS 116910 HQ231818
P. a ter CBS 400.34 LM652447
C. microsporum CBS 523.63 LN850967
P. nid icola CBS 197.61 LM652451
Scopulariopsis flava CBS 207.61 LM652493
G. madagascariense CMW30626 GQ200616
S. candida MUCL 40743 LM652484
P. musispora MF782715
P. putredinis CBS 108.10 HQ185347
Petriella sordida MF782721
Acaulium albonigrescens IHEM 18560 LM652389
S. candida MF782722
G. s implex CBS 546.69 LM652379
Graphiums p. 1 MF782695
Wardomyces humico la CBS 369.62 LN850993
S. candida MF782723
C. s temonitis CBS 103.19 LN850951
S. soppii UAMH 9169 LM652495
P. guttulata MF782708
Graphiums p. 2 MF782697
Petriella guttulata MF782707
W. hu micola CBS 487.66 LM652497
W. anomalus CBS 299.61 LN850992
L. fimet i CBS 129.78 AY879799
Lophotric hus sp . MF782703
P. pu tredin is CBS 127.84 AY228113
Graphiumbasitruncatum JCM 8083 AB038425
G. adansoniaeCMW30617 GQ200610
S. boydiiCBS 330.93 AY863196
C. verruc isporu m CBS 187.78 LN850986
Graphiums p. 2 MF782698
M. gra cilis CBS 369.70 LM652412
S. candida MF782726
G. fimbriaspo rumCMW5610 AY148176
A. albonigrescens MF782689
Acaulium sp. MF782691
C. dendrocephalum CBS 528.85 LN850966
Petriella seti fera CBS 559.80 AY882356
P. guttulataCBS 362.61 AY879800
S. candida MF782724
Fairmania singularis CBS 505.66 LN850988
P. musispora MF782712
S. cordiaeCBS 138129 LM652491
P. sordida CBS 297.58 AY882359
G. carbonariumCMW12420 FJ434979
C. cylin dricu m
CBS 448.51 LN850964
Pithoascus stov eri CBS 176.71 LM652453
W. ova lis CBS 234.66 LN850996
P. guttulata MF782709
S. asperula CBS 401.34 LM652463
W. giganteus CBS 746.69 LM652411
P. musispora MF782720
P. musispora MF782716
Lophotrichus cf. ampullus UAMH 11809 KM580494
Scopulariopsis cf.soppii MF782729
P. guttulata MF782710
P. s etif era CBS 265.64 AY882349
P. musispora MF782718
S. brevica ulis MUCL 40726 LM652465
S. candida MUCL 9007 LM652481
Graphiums p. 1 MF782694
P.sordida CBS 301.66 AY882354
P. exs ertus CBS 583.75 LM652448
Gams ia aggregata CBS 251.69 LM652378
M. macrosporus CBS 662.71 LM652423
P. s etif era CBS 347.64 AY882346
S. candida MF782725
Wardomycopsis inopinataFMR 10305 LM652498
Cephalotrichum hinnuleumCBS 289.66 LN850985
P. sordida CBS 385.87 AY882345
Scopulariopsis cf. soppii MF782728
C. purpureofuscum UAMH 9209 LN850971
P. musispora MF782719
G. laric is CMW5601 AY148183
Parascedosporiumputredinis CBS 102083 HQ185348
C. s temon itis MF782693
Graphiums p. 2 MF782696
G. euwallaceae UCRCFU12 KF540219
Petriella musispora MF782714
P. musispora MF782717
W. pulvinatus CBS 112.65 LN850997
A. cavia rifo rme CBS 536.87 LM652392
C. nanum CBS 191.61 LN850969
P. putredinis MF782705
G. ju mulu CBS 139898 KR476722
P. putredinis MF782704
M. alveo laris U THS C 0 5 -3416 LM652381
Acaulium sp. MF782692
Ps eu da lles ch eria ellips oid ea CBS 418.73 AJ888426
C. columnareCBS 159.66 LN850963
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ITS
Fig. 1 Phylogram obtained from maximum likelihood (ML) analysis of the internal transcribed spacer (ITS) region. e phylogram
shows the placement of isolates obtained from woodpecker nest cavities representing the order Microascales. Sequences obtained
during this study are presented in boldface. Bootstrap values >75% for ML and posterior probabilities >75% obtained from Bayesian
(BI) analysis are presented at nodes as follows: ML/BI. * Bootstrap values <75%.

