10.6092/issn.2531-7342/14474
The medicinal tinder polypore Fomes fomentarius is widely distributed in all floristic regions of Armenia on different woody substrates. The phylogenetic analysis of Armenian collections using ITS barcoding revealed that it is taxonomically complex species represented by sublineages A2 and B2 (in ratio 1:1) of European phylogenetic lineages A and B, which correspond to two cryptic sympatric species F. fomentarius sensu stricto and F. inzengae, respectively. These species are phylogenetically almost equidistant from Fomes fasciatus by the level of nucleotide divergence (6.75% and 7.17%, respectively). Nucleotide divergence between these two species is 1.85% which does not exceed the average level of intraspecific ITS variability in basidiomycetes fungi (3.33%). It is suggesting that F. fomentarius s.s. and F. inzengae are possibly not taxonomically separate species, but sympatric cryptic subspecies of F. fomentarius sensu lato. Both taxa significantly differ by their ecology and distribution: F. fomentarius s.s. is mainly found on Betula spp. trees and widespread in temperate forests, while F. inzengae has been recorded on Carpinus sp., Fagus sp., Populus sp. and other deciduous trees in subtropical latitudes. In Armenia, F. fomentarius s.s. was found on Fagus sp. and Quercus sp., while F. inzengae - on Carpinus sp., Juglans sp., Fagus sp., Populus sp., and Salix sp. trees. Although the species rank of Mediterranean subtropical species F. inzengae remains disputable it has been originally described for the mycobiota of Armenia.
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Research article

           The phylogenetic analysis of Armenian collections of
              medicinal tinder polypore Fomes fomentarius
                    (Agaricomycetes, Polyporaceae)

              Susanna M. Badalyan1, Elena V. Zhuykova2, Victor A. Mukhin2,3

1
  Laboratory of Fungal Biology and Biotechnology, Yerevan State University, Yerevan, Armenia
2
  Department of Vegetation and Mycobiota Biodiversity, Institute of Plant and Animal Ecology, the Ural Branch of the
Russian Academy of Sciences, Yekaterinburg, Russia; e.zhuykova@list.ru
3
  Department of Biodiversity and Bioecology, Ural Federal University, Yekaterinburg, Russia; V.A.Mukhin@urfu.ru

Corresponding author e-mail: s.badalyan@ysu.am


Article info
Received 28/02/2022; accepted 15/04/2022
http://doi.org/10.6092/issn.2531-7342/14474

Abstract
The medicinal tinder polypore Fomes fomentarius is widely distributed in all floristic regions of Armenia on
different woody substrates. The phylogenetic analysis of Armenian collections using ITS barcoding revealed
that it is taxonomically complex species represented by sublineages A2 and B2 (in ratio 1:1) of European
phylogenetic lineages A and B, which correspond to two cryptic sympatric species F. fomentarius sensu stricto
and F. inzengae, respectively. These species are phylogenetically almost equidistant from Fomes fasciatus by
the level of nucleotide divergence (6.75% and 7.17%, respectively). Nucleotide divergence between these two
species is 1.85% which does not exceed the average level of intraspecific ITS variability in basidiomycetes
fungi (3.33%). It is suggesting that F. fomentarius s.s. and F. inzengae are possibly not taxonomically separate
species, but sympatric cryptic subspecies of F. fomentarius sensu lato. Both taxa significantly differ by their
ecology and distribution: F. fomentarius s.s. is mainly found on Betula spp. trees and widespread in temperate
forests, while F. inzengae has been recorded on Carpinus sp., Fagus sp., Populus sp. and other deciduous
trees in subtropical latitudes. In Armenia, F. fomentarius s.s. was found on Fagus sp. and Quercus sp., while
F. inzengae - on Carpinus sp., Juglans sp., Fagus sp., Populus sp., and Salix sp. trees. Although the species
rank of Mediterranean subtropical species F. inzengae remains disputable it has been originally described for
the mycobiota of Armenia.

