10.6092/issn.2531-7342/16437
Laetiporus is a cosmopolitan genus of the "Antrodia clade" in the order Polyporales that causes brown rot in many hardwood trees and some conifers. In the current study, we examined specimens of Laetiporus zonatus collected from Quercus semecarpifolia (Fagaceae) at three different localities in the district Swat, KP, Pakistan. The specimen’s identity was determined through extensive morphoanatomical examination and molecular characterization. For reconstruction of the phylogenetic relationships of the species, the study applied three methods: maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses to the concatenated dataset (ITS + nrLSU + rpb2). The mean character difference approach was utilized to create a phenetic cladogram through morphometric analysis. Our sample sequence grouped together with Laetiporus zonatus showing significant support values of 87% for MP, 98% for ML and 1.0 for Bayesian analyses. The morphological data matrix showed a high degree of similarity (Bray-curtis similarity = 0.925, Euclidean distance = 3.81) between our specimen and L. zonatus. Our study also provides habitat characteristics and in vitro cultural characteristics of the isolates. Laetiporus zonatus is characterized by yellow pileal surface, become pale buff to creamy when dry and whitish cream pore surface, distinctly zonate, radially furrowed upper surface and undulating white margin, 2-3 pores mm-1, ellipsoid to ovoid basidiospores (5.6–8.7 × 4.2–5.9 μm) with Q value of 1.16-1.55 and is found exclusively on Quercus spp. in temperate forests.
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet
2531-7342
16437
en
Nausheen Nazir
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Italian Journal of Mycology
brown rot
<em>Laetiporus zonatus</em>: an addition to edible polypore fungi in Pakistan
Text.Serial.Journal
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Articles
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2023-02-08
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126-143
Ital J Mycol
Nausheen Nazir
2023/08/02
10.6092/issn.2531-7342/16437
126
Department of Biochemistrty, University of Malakand, Chakdara Dir Lower-18800, Khyber Pakhtunkhwa, Pakistan
2531-7342
brown rot
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White TJ, Bruns T, Lee SJWT, Taylor J, Innis MA, Gelfand DH, Sninsky JJ (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds). Academic press, New York, pp 315‒322. http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1
<em>Laetiporus zonatus</em>: an addition to edible polypore fungi in Pakistan
https://italianmycology.unibo.it/article/download/16437/16720
52
                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 126-143
                Journal homepage: https://italianmycology.unibo.it/

Research article

         Laetiporus zonatus: an addition to edible polypore fungi
                               in Pakistan

                     Shahid Hussain1*, Mohammad Nisar1*, Hassan Sher2,
                            Muhammad Zahoor3, Nausheen Nazir3
1
  Department of Botany, University of Malakand, Chakdara Dir Lower-18800, Khyber Pakhtunkhwa, Pakistan
2
  Center for Plant Sciences and Biodiversity, University of Swat, Swat, Khyber Pakhtunkhwa, Khyber Pakhtunkhwa,
Pakistan
3
  Department of Biochemistrty, University of Malakand, Chakdara Dir Lower-18800, Khyber Pakhtunkhwa, Pakistan

Corresponding author e-mail: shahid_sattar84@yahoo.com (SH); mnshaalpk@yahoo.com (MN)


Article info
Received 8/2/2023; accepted 23/6/2023
https://doi.org/10.6092/issn.2531-7342/16437

Abstract
Laetiporus is a cosmopolitan genus of the “Antrodia clade” in the order Polyporales that causes brown rot
in many hardwood trees and some conifers. In the current study, we examined specimens of Laetiporus
zonatus collected from Quercus semecarpifolia (Fagaceae) at three different localities in the district Swat,
KP, Pakistan. The specimen’s identity was determined through extensive morphoanatomical examination
and molecular characterization. For reconstruction of the phylogenetic relationships of the species, the study
applied three methods: maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses to the
concatenated dataset (ITS + nrLSU + rpb2). The mean character difference approach was utilized to create a
phenetic cladogram through morphometric analysis. Our sample sequence grouped together with Laetiporus
zonatus showing significant support values of 87% for MP, 98% for ML and 1.0 for Bayesian analyses. The
morphological data matrix showed a high degree of similarity (Bray-curtis similarity = 0.925, Euclidean
distance = 3.81) between our specimen and L. zonatus. Our study also provides habitat characteristics and
in vitro cultural characteristics of the isolates. Laetiporus zonatus is characterized by yellow pileal surface,
become pale buff to creamy when dry and whitish cream pore surface, distinctly zonate, radially furrowed
upper surface and undulating white margin, 2-3 pores mm-1, ellipsoid to ovoid basidiospores (5.6–8.7 × 4.2–5.9
μm) with Q value of 1.16-1.55 and is found exclusively on Quercus spp. in temperate forests.