11 of 20© The Author(s) 2019 Published by Polish Bota nical Society Act a Mycol 54(1) :1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
0.2
Lophotrichussp . MF782 701
G. penicillioides AF2 2250 0 C15 05
Wardomycopsishumicola LM652554 CBS 487.66
S. candidaH G38 0458 MUC L 407 43
CephalotrichumcylindricumL N851011 CB S 448 .51
Scedosporium aur antiacum E F15132 6 CBS 11691 0
S. brevicaulisH G380 440 MUC L 407 26
C. dendrocephalum LN851 013 C BS 5 28.8 5
Petriella musisporaM F78 272 0
Scopulariopsi s can dida LM6 52546 MUC L 9007
Gam sia aggregataL M6525 00 CB S 251. 69
Parascedosporium putredinisM F782 706
Pithoascus at er LM6 52526 C BS 4 00.34
C. stemonitis L N85095 2 CBS 103. 19
S. candida MF 78 2723
A. albonigrescens MF782688
P. intermedius LM65 2529 C BS 21 7.32
A. caviariformeL N85 1005 C BS 53 6.87
Petriella sor di da AF 27549 7 CBS 25 8.31
W. ovalis L N85105 0 CBS 234. 66
Graphium sp. 2 MF 782 697
P. musispora M F 782715
P. sordida MF7 82 721
S. fla va H G38 0464 C BS 20 7.61
C. nanum LN851 016 C BS 191 .61
Graphium sp. 2 MF782 696
C. verrucisporum L N851 033 CB S 1 87.78
C. purpureofuscum LN851 018 UA MH 92 09
G. fimbriasporum KM49 5388 C MW560 5
P. guttulata MF782707
P. musispora M F 782719
P. musispora M F 782714
Wardomyces giganteus L N851045 CBS 7 46.6 9
S. asperula H G38 0465 CB S 40 1.34
GraphiumpseudormiticumK M49539 0 CMW503
A. albonigrescens MF 7 826 90
G. basitruncatum H Q85774 7 R-461 3
C. brevistipitatum L N851031 C BS 1 57.57
S. candida MF 78 272 6
S. soppii LM6 52552 UAM H 91 69
Scopulariopsis cf. soppiiMF782 728
M. campaniformis HG 38049 5 CBS 13 8126
Wardomyces humicol aL N851 046 CB S 369.6 2
S. candida MF 78 2725
P. musispora M F 782713
Graphium sp. 1 MF 782 695
Lophotrichussp . M F782 700
W. inopinataL N851 054 FM R 10305
P. stov eri LM652532 CBS 176.71
Pseudallescheriae ll ipso idea EF151323 CBS 418.73
Graphium sp. 2 MF 782 699
Microascus mac rosp orus LM652517 CBS 662.71
Petriella guttulata AF27 5534 C BS 36 2.61
P. guttulata MF782711
P. guttulata MF782708
P. musispora M F 782716
P. musispora M F 782718
Acaulium sp. MF 7 826 91
P. guttulata MF782709
W. inflatus MF 782 730
S. cordiae HG 380499 CBS 1 38129
P. sordida AY2810 99 CC FC 16 2159a
Lophotrichussp . M F782 703
Pseudoscopulariopsis sc h umacher i L M652534 CBS 435 .86
Graphium sp. 1 MF 782 694
M. cirrosus HG 3804 29 CBS 217. 31
Lophotrichus plumbescens L C146 745 NB RC 308 64
W. inflatusL N851048 CBS 3 67.6 2
S. candida M F78 272 4
Acaulium acr emo ni um LN8510 03 MUCL 8409
Graphium sp. 2 MF 782 698
Scopulariopsis cf. soppiiMF782 729
Lophotrichussp . MF782 702
L. prolificans E F15 1329 C BS 45 2.89
P. putredinis M F78 270 5
P. exsert us LM6 525 27 CB S 583. 75
P. musispora M F 782712
M. gracilisH G380 467 CBS 3 69.7 0
C. stemonitis L N85101 9 CBS 180. 35
G. fabi for me KM4 95387 CMW3 0626
C. microsporum L N851014 CBS 5 23.6 3
P. setifer a AY882377 CBS 559.8
M. alveolarisH G380 482 UTH SC 04-1 534
Acaulium sp. MF 7 826 92
S.candida MF 782 727
C. hinnuleum LN8510 32 CB S 289. 66
A. albonigrescens MF782689
Fairmania singularis L N8510 36 CB S 505. 66
P. putredinis M F78 270 4
Parascedosporiumt ecto nae EF 1513 32 CBS 127.84
S. candida MF 78 2722
Petriellopsisafricana EF 15133 1 CBS 311.72
