Available via license: CC BY 4.0
Content may be subject to copyright.
357
BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM
Al Anbagi et al.
Bull. Iraq nat. Hist. Mus.
(2024) 18 (2): 357-366. https://doi.org/10.26842/binhm.7.2024.18.2.0357
ORIGINAL ARTICLE
INTRASPECIES GENETIC AND MORPHOLOGICAL ANALYSES OF THE NEW
COLLECTION OF MEDICINAL TINDER FOMES FOMENTARIUS (L.) FR.1849
(AGARICOMYCETES, POLYPORALES, POLYPORACEAE) FROM IRAQ
Rajaa Abdulrazzaq Al Anbagi*, Sara Q. Sulaiman **
and
Fakhir R. H. Alshuwaili***
* Department of Medical Biotechnology, College of Biotechnology, Al-Qasim Green
University, Babil, Iraq.
**Department of Biology, Collage of Science, Tikrit University, Tikrit, Iraq.
***Department of Applied Biotechnology, College of Biotechnology, Al-Qasim Green
University, Babil, Iraq.
Corresponding author E-mail: saraqahtan@tu.edu.iq
Received: 20 Nov. 2023, Revised: 18 Aug. 2024, Accepted:24 Aug. 2024, Published: 20 December 2024
This work is licensed under a Creative Commons Attribution 4.0 International License
ABSTRACT
Fomes fomentarius (L.) Fr., 1849 (Agaricomycetes, Polyporales, Polyporaceae) is a
significant fungal species widely used in traditional medicine. However, several studies have
noted there is a genetic variation among strains collected from different geographic regions.
The current study focused on identifying the genetic lineage of the first record of Iraqi strain
F. fomentarius which was collected from the dead trunk Ficus sycomorus L., 1753 in
Sulaylmaniyah Province. The study also and investigated the intraspecific genetic variability
of the polypore. After multiple DNA sequence alignment analysis with international
sequences, the study confirmed a genetic non-homogeneity of the presented species, and
revealed the Iraqi strain has a unique 7 pb signature sequence and belonged to the lineage B.
The Iraqi strain has only 2 base pairs differences from Slovak Southern European sequence
and 4-8 base pairs from China, Asia (lineage B) and North American. Characterizing the
genetic lineage of the current Iraqi strain is important to preserve its divers therapeutic
prosperities and biotechnological applications of this species.
Keywords: Bioinformatic analyses, Intraspecific genetic variability, Lineages, Polyporus
plorans, Rare species.
INTRODUCTION
The medicinal tinder polypore, Fomes fomentarius (L.) Fr., 1849 (Polyporaceae,
Polyporales) is widely distributed across Africa and Asia and throughout North America and
Europe. It flourishes in nearly all habitat zones in the forests of Eurasia and North America
(Judova et al., 2012). The species is popular in traditional Chinese medicine and still
extensively used today. Extensively and involved globally as a source of bioactivity and
metabolite production, medicinal complex materials, and therapeutic adjuvants (Chang and
BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM
Iraq Natural History Research Center & Museum, University of Baghdad
https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home
Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799-Print ISSN: 1017-8678
358
Bull. Iraq nat. Hist. Mus. 18 (2): 357-366.
Intraspecies genetic and morphological analyses
Wasser, 2012; Dresch et al., 2015). Ecologically, this species is diversely active with
secondary metabolites making the species one of the major decomposers of deciduous tree
debris; consequently, it plays a key role as a CO2, CO2 emitters in forest ecosystems, and
providies nesting cavities and shelter for animals and insects (Gilbertson, 1980; Judova et al.,
2012; Mukhin et al., 2021).
The tinder polypore is characterized by binding hyphae or branched skeletal hyphae, which
confer hardiness to the basidiomata that phylogenetically has been placed in a strongly
supported clade (98%) with Cryptoporus volvatus, and Datronia mollis (Hibbett et al., 2014).