Keywords
Armenia, cryptic, Fomes inzengae, Fomes fomentarius sensu stricto, ITS barcoding, polypore, sympatric


Introduction

The tinder polypore Fomes fomentarius (L.) Fr. (Polyporaceae, Agaricomycetes) is a very common
bracket fungus largely distributed and growing nearly all habitat zones in the forests of Eurasia and North
America (Gilbertson and Ryvarden, 1986; Ryvarden and Gilbertson, 1993; Mukhin and Votintseva,
2002). As a parasite, Fomes fomentarius sensu lato infects a very large number of deciduous, rarely
coniferous trees and continues to habitat them as a white-rot saprotrophic fungus (Větrovský et al.,
2011; Pristaš et al., 2013). In Eurasia, this fungus is predominantly reported on the remnants of Betula

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2022 The Authors. This work is licensed under CC BY 4.0
Badalyan, Zhuykova, Mukhin                                      Italian Journal of Mycology 51 (2022): 23-33

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sp. and Fagus sp. trees (Bondartsev, 1953; Jülich, 1984; Ryvarden and Gilbertson, 1993; Mukhin,
1993; Bondartseva, 1998), while in North America on Betula sp. and Alnus sp. trees (Gilbertson
and Ryvarden, 1986; Farr et al., 1989; McCormick et al., 2013; Gáper and Gáperová, 2014). Wide
ecological distribution and high occurrence of F. fomentarius s.l. makes it one of the main destructors
of deciduous tree debris and, accordingly, CO2 emitters in forest ecosystems (Mukhin et al., 2021).
    Tinder polypore is also regarded as a producer of several pharmacologically active compounds,
such as phenolics, flavonoids, polysaccharides, triterpenoids and ketones with antibacterial, antifungal,
antiviral, anticancer, immunomodulatory and antioxidant effects (Pеintner et al., 1998; Grienke et al.,
2014; Badalyan and Shahbazyan, 2015; Badalyan and Gharibyan, 2016; Gáper et al., 2016; Mukhin
et al., 2018; Badalyan et al., 2019a). However, there is insufficient fundamental knowledge on species
structure and genetic variability, pathways of biosynthesis of their bioactive molecules and genes
behind for biotechnological cultivation of medicinal mushrooms, including F. fomentarius s.l., to
develop biotech products and mycopharmaceuticals (Gáper et al., 2016; Kües and Badalyan, 2017).
    It has been described that F. fomentarius s.l. is genetically inhomogeneous species and consists
of several phylogenetic lineages (A, B) and sublineages (A1, A2, B1, B2) (Júdová et al., 2012;
McCormick et al., 2013; Pristaš et al., 2013; Gáper et al., 2016; Mukhin et al., 2018; Náplavová et
al., 2020). Moreover, phylogenetic lineages A and B were suggested as two sympatric cryptic species
(Júdová et al., 2012; Pristaš et al., 2013). The area of distribution of sublineage B2 is limited in the
European subcontinent (England, Italy, Latvia, Slovakia, and Slovenia) with an Eastern border in the
Urals (Mukhin et al., 2018), whereas sublineage A2 has a wider distribution and is found not only
in Europe (Gáper et al., 2016; Náplavová et al., 2020), but also in the Urals (border between the
European and Asian subcontinents) and Southern Siberia (Altai, Western Sayan and Baikal region)
(Mukhin et al., 2018). In Europe, F. fomentarius s.l. is considered a complex species, consisting of
two cryptic species: F. fomentarius sensu stricto and F. inzengae (Ces. & De Not.) Cooke (Pеintner et
al., 2019). Phylogenetic heterogeneity of this species has been also described in China (Gáper et al.,
2016; Mukhin et al., 2018; Pеintner et al., 2019).
    The Armenian highlands is mountainous region of Western Asia, one of biogeographically
interesting and important regions of speciation. F. fomentarius s.l. is one of the most widespread
polypore described in all floristic regions of Armenia (Takhtajyan, 1954) on Carpinus sp., Fagus sp.,
Quercus sp., and Populus sp. (Melik-Khachatryan and Martirosyan, 1971), Fraxinus sp., Junglans
regia L., Salix alba L., Ulmus sp. trees and other broad leaves substrates (Badalyan and Gharibyan,
2008; 2016; 2017). The current phylogenetic analysis of F. fomentarius s.l. in the territory of Armenia
has been carried out originally. This paper discusses the species structure and intraspecific genetic
variability of Armenian collections of medicinal tinder polypore fungus.