G. jumulu KR4 76757 CP C 246 39
P. guttulata MF782710
Graphiumsp . D Q26858 6
C. stemoni tis MF 782693
P. musispora M F 782717
P. nidicola L M6525 30 CBS 197. 61
C. asperulum L N85100 7 CB S 582. 71
C. columnareL N8 51010 CB S 159. 66
C. gorgonifer LN 85102 5 UAMH 3 585
Lophotrichus fi me t i AF02 7672 C BS 12 9.78
P. musi spo ra AF275537 CBS 745.69
Lomentospora prol ificans EF1 51330 C BS 4 67.74
S. boydii AY882372 CBS 330.93
W. pulvinatus L N85105 1 CB S 112. 65
A. albonigrescens LM 65250 2 IHEM 1 8560
G. columbinaLM 65250 1 CBS 54 6.69
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LSU
Fig. 2 Phylogram obtained from maximum likelihood (ML) analysis of the 28S large ribosomal subunit (LSU) region. e phylogram
shows the placement of isolates representing the order Microascales. Sequences obtained during this study are presented in boldface.
Bootstrap values >75% for ML and posterior probabilities >75% obtained from Bayesian (BI) analysis are presented at nodes as fol-
lows: ML/BI. * Bootstrap values <75%.

12 of 20© The Author(s) 2019 Published by Polish Bo tanical Society Ac ta Mycol 54 (1):1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
Spatial distribution of fungal communities within the cavity
excavated by white-backed woodpecker (D method)
We obtained 144 fungal isolates from wood samples collected from dierent sites of
the cavity. We isolated 30 fungal species: ve species of Basidiomycota, six species of
Microascales, and 19 species of other fungi order. Some of the fungi isolated in this
study, particularly Basidiomycota and Microascales, were not detected with the ND
method. Among the basidiomycetes, we could not detect Bjerkandera adusta, Cylin-
drobasidium evolvens, Ischnoderma benzoinum, and Trametes versicolor in the cavities
using the ND method. Additionally, two species of the Microascales order (Acaulium
albonigrescens and Acaulium sp.) were not detected in the same cavity using the ND
method. e most dominant species was Inonotus obliquus with an average isolation
frequency of 20.8%. e second-most dominant species was Acaulium albonigrescens,
which was isolated from 16.7% of the wood samples. Scopulariopsis candida was also
commonly isolated (15.8%) (Tab. 3).
We detected considerable variation in the fungal diversity upon comparison of the
data from the ve dierent sites of the cavity. e highest species richness was found
directly in the entrance and under the entrance of the cavity, followed by the central
parts of the cavity. e lowest species richness and diversity were at the cavity bottom
(Tab. 3). Most basidiomycete species were found at the cavity entrance (Bjerkandera
adusta, Ischnoderma benzoinum, Trametes versicolor), although they were isolated at
low frequencies (Tab. 3 ). e cavity entrance was dominated by various ascomycetes,
particularly by Phoma sp., Pseudocosmospora rogersonii, Pseudogymnoascus pannorum,
and Trichoderma spp. (Tab. 3 ). e wood underneath the entrance was most com-
monly colonized by the basidiomycete species Inonotus obliquus, which was isolated
from 62.3% of the wood samples. Besides Inonotus obliquus, Petriella spp. and Phoma
sp. were also frequently detected from the wood of the cavity (Tab. 3 ). e number of
P. v.
P. v.
D.me.