Keywords
brown rot, phylogeny, morphometrics, Laetiporus zonatus, edible mushroom


Introduction
Polyporales is an order of the phylum Basidiomycota that includes 285 genera, 18 families, and over
2,500 species (He et al., 2019). However, according to the formal classification by Justo et al. (2017),
37 families within the Polyporales are distributed among 18 distinct clades. One of the largest clades,
“Antrodia” was first recognized in the order by Hibbett and Donoghue (2001) and encompasses
approximately 26 genera (Ortiz-Santana et al., 2013). Some of the members within the clade are
edible and also have medicinal properties referred to as culinary-medicinal mushrooms (Gargano
et al., 2020). The species of Laetiporus sulphureus (Bull.:Fr.) Murrill complex are highly prized for
Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
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their fleshy textured and brightly colored aromatic fruiting bodies, commonly known as sulfur shelf
or chicken of the wood (Turkoglu et al., 2007). Proximate analysis reveals that these mushroooms
contain important nutrients, including carbohydrate (particularly polysaccharides), proteins, essential
amino acids, minerals, vitamins, polyunsaturated fatty acids and dietary fibre (Martinez et al., 2015;
Khatua et al., 2017). Additionally, their secondary bioactive constituents have numerous therapeutic
properties including anti-inflammatory, antimicrobial, antioxidant, anti-hyperglycemic, anti-tumor,
and immunomodulatory effects (Saba et al., 2015). Ecologically, some Laetiporus spp. cause brown
rotting in many deciduous and coniferous trees and are pathogenic to living trees in forests (Dai et al.,
2007; Banik et al., 2010). Hence, they play a very important role in forest ecosystems.
    Laetiporus Murrill 1904 is a cosmopolitan genus belonging to the Laetiporaceae Jülich. The
genus was first placed in the “Antrodia clade” of the Polyporales by Hibbett and Donoghue (2001).
The genus Laetiporus has a total of 40 specific or infraspecific records listed on Index Fungorum
(http://www.indexfungorum.org, accessed on March, 2023), while MycoBank database (http://www.
mycobank.org, accessed on March, 2023) displays a total of 22 legitimate names, with some of these
species having been reclassified under other genera. Currently, 15 well-defined species have been
recognized, of which 11 belong only to the L. sulphureus complex (Song et al., 2018). The genus has a
wide distribution in boreal to subtropical regions and has been reported from Europe, Asia, and North
America (Ota and Hattori, 2008). The Laetiporus spp. produce annual basidiocarps, which are sessile
or laterally substipitate, flabelliform, and yellow in color, with regular round or angular pores ranging
from 3‒5 mm-1. The hyphal system is dimitic, consisting of septate generative hyphae and binding
hyphae, lacking clamp connections. It has pyriform, 4-sterigmate, simple septate basidia that produce
broadly ovoid basidiospores and lacks cystidial elements (Gilbertson and Ryvarden, 1986; Ryvarden,
1991; Burdsall and Banik, 2001; Lindner et al., 2008; Ota et al., 2009).
    Several taxonomic researches have been conducted on the Laetiporus genus using a combination of
molecular, morphological, cultural, and ecological methods to determine phylogenetic relationships
and interspecific delimitation. Gilbertson (1981) reported that the genus was represented by two
species namely L. sulphureus and L. persicinus (Berk. & M. A. Curtis) Gilb. The latter species was
transferred to Berkcurtia persicina (Berk. & M.A. Curtis) Robledo & Campi, Lillo (Campi et al.,
2022). However, based on the morphological and multilocus molecular data of Paez et al. (2022),
L. persicinus was recently renamed as Kusaghiporia persicinus (Berk. & M.A. Curtis) C.A. Paez,
Kraisit. & M.E. Sm. Based on ITS sequence data and ecological relationships with hosts, European
isolates of L. sulphureus were classified into two major clades (Rogers et al., 1999). Further, mating
and molecular studies have been conducted on the genus from North America (Banik, 1999; Banik and
Burdsall, 2000; Burdsall and Banik, 2001). A study conducted by Burdsall and Banik (2001) identified
six distinct taxa, including L. cincinnatus (Morgan) Burds., Banik & Volk, L. conifericola Burds. &
Banik, L. gilbertsonii Burds., L. gilbertsonii var. pallidus Burds., L. huroniensis Burds. & Banik, and
L. sulphureus. This differentiation was largely based on the attachment of the stipe, the color of the
pileal and pore surfaces, and host association. Similarly, Tomsovsky and Jankovsky (2008) added
another species L. montanus Cerny ex Tomsovsky & Jankovsky from Europe, which was associated
with coniferous hosts. Similarly, Banik et al. (2012) described L. caribensis Banik & D. L. Lindner,
a new species found in the Caribbean basin associated with hardwood hosts. A molecular study from
Japan by Ota and Hattori (2008) identified three species of Laetiporus including L. cremeiporus Y.
Ota & T. Hatt., L. montanus, and L. versisporus (Lloyd) Imazeki, Bull. The phylogenetic relationships
among Euro-American and East Asian Laetiporus species were studied by Lindner and Banik (2008),
Ota et al. (2009), Banik et al. (2010), Song et al. (2014) and Song and Cui (2017). After analyzing
Chinese materials Song et al. (2014) described two species, L. versisporus and L. zonatus B.K. Cui &
J. Song, from Southwestern China. Subsequently, Song et al. (2018) described two additional species,

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L. medogensis J. Song & B.K. Cui and L. xinjiangensis J. Song, Y.C. Dai & B.K. Cui from the country.
A review of the literature showed that several researchers have thoroughly investigated the species
from different countries. However, South Asian countries have not been sampled or lack adequate
voucher materials for thorough taxonomic and biogeographic analyses of the genus in global context.
Therefore, the current study aims to examine collection from three different localities and infer the
phylogenetic relationship using both molecular and morphological data.