Materials and Methods

Fungal material
This work was performed on 11 dikaryotic strains of F. fomentarius s.l. from the fungal culture
collection of the Laboratory of Fungal Biology and Biotechnology of the Yerevan State University
(FCC-YSU, Yerevan, Armenia) (Badalyan and Gharibyan, 2017). The 11 original strains were isolated
from F. fomentarius s.l. basidiocarps collected from living and dead deciduous trees (Carpinus sp.,
Fagus sp., Juglans regia, Populus sp., Quercus sp., Salix alba) in 6 localities of Aparan, Ijevan, and
Yerevan floristic regions of Armenia during 2002-2016 (Table 1). The morphological identification and
species diagnosis of basidiocarps were performed using traditional taxonomic keys and microscopy
(Ryvarden and Gilbertson, 1993) (Fig. 1).



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   For molecular studies the cultures were grown on 1.5% malt-extract agar in Petri dishes during
10 days at 25 °C. The mycelial samples with agar were dried at room temperature (22 ± 2 °C) for
further usage in DNA isolation and phylogenetic analysis. The published sequences KJ857249 and
KJ857248 of strains Ff/1 and Ffa/2 were taken from the GenBank (Badalyan et al., 2015).




 Fig. 1 - Fomes inzengae: (a, b) basidiocarps (21×14 cm) in Ff/24 and (c) hymenium in Ff/15, (g) basidiospores in
 Ff/15 (× 600) and (h) in Ff/26 isolates (Size bar correspond 10 μm). Fomes fomentarius s.s.: (d, e) basidiocarps, (f)
 hymenium, (i) basidium and (j) basidiospores in Ff/18 isolates (× 600).


DNA isolation and PCR amplification

The extraction of DNA and PCR amplification with visualization were performed in the Laboratory
of Molecular Research of Plants and Fungi of the Institute of Natural Sciences and Mathematics of
the Ural Federal University (Yekaterinburg, Russia). DNA isolation was performed using a DNeasy
Plant Mini Kit (QIAGEN) without RNase treatment dissolving DNA in 100 μL Tris-EDTA buffer.
   Two primers, ITS1F and ITS4b (Gardes and Bruns, 1993), were used to amplify ITS regions
of nuclear ribosomal DNA by PCR which was carried out in the C1000 Touch Amplifier (Bio-Rad
Laboratories) in 25 µL reaction mixture containing deionized water, 1× buffer solution with 2.5 µM
MgCl2, 0.2 µM dNTP, 0.1 µM of each primer, 0.08 u µl-1 Taq DNA polymerase (Evrogen Joint Stock
Company) and 5 µl of DNA solution. The PCR program included a pre-denaturation at 95 °C for 5

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min, 35 cycles of a denaturation at 95 °C for 30 sec, an annealing at 55 °C for 45 sec, an elongation
at 72 °C for 45 sec, and a final elongation at 72 °C for 10 min. The reactions were monitored by
electrophoresis in a 1.2% agarose Tris-borate-EDTA gel stained with ethidium bromide. The length
of fragments was monitored by a 100 bp DNA ladder (Invitrogen). The gel was visualized with a Gel
Doc XR+ Gel Documentation System (Bio-Rad Laboratories).
   The enzymatic purification or purification by PCR products electrophoresis and sequencing in both
directions were performed at Sintol LLC (Moscow, Russia). The primary data processing was carried
out using Sequencing Analysis Software v. 5.3.1 (Applied Biosystems): Finch TV v. 1.4.0 (Geoprisa,
Inc.) and MEGA v. 7.0.18 (Kumar et al., 2016). Identification of sequences was performed with a
search for structurally similar sequences in the GenBank database using the Blast algorithm (blast.
ncbi.nlm.nih.gov). The obtained sequences were submitted to the GenBank database and accession
numbers were received (Table 1).