P. v. P. v .
D.m.
D.l. D .m.
P. t . D.m.
D.ma.
D.l.
D.m.
P. v.
D.m.
D.l.
P. t .
D.mi.
D.me.
D.m.
Petriella musispora
Phellinus igniarius
Chaetomium sp. 2
Paracremonium sp.
Chaetomium sp. 3
Petriella guttulata
Trichoderma harzianum
Lophotrichus sp.
Trametes versicolor
Inonotus obliquus
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
-0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2
PC2 (19.6%)
PC1 (22.3%)
Fig. 3 Biplot of the principal components analysis (PCA) with log-transformed frequencies of fungal species, woodpecker
species (D.l. – Dendrocopos leucotos; D.m. – Dendrocopos major; D.ma. – Dryocopus martius; D.me. – Dendrocopos medius;
D.mi. – Dendrocopos minor; P.t. – Picoides tridactylus; P.v. – Picus viridis) and cavity trees (diamond – Abies alba; triangle –
Acer pseudoplatanus; square – Alnus incana; ll square – Fagus sylvatica; plus – Malus domestica; star – Picea abies; x – Prunus
domestica; dot – Salix fragilis). Principal components were calculated from a covariance matrix for those fungal species for
which the total sample size exceeded 20 isolates.

13 of 20© The Author(s) 2019 Published by Polish B otanical Society Ac ta Mycol 54 (1):1126
Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
Tab. 3 Number of isolates and frequency of fungi (in parentheses*) isolated from the Fagus sylvatica cavity excavated by Dendrocopos
leucotos.
Fungal species
Cavity site
Tot a lEntrance Under entrance Central Under central Bottom
Basidiomycota
Bjerkandera adusta 2(8.3) Aa0 a0 a0 a0 a2(1.7)
Cylindrobasidium evolvens 0 a0 a0 a0 a2(8.3) a2(1.7)
Inonotus obliquus 0 c15(62.5) Aa8(33.3) Aab 2(8.3) bc 0 c25(20.8) A
Ischnoderma benzoinum 1(4.2) a0 a0 a0 a0 a1(0.8)
Trametes versicolor 2(8.3) A0 a0 a0 a0 a2(1.7)
Ascomycota: Microascales
Acaulium albonigrescens 0 b0 b0 b11(45.8) Aa9(37.5) Aa20(16.7) A
Acaulium sp. 0 a0 a0 a0 a1(4.2) a1(0.8)
Cephalotrichum stemonitis 0 a1(4.2) a0 a0 a0 a1(0.8)
Petriella guttulata 0 a2(8.3) a0 a0 a0 a2(1.7)
Petriella musispora 0 a2(8.3) a0 a0 a0 a2(1.7)
Scopulariopsis candida 0 b0 b0 b16(66.7) Aa3(12.5) b19(15.8) A
Ascomycota: other and Mucoromycotina
Alternaria arborescens 1(4.2) a1(4.2) a0 a0 a0 a2(1.7)
Alternaria sp. 2(8.3) A0 a0 a0 a0 a2(1.7)
Cadophora malorum 0 a0 a0 a1(4.2) a0 a1(0.8)
Chaetomium sp. 1 0 a0 a1(4.2) a0 a0 a1(0.8)
Chaetomium sp. 2 0 a1(4.2) a0 a0 a0 a1(0.8)
Chaetomium sp. 3 0 a0 a0 a2(8.3) a0 a2(1.7)
Epicoccum nigrum 0 a1(4.2) a0 a0 a0 a1(0.8)
Humicola fuscoatra 0 a0 a1(4.2) a0 a0 a1(0.8)
Mortierella cf. hyalina 1(4.2) a0 a0 a1(4.2) a0 a2(1.7)
Mucor hiemalis 1(4.2) a0 a0 a0 a0 a1(0.8)
Neonectria sp. 0 a1(4.2) a0 a0 a0 a1(0.8)
Penicillium brevicompactum 2(8.3) A0 a0 a0 a0 a2(1.7)
Phialemonium s p. 0 a1(4.2) a0 a0 a0 a1(0.8)
Phoma sp. 7(29.2) Aa7(29.2) Aa0 b0 b0 b14(11.7) A
Pseudocosmospora rogersonii 4(16.7) Aa1(4.2) ab 1(4.2) ab 0 b0 b6(5.0) A
Pseudogymnoascus pannorum 8(33.3) Aa0 b7(29.2) Aa0 b0 b15(12.5) A
Trichoderma longibrachiatum 0 a1(4.2) a0 a0 a0 a1(0.8)
Trichoderma olivascens 7(29.2) Aa0 a0 a0 a0 a7(5.8) A
Trichoderma trixiae 6(25.0) Aa0 a0 a0 a0 a6(5.0) A
No. of total fungal isolates 44 34 18 33 15 144
Species richness (S) 13 12 5 6 4 30