Materials and Methods

Sampling and morphological characterization
The present study analyzed voucher specimens collected from the temperate forest of the district
Swat, KP, Pakistan, located within the floristically diverse region of the Hindu-Kush mountain range.
Dominant tree species in the area are Picea smithiana (Wall.) Boiss, Abies pindrow Royle, Cedrus
deodara (Roxb. ex D. Don), G. Don, Pinus spp., and Quercus spp. Basidiocarps were photographed
and collected from three different localities. During the field examination, macro-morphological
features such as color changes, bruising, exposure to air and other ephemeral characters were recorded
in the field note book. Color terms were adopted from Petersen (1996), and the voucher specimens
were dried and deposited in the mycological section of the herbarium at the University of Malakand
(BGH), KP, Pakistan.
    In the present study, the basidiocarps were subjected to a detailed microanatomical examination.
Microscopic techniques and routine notations were performed following the guidelines of Han et
al. (2016), Song et al. (2018), and Ji (2022). Freehand anatomical sections were obtained from the
hymenial and context layers for microscopic analysis of hyphal system, septal features, hymenial
elements, including basidial and sterile elements, and spore characteristics using a camera-equipped
binocular microscope. Mounting and staining reagents such as 5% KOH, 2% congo red, lactophenol
cotton blue and Melzer’s reagent (IKI) were used. Staining was performed for 1‒3 hours, and the
reactions were classified as follows: amyloid or Melzer’s-positive (IKI+) stains blue or black, while
inamyloid or Melzer’s-negative (IKI-) showed no color change or became faintly yellow to brown.
The reaction with lactophenol cotton blue (CB) was used to assess the cyanophily of basidiospore
walls, with CB+ denoting cyanophilous and CB- denoting acyanophilous (Banik et al., 2012). On
average 32 measurements were taken and rounded to the nearest 0.5 µm. ± standard error, n (a/b) =
number of spores (a) measured from given number (b) of specimens (Ji, 2022). L = mean spore length
(arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q =
variation in the L/W ratios between the specimens studied, n = number of spores measured from given
number of specimens. The measurements were assessed with Image J (Wayne Rasband, National
Institute of Health, USA) software (Tsujikawa et al., 2003).

Morphometrics analyses
The morphological data matrix was generated, comprising of 58 morphological characters obtained
from 22 taxa. Some of these characters were binary, while others were multistate, extracted from the
original descriptions provided by the authors. Each character state was then scored for the presence
or absence, or assigned a specific score for multistate characters. The characters were unweighted and
unordered, and the gamma shape was set to 0.5. To show the average difference between the character
states of taxa a phenetic cladogram was constructed through the mean character difference method
using the neighbor-joining (NJ) algorithm in the software package Paup4 (Swofford, 2002).




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Cultural characterization
The isolation was done from the mycelial tissue (5 × 5 mm) taken from the context portion of the
fresh basidiocarps. Culturing techniques were adopted from Lindner and Banik (2011) and Virginia
and Catalin (2013). Mycelial tissue was inoculated onto 90 mm potato dextrose agar (PDA) and
malt extract agar (MEA) plates and observed after growth under a stereomicroscope for macro-
morphological features according to Stalpers (1978). The growth rate and mycelial density were
measured and recorded. Microscopic examination was done according to Peintner et al. (2019).

Molecular characterization
DNA extraction
DNA extraction was carried out using CTAB method (Murray and Thompson, 1980; Stirling, 2003)
with slight modification. About 25 mg of the material from pore surface was taken and homogenized
with multi-beads shocker at 3000 rpm/min using zirconia beads. Homogenization were done in 400
µl of 2% CTAB buffer and incubated at 65 °C for 50 min. The homogenate was added with 350 µl
of chloroform:isoamyl alcohol 24:1 for the purification then vortexed for one minute and centrifuged
for 20 min at 13,200 rpm at 4 ºC. The S/N or aqueous phase was transferred into another autoclaved
microtube. Overnight precipitation was done with 133 µl of ice cold iso-propanol and centrifuged for
20 min at 13,200 rpm at 4 ºC and S/N was discarded. The pellet was washed twice by adding 500 µl
of chilled ethanol by vortexing briefly and centrifuged for 3 min. The pellets were air dried and re-
suspended in distilled water and stored at -20 ºC.

