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Morphology and multigene phylogeny reveal two novel species and three new records of Polypores in Swat, Pakistan

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  • Received: 20 October 2023
    Revised: 21 March 2024
    Accepted: 03 April 2024
    Published online: 29 April 2024
    Studies in Fungi  9 Article number: e004 (2024)  |  Cite this article
  • Polypores, as wood-rotting fungi, play a vital ecological role in breaking down the wood substrate, releasing crucial nutrients into the soil, shaping the carbon dynamics, and contributing to the overall health of the forest ecosystem. Despite their significance, the fungal diversity in the Hindu Kush region remains inadequately explored. This study collected specimens from district Swat, Khyber Pakhtunkhwa Province, Pakistan, a part of the Hindu Kush region. After a rigorous examination of the collected specimens for the morphoanatomical characteristics, the concatenated sequence dataset (ITS + nrLSU) derived from generated sequences along with valid and published reference sequences was subjected to phylogenetic analyses using different methods: maximum parsimony, maximum likelihood, and Bayesian analyses. The study revealed two new species from the country, belonging to two polypores families i.e., Climacocystaceae and Fomitopsidaceae. Furthermore, the analysis confirmed the identification of Daedalea dickinsii Yasuda, Neoantrodia serialis (Fr.) Audet, and Rhodofomes roseus (Alb. & Schwein.) Kotl. & Pouzar as a new addition to the polypore inventory of Pakistan. These species received phylogenetic support and were proven to have corresponding morphological characteristics concerning pertinent original descriptions. The inclusion of these new wood-inhabiting fungi in the country's mycofloral list expands our understanding of fungal diversity, and distribution patterns, and contributes to global fungal biodiversity.
  • Salvia rosmarinus L. (old name Rosmarinus officinalis), common name Rosemary thrives well in dry regions, hills and low mountains, calcareous, shale, clay, and rocky substrates[1]. Salvia rosmarinus used since ancient times in traditional medicine is justified by its antiseptic, antimicrobial, anti-inflammatory, antioxidant, and antitumorigenic activity[1,2]. The main objective of the study is to evaluate the antimicrobial activity of different extracts of Salvia rosmarinus in vitro, and its compounds related to in silico targeting of enzymes involved in cervical cancer. Since the start of the 20th century, some studies have shown that microbial infections can cause cervical cancers worldwide, infections are linked to about 15% to 20% of cancers[3]. More recently, infections with certain viruses like Human papillomaviruses (HPV) and Human immunodeficiency virus (HIV), bacteria like Chlamydia trachomatis, and parasites like schistosomiasis have been recognized as risk factors for cancer in humans[3]. Then again, cancer cells are a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Many things are known to increase the risk of cancer, including dietary factors, certain infections, lack of physical activity, obesity, and environmental pollutants[4]. Some studies have found that unbalanced common flora Lactobacillus bacteria around the reproductive organ of females increases the growth of yeast species (like Candida albicans) and some studies have found that women whose blood tests showed past or current Chlamydia trachomatis infection may be at greater risk of cervical cancer. It could therefore be that human papillomavirus (HPV) promotes cervical cancer growth[3]. Salvia rosmarinus is traditionally a healer chosen as a muscle relaxant and treatment for cutaneous allergy, tumors, increases digestion, and the ability to treat depressive behavior; mothers wash their bodies to remove bacterial and fungal infections, promote hair growth, and fight bad smells[5] .

    The study of plant-based chemicals, known as phytochemicals, in medicinal plants is gaining popularity due to their numerous pharmacological effects[6] against drug resistance pathogens and cancers. The causes of drug resistance to bacteria, fungi, and cancer are diverse, complex, and only partially understood. The factors may act together to initiate or promote infections and carcinogenesis in the human body is the leading cause of death[7]. Antimicrobial medicines are the cornerstone of modern medicine. The emergence and spread of drug-resistant pathogens like bacteria and fungi threaten our ability to treat common infections and to perform life-saving procedures including cancer chemotherapy and cesarean sections, hip replacements, organ transplantation, and other surgeries[7]. On the other hand, information about the current magnitude of the burden of bacterial and fungal drug resistance, trends in different parts of the world, and the leading pathogen–drug combinations contributing to the microbial burden is crucial. If left unchecked, the spread of drug resistance could make many microbial pathogens much more lethal in the future than they are today. In addition to these, cancers can affect almost any part of the body and have many anatomies and molecular subtypes that each require specific management strategies to avoid or inhibit them. There are more than 200 different types of cancer that have been detected. The world's most common cancers affecting men are lung, prostate, colorectal, stomach, and liver cancers[8]. While breast, cervix, colorectal, lung, and stomach cancers are the most commonly diagnosed among women[8]. Although some cancers said to be preventable they seem to still be one of the causes of death to humans, for example cervical cancer. The need to fill the gap to overcome the problem of searching for antimicrobials and anticancers from one source of Salvia rosmarinus is of importance.

    Cervical cancer is a common cancer in women and a prominent cause of death[9]. In Ethiopia, cervical cancer is a big deal for women aged 15 to 44, coming in as the second most common cancer[9]. Globally, it's the fourth most common prevalent disease for women[10]. Aberrant methylation of tumor-suppressor genes' promoters can shut down their important functions and play a big role in causing cervical tumors[10]. There are various cervical cancer repressor genes (proteins turn off or reduce gene expression from the affected gene), such as CCNA1, CHF, HIT, PAX1, PTEN, SFRP4, and TSC1. The genes play a crucial role in causing cervical cancer by regulating transcription and expression through promoter hypermethylation, leading to precursor lesions during cervical development and malignant transformation[11]. The process of DNA methylation is primarily carried out by a group of enzymes known as DNA methyltransferases (DNMT1). It has been reported that DNMT1 (PDB ID: 4WXX), a protein responsible for DNA methylation can contribute to the development of cervical cancer. DNMT1 inhibits the transcription of tumor suppressor genes, facilitating tumorigenesis, which finally develops into cervical cancer. Tumor suppressor gene transcription is inhibited by DNMT1, which helps cancer grow and eventually leads to cervical cancer. Repressive genes' hypermethylation may be decreased, their expression can be increased, and the phenotype of malignant tumors can be reversed by inhibiting the DNMT1 enzyme.

    On the other hand, infection by the human papilloma virus (HPV) phenotype 16, enzyme 6 (PDB ID: 4XR8) has been correlated with a greatly increased risk of cervical cancer worldwide[12]. Based on variations in the nucleotide sequences of the virus genome, over 100 distinct varieties of the human papilloma virus (HPV) have been identified (e.g. type 1, 2 etc.). Genital warts can result from certain types 6 and 11 of sexually transmitted HPVs. Other HPV strains, still, that can infect the genitalia, do not show any symptoms of infection[8]. Persistent infection with a subset of approximately 13 so-called 'high-risk' sexually transmitted HPVs, including such as types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 different from the ones that cause warts may lead to the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). These are precancerous lesions and can progress to invasive cancer. Almost all occurrences of cervical cancer have HPV infection as a required component[13]. Superfluous infection by HPV type 16 E6 (PDB ID: 4XR8) has been correlated with a greatly increased genital risk of precursor cervical cancer worldwide[11]. Scholars more defined in major biochemical and biological activities of HPV type 16 E6 (PDB ID: 4XR8) in high-risk HPV oncogenes and how they may work together in the development of cervical disease and cancer[13].

    One potential approach to treat cervical cancer is to inhibit the activity of the DNMT1 and HPV type 16 E6 enzymes specifically[1316]. Over 50% of clinical drug forms worldwide originate from plant compounds[17]. In the past, developing new drugs was a lengthy and costly process. However, with the emergence of bioinformatics, the use of computer-based tools and methods have become increasingly important in drug discovery. One such method is molecular docking and ADMET profiling which involves using the structure of a drug to screen for potential candidates. This approach is known as structure-based drug design and can save both time and resources during the research process[15]. Structural-based drug designing addresses ligand binding sites with a known protein structure[15]. Using free binding energies, a computational method known as docking examines a large number of molecules and suggests structural theories for impeding the target molecule[17]. Nowadays, due to increasing antibiotic resistance like bacteria, fungi, and cancer cells, natural products remain an important source for discovering antimicrobial compounds and novel drugs for anti-cancers like cervical cancers. Therefore, the purpose of this research is to assess the antimicrobial activity of extracts, molecular docking, ADMET profiling in anticancer properties of compounds isolated from Salvia rosmarinus, on a targeting DNMT1 and HPV type 16 E6 in human cervical cancer. In the present study, various solvent crude extracts obtained from Salvia rosmarinus were used for antimicrobial activity and the isolated compounds 1 and 2 were submitted for in silico study to target the DNMT1 and HPV type 16 E6 enzymes to inhibit the growth of human cervical cancer cells.

    Healthy Salvia rosmarinus leaves were collected in Bacho district, Southwest Showa, Oromia, Ethiopia, during the dry season of November 2022. The plant materials were authenticated by Melaku Wondafrish, Natural Science Department, Addis Ababa University and deposited with a voucher number 3/2-2/MD003-80/8060/15 in Addis Ababa University's National Herbarium.

    The most common organic solvent used in extractions of medicinal plants is 2.5 L of petroleum ether, chloroform/methanol (1:1), and methanol. The test culture medium for microbes was used and performed in sterile Petri dishes (100 mm diameter) containing sterile Muller–Hinton Agar medium (25 mL, pH 7) and Sabouraud Dextrose Agar (SDA) for bacteria and fungi, respectively. A sterile Whatman filter paper (No. 1) disc of 6 mm diameter was used to determine which antibiotics an infective organism is sensitive to prescribed by a minimum zone of inhibition (MZI). Ciprofloxacin antibiotic reference (manufactured by Wellona Pharma Ciprofloxacin tablet made in India) and Ketoconazole 2% (made in Bangladesh) were used as a positive controls for antibacterial and antifungal, respectively and Dimethyl sulfoxide (DMSO) 98.9% was used as a negative control for antimicrobial tests. In the present study, the height of the column was 650 mm and the width was 80 mm. Several studies by previous researchers showed the acceptable efficiency of column chromatography (up to 43.0% w/w recovery) in the fractionation and separation of phenolic compounds from plant samples[18]. In column chromatography, the ideal stationary phase used silica gel 60 (0.200 mm) particles. The 1H-NMR spectrums of the compounds were analyzed using a 600 MHz NMR machine and 150 MHz for 13C NMR. The compounds were dissolved in MeOD for compound 1 and in DMSO for compound 2 for NMR analysis. On the other hand, UV spectroscopy (made in China) used 570 nm ultraviolet light to determine the absorbency of flavonoids (mg·g−1) phytochemicals.