Camargo’s index (1/S) 0.08 0.08 0.20 0.17 0.25 0.03
Simpson’s index (D) 0.12 0.25 0.36 0.36 0.42 0.10
Simpson’s index of diversity [SID; SID
= (1 − D)]
0.88 0.75 0.64 0.64 0.58 0.90
Shannon index of diversity (H) 2.29 1.85 1.21 1.27 1.08 2.73
Percentage of sterile fragments MEA/
CMEA/BMEA
38/100/83 50/100/38 71/100/67 25/100/92 67/100/92 48/100/55
No. of investigated samples MEA/
CMEA/BMEA
24/24/24 24/24/24 24/24/24 24/24/24 24/24/24 120/120/120
* e isolation frequency = (No. of hole fragments, from which a particular fungus was isolated / Total No. of hole fragments) × 100. e isolation frequency was calculated
using isolation results obtained individually from selective media: CMEA for Ophiostoma/Leptographium spp., BMEA for basidomyceteous fungi, and MEA for other
fungi. A – dominant species. Within rows, values with dierent subscript (small letters) are statistically dierent (p < 0.05) according to post hoc multiproportions test
using the Marascuilo procedure.

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fungal species in the central part of the cavity was considerably lower than that in the
entrance and underneath the entrance of the cavity. e wood of the central parts of
the cavity was most frequently colonized by Inonotus obliquus and Pseudogymnoascus
pannorum (Tab . 3 ). e microascalean fungi was abundant in the wood surrounding the
lower part of the cavity along with the two species of basidiomycetes, Cylindrobasidium
evolvens and Inonotus obliquus (Tab. 3). e central and the bottom parts of the cavity
were most commonly colonized by Acaulium albonigrescens, which was isolated from
45.8% and 37.5% of samples, respectively. Scopulariopsis candida was also commonly
obtained from the wood located underneath the central part of the cavity (Tab. 3).
e fungal diversity values varied widely along the cavity sites. e fungal community
associated with the cavity entrance exhibited the highest diversity (D = 0.88), while that
associated with the cavity bottom exhibited the lowest diversity (D = 0.58). e highest
species-richness values were associated with the fungal community at the entrance (S
= 13 species), while the lowest species-richness values were associated with the fungal
community at the bottom of the cavity (S = 4 species) (Tab. 3 ).
Discussion
The results of this study provide preliminary evidence that the woodpecker nest
cavity serves as a fungal diversity hotspot in the temperate forests. In this study, we
surveyed seven woodpecker species cavities in eight dierent host tree species. Our
data revealed the presence of complex fungal communities in this niche. We isolated
69 fungal species representing at least 12 orders of Ascomycota, Basidiomycota, and
Mucoromycotina. Ascomycota was the predominant phylum, comprising 74% of
the isolates and represented by 51 species. Among the 51 species, Petriella musispora
was the most common species. Another group of fungi that commonly colonize the
woodpecker nest cavities were basidiomycetes, which were represented by nine taxa.
Both ascomycetes and basidiomycetes are frequently detected in the dead wood and
are wood-decaying or saprophytic fungi [30,54–57].