Table 1 - List of studied dikaryotic collections of Fomes fomentarius s.l.
                                                                                                     Genetically
 Catalogue               Location/Date of
               Isolate                             Host tree        GenBank code       Sublineage    identified
 number                  collection
                                                                                                     species
                                                                                                     F. fomentarius
 5201          Ff/1      Yerevan, 2002; YR         Quercus sp.      KJ857249           A2
                                                                                                     s.s.
                                                                                                     F. fomentarius
 5204          Ffa/2     Berdavan, 2011; IJ        Fagus sp.        KJ857248           A2
                                                                                                     s.s.

 5206          Ff/8      Berdavan, 2014; IJ        J. regia         OL583665           B2            F. inzengae

                                                                                                     F. fomentarius
 5207          Ff/11     Berdavan, 2014; IJ        Fagus sp.        OL583666           A2
                                                                                                     s.s.
                                                                                                     F. fomentarius
 5208          Ff/12     Berdavan, 2014; IJ        Fagus sp.        OL583667           A2
                                                                                                     s.s.

 5215          Ff/15     Solak, 2015; AP           S. alba stump    OL583668           B2            F. inzengae

 5216          Ff/16     Solak, 2015; AP           S. alba stump    OL583669           B2            F. inzengae

                                                   Deciduous                                         F. fomentarius
 5218          Ff/18     Marmarik, 2015; AP                         OL583670           A2
                                                   tree                                              s.s.

 5221          Ff/24     Baghanis, 2016; IJ        Fagus sp.        OL583671           B2            F. inzengae

 5222          Ff/25     Yerevan, 2016; YR         Populus sp.      OL583672           B2            F. inzengae

 5223          Ff/26     Dilijan, 2016; IJ         Carpinus sp.     OL583673           B2            F. inzengae

(AP) – Aparan, (IJ) – Ijevan, (YR) - Yerevan floristic regions of Armenia (Takhtajyan, 1954)

Phylogenetic and statistical analysis

Two groups of sequences were selected from the GenBank database (www.ncbi.nlm.nih.gov/genbank/)
for phylogenetic analysis. The first, reference for F. fomentarius s.s. and F. inzengae according to
Peintner et al. (2019). From the sequences belonging to the F. fomentarius s.s. and F. inzengae,


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epitypes and isolates close to them were selected. The clade “Fomes sp. Asia” and “F. fomentarius II”
were fully included in the final array. The analysis included a total of 23 ITS sequences. The second
group included sequences reference for sublineages A2 and B2. They were generated from 20 ITS1-
5.8S-ITS2 sequences according to Gáper et al. (2016) and Mukhin et al. (2018) and deposited in the
GenBank. The sequences from Armenia were added to reference groups and aligned using the Muscle
Algorithm with manual verification of obtained results. The Fomes fasciatus (Sw.) Cooke sequence
was selected as an outgroup.
    Phylogenetic trees were constructed using the maximum likelihood method based on the Hasegawa-
Kishino-Yano model (Hasegawa et al., 1985). All positions with site coverage less than 90% were
excluded. The trees are drawn to scale with branch lengths in the same units as the evolutionary
distances used to derive the phylogenetic tree. The evolutionary analysis was carried out in MEGA7
(Kumar et al., 2016). The database for calculating DNA polymorphism included 11 sequences obtained
in this study and 5 F. fasciatus sequences (Peintner et al., 2019). The alignment was performed by the
Muscle Algorithm with manual verification of results. The number of single nucleotide substitutions,
deletions and insertions in pairwise sequence comparisons of both individual lineages and between
them was manually calculated. The nucleotide diversity (π) and divergence (average number of
nucleotide substitutions per site between species, Dxy) were calculated using the DnaSP v. 6.12 (Rozas
et al., 2017).