Historically, the species strains also have been questioned their separation from isolates of
Fomes fasciatus (Sw.) Cooke, 1885 due to their limited morphological features (Gilbertson
and Ryvarden, 1986). However, with limited molecular and physiological data available, it
has been confirmed that F. fasciatus and fomentarius are two distinctive morphological
species with some cryptic species within this group of strains (Gáper et al., 2016).
The ITS rDNA sequence studies of F. fomentarius have shown that the species are
genetically heterogenic and previously existed in two lineages (lineages A and B) within
European strains (Judova et al., 2012) and recently in four sublineages (A1, A2, B1, B2)
(Mukhin et al., 2018; 2021). Therefore, several studies have been concluded that F.
fomentarius possibly likely contains cryptic species based on phylogenetic analyses using ITS
or multiple genetic markers (Pristaš et al., 2013).
Therapeutically, F. fomentarius has a long history of therapeutic uses in Hungary, China,
and India where it is regarded as a producer of several pharmacologically active compounds.
Both F. fomentarius basidiomata and cultured mycelia have been recognized to be effective
antitumor, glycemic, antiviral, and antimicrobial substances (Huang et al., 2012; Gáper et al.,
2016). However, the results suggested that metabolic production and bioactivates are strongly
triggered by fungal host or substrates and the geographic locations of isolates (Dresch et al.,
2015).
In Iraq, several studies have reported the first records of medical macrofungi to date
(Suliaman et al., 2022; Al Anbagi and Al-Khesraji, 2022; Marie et al., 2023). species of the
genera Populus (poplars) along with Salix (willows), both related to the Malpighiales,
Salicaceae, along with other subspecies are widely distributed across different geographic
regions and are ecologically and economically important landscape trees in Iraq (FAO, 2009;
Mustafa, 2018). Consequently, many Basidiomyceres species are expected to be associated
with these plants or their microhabitats (Suliaman et al., 2017; Alshuwaili et al., 2021; Al
Anbagi and AL-Khesraji, 2022). Recently, the polypore F. fomentarius was collected from
the dead trunk Ficus sycomorus in Sulaylmaniyah Province (Marie, 2022). This important
species requires genetic characterisation and detection or investigation of its antimicrobial
activities. Thus, the aim of the present study is to analyse the intraspecific variability and
determine the genetic lineage of F. fomentarius for the first time using phylogenetic species
analyses based on available ITS sequences.
359
BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM
Al Anbagi et al.
MATERIALS AND METHODES
Specimens' information: The basidiocarps were collected from the dead tree trunk Ficus
sycomorus using a clean knife during a mushroom survey in 2021 in selected parts of
Sulaymaniyah Province, North Iraq. The phenotypic characteristics of the fruiting body were
described including macro and microscopic characteristics, as documented by AL-Obaidy
(2023). The current scientific name and taxonomic position of species follow the recent
edition of Authors of fungal names available in the international Index Fungorum website
www.indexfungorum.org.
The extracted DNA of F. fomentarius was amplified and sequenced using ITS1 and ITS4
primers (White et al., 1990) with a Thermal Cycler (Gene Amp, PCR system 9700; Applied
Biosystem, the newly generated sequence was later submitted into the GenBank database
under the genetic code OQ109278.
Phylogenetic and statistical analysis: The submitted sequence was reverified and reanalyzed
using the Basic Local Alignment Search Tool (BLAST) accessible through the National
Center for Biotechnology Information (NCBI) Gen Bank (www.ncbi.nlm.nih.gov/genbank/).
The reiterating of sequence similarity searching was applied to detect the closest sequences to
the Iraqi strain for subsequent genetic analyses (Tab. 1).
Phylogenetic analyses were achieved using 15 rDNA ITS sequences obtained. These
included the Iraqi strain of F. fomentarius and selected sequences available in the GenBank
database (http://www.ncbi.nlm.nih.gov/genbank/, Tab. 2). According to Gáper et al. (2016)
and Mukhin et al., (2018), the selected sequences served as references for sublineages A1,
A2, B1and B2. These sequences were added and multiple aligned using MAFFT Algorithm
with manual verification of the obtained results. The 7 bp signature sequence TCGTTTG.