    The samples (extracts) were analyzed to detect the presence of certain chemical compounds such as alkaloids (tested using Wagner's reagents), saponins (tested using the froth test), steroids (tested with Liebermann Burchard's tests), terpenoids (tested with Lidaebermann Burchard's tests), quinones, and flavonoids (tested using Shinoda tests)[19].

    The leaves of Salvia rosmarinus (500 g) were successively extracted using maceration using petroleum ether, chloroform/methanol (1:1), and methanol, every one 2.5 L for 72 h to afford 3.6, 6, and 53 g crude extracts, respectively. The methanol/chloroform (1:1) extract (6 g) was loaded to silica gel (150 g) column chromatography using the increasing polarity of petroleum ether, methanol/chloroform (1:1) solvent system to afford 80 fractions (100 mL each). The fraction obtained from chloroform/methanol 1:1 (3:2) after repeated column chromatography yielded compound 1 (18 mg). Fractions 56-65, eluted with chloroform/methanol (1:1) were combined and purified with column chromatography to give compound 2 (10 mg).

    The microorganisms were obtained from the Ethiopia Biodiversity Institution (EBI). Two gram-positive bacteria namely Staphylococcus aureus serotype (ATCC 25923) and Streptococcus epidermidis (ATCC14990); and three gram-negative bacteria, namely Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 5702), and Klebsiella pneumonia (ATCC e13883) were inoculated overnight at 37 °C in Muller–Hinton Agar/MHA culture medium and two fungus strains of Candida albicans (ATCC 16404) and Aspergillus niger (ATCC 11414) were inoculated overnight at 27−30 °C in Sabouraud Dextrose Agar/SDA culture medium[20].

    The antibacterial and antifungal activities of different crude extracts obtained from Salvia rosmarinus plant leaves were evaluated by the disk diffusion method (in accordance with the 13th edition of the CLSI M02 document on hardydiagnostics.com/disk-diffusion). Briefly, the test was performed in sterile Petri dishes (100 mm diameter) containing solid and sterile Muller–Hinton Agar medium (25 mL, pH 7) and Sabouraud Dextrose Agar (SDA) for bacteria and fungi, respectively. The extracts were placed on the surface of the media that had previously been injected with a sterile microbial suspension (one microbe per petri dish) after being adsorbed on sterile paper discs (5 μL per Whatman disc of 6 mm diameter). To prevent test samples from eventually evaporating, all Petri dishes were sealed with sterile laboratory films. They were then incubated at 37 °C for 24 h, and the zone diameter of the inhibition was measured and represented in millimeters. Ciprofloxacin antibiotic reference (manufactured by Wellona Pharma Ciprofloxacin tablet, India) was used as a positive control and DMSO was used as a negative control for antibacterial activity test while Ketoconazole 2% (Bangladesh) was used as a positive control and 10 μL of 0.2% agar as a negative control for antifungal activity tests[20]. The term 'inhibitory concentration' refers to the minimum sample concentration required to kill 99.9% of the microorganisms present[21]. Three repetitions of the crude extract sample were used to precisely measure the inhibitory halo diameter (in mm), which was then expressed as mean ± standard deviation to assess the anti-microbial activity.

    Cervical cancer-causing protein was identified through relevant literature. The protein molecule structure of DNA (cytosine-5)-methyltransferase 1 (DNMT1) (PDB ID: 4WXX)[21] and HPV type 16 E6 (PDB ID: 4XR8)[21] - a protein known to cause cervical cancer - were downloaded from the Protein Data Bank[22]. The stability of the protein molecule was assessed using Rampage[23].

    Phytochemical constituents of Salvia rosmarinus plant leaves were used to select a source of secondary metabolites (ligands). Ligand molecules were obtained through plant extraction, and isolation, and realized with PubChem (https://pubchem.ncbi.nlm.nih.gov/). The ligands were downloaded in Silver diamine fluoride format (SDF) and then converted to PDB format using an online SMILES translator (https://cactus.nci.nih.gov/translate/). The downloaded files were in PDB format, which was utilized for running various tools and software[24].

    The Biovia Discovery Studio Visualizer software was used to analyze the protein molecule. The protein molecule was converted into PDB format and its hierarchy was analyzed by selecting ligands and water molecules. Both the protein molecule and the water molecules lost their attached ligands during the analysis. Finally, the protein's crystal structure was saved in a PDB file[25].

    PyRx software was utilized to screen secondary metabolites and identify those ligands with the lowest binding energy to the protein target. The ligands with the lowest binding energy were further screened for their drug-likeliness property through analysis. It is worth noting that PyRx runs on PDBQT format. To begin using PyRx, it needs to load a protein molecule. This molecule should be converted from PDB to the protein data bank, partial charge (Q), and Atom Type (PDBQT) format. Once the protein molecule is loaded, it can import ligands from a specific folder in Silver diamine fluoride format. The ligand energy was minimized and changed to PDBQT format. The protein was docked with the ligand and screened based on minimum binding energy (https://cactus.nci.nih.gov/translate/).

    The optimal ligand was selected for final docking using AutoDock Vina and Biovia by modifying the reference of Discovery Studio Client 2021 (https://cactus.nci.nih.gov/translate/).

    The protein target from the Protein Data Bank (PDB) was loaded onto the graphical interface of AutoDock Vina. To prepare the protein for docking, water molecules were removed, hydrogen polar atoms were added, and Kollman charges were assigned to the protein molecule. Ultimately, PDBQT format was used to store the protein. After being imported in PDB format, the Ligand molecule was transformed to PDBQT format. Next, a grid box was chosen to represent the docked region. The command prompt was used to run AutoDock Vina and the outcomes were examined (https://cactus.nci.nih.gov/translate/).

    Docking the ligand with the protein target DNMT1(PDB ID: 4WXX)[22] and HPV type 16 E6 (PDB ID: 4XR8)[21] enzymes were performed using Biovia Discovery Studio Client 2021 by loading the protein target first followed by the ligand in PDB format. The charges were attached to the protein molecule, and the energy was minimized for the ligands. Both the protein and ligand molecules were prepared for docking. Once the docking process was complete, the results were analyzed based on several parameters, including absolute energy, clean energy, conf number, mol number, relative energy, and pose number. The interaction between the protein and ligand was analyzed using structure visualization tools, such as Biovia Discovery Studio Visualizer and PyMol (https://cactus.nci.nih.gov/translate/).

    The process of visualizing the structure was carried out using the PyMol tool. PyMol is a freely available software. Firstly, the protein molecule in PDBQT form was loaded on the PyMol graphical screen. Then, the output PDBQT file was added. The docked structure was visualized and the 'molecule' option was changed to 'molecular surface' under the 'shown as' menu (https://cactus.nci.nih.gov/translate/).

    Drug likeliness properties of the screened ligands were evaluated using the SwissADME online server. SMILE notations were obtained from PubChem and submitted to the SwissADME web server for analysis. The drugs were subjected to Lipinski's rule of five[20] for analysis. Lipinski's rules of five were selected for final docking through AutoDock Vina and Biovia Discovery Studio Client 2021. Ligands 1 and 2 were analyzed using Lipinski's rule of five for docking with AutoDock Vina and Biovia Discovery Studio Client 2021.

    The antimicrobial analysis data generated by triplicate measurements reported as mean ± standard deviation, and a bar graph also generated by GraphPad Prism version 8.0.1 (244) for Windows were used to perform the analysis. GraphPad Prism was used and combined with scientific graphing, comprehensive bar graph fitting (nonlinear regression), understandable statistics, and data organization. Prism allows the performance and modification of basic statistical tests commonly used and determined through the statistical applications in microbiology labs (https://graphpad-prism.software.informer.com/8.0/).

    Phytochemical screening of the different extracts for the presence (+) and absence (−) of alkaloids, steroids, glycosides, coumarins, terpenoids, flavonoids, carbohydrates, tannins, and saponins were done. The present study showed that alkaloids, terpenoids, flavonoids, and tannins tests in S. rosmarinus leaves of petroleum ether, chloroform/methanol (1:1), and methanol extracts were high whereas glycoside, coumarins, and carbohydrates had a moderate presence. The extract of S. rosmarinus leaves contain commonly bioactive constituents such as alkaloids, steroids, terpenoids, flavonoids, tannins, and saponins. These bioactive chemicals have active medicinal properties. Phytochemical compounds found in S. rosmarinus leaves have the potential to treat cancer cells and pathogens. The study also found that these flavonoids are related to natural phenolic compounds with anticancer and antimicrobial properties in the human diet (Table 1).