Results and Discussion

The phylogenetic analysis of Armenian collections showed that 5 out of 11 isolates are included in
the cluster with reference sequences for A2, and 6 in the cluster with the reference sequences for
sublineages B2 (Fig. 2). Thus, the phylogenetic sublineages A2 and B2 of F. fomentarius s.l. are
distributed in 1:1 ratio in all studied floristic regions in Armenia.
    The Armenian isolates that cluster with sublineage A2 reference sequences are combined at the
same time into one group with reference sequences for F. fomentarius s.s., and isolates cluster with
sublineage B2 reference sequences are included in the same group with the reference ones for F.
inzengae (Figs 2‒3). Thus, in Armenia both phylogenetic sublineages are represented, described for
the tinder fungus in Europe and considered by Pеintner et al. (2019) as two cryptic sympatric species:
F. fomentarius s.s. and F. inzengae (Ces. & De Not.) Cooke. They are the very close cryptic species
regarding to basidiocarp morphology. However, basidiocarps of F. inzengae differ from F. fomentarius
s.s. by smaller hymenial pores (0.31 mm vs 0.36 mm in F. fomentarius s.s.), therefore by their large
number (31-34 pores / cm vs 27-30 pores / cm in F. fomentarius s.s.). Fomes inzengae also differs by
smaller size of basidiospores (9-12.5 × 3-4 μm vs 13.5-18 × 4.5-6.5 μm in F. fomentarius s.s.) and
thicker skeletal hyphae (3.2-6.9 µm) compared to F. fomentarius s.s. (3.0-6.4 µm).
    F. inzengae has been observed in Great Britain, Italy, Slovakia, Slovenia, Switzerland, France,
Iran, and China. However, it is widely distributed in the forests of subtropical latitudes of the
Mediterranean region (Peintner et al., 2019). The genus Fomes is represented only by F. inzengae
species in the Iberian Peninsula (Garrido-Benavent et al., 2020). Moreover, 35 out of 36 DNA isolates
obtained from F. fomentarius s.l. basidiocarps collected in Greece, Italy, Spain, and Portugal belong
to the phylogenetic lineage B (F. inzengae), and only one from Northern Greece – to sublineage A2
(F. fomentarius s.s.) (Náplavová et al., 2020).
    Previous studies have shown that all Fomes strains grow well at temperatures 25-30 °C and do
not show any significant differences, particularly at 25 °C. However, F. inzengae (sublineage B2)
have more than 30 °C optimal growth temperature range and grew significantly faster at higher
temperature, whereas mycelia of F. fomentarius s.s. (sublineage A2) a fungus of northern, temperate

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Fig. 2 - Phylogenetic tree of Fomes fomentarius s.l. isolates from Armenia and reference for phylogenetic sublineages A2
and B2 based on 521 positions of ITS sequences. The bootstrap values are greater than 50% next to the nodes.

latitudes, grow faster at lower temperatures (10 and 20 °C) (Peintner et al., 2019). The southern
distribution of F. inzengae and its adaptation to warm and dry climate conditions are also evidenced
by substrate spectrum (Carpinus sp., Castanea sp., Quercus sp., Platanus sp., Populus sp., and rarely
Cerasium sp. and Abies sp. trees) and form of basidiocarps with increased water absorption ability
from the air (Peintner et al., 2019). A distinctive feature of substrate spectrum of F. fomentarius s.s. is
the presence of Betula sp. tree which is absent in the substrate spectrum of F. inzengae (Mukhin et al.,
2018). In Europe, the southern boundary of F. fomentarius s.s. (sublineage A2) distribution is possibly
determined by the corresponding distribution boundary of Betula pendula Roth (Náplavová et al.,
2020). The characteristics of substrate spectra and preferences provided by F. fomentarius s.s. and
F. inzengae are the possibility of sympatric existence where their ranges overlap. In most locations
of Central Europe, phylogenetic sublineages A2 (F. fomentarius s.s.) and B2 (F. inzengae) occur
simultaneously on substrates different to sympatric species (Júdová et al., 2012; Peintner et al., 2019).
    According to our data, these two taxa are similarly distributed in Aparan, Ijevan and Yerevan
floristic regions of Armenia. The studied collections, identified as F. inzengae, have been isolated