PCR and Sanger sequencing
Amplification of ITS1-5.8S-ITS2 region of rDNA was carried out using ITS1-ITS4 primer pair (White
et al. 1990; Gardes and Bruns, 1993), nrLSU region using LR0R-LR5 primer pair (Vilgalys and
Hester, 1990; Moncalvo et al., 2000) and partial region of second largest subunit RNA polymerase-II
using RPB2-6R-RPB2-7F primer pair (Liu et al., 1999). Genes were PCR-amplified through PCR
(Applied Biosystems Veriti thermal cycler). PCR reaction (20 μl in volume) mixtures contained: 2
μl of gDNA (about 100 ng), 0.5 μl of each primer (10 µl), 14.17 μl of sterile deionized water (Fisher
Scientific, Waltham, USA), 26 μl of 2×Taq PCR Mastermix, 0.5 μl Taq DNA polymerase (Takara
BIO INC., Shiga, Japan), 2.0 µl MgCl2, 0.5 µl dNTPs (Promega, Madison, Wisconsin). PCR reactions
were carried out using following temperature regime: 95 °C for 3 min, then 35 cycles of 95 °C for 30
s, 55 °C for 30 s and 72 °C for 30 s. The final extension was kept as 72 °C for 8 min (Schoch et al.,
2012). The amplified PCR bands were visualized using 1% agarose gel (Fisher Scientific, Waltham,
MA, USA) prepared in 0.5× TAE buffer, stained with ethidium bromide (Stefanova et al., 2022). One
Kb ladder (Promega, Durham, NC, USA) was used as standard marker.
    Amplicons were purified by using the total fragment DNA purification Kit (MEGA quick-spinTM
PLUS (Thermo Fisher Scientific, Seoul, Korea) following manufacturer instructions. The purified
samples were sequenced through automated Sanger sequencing by submitting samples to the
Macrogen Inc. Seoul (https://dna.macrogen.com) for sequencing facility, South Korea.

Phylogenetic analyses
To verify identity level, query sequences were blasted in Basic Local Alignment Search Tool
(BLAST) in NCBI-GenBank (Altschul et al., 1990). For correct identification and to retrieve reference
sequences, protocols recommended by Nilsson et al. (2012) and Schoch et al. (2014) were followed.
Furthermore, the taxonomic annotations of the included sequences were carefully checked to ensure
they were authentic and published, while uncultured and environmental sequences were omitted.
The query coverage was set at 80% and sequence similarity at 97–100%, according to Raja et al.

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(2017). The sequences, based on vouchered herbarium samples, are available in GenBank under the
specified accession numbers in the Table 1. The sequences were aligned using the MAFFT 7 online
multiple sequence alignment program (https//mafft.cbrc.jp/ alignment/server/; Katoh et al., 2019).
The alignment was reviewed and manual adjustments were made at misaligned sites. Ambiguous sites
at both ends were excluded using BioEdit 7.2.5 (Hall et al., 2011). Most of the gaps were removed
and the remaining gaps were treated as missing data in all analyses (Shen et al., 2002). Wolfiporia
dilatohypha (FP72162) was used as the outgroup (Song et al., 2014). Three different phylogenetic
analyses were performed on the combined ITS + nLSU + rpb2 data set. The best-fit substitution
model was found using jModelTest2 (Darriba et al., 2012) based on different criteria.
   The parsimony analyses (MP) were conducted using PAUP v.4.0.b10 (Swofford, 2002) with equal
weighting. The analysis involved 1,000 heuristic search replicates, utilizing random taxon addition
searches and tree-bisection-reconnection (TBR) branch swapping, with an unrestricted number of
trees. A 50% majority-rule consensus tree was generated from the remaining trees, and the tree’s
topology was evaluated by calculating various indices such as tree length, consistency index (CI),
homoplasy index (HI), and retention index (RI) (Justo and Hibbett, 2011). The Maximum Likelihood
(ML) estimates were computed on the three partitioned data sets (ITS, nLSU, and rpb2) using IQ-TREE
version 1.6.12 (Nguyen et al., 2015), with the substitution models of TIM2ef+G, TrN+I, and TrN+I
selected for the respective partitions (Chernomor et al., 2016). The branch support was determined
through bootstrapping with 1,000 replicates (Hoang et al., 2018). Bayesian Metropolis-coupled
Markov Chain Monte Carlo (MCMCMC) inference was performed on the same three partitioned
data sets using MrBayes version 3.1 (Ronquist and Huelsenbeck, 2003). The substitution models for
each partition were specified as nst = 6, rates = gamma (ITS) and nst = 6, rates = propinv (nLSU and
rpb2). The base frequencies, substitution rates, gamma shape, and p.inv. parameters were set based
on the best-fit models obtained through the Akaike Information Criterion (AIC) as determined by
JModelTest2. The analysis was run for 2 million generations with four chains and trees were sampled
every 100 generations. The first 5,000 trees (25% of the total) were excluded as burn-in and were not
used in constructing the consensus tree. The stop rule was set at stopval = 0.01 (Lindner and Banik,
2008). The trees were analyzed using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and
later edited. The optimal topologies from maximum likelihood (ML) analyses were displayed and
validated based on the ML-BS score (≥75), MP-BS score (≥50), and BPPs (≥0.95) (Ji, 2022).

Results
In the current study, morpho-anatomical characteristic of the three specimens (MUBS80, MUSL21-
44, MUMnd301) showed resemblance with L. zonatus, particularly in the color of pore and pileal
surface, pore size, thickness of contextual binding hyphae, basidiospore dimension, spore quotient,
and host plants. Similarly, the phylogenetic assessment of concatenated dataset from three different
genetic markers (ITS, nrLSU, rpb2) including sequence data from the voucher MUBS80 indicated
that our sample sequence clustered together with L. zonatus, with highly significant support values.
Additionally, a morphometric analysis yielded similar results, corroborating the resemblance between
our specimen and the L. zonatus described by Song et al. (2014).