    Table 1.  Phytochemical screening tests result of petroleum ether, chloroform/methanol (1:1) and methanol extracts of Salvia rosmarinus leaves.
    Botanical name Phytochemicals Phytochemical screening tests Different extracts
    Petroleum ether Chloroform/methanol (1:1) Mehanol
    Salvia rosmarinus Alkaloids Wagner's test ++ ++ ++
    Steroids Libermann Burchard test ++ + ++
    Glycoside Keller-Killiani test +
    Coumarins Appirade test + +
    Terpenoids Libermann Burchard test ++ ++ ++
    Flavonoids Shinoda test ++ ++ ++
    Carbohydrate Fehling's test ++ ++
    Tannins Lead acetate test ++ ++ ++
    Saponins Foam test + + +
    + indicates moderate presence, ++ indicates highly present, − indicates absence.
     | Show Table
    DownLoad: CSV

    Two compounds were isolated and characterized using NMR spectroscopic methods (Fig. 1 & Supplementary Fig. S1ac). Compound 1 (10 mg) was isolated as yellow crystals from the methanol/chloroform (1:1) leaf extract of Salvia rosmarinus. The TLC profile showed a spot at Rf 0.42 with methanol/chloroform (3:2) as a mobile phase. The 1H-NMR spectrum (600 MHz, MeOD, Table 2, Supplementary Fig. S1a) of compound 1 showed the presence of one olefinic proton signal at δ 5.3 (t, J = 3.7 Hz, 1H), two deshielded protons at δ 4.7 (m, 1H), and 4.1 (m, 1H) associated with the C-30 exocyclic methylene group, and one O-bearing methine proton at δH 3.2 (m, 1H), and six methyl protons at δ 1.14 (s, 3H), 1.03 (d, J = 6.3 Hz, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.87 (s, 3H), and 0.80 (s, 3H). A proton signal at δ 2.22 (d, J = 13.5 Hz, 1H) was attributed to methine proton for H-18. Other proton signals integrate for 20 protons were observed in the range δ 2.2 to 1.2. The proton decoupled 13C-NMR and DEPT-135 spectra (151 MHz, MeOD, Supplementary Fig. S1b & c) of compound 1 revealed the presence of 30 well-resolved carbon signals, suggesting a triterpene skeleton. The analysis of the 13C NMR spectrum displayed signals corresponding to six methyl, nine methylene, seven methine, and eight quaternary carbons. Among them, the signal observed at δ 125.5 (C-12) belongs to olefinic carbons. The methylene carbon showed signals at δC 39.9, 28.5, 18.1, 36.7, 23.9, 30.4, 26.5, 32.9, and 38.6. The quaternary carbons showed a signal at δC 39.4, 41.9, 38.4, 138.2, 41.8, and 47.8. The signals of exocyclic methylene carbon signals appeared at δ 153.1 and 103.9. The spectrum also showed sp3 oxygenated methine carbon at δ 78.3 and carboxyl carbon at δ 180.2. The spectrum revealed signals due to methyl groups at δC 27.4, 16.3, 15.0, 20.2, 22.7, and 16.4. The remaining carbon signals for aliphatic methines were shown at δC 55.3, 55.2, 53.0, and 37.1. The NMR spectral data of compound 1 is in good agreement with data reported for micromeric acid, previously reported from the same species by Abdel-Monem et al.[26]. (Fig. 1, Table 2).

    Figure 1.  Structure of isolated compounds from the leaves of Salvia rosmarinus.
    Table 2.  Comparison of the 13C-NMR spectral data of compound 1 and micromeric acid (MeOD, δ in ppm).
    Position NMR data of compound 1 Abdel-Monem
    et al.[26]
    1H-NMR 13C-NMR 13C-NMR
    1 38.60 39.9
    2 27.8 28.5
    3 3.2 (m, 1H) 78.3 80.3
    4 39.4 39.9
    5 55.3 56.7
    6 18.1 18.3
    7 36.7 34.2
    8 41.9 40.7
    9 53 48.8
    10 38.4 38.2
    11 23.9 24.6
    12 5.3 (t, J = 3.7 Hz, 1H) 125.5 127.7
    13 138.2 138
    14 41.8 43.3
    15 30.4 29.1
    16 26.5 25.6
    17 47.8 48
    18 δ 2.22 (d, J = 13.5 Hz, 1H) 55.2 56.1
    19 37.1 38.7
    20 153.1 152.8
    21 32.9 33.5
    22 39.0 40.1
    23 27.4 29.4
    24 16.3 16.9
    25 15.0 16.6
    26 20.2 18.3
    27 22.7 24.6
    28 180.2 177.8
    29 16.4 17.3
    30 4.7 (m, 1H), and 4.1 (m, 1H) 103.9 106.5
     | Show Table
    DownLoad: CSV

    Compound 2 (18 mg) was obtained as a white amorphous isolated from 40% methanol/chloroform (1:1) in petroleum ether fraction with an Rf value of 0.49. The 1H NMR (600 MHz, DMSO, Supplementary Fig. S2a) spectral-data showed two doublets at 7.79 (d, J = 8.7 Hz, 2H), and 6.90 (d, J = 8.7 Hz, 2H) which are evident for the presence of 1,4-disubstituted aromatic group. The oxygenated methylene and terminal methyl protons were shown at δ 4.25 (q, J = 7.1 Hz, 2H) and 1.29 (t, J = 7.1 Hz, 3H), respectively. The13C-NMR spectrum, with the aid of DEPT-135 (151 MHz, DMSO, Table 3, Supplementary Fig. S2b & c) spectra of compound 2 confirmed the presence of well-resolved seven carbon peaks corresponding to nine carbons including threee quaternary carbons, one oxygenated methylene carbon, one terminal methyl carbon, and two symmetrical aromatic methine carbons. The presence of quaternary carbon signals was shown at δ 120.9 (C-1), 148.2 (C-4), and ester carbonyl at δ 166.0 (C-7). The symmetry aromatic carbons signal was observed at δ 131.4 (C-2, 6), and 116.8 (C-3, 5). The oxygenated methylene and terminal methyl carbons appeared at δC 60.4 (C-8) and 14.7 (C-9), respectively. The spectral results provided above were in good agreement with those for benzocaine in the study by Alotaibi et al.[27]. Accordingly, compound 2 was elucidated to be benzocaine (4-Aminobenzoic acid-ethyl ester) (Table 3, Fig. 1, Supplementary Fig. S2ac), this compound has never been reported before from the leaves of Salvia rosmarinus.

    Table 3.  Comparison of the 1H-NMR, and 13C-NMR spectral data of compound 2 and benzocaine (DMSO, δ in ppm).
    Position NMR data of compound 2 Alotaibi et al.[27]
    1H-NMR 13C-NMR 1H-NMR 13C-NMR
    1 120.9 119
    2 7.79 (d, J = 8.7 Hz, 2H) 131.4 7.86 (d, J = 7.6 Hz) 132
    3 6.90 (d, J = 8.7 Hz, 2H) 116.8 6.83 (d, J = 7.6 Hz) 114
    4 148.2 151
    5 6.90 (d, J = 8.7 Hz, 2H) 116.8 6.83 (d, J = 7.6 Hz) 114
    6 7.79 (d, J = 8.7 Hz, 2H) 131.4 7.86 (d, J = 7.6 Hz) 132
    7 166.0 169
    8 4.3 (q, J = 7.1 Hz, 2H) 60.4 4.3 (q, J = 7.0 Hz) 61
    9 1.3 (t, J = 7.1 Hz, 3H) 14.7 1.36 (t, J = 7.0 Hz) 15
     | Show Table
    DownLoad: CSV

    The extracts and isolated compounds from Salvia rosmarinus were evaluated in vitro against microbes from gram-positive bacteria (S. aureus and S. epidermidis), gram-negative bacteria (E. coli, P. aeruginosa, and K. pneumoniae) and fungi (C. albicans and A. Niger) (Table 4). The petroleum ether extracts exhibited significant activity against all the present study-tested microbes at 100 μg·mL−1, resulting in an inhibition zone ranging from 7 to 21 mm. Chloroform/methanol (1:1) and methanol extracts demonstrated significant activity against all the present study-tested microbes at 100 μg·mL−1 exhibiting inhibition zones from 6 to 14 mm and 6 to 13 mm, respectively (Table 4). The chloroform/methanol (1:1) extracts were significantly active against bacteria of E. coli and K. pneumonia, and A. Niger fungi at 100 μg·mL−1. On the other hand, chloroform/methanol (1:1) extracts were significantly inactive against the S. rosmarinus and P. aeruginosa of bacteria and C. albicans of fungi, and again chloroform/methanol (1:1) extracts overall significantly active produced an inhibition zone of 12 to 14 mm (Table 4). Methanol extracts exhibited significant activity against S. aureus, E. coli bacteria, and A. Niger fungi at 100 μg·mL−1. The inhibition zone was recorded to be 11 to 13 mm. However, methanol extracts exhibited significant inactivity against K. pneumoniae (Table 4). The overall result of our studies shows that Salvia rosmarinus was extracted and evaluated in vitro, exhibiting significant antibacterial and antifungal activity, with inhibition zones recorded between 6 to 21 mm for bacteria and 5 to 21 mm for fungi. In our study, the positive control for ciprofloxacin exhibited antibacterial activity measured at 21.33 ± 1.15 mm, 15.00 ± 0.00 mm, and 14.20 ± 0.50 mm for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. Similarly, the positive control for ketoconazole demonstrated antifungal activity of 22.00 ± 1.00 mm, 13.67 ± 0.58 mm, and 15.00 ± 0.58 mm for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. Additionally, our findings indicated that the mean values of flavonoids (mg/g) tested were 92.2%, 90.4%, and 94.0% for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. This suggests that the groups of phenolic compounds evaluated play a significant role in antimicrobial activities, particularly against antibiotic-resistant strains.