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Fig. 3 - Phylogenetic tree of Fomes fomentarius s.l. isolates from Armenia and reference for F. fomentarius s.s. and F.
inzengae based on 516 positions of ITS sequences. The bootstrap values are above 50% next to the nodes.
from basidiocarps growing on Carpinus sp., Juglans sp., Fagus sp., Populus sp., and Salix sp. trees,
while F. fomentarius s.s. on Fagus sp. – one of the host trees in Europe (Peintner et al., 2019) and
Quercus sp. (Table 1).
    The sympatric and cryptic characters of F. fomentarius s.s. and F. inzengae are beyond doubt, but
their species rank, in our opinion, is disputable. The phylogenetic divergences between Armenian
isolates of F. fomentarius s.s. and F. inzengae species and F. fasciatus species constitute 35-37 bp
(6.75%) and 36-38 bp (7.17%), respectively. The genetic divergence between F. fomentarius s.s. and
F. inzengae is significantly smaller (9-11 bp or 1.85% of nucleotide substitutions per site on average)
which is significantly higher compared to individual differences in the nucleotide diversity of F.
fomentarius s.s. and F. inzengae from Armenia (0˗0.158%) but does not exceed the average level of
intraspecific ITS variability for basidiomycetes fungi (3.33%) (Nilsson et al., 2008).
    Similar data were reported by Dresch et al. (2015) and Peintner et al. (2019). The authors of this
study have revealed genetic differences between F. fasciatus, F. fomentarius s.s. and F. inzengae in
Europe is 41-62 bp, between F. fomentarius s.s. and F. inzengae is 9-18 bp, and between individual
strains of F. inzengae and F. fomentarius s.s. are 0-1 bp and 0-3 bp, respectively. In our opinion, these


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data show the absence of phylogenetic differences between Armenian isolates of F. fomentarius s.s.
and F. inzengae at the species level and, may therefore, represent cryptic subspecies of one taxonomic
species F. fomentarius s.l.
    The cultural studies of dikaryotic Armenian collections support data of current phylogenetic
analysis (Badalyan et al., 2019b). The results showed that F. fomentarius s.s. strains grow in up to
35 °C (only on inoculum) with an optimum growth temperature 25 °C (3.0–4.4 mm d-1 and 2.1–4.2
mm d-1 on MEA - Malt Extract Agar - and PDA - Potato Dextrose Agar -, respectively), meanwhile F.
inzengae strains tolerate temperature 35 ºC and above (up to 40 °C, only on inoculum) with optimum
30 °C and higher growth rate (4.8-6.0 mm d-1 and 4.0-5.3 mm d-1, on MEA and PDA, respectively)
(Badalyan et al., 2019b). This corresponds to similar data reported by Peintner et al. (2019), according
to which the highest growth rate of F. inzengae isolates was 5.50 mm d-1 and of F. fomentarius s.s. –
4.25 mm d-1 on 3% MEA at 30 °C. Peintner et al. (2019) have also shown that all Fomes sp. strains
grow well at temperatures 25-30 °C and do not show any significant differences, particularly at 25 °C.
However, F. inzengae (sublineage B2) have more than 30 °C optimal growth temperature range and
grew significantly faster at higher temperature, whereas mycelia of F. fomentarius s.s. (sublineage
A2) a fungus of northern, temperate latitudes, grow faster at lower temperatures (10 and 20 °C). The
morphological differences between colonies of F. inzengae and F. fomentarius s.s. have also been
revealed. Asexual chlamydospores were described in cultures of both species, while oidia – mainly in
F. fomentarius s.s. strains (Badalyan et al., 2019a). Further morphological observation of Armenian
Fomes sp. collections is warranted.

Conclusion

The tinder polypore F. fomentarius (L.) Fr. in the territory of Armenia was described as a taxonomically
complex species represented by two cryptic sympatric phylogenetic sublineages (A2 and B2)
corresponding to two species: F. fomentarius s.s. and F. inzengae. F. inzengae is a Mediterranean,
subtropical species which was originally described for the mycobiota of Armenia although its
species rank is remaining disputable. Both taxa F. inzengae and F. fomentarius s.s. could be possibly
represented as cryptic sympatric subspecies evolved from F. fomentarius s.l.

Acknowledgments

This research was funded by RFBR and Government of the Sverdlovsk region (project 20-44-660012),
RFBR (project 20-34-90054), State Assignment of Institute of Plant and Animal Ecology UB RAS
(122021000092-9), as well as a grant project 21T-1F228 by the Scientific Committee of MESCS of
the Republic of Armenia.


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