From the aligned sequences was detected to discriminate F. fomentarius genotypes using
Genoius program (Judova et al., 2012). The sequence of F. fasciatus was selected as an
outgroup (Badalyan et al., 2022). The RAxML analyses were performed after determining the
best ML model and a tree was constructed using RAxML 7.2.8. Bootstrap values were
calculated using Maximum Parsimony analyses with 1000 bootstrap replicates.
Table (1): The blast results of the compared Iraqi sequence Fomes fomentarius with NCBI
isolates.
Species
Identification
%
Host tree
Country
GenBank
code
Fomes
fomentarius
99.69
Fagus
sylvatica
Serbia
MW327504
F. fomentarius
99.53
Acer
platanoides
Asian part of
Russia
MF563981
F. fomentarius
99.53
Carpinus sp.
Armenia
OL583673
F. fomentarius
99.53
Fagus sp.
Armenia
OL583671
F. fomentarius
99.53
Salix alba
Armenia
OL583669
360
Bull. Iraq nat. Hist. Mus. 18 (2): 357-366.
Intraspecies genetic and morphological analyses
Table (2): Overview of polypore strains with isolates in Table (1) used for phylogenetic
analyses including GenBank accession number, natural substrate (host), and
provenance.
Species
Host tree
Country
GenBank code
F. fomentarius
Salix spp.
Iran
KM433840
F. fomentarius
Populus tremula
Slovakia
GQ184600
F. fomentarius
Fagus sp.
Armenia
OL583667
F. fomentarius
Picea abies
Austria
KM360128
F. fomentarius
Betula papyrifer
USA
JX126893
F. fomentarius
Betula papyrifera
USA
JX183708
F. fomentarius
Unspecified
China
JX290073
F. fomentarius
Unspecified
China
EU273503
F. fasciatus
Platanus occidenttalis
USA
JX126900
RESULTS AND DISCUSSION
The newly submitted sequence was 723 continuous base pairs with 48% of GC content.
The molecular identification of the Iraqi query sequence confirmed the morphological species
affinity and identified it as F. fomentarius. The BLAST results in NCBI databases showed a
sequence similarity, the value of the newly sequence 99.69% and query coverage of 100%
with the F. fomentarius strain TMF2 which was isolated from the deciduous forest located on
mountain Avala, Serbia, from a beech Fagus sylvatica tree that has accession number
MW327504. This newly generated ITS sequence also showed 99.53% similarity with other
isolates highlighted with blue arrows in Diagram (1). The ITS regions in particular are useful
for taxonomic reasons due to being highly variables between species and their conserved with
intraspecific similarities usually higher than 99% (White et al., 1990). The currently
generated ITS sequence is the first deposited sequences in Iraq and the third sequence from
Asia, the world’s largest continent, submitted to the GenBank. According to our current
knowledge, the species has been isolated from only a few strains in China and two strains in
Iran representing the Asia isolates sublineage.
Diagram (1): The heatmap and number of bases/ residues which are not identical of the
Fomes fomentarius Iraq strain (violate color) compared with some
international strains in the DNA alignment of partial internal transcribed
spacer (ITS) sequences. The Iraqi strain was different in a single nitrogen
base from the bases found in DNA isolates in the column 4 (KM433840)
to the column 9 (MW327504).
361
BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM
Al Anbagi et al.
The current results from the heatmap differences displayed that sequence divergence
between the Iraqi strain of F. fomentarius and the outgroup F. fasciatus was 100 base pairs in
agreement with the findings of Gáper et al. (2016). The sequence of the Iraqi strain is highly
homologous with only one base difference per sequence due to the insertion of a single
adenine (A) base into its sequence in the site 23 Compared to the closest international
sequences (Diag. 1). The number of base differences between the Iraqi sequence (OQ109278)
and the Slovak Southern European sequence, (GQ184600) was 2 base pairs while differences
with other sequences from China, Asia (lineage B), North American, and Northern European
(both lineage A) strains were 4-8 and 20-22 base pairs respectively. These results concurred
with previous results which revealed there were base sequence between lineages A and B of
F. fomentarius nearly 20 times higher (Gáper et al., 2016).
The sequence genotype of the Iraqi strain was classified as genotype B based on the
presence of the 7 bp signature sequence TCGTTTG in both the ITS1 region of the isolate
(data not shown) and alignment sequences of the investigated strains showed in Diagram (2).