    Table 4.  Comparison of mean zone of inhibition (MZI) leaf extracts of Salvia rosmarinus.
    Type of specimen, and standard antibiotics for
    each sample
    Concentration (μg·mL−1) of extract
    in 99.8% DMSO
    Average values of the zone of inhibition (mm)
    Gram-positive (+) bacteria Gram-negative (−) bacteria Fungai
    S. aurous S. epidermidis E. coli P. aeruginosa K. pneumoniae C. albicans A. niger
    Petroleum ether extracts
    S. rosmarinus 50 18.50 ± 0.50 15.33 ± 0.58 0.00 ± 0.00 0.00 ± 0.00 10.00 ± 0.00 15.93 ± 0.12 4.47 ± 0.50
    75 19.87 ± 0.06 17.00 ± 0.00 9.33 ± 0.29 10.53 ± 0.50 10.93 ± 0.12 18.87 ± 0.23 5.47 ± 0.50
    100 21.37 ± 0.78 17.50 ± 0.50 11.47 ± 0.50 13.17 ± 0.29 12.43 ± 0.51 20.83 ± 0.76 6.70 ± 0.10
    Standard antibiotics Cipro. 21.33 ± 1.15 18.33 ± 0.58 9.33 ± 0.58 12.30 ± 0.52 15.00 ± 0.00
    Ketocon. 22.00 ± 1.00 10.67 ± 0.58
    Chloroform/methanol (1:1) extracts
    50 5.47 ± 0.42 0.00 ± 0.00 10.33 ± 0.00 0.00 ± 0.00 9.70 ± 0.00 0.00 ± 0.12 8.47 ± 0.50
    S. rosmarinus
    75 5.93 ± 0.06 0.00 ± 0.00 11.33 ± 0.29 0.00 ± 0.50 12.50 ± 0.12 0.00 ± 0.23 10.67 ± 0.50
    100 6.47 ± 0.06 0.00 ± 0.00 14.17 ± 0.50 7.33 ± 0.29 14.17 ± 0.51 0.00 ± 0.76 12.67 ± 0.10
    Standard antibiotics Cipro. 15.00 ± 0.00 11.00 ± 1.00 11.33 ± 0.58 10.00 ± 0.52 12.67 ± 0.00
    Ketocon. 7.00 ± 1.00 13.67 ± 0.58
    Methanol extracts
    50 9.17 ± 0.29 5.50 ± 0.50 0.00 ± 0.00 7.50 ± 0.00 0.00 ± 0.00 6.57 ± 0.12 0.00 ± 0.50
    S. rosmarinus
    75 9.90 ± 0.10 6.93 ± 0.12 9.33 ± 0.29 8.50 ± 0.50 0.00 ± 0.00 8.70 ± 0.23 0.00 ± 0.50
    100 11.63 ± 0.55 7.97 ± 0.06 11.47 ± 0.50 9.90 ± 0.10 0.00 ± 0.00 10.83 ± 0.76 13.13 ± 0.10
    Standard antibiotics Cipro. 13.00 ± 0.00 11.50 ± 0.50 14.20 ± 0.58 13.33 ± 0.29 10.00 ± 0.00
    Ketocon. 12.00 ± 1.00 15.00 ± 0.58
    Mean values of flavonoids (mg·g−1) by 570 nm
    S. rosmarinus
    Petroleum ether extracts Chloroform/methanol (1:1) extracts Methanol extracts
    50 0.736 0.797 0.862
    75 0.902 0.881 0.890
    100 0.922 0.904 0.940
    Samples: Antibiotics: Cipro., Ciprofloxacin; Ketocon., ketoconazole (Nizoral); DMSO 99.8%, Dimethyl sulfoxide.
     | Show Table
    DownLoad: CSV

    Determining the three solvent extracts in S. rosmarinus plants resulted in relatively high comparable with positive (+) control. Especially, the S. rosmarinus petroleum ether leaf extracts against drug resistance human pathogenic bacteria S. aureus, S. epidermidis, E. coli, P. aeruginosa, and K. pneumoniae were minimum zone of inhibition (MZI) recorded that 21.37 ± 0.78, 17.50 ± 0.50, 11.47 ± 0.50, 13.17 ± 0.29, and 12.43 ± 0.51 mm, respectively and against human pathogenic fungi C. albicans and A. niger were minimum zone of inhibition (MZI) recorded that 20.83 ± 0.76 and 6.70 ± 0.10 mm, respectively which was used from bacteria against S. aureus MZI recorded that 21.37 ± 0.78 mm higher than the positive control (21.33 ± 1.15 mm). The S. rosmarinus of chloroform/methanol (1:1) extracts were found to be against E. coli (14.17 ± 0.50 mm) and K. pneumoniae (14.17 ± 0.51 mm) higher than the positive control 11.33 ± 0.58 and 12.67 ± 0.00 mm, respectively. The methanol extracts of leaves in the present study plants were found to have overall MZI recorded less than the positive control. The Salvia rosmarinus crude extracts showed better antifungal activities than the gram-negative (−) bacteria (Table 4, Fig 2, Supplementary Fig. S3). Therefore, the three extracts, using various solvents of different polarity indexes, have been attributed to specific biological activities. For example, the antimicrobial activities of Salvia rosmarinus extracts may be due to the presence of alkaloids, terpenoids, flavonoids, tannins, and saponins in natural products (Table 1).

    Figure 2.  Microbes' resistance with drugs relative to standard antibiotics in extracts of Salvia rosmarinus. The figures represent understudy of three extracts derived from Salvia rosmarinus. (a) Petroleum ether, (b) chloroform/methanol (1:1), and (c) methanol extracts tested in Salvia rosmarinus.

    Compounds 1 and 2 were isolated from chloroform/methanol (1:1) extract of Salvia rosmarinus (Fig. 1, Tables 2 & 3). The plant extract exhibited highest antibacterial results recorded a mean inhibition with diameters of 21 and 14 mm at a concentration of 100 mg·mL−1 against S. aureus and E. coli/K. pneumoniae, respectively. After testing, overall it was found that the highly active petroleum ether extract of Salvia rosmarinus was able to inhibit the growth of S. aureus and C. albicans, with inhibition zones of 21 and 20 mm, respectively. The petroleum ether extracts showed good efficacy against all tested microbes, particularly gram-positive bacteria and fungi (Table 4). This is noteworthy because gram-negative bacteria generally exhibit greater resistance to antimicrobial agents. Petroleum ether and chloroform/methanol (1:1) extracts of the leaves were used at a concentration of 100 mg·mL−1, resulting in impressive inhibition zone diameters of 11 and 14 mm for E. coli, 13 and 7 mm for P. aeruginosa, and 12 and 14 mm for K. pneumoniae, respectively.

    The present study found that at a concentration of 50 μg·mL−1, petroleum ether, chloroform/methanol (1:1), and MeOH extracts did not display any significant inhibition zone effects against the tested microbes. This implies that the samples have a dose-dependent inhibitory effect on the pathogens. The leaves of Salvia rosmarinus have been found to possess remarkable antimicrobial properties against gram-negative bacteria in different extracts such as E. coli, P. aeruginosa, and K. pneumoniae with 14.17 ± 0.50 in chloroform/methanol (1:1), 13.17 ± 0.29 in petroleum ether and 14.17 ± 0.51 in chloroform/methanol (1:1), respectively. However, in the present study, Salvia rosmarinus was found to possess remarkable high zones of inhibition with diameters of 21.37 ± 0.78 and 17.50 ± 0.50 mm antimicrobial properties against S. aureus, and S. epidermidis of gram-positive bacteria, respectively (Supplementary Fig. S3). The results are summarized in Fig. 2ac.

    The crystal structure of human DNMT1 (351-1600), classification transferase, resolution: 2.62 Å, PDB ID: 4WXX. Active site dimensions were set as grid size of center X = −12.800500 Å, center Y = 34.654981 Å, center Z = −24.870231 Å (XYZ axis) and radius 59.081291. A study was conducted to investigate the binding interaction of the isolated compounds 1 and 2 of the leaves of Salvia rosmarinus with the binding sites of the DNMT1 enzyme in human cervical cancer (PDB ID: 4WXX), using molecular docking analysis.

    The study also compared the results with those of standard anti-cancer agents Jaceosidin (Table 5 & Fig. 3). The compounds isolated had a final fixing energy extending from −5.3 to −8.4 kcal·mol−1, as shown in Table 4. It was compared to jaceosidin (–7.8 kcal·mol−1). The results of the molecular docking analysis showed that, compound 1 (−8.4 kcal·mol−1) showed the highest binding energy values compared with the standard drugs jaceosidin (–7.8 kcal·mol−1). Compound 2 has shown lower docking affinity (–5.3 kcal·mol−1) but good matching amino acid residue interactions compared to jaceosidin. After analyzing the results, it was found that the isolated compounds had similar residual interactions and docking scores with jaceosidin.

    Table 5.  Molecular docking results of ligand compounds 1 and 2 against DNMT1 enzyme (PDB ID: 4WXX).
    Ligands Binding affinity

    ( kcal·mol−1)
    H-bond Residual interactions
    Hydrophobic/electrostatic Van der Waals
    1 −8.4 ARG778 (2.85249), ARG778 (2.97417), VAL894 (2.42832) Lys-889, Pro-879, Tyr-865, His-795, Cys-893, Gly-760, Val-759, Phe-892, Phe-890, Pro-884, Lys-749
    2 −5.3 ARG596 (2.73996), ALA597 (1.84126), ILE422 (2.99493), THR424 (2.1965), ILE422 (2.93653) Electrostatic Pi-Cation-ARG595 (3.56619), Hydrophobic Alkyl-ARG595 (4.15839), Hydrophobic Pi-Alkyl-ARG595 (5.14967) Asp-423, Glu-428, Gly-425, Ile-427, Trp-464, Phe-556, Gln-560, Gln-594, Glu-559, Gln-598, Ser-563
    Jaceosidin −7.8 ASP571 (2.93566), GLN573 (2.02126), GLU562 (2.42376), GLN573 (3.49555), GLU562 (3.46629) Hydrophobic Alkyl-PRO574 (4.59409), Hydrophobic Alkyl-ARG690 (5.09748), Hydrophobic Pi-Alkyl-PHE576 (5.1314), Hydrophobic Pi-Alkyl-PRO574 (4.97072), Hydrophobic Pi-Alkyl-ARG690 (5.07356) Glu-698, Cys-691, Ala-695, Pro-692, Val-658, Glu-566, Asp-565
     | Show Table
    DownLoad: CSV
    Figure 3.  The 2D and 3D binding interactions of compounds against DNMT1 enzyme (PDB ID: 4WXX). The 2D and 3D binding interactions of compound 1 and 2 represent against DNMT1 enzyme, and jaceosidin (standard) against DNMT1 enzyme.

    Hence, compound 1 might have potential anti-cancer agents. However, anti-cancer in vitro analysis has not yet been performed. Promising in silico results indicate that further research could be beneficial. The 2D and 3D binding interactions of compounds 1 and 2 against human cervical cancer of DNMT1 enzyme (PDB ID: 4WXX) are presented in Fig. 3. The binding interactions between the DNMT1 enzyme (PDB ID: 4WXX), and compound 1 (Fig. 3) and compound 2 (Fig. 3) were displayed in 3D. Compounds and amino acids are connected by hydrogen bonds (green dash lines) and hydrophobic interactions (non-green lines).