Taxonomy

Laetiporus zonatus B.K. Cui & J. Song, Mycologia 106 (5): 1042 (2014)
MB#808204
Basidiocarps large sized, annual, solitary pileate or imbricate, flabellate or dimidiate to applanate,
sessile to laterally substipitate, thick fleshy textured, become crumbly and lightweight when dry,

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having a distinct pungent odor and acidic flavor. Pilei projecting 8-15 cm., 10‒20 cm. wide, 2‒3 cm.
thick at the base; upper surface orange yellow, become pale buff to creamy when dry, distinctly zonate,
concentric zonation in the growing margin are distinct, deeply furrowed along the radii, completely
glabrous become dull brown or buff brown when dried. Margin: whitish or pale, sterile, smoothly
rounded and blunt and undulating or profusely wavy, up to 3‒4 mm thick, fading to brownish colored
when dry. Stipe almost absent but stout or robust attachment to the bark can be observed. Pore surface
whitish cream or light yellow when fresh, brown when become dried; pores mostly angular and
irregularly distributed. Pore lining rounded later become angular when dry, number of pores 2‒4 or
usually 3 per mm; dissepiment thin uniform in thickness 0.1‒0.2 mm, and usually lacerate. Tube layer
is tightly affixed to the context, whitish to pale colored about 1‒3.5 (2) mm in thickness, concolorous
with pore surface and context which become distinct in color when dry. Context is differentiated from
the upper pileal cover whitish cottony becoming light brown and spongy on drying, 3-7 mm at the
widest point, context to tube layer ratio is 2-5.5:1 (Fig. 1).
    Hyphal system dimitic, skeletal hyphae in the tube layer and binding hyphae from the context layer
dissolve in 2% KOH. The contextual binding hyphae are dominant characterized by light yellow,
usually branched, laterally interwoven, septate (wavy septae) hyphae showing parallel or subparallel
arrangement and give up frequently lateral tapering branches. Hyphal thickness range 7.5‒20 (15) μm
with wall thickness 0.6‒3 μm, while the lateral hyphae vary in thickness from 3‒4.2 μm. Generative
hyphae rarely observed in the context. Tramal layer dimetic consisting of predominantly generative
hyphae, profusely septated, 2.5‒8.7 (5.0) µm in diameter, thin walled, regularly arranged, yellowish
in color. Skeletal hyphae from the trama (subhymenium) rare, usually hyaline, thick walled less
frequently branched and septated. Basidia are clavate, with short sterigmata and a simple septum
at the base without clamp connection, 11.5–21.6 (15.6) × 5.5–8.2(6.4) μm, containing a large sized
guttule. Basidioles frequent small sized, pyriform to globose shaped 7–10(8.5) × 5.5–6.8(5.7) μm.
Cystidial elements are absent. Large sized diffused crystals were also observed. Basidiospores are
ellipsoid to ovoidal shaped, thin walled, non-dixtrinoid, acyanophilous, 5.6–8.7 × 4.2–5.9 μm, L = 6.5
± 0.21 μm, W = 5.0 ± 0.14 μm, Q = 1.16-1.55 (n = 32/3) (Fig. 2).

Materials examined: PAKISTAN, KP PROVINCE: (i) Laetiporus zonatus voucher no. MUBS80,
Sailand, district Swat, (34º59’45’’ N and 72º11’08’’, 2797 m asl), mixed coniferous forest on living
trees and dead tree stumps of Quercus semecarpifolia, August, 2020, (BGH000303; BGH); (ii)
Laetiporus zonatus voucher no. MUSL21-44, Lalku, district Swat, (34º09’41’’ N and 72º25’03’’
E 2755 m asl), in mixed coniferous forest on Q. semecarpifolia, September, 2021, (BGH000304;
BGH); (iii) Laetiporus zonatus voucher no. MUMnd301, Miandam, district Swat, (35º01’14’’ N and
72º35’06’’ E 2876 m asl), in mixed coniferous forest on living trees of Quercus sp., September, 2020,
(BGH000305; BGH).

Rot type: brown rotting
Cultural characteristics (Fig. 3): mycelial mat was irregular to circular shaped, consisting of sparsely
and slow grown interwoven hyphae. Mat was raised with abundant aerial hyphae, showed azonate
growth and slowly becoming moderately dense at the center; advancing edge: raised, uneven or irregular
margin with sparse hyphal extension; colony odour: fruit or mushroom like; exudates: not found;
front color: white to brown yellowish; obverse color: pale whitish; in vitro teleomorph formations:
not found; KOH test: brown; growth rate: the average GR was measured as 4.7 (± 0.5) mm day-1.
The colony achieved the total radial growth of 42.7 mm on day 10 at 25 oC; hyphal characteristics:
the aerial hyphae were usually thick, pale yellow, branched, non-clamped, septate (ring like), and
diameter was 3‒8.4 μm, producing lateral branches (dia. 2.5-3 μm). The conidiophores were showing

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racemose branching terminated on oval, spherical or nearly lemon shaped dense blastoconidia (8.5‒10
× 10–12.5 µm) which are separated by double or single septum. Submerged hyaline hyphae were
branched and septate lacking clamp connection. Spherical or subglobose shaped chlamydospores
were frequently observed. Comments: colony initiation and establishment was slightly slow. Success
rate of isolation was high on both on PDA (4/5) and MEA (4/5). Culture viability lost after few sub-
culturing.