According to Judova et al. (2012), the presence of 7 bp signature sequence in the ITS1 region
is used to distinguish F. fomentarius genotypes in which all strains are classified as genotype
B when that sequence presence while strains are classified as genotype A, when strains
lacking sequence as being shown in Diagram (2). Other researchers have confirmed this
signature and use it to differentiate isolates in into lineages A and B or A1, A2 and B for
strains collected from (Gáper et al., 2016; Náplavová et al., 2019). This evidence suggests
that F. fomentarius may include several sympatric cryptic species and multifaceted genetic
structure of F. fomentarius population presenting different host preferences (Judova et al.,
2012; Gáper et al., 2016).
Diagram (2): The DNA alignment of partial internal transcribed spacer (ITS) sequences
of Fomes fomentarius Iraq strain (pointed in the green arrow) with some
international strains. The strains were divided into two variable lineages:
lineage A strains (GenBank accession nos. starting with JX126893 and
ending with OL583667) and lineage B strains (GenBank accession nos.
starting with EU273503 and ending with MW3275O4) based on Judova et
al. (2012). Lineage specific residue is surrounding by two green lines.
Unidentical nucleotide residues are colored by different.
B
A
362
Bull. Iraq nat. Hist. Mus. 18 (2): 357-366.
Intraspecies genetic and morphological analyses
The phylogeny tree of the current results supports the separation of F. fomentarius from
Iraq and other international species sequences from the sequence of F. fasciatus as distinct
phylogenetic species (Diag. 3) in agreement with the previous studies (McCormick and
Grand, 2013; Badalyan et al., 2022). The investigated isolates in the presented tree clearly
indicate the presence of four clades within F. fomentarius with Bootstrap values >70%. The
tree separated clades based on the geographic origin and the substrate. Both these features are
essential factors driving speciation in this genus (Dresch et al., 2015). The topology of the
tree in the current study aligns with Dresch et al. (2015) who inferred four clades of F.
fomentarius corresponding to North European, North American and East Asian, Chinese, and
South European clades using the Maximum likelihood method. The strains of these distinct
lineages grow on different host tree species North European (Austria), and Chinese isolates
and another clade compressing a mixture of isolates from Iraq (the current strain), Iran,
Sothern European (Slovakia) and Asia parts of Russia (Southern Urals). The Iraqi sequence
clustered with the Iranian and Southern European strains (sequences) which were sister clade
to Chinese strains.
Diagram (3): Phylogenetic placement of the F. fomentarius Iraqi strain and some
international strains from ITS data inferred by using the Maximum
Likelihood method.
Unlike the findings of Badalyan et al. (2022), the current results separated the Iranian and
Chinese strains from each other into different groups. The presented results support the recent
363
BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM
Al Anbagi et al.
proposal that F. fomentarius is a taxonomically complex species (Badalyan et al., 2022).
Within a single lineage, species strains isolated from distinctive substrates exhibited
significant variation in their optimal growth temperatures, secondary metabolites, and
bioactivities of fruit body extracts. Therefore, understanding the intraspecies genetic relations
between strains connecting with other data such as species host, geographic location, and
physiological properties along with other related factors provides valuable insights into the
medicinal F. fomentarius lineages/sublineages (Dresch et al., 2015).
CONCLUSIONS
In conclusion, the current results reveal the interspecies genetic variation of Fomes
fomentarius from the north part of Iraq (a new record) in comparison to strains from other
parts of the world. Further studies are needed to collect isolates of the species from other parts
of Iraq to detect whether there are other lineages/sublineages and/or even other species related
to the genus. Species delimitation is a significant issue in medicinal fungi. Therefore,
detecting intraspecies genetic variability of F. fomentarius strains is essential due to their
diverse therapeutic prosperities. That is vital for improving biological features of species
strains as enhanced bioactive materials leading to improve the medicinal value or
biotechnological applications of this species.