    Crystal structure of the HPV16 E6/E6AP/p53 ternary complex at 2.25 Å resolution, classification viral protein, PDB ID: 4XR8. Active site dimensions were set as grid size of center X = −43.202782 Å, center Y = −39.085513 Å, center Z = −29.194115 Å (XYZ axis), R-value observed 0.196, and Radius 65.584122. A study was conducted to investigate the binding interaction of the isolated compounds 1 and 2 of the leaves of Salvia rosmarinus with the binding sites of the enzyme of human papilloma virus (HPV) type 16 E6 (PDB ID: 4XR8), using molecular docking analysis software. The study also compared the results with those of standard anti-cancer agents jaceosidin (Table 6 & Fig. 4). The compounds isolated had a bottom most fixing energy extending from −6.3 to −10.1 kcal·mol−1, as shown in Table 6. It was compared to jaceosidin (–8.8 kcal·mol−1). The results of the molecular docking analysis showed that, compound 1 (−10.1 kcal·mol−1) showed the highest binding energy values compared with the standard drugs jaceosidin (–8.8 kcal·mol−1). Compound 2 has shown lower docking affinity (–6.3 kcal·mol−1) but good matching amino acid residue interactions compared to jaceosidin. After analyzing the results, it was found that the isolated compounds had similar residual interactions and docking scores with jaceosidin.

    Table 6.  Molecular docking results of ligand compounds 1 and 2 against HPV type 16 E6 (PDB ID: 4XR8).
    Ligands Binding affinity
    (kcal·mol−1)
    H-bond Residual interactions
    Hydrophobic/electrostatic Van der Waals
    1 −10.1 ASN101 (2.25622), ASP228 (2.88341) Asp-148, Lys-176, Lys-180, Asp-178, Ile-179, Tyr-177, Ile-334, Glu-382, Gln-336, Pro-335, Gln-73, Arg-383, Tyr-100
    2 −6.5 TRP63 (1.90011), ARG67 (2.16075), ARG67 (2.8181) Hydrophobic Pi-Sigma-TRP341 (3.76182), Hydrophobic Pi-Pi Stacked-TYR156 (4.36581), Hydrophobic Pi-Pi T-shaped-TRP63 (5.16561), Hydrophobic Pi-Pi T-shaped-TRP63 (5.44632), Hydrophobic Alkyl-PRO155 (4.34691), Hydrophobic Pi-Alkyl-TRP341 (4.11391), Hydrophobic Pi-Alkyl-ALA64 (4.61525) Glu-154, Arg-345, Asp-66, Met-331, Glu-112, Lys-16, Trp-231
    Jaceosidin −8.8 ARG146 (2.06941), GLY70 (3.49991), GLN73 (3.38801) Electrostatic Pi-Cation-ARG67 (3.93442), Hydrophobic Pi-Alkyl-PRO49 (5.40012) Tyr-342, Tyr-79, Ser-338, Arg-129, Pro-335, Leu-76, Tyr-81, Ser-74, Tyr-71, Ser-80, Glu-46
     | Show Table
    DownLoad: CSV
    Figure 4.  The 2D and 3D binding interactions of compounds against HPV type 16 E6 (PDB ID: 4XR8). The 2D and 3D binding interactions of compound 1 and 2 represent against HPV type 16 E6 enzyme, and jaceosidin (standard) against HPV type 16 E6 enzyme.

    Hence, compounds 1 and 2 might have potential anti-cancer agents of HPV as good inhibitors. However, anti-cancer in vitro analysis has not been performed yet on HPV that causes cervical cancer agents. Promising in silico results indicate that further research could be beneficial. The 2D and 3D binding interactions of compounds 1 and 2 against human papilloma virus (HPV) type 16 E6 enzyme (PDB ID: 4XR8) are presented in Fig. 4. The binding interactions between the HPV type 16 E6 enzyme (PDB ID: 4XR8) and compound 1 (Fig. 4) and compound 2 (Fig. 4) were displayed in 3D. Compounds and amino acids are connected by hydrogen bonds (magenta lines) and hydrophobic interactions (non-green lines).

    In silico bioactivities of a drug, including drug-likeness and toxicity, predict its oral activity based on the document of Lipinski's Rule[25] was stated and the results of the current study showed that the compounds displayed conform to Lipinski's rule of five (Table 7). Therefore, both compounds 1 and 2 should undergo further investigation as potential anti-cancer agents. Table 8 shows the acute toxicity predictions, such as LD50 values and toxicity class classification (ranging from 1 for toxic, to 6 for non-toxic), for each ligand, revealing that none of them were acutely toxic. Furthermore, they were found to be similar to standard drugs. Isolated compound 1 has shown toxicity class classification 4 (harmful if swallowed), while 2 showed even better toxicity prediction giving results of endpoints such as hepatotoxicity, mutagenicity, cytotoxicity, and irritant (Table 8). All the isolated compounds were predicted to be non-hepatotoxic, non-irritant, and non-cytotoxic. However, compound 1 has shown carcinogenicity and immunotoxicity (Table 9). Hence, based on ADMET prediction analysis, none of the compounds have shown acute toxicity, so they might be proven as good drug candidates.

    Table 7.  Drug-likeness predictions of compounds computed by Swiss ADME.
    Ligands Formula Mol. Wt. (g·mol−1) NRB NHA NHD TPSA (A°2) Log P (iLOGP) Log S (ESOL) Lipinski's rule of five
    1 C30H46O3 454.68 1 3 2 57.53 3.56 −6.21 1
    2 C 9H11NO2 165.19 3 2 1 52.32 1.89 −2.21 0
    Jaceosidin C17H14O7 330.3 3 7 3 105 1.7 1 0
    NHD, number of hydrogen donors; NHA, number of hydrogen acceptors; NRB, number of rotatable bonds; TPSA, total polar surface area; and log P, octanol-water partition coefficients; Log S, turbid metric of solubility.
     | Show Table
    DownLoad: CSV
    Table 8.  Pre ADMET predictions of compounds, computed by Swiss ADME.
    Ligands Formula Skin permeation value
    (logKp - cm·s−1)
    GI
    absorption
    Inhibitor interaction
    BBB permeability Pgp substrate CYP1A2 inhibitor CYP2C19 inhibitor CYP2C9 inhibitor CYP2D6 inhibitor
    1 C30H46O3 −4.44 Low No No No No No No
    2 C 9H11NO2 −5.99 High Yes No No No No No
    Jaceosidin C17H14O7 −6.13 High No No Yes No Yes Yes
    GI, gastrointestinal; BBB, blood brain barrier; Pgp, P-glycoprotein; and CYP, cytochrome-P.
     | Show Table
    DownLoad: CSV
    Table 9.  Toxicity prediction of compounds, computed by ProTox-II and OSIRIS property explorer.
    Ligands Formula LD50
    (mg·kg−1)
    Toxicity
    class
    Organ toxicity
    Hepatotoxicity Carcinogenicity Immunotoxicity Mutagenicity Cytotoxicity Irritant
    1 C30H46O3 2,000 4 Inactive Active Active Inactive Inactive Inactive
    2 C 9H11NO2 NA NA Inactive Inactive Inactive Inactive Inactive Inactive
    Jaceosidin C17H14O7 69 3 Inactive Inactive Inactive Inactive Inactive Inactive
    NA, not available.
     | Show Table
    DownLoad: CSV

    Rosemary is an evergreen perennial plant that belongs to the family Lamiaceae, previously known as Rosmarinus officinalis. Recently, the genus Rosmarinus was combined with the genus Salvia in a phylogenetic study and became known as Salvia rosmarinus[28,29] and it has been used since ancient times for various medicinal, culinary, and ornamental purposes. In the field of food science, rosemary is well known as its essential oil is used as a food preservative, thanks to its antimicrobial and antioxidant properties, rosemary has many other food applications such as cooking, medicinal, and pharmacology uses[30]. According to the study, certain phytochemical compounds found in Salvia rosmarinus leaves have the potential to halt the growth of cancer cells, and pathogens or even kill them[31]. In literature, alkaloids are found mostly in fungi and are known for their strong antimicrobial properties, which make them valuable in traditional medicine[32,33]. However, in the present study, S. rosmarinus species have been shown to possess alkaloids. Most alkaloids have a bitter taste and are used to protect against antimalarial, antiasthma, anticancer, antiarrhythmic, analgesic, and antibacterial[33] also some alkaloids containing nitrogen such as vincristine, are used to treat cancer.

    Steroids occur naturally in the human body. They are hormones that help regulate our body's reaction to infection or injury, the speed of metabolism, and more. On the other hand, steroids are reported to have various biological activities such as chronic obstructive pulmonary disease (COPD), multiple sclerosis, and imitate male sex hormones[34]. It is a natural steroid compound occurring both in plants and animals[35]. Thus, were found in the present study. Terpenoids are derived from mevalonic acid (MVA) which is composed of a plurality of isoprene (C5) structural units. Terpenoids, like mono-terpenes and sesquiterpenes, are widely found in nature and more than 50,000 have been found in plants that reduce tumors and cancers. Many volatile terpenoids, such as menthol and perillyl alcohol, are used as raw materials for spices, flavorings, and cosmetics[36]. In the present study, high levels of these compounds were found in Salvia rosmarinus leaves.

    Flavonoids are a class of phenolic compounds commonly found in fruits and vegetables and are considered excellent antioxidants[37]. Similarly, the results of this study revealed that S. rosmarinus contain flavonoids. According to the literature, these flavonoids, terpenoids, and steroid activities include anti-diabetic, anti-inflammatory, anti-cancer, anti-bacterial, hepatic-protective, and antioxidant effects[36]. Tannins are commonly found in most terrestrial plants[38] and have the potential to treat cancer, and HIV/AIDS as well as to treat inflamed or ulcerated tissues. Similarly, in the present study, tannins were highly found in the presented plant. On the other hand, due to a sudden rise in the number of contagious diseases and the development of antimicrobial resistance against current drugs, drug development studies are vital to discovering novel medicinal compounds[30] and add to these cancer is a complex multi-gene disease[39] as in various cervical cancer repressor genes[11] that by proteins turn off or reduce gene expression from the affected gene to cause cervical cancer by regulating transcription and expression through promoter hypermethylation (DNMT1), leading to precursor lesions during cervical development and malignant transformation.