          Fig. 1 - Basidiocarps of Laetiporus zonatus (a,b,c,f) from natural habitat focusing pileal and pore
          surfaces, margin; d) pores surface (fresh); e) pore surface (dried); g) cross section of fresh specimen;
          h) cross section of dried specimens. Bars: a,b = 3 cm, c,f = 2 cm, d,e,g,h = 3 mm.




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   Fig. 2 – Microscopic structures drawn from voucher specimens of Laetiporus zonatus. a, b) binding hyphae of
   context showing branches; c) generative and skeletal hyphae from trama and hymenium structure; d) basidia; e)
   basidioles; f) basidiospores. Scale bars: a,b,c,d,e = 10 µm f = 5 µm.

Phylogenetic analyses
The phylogenetic relationships of the species were reconstructed by utilizing three methods: MP, ML,
and Bayesian analyses. The concatenated dataset comprised of 75 sequence variants, including 29 ITS,
28 nrLSU, and 19 RPB2 sequences, including our subject sequences from the representative voucher
sample (MUBS80) (Table 1). The topology of the ML analysis depicting the bootstrap proportion
(MP), bootstrap maximum likelihood (ML), and Bayesian posterior probability (BPP) values, was
shown in Fig. 4. Topologically, the inferred trees of MP, ML and Bayesian analyses on each region
were more or less congruent, hence combined matrix was developed to undertake the analyses. A
total of 13 species were included in the matrix i.e., L. cincinnatus, L. conifericola, L. gilbertsonii,
L. huroniensi, L. sulphureus, L. versisporus, L. ailaoshanensis, L. zonatus, L. cremeiporus, L.
caribensis, L. medogensis, L. montanus, and Wolfiporia dilatohypha Ryvarden & Gilb. (as outgroup).
The aligned data matrix consisted of 1,547 nucleotide characters, including gaps, of which 86.3%
were constant sites, 4.91% were variable characters that were parsimoniously uninformative, and
8.8% were parsimoniously informative characters with 222 distinct patterns.

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   Fig. 3 - Cultural characteristics; a) Laetiporus zonatus MEA grown culture (90 mm); b) margin of distant and
   branched hyphal extension; c) conidiophore producing terminal blastoconidia; d) submerged hyaline hyphae; e)
   aerial hyphae. Scale in c,d,e. 1 division = 1.16 µm (100×).


   The heuristic search of MP analysis resulted in 100 equally parsimonious trees on one island using
tree-bisection-reconnection (TBR) branch swapping. A 50% majority rule consensus parsimonious
tree was produced with the with tree description: tree length = 330, consistency index = 0.709, retention
index = 0.773, rescaled consistency index = 0.548, and homoplasy index = 0.2994. The ML analysis

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was conducted on a three-partition dataset (ITS + nLSU + RPB2) with an initial log-likelihood of
-3955.6186. The best evolutionary models were found to be time-independent model (TIM2ef) +G
(with estimated gamma shape 0.26721) for the ITS region, Tamura-Nei (TrN)+I (with estimated
p-invariance 0.894899) for the LSU region, and TrN+I (with estimated p-invariance 0.796262) for
the RPB2 region. A Bayesian analysis with lset nst = 6 was applied to all three partitions, converging
at ngen=20,000,000 with a standard deviation of split frequencies of 0.048481. The results of the
Bayesian and likelihood analysis showed a similar topology and clade distribution. The data set
revealed lineages mostly supporting species-level clades. Our sample sequence grouped together
with L. zonatus, showing significant support values (87% MP, 98% ML and 1.00 BPP) showed sister
relationship with L. ailaoshanensis, L. medogensis (branches were poorly resolved in this group) and
distantly related to L. cincinnatus, L. sulphureus and L. cremeiporus (Fig. 4).




     Fig. 4 - The strict consensus tree determined through maximum likelyhood depicts the phylogenetic
     relationships among Laetiporus spp., (including Laetiporus zonatus placed in box) inferred from ITS + nLSU
     + RPB2 sequences. Branch support values indicated before the corresponding node bootstrap MP/ bootstrap
     ML/ posterior probabilities BPP. Tip labels after taxa name designate voucher specimen number.