Our data revealed that woodpeckers preferentially excavate cavities in trees having
decay caused by basidiomycetes, although these fungi were present in only half of the
studied cavities. e correlation between wood-decaying fungi and woodpeckers has
been demonstrated in several studies [6–8]. Our investigation revealed that European
woodpeckers excavate nest cavities in wood exhibiting clear signs of decay. Recently,
Jusino et al. [13] indicated that interactions between fungi and primary woodpecker
species are likely to be more complex. e study demonstrated that red-cockaded
woodpecker may facilitate the dispersal of Basidiomycota, which helps the excavator
by soening the wood. e basidiomycetous species found in our study are common
rot fungi inhabiting various hardwood species in Poland [58,59].
e cavities were also commonly colonized by members of the Microascales order
belonging to eight genera within the Graphiaceae family (Graphium) and the Micro-
ascaceae family (Acaulium, Cephalotrichum, Lophotrichus, Parascedosporium, Petriella,
Scopulariopsis, and Wardomyces). e Microascaceae family includes many ecologically
important species, comprising saprobic fungi mostly found in air, soil, plant material,
and urban environment. Some species of the Microascaceae are opportunistic patho-
gens of animals, including humans [60–65]. e members of Microascaceae isolated
from the woodpecker nest cavity represented species with dierent ecological roles
and interactions. Among them, the Graphium species represent wound-inhabiting
saprobes. e vectors of Graphium species are insects [66–68] and it is likely that the
two unknown species found in this study could have been introduced into the cavities
by dierent insects. Moreover, woodpeckers could transfer the Graphium species into
the cavity. As these birds forage commonly on bark and wood-boring beetles during
the breeding season, they may transfer insects (which carry fungal spores) as a food
for the ospring. While feeding some food items could accidentally fall out of the bill,
or spores could be transmitted through excrements into the cavity.
In this study, the dominant species detected in the cavities was Petriella musispora.
is species was strongly associated with the Salix fragilis cavities excavated by Picus
viridis. e presence of Petriella musispora and the two other representatives of the

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Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
genus Petriella (Petriella guttulata and Petriella sordida) may be transferred through
the bird digestive system and feces. e members of this genus tend to grow on dung
or in environments enriched by animal feces [69]. Although the parent woodpeckers
remove the ospring’s feces from cavities [22,23], our study demonstrated that nestlings
probably live in environment contaminated by bird excrements, which can be utilized
by Petriella fungi. It is possible that other Microascales, including Lophotrichus, Micro-
ascus, and Scopulariopsis may also utilize bird feces in cavities. However, excrements
are a source of potential pathogenic microorganism, which may play an important
role in breeding success and survival [70]. Some species of the Microascales can cause
diseases in young nestlings [41,63,64,71]. Some species of Scopulariopsis, including
Scopulariopsis candida, are known as opportunistic pathogens, mainly causing supercial
tissue infections, and are associated with nondermatophytic onychomycosis [37,64].
Additionally, Lophotrichus and Petriella species have been reported from supercial
tissue [63] and can infect humans and other animals. Our study demonstrated that
potentially pathogenic fungi can grow in the cavities and probably survive until the
following breeding season. It appears that these fungi can be particularly dangerous in
secondary cavity nesting birds. However, many aspects of the woodpeckers–pathogenic
fungi association are poorly understood, which require further studies.
Interestingly, we detected the occurrence of two members of the Ophiostomatales
in the cavities. Due to their morphological and ecological similarities, fungi from the
orders Ophiostomatales and Microascales have been designated as ophiostomatoid
fungi [72]. One of the species reported in this study, Ophiostoma exuosum, has been
reported in only one previous study in Poland. Jankowiak [73] detected this species
from the galleries of Ips typographus on Norway spruce (Picea abies). e presence of
Ophiostoma exuosum in silver r (Abies alba) is reported here for the rst time, sug-
gesting that this species may have a wider host distribution. A second ophiostomatoid
species was tentatively identied as an unknown Sporothrix species, which is closely
related to Sporothrix polyporicola.