    In a previous study[40], a good antibacterial result was recorded at a median concentration (65 μg·mL−1). Methanol extract showed a maximum and minimum zone antibacterial result against negative bacteria E. coli 14 + 0.71 and most of the petroleum ether tests show null zone of inhibition. However, in the present study at a concentration of 100 μg·mL−1, the methanol extract demonstrated both maximum and minimum antibacterial zones against E. coli 11.47 ± 0.50. Conversely, the test conducted with petroleum ether exhibited a good zone of inhibition by increasing concentration. Further research may be necessary to determine the optimal concentration for this extract to maximize its efficacy. The results obtained in gram-negative bacteria such as E. coli, P. aeruginosa, and K. pneumoniae are consistent with previous research findings[41]. However, in the present study, Salvia rosmarinus has been found to possess high zones of inhibition with diameters of 21.37 ± 0.78 and 17.50 ± 0.50 mm antimicrobial properties against S. aureus, and S.epidermidis of gram-positive bacteria, respectively (Table 4 & Fig. 2, Supplementary Fig. S3). According to a previous study[42], the ethanolic leaf extract of Salvia rosmarinus did exhibit activity against C. albicans strains. In the present study, the antifungal activity of petroleum ether extracts from Salvia rosmarinus were evaluated against two human pathogenic fungi, namely C. albicans and A. niger. The findings showed that at a concentration of 100 μg·mL−1, the extracts were able to inhibit the growth of C. albicans 20.83 ± 0.76 resulting in a minimum zone of inhibition.

    Antimicrobial agents can be divided into groups based on the mechanism of antimicrobial activity. The main groups are: agents that inhibit cell wall synthesis, depolarize the cell membrane, inhibit protein synthesis, inhibit nucleic acid synthesis, and inhibit metabolic pathways in bacteria. On the other hand, antimicrobial resistance mechanisms fall into four main categories: limiting the uptake of a drug; modifying a drug target; inactivating a drug; and active drug efflux. Because of differences in structure, etc., there is a variation in the types of mechanisms used by gram-negative bacteria vs gram-positive bacteria. Gram-negative bacteria make use of all four main mechanisms, whereas gram-positive bacteria less commonly use limiting the uptake of a drug[43]. The present findings showed similar activity in chloroform/methanol (1:1) and methanol extracts of leaves of Salvia rosmarinus than gram-negative bacteria like P. aeruginosa and Klebsiella pneumoniae. However, Staphylococcus epidermidis of gram-positive bacteria under chloroform/methanol (1:1) extracts have similarly shown antimicrobial résistance. This occurred due to intrinsic resistance that may make use of limiting uptake, drug inactivation, and drug efflux that need further study. The structure of the cell wall thickness and thinners of gram-negative and gram-positive bacteria cells, respectively when exposed to an antimicrobial agent, there happen two main scenarios may occur regarding resistance and persistence. In the first scenario, resistant cells survive after non-resistant ones are killed. When these resistant cells regrow, the culture consists entirely of resistant bacteria. In the second scenario, dormant persistent cells survive. While the non-persistent cells are killed, the persistent cells remain. When regrown, any active cells from this group will still be susceptible to the antimicrobial agent.

    Ferreira et al.[44] explained that with molecular docking, the interaction energy of small molecular weight compounds with macromolecules such as target protein (enzymes), and hydrophobic interactions and hydrogen bonds at the atomic level can be calculated as energy. Several studies have been conducted showing natural products such as epigallocatechin-3-gallate-3-gallate (EGCG), curcumin, and genistein can be used as an inhibitor of DNMT1[4547] . In the literature micromenic (1) is used for antimicrobial activities and for antibiotic-resistance like methicillin-resistant Staphylococcus aureus (MRSA)[48], and benzocaine (2) is used to relieve pain and itching caused by conditions such as sunburn or other minor burns, insect bites or stings, poison ivy, poison oak, poison sumac, minor cuts, or scratches[49]. However, in the present study, Salvia rosmarinus was used as a source of secondary metabolites (ligands) by using chloroform/methanol (1:1) extract of the plant leaves yielded to isolate micromeric (1) and benzocaine (2) in design structure as a candidate for drugs as inhibitors of the DNMT1 enzyme by inhibiting the activity of DNMT1 that prevent the formation of cervical cancer cells.

    Cervical cancer is one of the most dangerous and deadly cancers in women caused by Human papillomaviruses (HPV). Some sexually transmitted HPVs (type 6 owner of E6) may cause genital warts. There are several options for the treatment of early-stage cervical cancer such as surgery, nonspecific chemotherapy, radiation therapy, laser therapy, hormonal therapy, targeted therapy, and immunotherapy, but there is no effective cure for an ongoing HPV infection. In the present study, Salvia rosmarinus leaves extracted and isolated compounds 1 and 2 are one of the therapeutic drugs design structure as a candidate drug for inhibiting HPV type 16 E6 enzyme. Similarly, numerous researchers have conducted studies on the impact of plant metabolites on the treatment of cervical cancer. Their research has demonstrated that several compounds such as jaceosidin, resveratrol, berberin, gingerol, and silymarin may be active in treating the growth of cells[47].

    Small-molecule drugs are still most commonly used in the treatment of cancer[50]. Molecular docking in in silico looks for novel small-molecule (ligands) interacting with genes or DNA or protein structure agents which are still in demand, newly designed compounds are required to have a specific even multi-targeted mechanism of action to anticancer and good selectivity over normal cells. In addition to these, in the literature, anti-cancer drugs are not easily classified into different groups[51]. Thus, drugs have been grouped according to their chemical structure, presumed mechanism of action, and cytotoxic activity related to cell cycle arrest, transcription regulation, modulating autophagy, inhibition of signaling pathways, suppression of metabolic enzymes, and membrane disruption[52]. Another problem for grouping anticancers often encountered is the resistance that may emerge after a brief period of a positive reaction to the therapy or may even occur in drug-naïve patients[50]. In recent years, many studies have investigated the molecular mechanism of compounds affecting cancer cells and results suggest that compounds exert their anticancer effects by providing free electron charge inhibiting some of the signaling pathways that are effective in the progression of cancer cells[53] and numerous studies have shown that plant-based compounds such as phenolic acids and sesquiterpene act as anticancer agents by affecting a wide range of molecular mechanisms related to cancer[53]. The present investigations may similarly support molecular mechanisms provided for the suppression of metabolic enzymes of cervical cancer.

    The main aim of the study was to evaluate the antimicrobial activity of different extracts of Salvia rosmarinus in vitro, and its compounds related to in silico targeting of enzymes involved in cervical cancer. The phytochemical screening tests indicated the presence of phytochemicals such as alkaloids, terpenoids, flavonoids, and tannins in its extracts. The plant also exhibited high antimicrobial activity, with varying efficacy in inhibiting pathogens in a dose-dependent manner (50−100 μg·mL−1). However, this extract exhibited a comparatively high inhibition zone in gram-positive and gram-negative bacteria had lower inhibition zones against E. coli, P. aeruginosa, and K. pneumoniae, respectively, and stronger antifungal activity 20.83 ± 0.76 mm inhibition zone against C. albicans fungi. Molecular docking is a promising approach to developing effective drugs through a structure-based drug design process. Based on the docking results, the in silico study predicts the best interaction between the ligand molecule and the protein target DNMT1 and HPV type 16 E6. Compound 1 (–8.3 kcal·mol−1) and 2 (–5.3 kcal·mol−1) interacted with DNMT1 (PDB ID: 4WXX) and the same compound 1 (–10.1 kcal·mol−1) and 2 (–6.5 kcal·mol−1) interacted with HPV type 16 E6 (PDB ID: 4XR8). Compounds 1 and 2 may have potential as a medicine for treating agents of cancer by inhibiting enzymes DNMT1 and HPV type 16 E6 sites, as well as for antimicrobial activities. None of the compounds exhibited acute toxicity in ADMET prediction analysis, indicating their potential as drug candidates. Further studies are required using the in silico approach to generate a potential drug through a structure-based drug-designing approach.

  • The authors confirm contribution to the paper as follows: all authors designed and comprehended the research work; plant materials collection, experiments performing, data evaluation and manuscript draft: Dejene M; research supervision and manuscript revision: Dekebo A, Jemal K; NMR results generation: Tufa LT; NMR data analysis: Dekebo A, Tegegn G; molecular docking analysis: Aliye M. All authors reviewed the results and approved the final version of the manuscript.

  • All data generated or analyzed during this study are included in this published article.

  • This work was partially supported by Adama Science and Technology University under Grant (ASTU/SP-R/171/2022). We are grateful for the fellowship support from Adama Science and Technology University (ASTU), the identification of plants by Mr. Melaku Wendafrash, and pathogenic strain support from the Ethiopian Biodiversity Institute (EBI). We also thank the technical assistants of the Applied Biology and Chemistry departments of Haramaya University (HU) for their help.

  • The authors declare that they have no conflict of interest.

  • [1]

    Runnel K, Miettinen O, Lõhmus A. 2021. Polypore fungi as a flagship group to indicate changes in biodiversity – a test case from Estonia. IMA Fungus 12:2

    doi: 10.1186/s43008-020-00050-y

    CrossRef   Google Scholar

    [2]

    Moose RA, Schigel D, Kirby LJ, Shumskaya M. 2019. Dead wood fungi in North America: an insight into research and conservation potential. Nature Conservation 7(32):1−7

    doi: 10.3897/natureconservation.32.30875

    CrossRef   Google Scholar

    [3]

    Liu S, Zhou JL, Song J, Sun YF, Dai YC, et al. 2023. Climacocystaceae fam. nov. and Gloeoporellaceae fam. nov., two new families of Polyporales (Basidiomycota). Frontiers in Microbiology 14:1115761

    doi: 10.3389/fmicb.2023.1115761

    CrossRef   Google Scholar

    [4]

    Ortiz-Santana B, Lindner DL, Miettinen O, Justo A, Hibbett DS. 2013. A phylogenetic overview of the antrodia clade (Basidiomycota, Polyporales). Mycologia 105:1391−1411

    doi: 10.3852/13-051

    CrossRef   Google Scholar

    [5]

    Han ML, Chen YY, Shen LL, Song J, Vlasák J, et al. 2016. Taxonomy and phylogeny of the brown-rot fungi: Fomitopsis and its related genera. Fungal Diversity 80:343−373

    doi: 10.1007/s13225-016-0364-y

    CrossRef   Google Scholar

    [6]

    Audet S. 2017. New genera and new combinations in Antrodia sl. Mushrooms Nomenclatural Novelties. pp. 1–9. https://sergeaudet myco.com/antrodia/

    [7]