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Morphometric analyses
Analysis of 58 morphological characters from 22 taxa, using Euclidean distance and other indices
of similarity showed that our specimens were clustered with L. zonatus B.K. Cui and J. Song (Bray-
curtis similarity = 0.925, Euclidean distance = 3.81). By comparing morphological characters, the
specimens shared several characteristics with other Laetiporus spp., especially L. zonatus and as well
as with the L. xinjiangensis, L. versisporus and L. medogenesis (Fig. 5). Resemblance was found
particularly in color of pore surface (white cream), and pileal surface, pore size, contextual binding
hyphae thickness, basidial length, basidiospore size, spore quotient (Q) value (1.2‒1.5), distribution
(temperate, high altitude forest), and host plants (associated with Quercus spp. only).
   The morphological character-matrix revealed that all of the described species exhibited a high degree
of morphological similarity (Supplementary Material S1). The largest basidiocarps were observed in
the L. cremeiporus, L. sulphureus, and L. persicinus (length > 25 cm). All species were flabelliform,
dimidiate shaped except for L. versisporus, L. discolour var. pallidus, L. persincinus, which appeared
in rosette or singly. Laetiporus cincinnatus and L. persincinus were observed to produce centrally
or excentrically stipitate basidiocarps, while the other species were sessile to laterally substipitate.
Similarly, L. persincinus and L. cincinnatus also had hyphal extension or tomentose pileal surfaces,
whereas the other species were glabrous. Polyporus rubricus Berk. [syn. L. sulphureus], L. discolor
var. diffluens, and L. miniatus had an azonate surface, while the other species exhibited some degree
of zonation. Laetiporus persincinus and P. rubricus had a brown pileal surface, while L. versisporus,
L. discolour var. pallidus, L. discolor var. brunnescens had a white surface, and L. medogensis and L.
xinjiangensis had a pink-buff or pale-buff pileal surface. The remaining species had pileal surfaces in
shades of yellow, orange, ochraceous, sulfur buff, and cream. Further, the pore surface of L. zonatus
and L. persincinus was white cream-colored, while the other species had pore surfaces in shades of
sulfur, yellow, orange, ochraceous, and pale when fresh.
   The morphological analysis further revealed that L. zonatus and L. xinjiangensis produced the
largest pores, measuring up to 3 pores mm-1, while smaller pores were recorded for L. cincinnatus, L.
sulphureus (E), L. ailaoshanensis, L. caribensis, and L. versisporus, with a measurement of up to 6
pores mm-1. The smallest pore size, measuring up to 7 pores mm-1, was recorded for L. discolour. The
data matrix also showed that the color of the tube layer was different from the pore surface in two
species, L. cremeiporus and L. versisporus, while the others were concolorous. The contextual binding
hyphae thickness was higher than 15 µm in species such as L. cremeiporus, L. montanus, L. discolour
var. pallidus, L. discolor var. brunnescens, L. versisporus, and L. sulphureus (E). The study observed
that three species, L. discolour var. pallidus, L. discolor var. diffluens, and P. miniatus, had tubular-
shaped basidia, while the others possessed clavate basidia. The morphological analysis revealed
that L. montanus, L. discolour var. pallidus, L. sulphureus (E), L. medogensis, L. xinjiangensis, L.
persincinus, and L. zonatus possessed basidia with lengths exceeding 20 µm, while the others were
less than 20 µm in length. Furthermore, L. huroniensis had the highest spore quotient (Q = 1.5‒2)
among the studied species, while the quotients of L. conifericola, L. cremeiporus, L. gilbertsonii var.
gilbertsonii, L. gilbertsonii var. pallidus, L. montanus, L. sulphureus (A), L. sulphureus (E), and L.
discolour spp. ranged from Q = 1.2‒>1.4.




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Table 1 - List of species specimens and accession of GenBank used in the phylogenetic analyses

 Species             Voucher          Country    ITS         nLSU          RPB2          Reference
 L. versisporus      Yuan 6319        China      KX354475    KX354503      KX354670      Song et al. 2018
 L. versisporus      Cui 7882         China      KF951269    KF951323      KT894783      Song et al. 2014
 L. versisporus      Dai 13160        China      KF951266    KF951320      KT894785      Song et al. 2014
 L. ailaoshanensis   Dai 13567        China      KX354470    KX354498      KX354665      Song et al. 2018
 L. ailaoshanensis   Dai 13256        China      KF951289    KF951317      KT894786      Song et al. 2018
 L. ailaoshanensis   HKAS 52508       China      NR154610    NG059499      -             Song et al. 2014
 L. zonatus          HKAS 71806       China      KF951284    KF951310      KT894796      Song et al. 2014
 L. zonatus          Cui 10403        China      KF951282    KF951307      ON424779      Song et al. 2014
 L. zonatus          Cui 10404        China      KF951283    KF951308      -             Song et al. 2014
 L. conifericola     CA-8             USA        EU402575    EU402523      -             Lindner and Banik
                                                                                         2008
 L. conifericola     JV 0709/81J      China      KF951292    KF951327      KX354683      Song et al. 2018
 L. huroniensis      HMC-3            USA        EU402571    EU402540      -             Lindner and Banik
                                                                                         2008
 L. huroniensis      MI-14            China      EU402573    EU402539      -             Song et al. 2018
 L. sulphureus       Cui 12389        China      KR187106    KX354487      KX354653      Song et al. 2018
 L. sulphureus       Dai 12154        China      KF951295    KF951302      KX354655      Song et al. 2014
 L. cincinnatus      Dai 12811        China      KF951291    KF951304      KT894788      Song et al. 2014
 L. cincinnatus      DA-37            USA        EU402557    EU402521      -             Lindner and Banik
                                                                                         2008
 L. cremeiporus      Dai 10107        China      KF951281    KF951301      KX354650      Song et al. 2014
 L. cremeiporus      Cui10991         China      KF951279    KF951298      KX354679      Song et al. 2014
 L. gilbertsonii     JV 1109/31       China      KF951293    KF951306      KX354671      Song et al. 2014
 L. gilbertsonii     TJV2000-101      USA        EU402553    EU402528      -             Lindner and Banik
                                                                                         2008
 L. caribensis       PR6583           USA        JN684766    -             -             Lindner et al. 2012
 L. caribensis       PR914            USA        JN684762    EU402526      -             Lindner et al. 2012
 L. medogensis       Cui 12219        China      KX354472    KX354500      KX354667      Song and Cui 2017
 L. medogensis       Cui 12240        China      KX354473    KX354501      KX354668      Song and Cui 2017
 L. montanus         Dai 15888        China      KX354466    KX354494      KX354662      Song and Cui 2017
 L. montanus         Cui 10011        China      KF951274    KF951315      KT894790      Song and Cui 2017
 L. zonatus          MUBS80           Pakistan   OQ300108    OQ300110      OQ320751      This study
 Wolfiporia          FP72162          USA        EU402556    EU402517      -             Lindner et al. 2012
 dilatohypha