e fungal isolations using a destructive method (D) in this study were limited to a
single cavity, and thus, provide only preliminary information. Additionally, we revealed
some variations in the spatial composition of fungal communities in the cavity excavated
by white-backed woodpecker. In our study, Alternaria, Penicillium, Mucor, Mortierella,
and Trichoderma species, known as typical invaders of wood [74,75], were mainly detected
in the wood surrounding the entrance to the cavity. e wood surrounding the entrance
to the cavity was also dominated by basidiomycetous species, such as Bjerkandera
adusta, Ischnoderma benzoinum, and Trametes versicolor, which did not occur in deeper
parts of the cavity. is suggested that these decay fungi are not involved in the wood
decay of the cavity. e wood was also oen colonized by Penicillium brevicompactum,
Pseudocosmospora rogersonii, Pseudogymnoascus pannorum, and Trichoderma spp. e
occurrence of Pseudocosmospora rogersonii was unexpected, as it is reported to be a
parasite of only Eutypella sp. (Ascomycota: Xylariales) [76]. In this study, we reported
the presence of Pseudocosmospora rogersonii for the rst time outside of the USA. e
middle part of the cavity was dominated by Inonotus obliquus. is indicates that this
fungus is mainly responsible for the wood decay of the cavity. Interestingly, in this part
of the cavity, the wood was also oen colonized by microascalean species. e dominant
species that colonized the deepest parts of the cavity were Acaulium albonigrescens and
Scopulariopsis candida. Acaulium albonigrescens is a well-circumscribed species detected
in soil, dung, and wood in Scandinavia, northern North America, and Japan [60,61].
We detected this species in Central Europe for the rst time. e abundant presence
of Acaulium albonigrescens and Scopulariopsis candida may be associated with specic
and a nutrient-rich microhabitat in the bottom of the cavity.
e dierence in the composition of fungal communities was highly dependent
on the sampling method. We extensively studied only one cavity using the D method
(C16). However, the number of fungal species obtained in this method was markedly
higher than that obtained by the ND method. We detected 30 fungal species using the
D method, while only ve species were detected by the ND method. We believe that
the ND sampling is extremely imprecise to detect the mycobiota of woodpecker nest
cavities. Our study clearly determined the spatial composition of fungal mycobiota in
the woodpecker nest cavities. e use of a special tool to excise a single sample from
the interior of cavity resulted in the omission of majority part of the cavity. Further

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Jankowiak et al . / Fungi associated with woo dpecker nest cavitie s in Poland
comparative studies are required to determine the ecacy of determining the fungal
composition of wood cavities between nondestructive and destructive sampling
method.
PCA analysis revealed that there was no correlation between the woodpecker
species and the fungal community structure in the cavities, although species such as
Petriella musispora, Paracremonium sp., Lophotrichus sp., and Phellinus igniarius were
strongly associated with the Salix fragilis cavities excavated by Picus viridis. e fungal
communities associated with the cavities excavated by Dendrocopos medius, Dryocopus
martius, and Piocoides tridactylus were distant from those associated with the cavities
excavated by Picus viridis. However, we evaluated only a small number of cavities in
each category (woodpecker species / tree species) in this study. erefore, we could not
evaluate various aspects of woodpecker nest cavity–fungi association, which requires
further studies.
In conclusion, the results of this study provided insight into the fungal communities
associated with woodpecker nest cavities in Poland. Some of the detected species are
reported to be wood-decay fungi, but several species remain unidentied. Our results
indicated that Microascales and Basidiomycota dominate the wood-inhabiting fungal
communities of woodpecker nest cavities. Additionally, our study clearly demonstrated
that the fungi exhibit a dierential spatial distribution within the woodpecker nest
cavity.
Acknowledgments
We wish to express our gratitude to Arkadiusz Fröhlich, Tomasz Baziak, Mateusz Albrycht, and
Katarzyna Paciora for their help with the woodpecker survey and cavity searching. We wish
to sincerely thank Marcin Trybała, whose tree-climbing experience was invaluable during the
cavity sampling.
Supplementary material
e following supplementary material for this article is available at http://pbsociety.org.pl/
journals/index.php/am/rt/suppFiles/am.1126/0:
Tab. S1 Information on loci used in the phylogenetic analyses.
Tab. S2 Fungi isolated from woodpecker holes.
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