    Binder M, Justo A, Riley R, Salamov A, Lopez-Giraldez F, et al. 2013. Phylogenetic and phylogenomic overview of the Polyporales. Mycologia 105:1350−73

    doi: 10.3852/13-003

    CrossRef   Google Scholar

    [8]

    Justo A, Miettinen O, Floudas D, Ortiz-Santana B, Sjökvist E, et al. 2017. A revised family-level classification of the Polyporales (Basidiomycota). Fungal Biology 121:798−824

    doi: 10.1016/j.funbio.2017.05.010

    CrossRef   Google Scholar

    [9]

    Liu S, Song CG, Xu TM, Ji X, Wu DM, et al. 2022. Species diversity, molecular phylogeny, and ecological habits of Fomitopsis (Polyporales, Basidiomycota). Frontiers in Microbiology 13:859411

    doi: 10.3389/fmicb.2022.859411

    CrossRef   Google Scholar

    [10]

    Kotlába F, Pouzar Z. 1958. Polypori novi vel minus cogniti Cechoslovakiae III. Ceská Mykologie 12:95−104

    Google Scholar

    [11]

    Song J, Chen YY, Cui BK. 2014. Phylogeny and taxonomy of Climacocystis (Polyporales) in China. Cryptogamie, Mycologie 35(3):221−31

    doi: 10.7872/crym.v35.iss3.2014.221

    CrossRef   Google Scholar

    [12]

    Ryvarden L, Gilbertson RL. 1993. European Polypores, Part 1: Synopsis Fungorum 6. Norway: Fungiflora, Oslo. pp. 1–387.

    [13]

    Núñez M, Ryvarden L. 2001. East Asian polypores 2: Synopsis Fungorum 14: 165e522. Norway: Fungiflora, Oslo. pp. 1–280.

    [14]

    Dai YC. 2012. Polypore diversity in China with an annotated checklist of Chinese polypores. Mycoscience 53:49−80

    doi: 10.1007/s10267-011-0134-3

    CrossRef   Google Scholar

    [15]

    Miettinen O, Larsson E, Sjökvist E, Larsson KH. 2012. Comprehensive taxon sampling reveals unaccounted diversity and morphological plasticity in a group of dimitic polypores (Polyporales, Basidiomycota). Cladistics 28:251−70

    doi: 10.1111/j.1096-0031.2011.00380.x

    CrossRef   Google Scholar

    [16]

    Ryvarden L, Johansen I. 1980. A preliminary polypore flora of East Africa. Fungiflora, Oslo, Norway. 636 pp.

    [17]

    Gilbertson RL, Ryvarden L. 1986. North American Polypores 1: Abortiporus – Lindtneria. Norway: Fungiflora, Oslo. pp. 1–433.

    [18]

    Hattori T, Sotome K. 2013. Type studies of the polypores described by E.J.H. Corner from Asia and West Pacific areas VIII. Species described in Trametes (2). Mycoscience 54:297−308

    doi: 10.1007/s10267-005-0250-z

    CrossRef   Google Scholar

    [19]

    Li HJ, Han ML, Cui BK. 2013. Two new Fomitopsis species from southern China based on morphological and molecular characters. Mycological Progress 12:709−18

    doi: 10.1007/s11557-012-0882-2

    CrossRef   Google Scholar

    [20]

    Han ML, Song J, Cui BK. 2014. Morphology and molecular phylogeny for two new species of Fomitopsis (Basidiomycota) from South China. Mycological Progress 13:905−14

    doi: 10.1007/s11557-014-0976-0

    CrossRef   Google Scholar

    [21]

    Han ML, Cui BK. 2015. Morphological characters and molecular data reveal a new species of Fomitopsis (Polyporales) from southern China. Mycoscience 56(2):168−176

    doi: 10.1016/j.myc.2014.05.004

    CrossRef   Google Scholar

    [22]

    Soares AM, Nogueira-Melo G, Plautz HL Jr, Gibertoni TB. 2017. A new species, two new combinations and notes on Fomitopsidaceae (Agaricomycetes, Polyporales). Phytotaxa 331:75−83

    doi: 10.11646/phytotaxa.331.1.5

    CrossRef   Google Scholar

    [23]

    Haight JE, Laursen GA, Glaeser JA, Taylor DL. 2016. Phylogeny of Fomitopsis pinicola: a species complex. Mycologia 108:925−38

    doi: 10.3852/14-225r1

    CrossRef   Google Scholar

    [24]

    Haight JE, Nakasone KK, Laursen GA, Redhead SA, Taylor DL, et al. 2019. Fomitopsis mounceae and F. schrenkii—two new species from North America in the F. pinicola complex. Mycologia 111:339−57

    doi: 10.1080/00275514.2018.1564449

    CrossRef   Google Scholar

    [25]

    Liu S, Song CG, Cui BK. 2019. Morphological characters and molecular data reveal three new species of Fomitopsis (Basidiomycota). Mycological Progress 18:1317−27

    doi: 10.1007/s11557-019-01527-w

    CrossRef   Google Scholar

    [26]

    Liu S, Han ML, Xu TM, Wang Y, Wu DM, et al. 2021. Taxonomy and phylogeny of the Fomitopsis pinicola complex with descriptions of six new species from east Asia. Frontiers in Microbiology 12:644979

    doi: 10.3389/fmicb.2021.644979

    CrossRef   Google Scholar

    [27]

    Zhou M, Wang CG, Wu YD, Liu S, Yuan Y. 2021. Two new brown rot polypores from tropical China. MycoKeys 82:173−97

    doi: 10.3897/mycokeys.82.68299

    CrossRef   Google Scholar

    [28]

    Binder M, Hibbett DS, Larsson KH, Larsson E, Langer E, et al. 2005. The phylogenetic distribution of resupinate forms across the major clades of mushroom-forming fungi (Homobasidiomycetes). Systematics and Biodiversity 3:113−57

    doi: 10.1017/S1477200005001623

    CrossRef   Google Scholar

    [29]

    Ali K, Khan N, Rahman IU, Ahmad H, Jury S. 2015. Multivariate analysis and vegetation mapping of a biodiversity hotspot in the Hindu Kush Mountains. International Journal of Advanced Research 3(6):990−1006

    Google Scholar

    [30]

    Razaq A, Shahzad S. 2017. Additions to the diversity of mushrooms in Gilgit-Baltistan, Pakistan. Pakistan Journal of Botany 49:305−9

    Google Scholar

    [31]

    Aman N, Khalid AN, Moncalvo JM. 2022. A compendium of macrofungi of Pakistan by ecoregions. MycoKeys 89:171−233

    doi: 10.3897/mycokeys.89.81148

    CrossRef   Google Scholar

    [32]

    Petersen JH. 1996. Farvekort. The Danish mycological society's colour-chart. Greve, Denmark: Foreningen til Svampekundskabens Fremme. pp. 1−6.

    [33]

    Hu Y, Karunarathna SC, Li H, Galappaththi MCA, Zhao CL, et al. 2022. The impact of drying temperature on basidiospore size. Diversity 14:239

    doi: 10.3390/d14040239

    CrossRef   Google Scholar

    [34]

    Ji X, Zhou JL, Song CG, Xu TM, Wu DM, et al. 2022. Taxonomy, phylogeny and divergence times of Polyporus (Basidiomycota) and related genera. Mycosphere 13:1−52

    doi: 10.5943/mycosphere/13/1/1

    CrossRef   Google Scholar

    [35]

    Banik MT, Lindner DL, Ortiz-Santana B, Lodge DJ. 2012. A new species of Laetiporus (Basidiomycota, Polyporales) from the Caribbean basin. Kurtziana 37:15−21

    Google Scholar

    [36]

    Tsujikawa K, Kanamori T, Iwata Y, Ohmae Y, Sugita R, et al. 2003. Morphological and chemical analysis of magic mushrooms in Japan. Forensic Science International 138:85−90

    doi: 10.1016/j.forsciint.2003.08.009

    CrossRef   Google Scholar

    [37]

    Murray MG, Thompson WF. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8:4321−26

    doi: 10.1093/nar/8.19.4321

    CrossRef   Google Scholar

    [38]

    Stirling D. 2003. DNA extraction from fungi, yeast, and bacteria. In PCR Protocols: Methods in Molecular Biology, eds. Bartlett JMS, Stirling D. vol. 226. Totowa, New Jersey: Humana Press. pp. 53–54. https://doi.org/10.1385/1-59259-384-4:53

    [39]

    Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology 2:113−118

    doi: 10.1111/j.1365-294X.1993.tb00005.x

    CrossRef   Google Scholar

    [40]

    White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: a guide to methods and applications, eds. Innis MA, Gelfand DH, Sninsky JJ, White TJ. New York: Academic Press. pp. 315–22. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

    [41]

    Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172:4238−46

    doi: 10.1128/jb.172.8.4238-4246.1990

    CrossRef   Google Scholar

    [42]

    Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, et al. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, 109:6241−46

    doi: 10.1073/pnas.1117018109

    CrossRef   Google Scholar

    [43]

    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403−10

    doi: 10.1016/S0022-2836(05)80360-2

    CrossRef   Google Scholar

    [44]

    Karsch-Mizrachi I, Takagi T, Cochrane G. 2018. The international nucleotide sequence database collaboration. Nucleic Acids Research 46:D48−D51

    doi: 10.1093/nar/gkx1097

    CrossRef   Google Scholar

    [45]

    Nilsson RH, Tedersoo L, Abarenkov K, Ryberg M, Kristiansson E, et al. 2012. Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences. MycoKeys 4:37−63

    doi: 10.3897/mycokeys.4.3606

    CrossRef   Google Scholar

    [46]

    Schoch CL. Robbertse B, Robert V, Vu D, Cardinali G, et al. 2014. Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database 2014:bau061

    doi: 10.1093/database/bau061

    CrossRef   Google Scholar

    [47]

    Katoh K, Rozewicki J, Yamada KD. 2019. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20:1160−66

    doi: 10.1093/bib/bbx108

    CrossRef   Google Scholar

    [48]

    Hall T. 2011. BioEdit: an important software for molecular biology. GERF Bulletin of Biosciences 2:60−61

    Google Scholar

    [49]