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  Fig. 5 - Phenetic cladogram calculated through the mean character difference method (characters = unweighted,
  gamma shape = 0.5 using NJ algorithm) using Paup4 showing relation between different species in Laetiporus based
  on the degree of similarity in their morphological inferred from the morphological data matrix created from original
  species description

Discussion
Taxonomically, the genus Laetiporus has been the subject of much revision in the past (Ota and
Hattori, 2008; Ota et al., 2009; Banik et al., 2012; Pires et al. 2016; Song et al., 2018; Paez et al.,
2022). Lack of distinctive morphological characters, many of those which are overlapping and
confusing to hinder the classification of the genus (Lindner and Banik, 2011). In the current study
we created replicable data set of the morphological characters based on the original descriptions to
establish phenetic relationships among the species. The morphological character-matrix revealed a
high degree of morphological similarity among the described species (Lindner and Banik, 2011). It
seems reasonable that inference based on this morphological data set was found to be concordant
to some extent with the molecular data. However, it was recognized that some of the species were
showing minimal description or lacking some important morphological characters and was assigned
as missing data. The morphoanatomical characteristics of three specimens of L. zonatus were found
similar to majority of other species in the genus, with the highest level of similarity (Bray-Curtis
similarity = 0.925) noted between L. zonatus from Southwestern China reported by Song et al.
(2014). Additionally, this study has shown some interesting anamorphic characteristics not previously
documented (Song et al., 2014).
   In terms of host association, similar results were observed with those of the previous studies
(Burdsall and Banik, 2001; Tomsovsky and Jankovsky, 2008; Ota et al., 2009). Unlike, L.
ailaoshanensis recorded from subtropical Lithocarpus spp. (Fagaceae), L. zonatus in this study and
Song et al. (2014) were collected from high-altitude temperate forests associated with Quercus spp.


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(Fagaceae). When considering basidiocarp size, pores size and spore dimension the L. zonatus is
more closely related to the L. xinjiangensis, L. versisporus and L. medogenesis than to L. conifericola,
and L. montanus (Song et al., 2014). However, comparing stipe attachment, context thickness, pore
size, decurrent pores, fruiting behaviors L. caribensis, L. cincinnatus, L. gilbertsonii, L. cremeiporus,
and L. huroniensis were distantly related and belong to the geographically different region (Burdsall
and Banik, 2001; Ota et al., 2009; Banik et al., 2012). L. persicinus was found to be entirely different
in many characteristics and greatly differ in ITS sequence (Burdsall and Banik, 2001).
    The outcomes of the phylogenetic analysis were consistent with previous studies conducted by
Lindner and Banik (2008), Ota et al. (2009), and Banik et al. (2010) and Song et al. (2014). The
current analysis failed to properly resolve the species complex of L. huroniensis and L. medogenesis.
Hence, further research is required to clarify the species complex in the genus as identified by Lindner
and Banik (2008). The rest of the species were successfully assigned into their respective position
in phylogenetic tree. The clade of L. zonatus was strongly supported by high level of bootstraps
and Bayesian posterior probability values (MP 87.7 - ML 95 - BPP 0.97) and was determined to be
distinct from other species. L. versisporus and L. montanus formed a strongly supported clade (MP
77.7 - ML 77 - BPP 0.9). The phylogenetic study showed that L. zonatus appear to be significantly
different from other species, such as L. versisporus, L. cremeiporus, L. caribensis, L. gilbertsonii, L.
cincinnatus, and L. sulphureus, which are otherwise well-supported clades.
    ITS based sequence data is best to determine species delimitation in the genus Laetiporus (Banik et
al., 2010). The genus is believed to possess distinct ecological, morphological and also several cryptic
species (Burdsall and Banik, 2001; Ota et al., 2009). Many of which were identified from Chinese,
Japanese and American collections by Tomsovsky and Jankovsky (2008), Ota et al. (2009) and Song
et al. (2014). However, recent studies showed that there is a high degree of genetic diversity within
the genus. Many populations showing significant genetic variation possibly due to intragenomic and
intraspecies variation within the ITS region even at the level of local population. This issue presents
a formidable challenge for correct species identification and phylogenetic analyses in the genus
(Lindner and Banik, 2011). There is a significant amount of diversity that has yet to be described and
resolved. To fully comprehend the relationships, it is crucial to conduct more in-depth studies that use
a multiple phylogenetic approach and a larger sample size from a broader geographical area.

Acknowledgments: This study was conducted with support of the laboratories, at the Center for
Plant Sciences and Biodiversity, University of Swat and Department of Botany, at the University of
Malakand.

Conflict of Interest: All authors of this study declare that they have no conflict of interest or affiliations
or involvement of any kind of personal or professional, financial or non-financial with anybody else
or any institution in the contents of this submitted manuscript.

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