CONFLICT OF INTEREST STATEMENT
"The authors declare no conflict of interest to declare".
LITEREATURE CITED
Al Anbagi, R. A. and AL-Khesraji, T. O. 2022. Morchella conica Pres., 1818 (Peziziales,
Morchellaceae): a new record from Iraq. Bulletin of the Iraq Natural History Museum,
17(1): 89-101. [CrossRef]
Al-Obaidy, S. M. M. 2023. Phenotypic and molecular diagnosis of some species of
Basidiomycete fungi and detection of the inhibitory effect of silver nanoparticles
manufactured from them against some species of pathogenic bacteria and fungi. M.Sc.
thesis, College of Science, University of Tikrit, 209 pp.
Alshuwaili, F. E., Al Anbagi, R. A. and Stephenson, S. L. 2021. Mycobiome sequencing and
analysis of the assemblages of fungi associated with leaf litter on the Fernow
Experimental Forest in the Central Appalachian Mountains of West Virginia. Current
Research in Environmental and Applied Mycology (Journal of Fungal Biology), 11(1):
315-332. [CrossRef]
Badalyan, S. Z. E. and Mukhin, V. 2022. The phylogenetic analysis of Armenian collections
of medicinal tinder polypore Fomes fomentarius (Agaricomycetes,
Polyporaceae). Italian Journal of Mycology, 51(1): 23-33. [CrossRef]
364
Bull. Iraq nat. Hist. Mus. 18 (2): 357-366.
Intraspecies genetic and morphological analyses
Chang, S. T. and Wasser, S. P. 2012. The role of culinary-medicinal mushrooms on human
welfare with a pyramid model for human health. International Journal of Medicinal
Mushrooms, 14(2): 95-134. [CrossRef]
Dresch, P., D'Aguanno, M. N., Rosam, K., Grienke, U., Rollinger, J. M. and Peintner, U.
2015. Fungal strain matters: colony growth and bioactivity of the European medicinal
polypores Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus. AMB
Express, 5(1): 4. [CrossRef]
FAO. 2009. International workshop improves the contribution poplars and willows in meeting
sustainable livelihoods and land-use in selected Mediterranean and central Asian
countries. FAO Project GCP/INT/059/ITA, Izmit, Turkey. [Click here]
Gáper, J., Gáperová S., Pristas, P. and Náplaková, K. 2016. Medicinal value and taxonomy of
the tinder polypore, Fomes fomentarius (Agaricomycetes): a review. International
Journal of Medicinal Mushrooms, 18(10): 851-859. [CrossRef]
Gilbertson, R. L. 1980. Wood-rotting fungi of North America. Mycologia, 72(1): 1-49.
[CrossRef]
Gilbertson, R. L. and L. Ryvarden. 1986. North American polypores. Vol. I. Abortiporus-
Lindtneria. Fungiflora Press, Oslo, Norway, 433 pp.
Hibbett, D. S., Bauer, R., Binder, M., Giachini, A. J., Hosaka, K., Justo, A., Larsson, E.,
Larsson, K. H., Lawrey, J.D., Miettinen, O., Nagy, L. G., Nilsson, R. H., Weiss, M.
and Thorn, R. G. 2014. Agaricomycetes. In: McLaughlin, D. J. and Spatafora R. J.
(eds). The mycota: systematics and evolution, part A. VII. 2nd ed. Heidelberg
(Germany), Springer, p. 373-429.
Huang, T. Z., Du, D. Y., Chen, Y. Q., Yuan, B., Ju, X. Y., Feng, Y. J. and Jiang, J. H. 2012.
Chemical constituents and antitumor activity of fruiting body of Fomes
fomentarius. Mycosystema, 31(5): 775-783. [Click here]
Judova, J., Dubikova, K., Gaperova, S., Gaper, J. and Pristas, P. 2012. The occurrence and
rapid discrimination of Fomes fomentarius genotypes by ITS-RFLP analysis. Fungal
Biology, 116(1): 155-160. [CrossRef]
Marie, S. M. 2022. Phenotypic and molecular diagnosis of macrofungal and detection of
antimicrobial and antioxidant activity of silver nanoparticles prepared from some
species. M.Sc. thesis in Biology, College of Science, Department of Biology,
University of Tikrit, 209 pp.
Marie, S. M., Al Anbagi R. A. and Suliaman, S. Q. 2023. Silver nanoparticles synthesized
from Polyporus plorans, a wild mushroom with detecting its physio-chemical
365
BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM
Al Anbagi et al.
characterization and antimicrobial activities. Egyptian Academic Journal of Biological
Sciences, 15(1): 43-59. [CrossRef]
McCormick, M. A., Grand, L. F., Post, J. B. and Cubeta, M. A. 2013. Phylogenetic and
phenotypic characterization of Fomes fasciatus and Fomes fomentarius in the United
States. Mycologia, 105(6): 1524-1534. [CrossRef]
Mukhin, V. A., Diyarova, D. K., Gitarskiy, M. L. and Zamolodchikov, D. G. 2021. Carbon
and oxygen gas exchange in woody debris: The process and climate-related
drivers. Forests, 12(9): 1156. [CrossRef]
Mukhin, V. A., Zhuykova, E. V. and Badalyan, S. M. 2018. Genetic variability of the
medicinal tinder bracket polypore, Fomes fomentarius (Agaricomycetes), from the
Asian part of Russia. International Journal of Medicinal Mushrooms, 20(6): 561-568.
[CrossRef]
Mustafa, S. A. 2018. A study biological and ecological of poplar leaf beetle, Chrysomela
populi L. (Coleoptera: Chrysomelidae) in Kirkuk Governorate. Kirkuk University
Journal for Agricultural Sciences, 9(4): 24-29. [CrossRef]
Náplavová, K., Gáper, J., Gáperová, S., Beck, T., Pristaš, P., Soares, C. and Lima, N. 2019.
Genetic and plant host differences of Fomes fomentarius in selected parts of Southern
Europe. Plant Biosystems, 154(1): 125-127. [CrossRef]
Pristaš, P., Gáperová, S., Gáper, J. and Júdová, J. 2013. Genetic variability in Fomes
fomentarius reconfirmed by translation elongation factor 1-α DNA sequences and 25S
LSU rRNA sequences. Biologia, 68(5): 816-820. [CrossRef]
Suliaman, S. Q., AL-Khesraji, T. O. and Hassan, A. A. 2017. New records of
basidiomycetous macrofugi from Kurdistan region-Northern Iraq. African Journal of
Plant Science, 11(6): 209-219. [CrossRef]
Suliaman, S.Q., Alsugmiany, R. Z. M. and Abed, S. A. 2022. Inhibitory effectiveness of the
extract of fungus Ganoderma adspersum against six species of Candida.
NeuroQuantology, 20 (5): 1631-1640. [CrossRef]
White, T. J., Bruns, T. D., Lee, S. B. and Taylor, J. W. 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for Phylogenetics. In: Innis, M. A.,
Gelfand, D. H., Sninsky, J. J. and White, T. J.(eds.), PCR protocols: a guide to
Methods and Applications, Academic Press, New York, p. 315-322. [CrossRef]
366
Bull. Iraq nat. Hist. Mus. 18 (2): 357-366.
Intraspecies genetic and morphological analyses
Bull. Iraq nat. Hist. Mus.
(2024) 18 (2): 357-366.
Fomes fomentarius
(L.) Fr., 1849
Agaricomycetes, Polyporales, Polyporaceae
2024
Fomes fomentarius
(L.) Fr., 1849 Agaricomycetes
Polyporales Polyporaceae
Genetic lineage
F. fomentarius
Ficus
sycomorus
L., 1753
Polypore.
pb B.
B)