    Shen Q, Geiser DM, Royse DJ. 2002. Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition separating eastern North American and Asian isolates. Mycologia 94:472−82

    doi: 10.1080/15572536.2003.11833212

    CrossRef   Google Scholar

    [50]

    Shen LL, Wang M, Zhou JL, Xing JH, Cui BK, et al. 2019. Taxonomy and phylogeny of Postia. Multi-gene phylogeny and taxonomy of the brown-rot fungi: Postia (Polyporales, Basidiomycota) and related genera. Persoonia - Molecular Phylogeny and Evolution of Fungi 42:101−26

    doi: 10.3767/persoonia.2019.42.05

    CrossRef   Google Scholar

    [51]

    Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32:268−74

    doi: 10.1093/molbev/msu300

    CrossRef   Google Scholar

    [52]

    Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9:772

    doi: 10.1038/nmeth.2109

    CrossRef   Google Scholar

    [53]

    Hoang DT, Chernomor O, Von Haeseler A, Minh BQ, Vinh LS. 2018. UFBoot2: improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35:518−22

    doi: 10.1093/molbev/msx281

    CrossRef   Google Scholar

    [54]

    Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572−74

    doi: 10.1093/bioinformatics/btg180

    CrossRef   Google Scholar

    [55]

    Lindner DL, Banik MT. 2008. Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America. Mycologia 100:417−30

    doi: 10.3852/07-124R2

    CrossRef   Google Scholar

    [56]

    Li HJ, Cui BK. 2013. Two new Daedalea species (Polyporales, Basidiomycota) from South China. Mycoscience 54:62−68

    doi: 10.1016/j.myc.2012.07.005

    CrossRef   Google Scholar

    [57]

    Cristaldo Centurión E, Kossmann T, Campi M, Maubet Y, Costa-Rezende D, et al. 2022. Neotropical Daedalea (Basidiomycota, Fomitopsidaceae) revisited: Daedalea rajchenbergiana sp. nov. from Brazil. Lilloa 59:273−89

    doi: 10.30550/j.lil/2022.59.S/2022.09.16

    CrossRef   Google Scholar

    [58]

    Decock CA, Ryvarden L, Amalfi M. 2022. Niveoporofomes (Basidiomycota, Fomitopsidaceae) in Tropical Africa: two additions from Afromontane forests, Niveoporofomes oboensis sp. nov. and N. widdringtoniae comb. nov. and N. globosporus comb. nov. from the Neotropics. Mycological Progress 21:29

    doi: 10.1007/s11557-022-01787-z

    CrossRef   Google Scholar

    [59]

    Kim KM, Lee JS, Jung HS. 2007. Fomitopsis incarnatus sp. nov. based on generic evaluation of Fomitopsis and Rhodofomes. Mycologia 99:833−41

    doi: 10.1080/15572536.2007.11832515

    CrossRef   Google Scholar

    [60]

    Yasuda A. 1922. Notes on Fungi. The Botanical Magazine. 36:128

    [61]

    Han ML, Vlasak J, Cui BK. 2015. Daedalea americana sp. nov. (Polyporales, Basidiomycota) evidenced by morphological characters and phylogenetic analysis. Phytotaxa 204:277−86

    doi: 10.11646/phytotaxa.204.4.4

    CrossRef   Google Scholar

    [62]

    Kotlaba F, Pouzar Z. 1990. Type studies of polypores described by A. Pilát-III. Type studies of polypores described by A. Pilát-III. 44:228–37

    [63]

    Donk MA. 1966. Notes on European polypores-I. Persoonia-Molecular Phylogeny and Evolution of Fungi 4:337−43

    Google Scholar

    [64]

    Carranza-Morse J, Gilbertson RL. 1986. Taxonomy of the Fomitopsis rosea complex (Aphyllophorales; Polyporaceae). Mycotaxon 25:469−86

    Google Scholar

  • Cite this article

    Hussain S, Nisar M, Lim YW, Cho Y, Sher H, et al. 2024. Morphology and multigene phylogeny reveal two novel species and three new records of Polypores in Swat, Pakistan. Studies in Fungi 9: e004 doi: 10.48130/sif-0024-0005
    Hussain S, Nisar M, Lim YW, Cho Y, Sher H, et al. 2024. Morphology and multigene phylogeny reveal two novel species and three new records of Polypores in Swat, Pakistan. Studies in Fungi 9: e004 doi: 10.48130/sif-0024-0005

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Morphology and multigene phylogeny reveal two novel species and three new records of Polypores in Swat, Pakistan

Studies in Fungi  9 Article number: e004  (2024)  |  Cite this article

Abstract: Polypores, as wood-rotting fungi, play a vital ecological role in breaking down the wood substrate, releasing crucial nutrients into the soil, shaping the carbon dynamics, and contributing to the overall health of the forest ecosystem. Despite their significance, the fungal diversity in the Hindu Kush region remains inadequately explored. This study collected specimens from district Swat, Khyber Pakhtunkhwa Province, Pakistan, a part of the Hindu Kush region. After a rigorous examination of the collected specimens for the morphoanatomical characteristics, the concatenated sequence dataset (ITS + nrLSU) derived from generated sequences along with valid and published reference sequences was subjected to phylogenetic analyses using different methods: maximum parsimony, maximum likelihood, and Bayesian analyses. The study revealed two new species from the country, belonging to two polypores families i.e., Climacocystaceae and Fomitopsidaceae. Furthermore, the analysis confirmed the identification of Daedalea dickinsii Yasuda, Neoantrodia serialis (Fr.) Audet, and Rhodofomes roseus (Alb. & Schwein.) Kotl. & Pouzar as a new addition to the polypore inventory of Pakistan. These species received phylogenetic support and were proven to have corresponding morphological characteristics concerning pertinent original descriptions. The inclusion of these new wood-inhabiting fungi in the country's mycofloral list expands our understanding of fungal diversity, and distribution patterns, and contributes to global fungal biodiversity.

    • Polyporales Gäum represents a taxonomically diverse group of fungi that have received much scientific attention due to their fundamental ecological functions in the forest ecosystem. Because of their lignicolous habit, they are associated with different types of forest woods and cause different types of rot in both living and dead tree species[1]. They are responsible for the disintegration of wood components and help humification and mineralization processes[2]. Based on phylogenetic analyses of nrRNA genes and other protein-coding genetic markers, 29 lineages at the family level have been revealed in this group[3]. One important family found in the Antrodia clade of Polyporales is Fomitopsidaceae Jülich 1981, which consists of brown-rotting genera, such as Antrodia P. Karst., Daedalea Pers., and Fomitopsis P. Karst.[46]. Recently, many authors have undertaken consistent taxonomic revisions within Polyporales. For instance, Binder et al.[7] and Justo et al.[8] specified that the genera Climacocystis Kotl. & Pouzar and Diplomitoporus Domański exhibit an independent status in the phylogenetic analyses, and their familial placement remained uncertain[9]. In a subsequent study by Liu et al. a new family named Climacocystaceae was proposed to accommodate these genera[3].

      Climacocystis Kotl. & Pouzar was introduced as a monotypic genus comprising only the type species Climacocystis borealis (Fr.) Kotl. & Pouzar[10]. This genus belongs to the residual polyporoid clade and shares a close phylogenetic relationship with Diplomitoporus Domański, Physisporinus P. Karst., and Steccherinum Gray[11]. The genus is characterized by annual, pileate basidiocarps, a monomitic hyphal system with clamped generative hyphae, thick-walled ventricose cystidia, thin-walled, broadly ellipsoid basidiospores that do not react to Melzer's reagent. Climacocystis is rarely distributed in the northern hemisphere and is known to cause white rot in gymnosperms[7,1215]. In 2014, Climacocystis montana B.K. Cui & J. Song was added to the genus as a new species from high-altitude temperate forests in China[11].

      Among the brown-rotting polypores, Fomitopsis P. Karst. sensu lato (s.l.) is the largest genus in terms of species diversity. The genus is characterized by annual to perennial, sessile to effused-reflexed, woody, white or pinkish poroid basidiocarps, possessing dimitic to trimitic hyphal system consisting of clamped generative hyphae, presence or absence of cystidial elements, and ellipsoid to subglobose thin-walled hyaline basidiospores, showing negative reaction to Melzer's reagent, it mainly causes a brown rot to a variety of hosts[5,12,16,17]. Based on Hattori & Sotome, approximately 40 distinct species have been identified within Fomitopsis[18]. Many species are found in North America, Europe, and East Asia[9,1927]. The phylogeny of Fomitopsis has long been debated, resulting in multiple taxonomic revisions and extensive investigations. In a comprehensive study conducted by Han et al.[20], it was discovered that Fomitopsis s.l. does not form a monophyletic group. Instead, numerous species within this group exhibit a close relationship with the brown-rotting species of Antrodia and Daedalea[4,28]. Such observations prompted a reevaluation, leading to the recognition of distinct genera of brown-rotting fungi. The 'rosea clade', primarily comprising Fomitopsis rosea (Alb. & Schwein.) P. Karst. and Fomitopsis cajanderi (P. Karst.) Kotl. & Pouzar, were reclassified under a new genus named Rhodofomes Kotl. & Pouzar[4]. Additionally, other significant genera, such as Fragifomes B.K. Cui, M.L. Han & Y.C. Dai, Niveoporofomes B.K. Cui, M.L. Han & Y.C. Dai, Rhodofomitopsis B.K. Cui, M.L. Han & Y.C. Dai, Rubellofomes B.K. Cui, M.L. Han & Y.C. Dai, and Ungulidaedalea B.K. Cui, M.L. Han & Y.C. Dai, were recognized within Fomitopsis s.l. by Han et al.[5].

      The Hindu Kush Mountain range in Pakistan is a biodiversity hotspot, which is poorly documented[29]. Recently, a variety of mushrooms have been reported[30,31], but their molecular data is still scarce. We conducted a detailed examination of wood-rotting specimens belonging to two families, collected from Hindu Kush Mountain in Pakistan, based on morphological observations and two genetic markers analysis (internal transcribed spacer — ITS and nuclear large subunit ribosomal DNA — nrLSU). Two species new to science and three new records in Pakistan are proposed in this study.

    • The current research examined voucher specimens obtained from high-altitude temperate forests in the Swat district, Khyber Pakhtunkhwa (KP) Province, Pakistan. This district is located within the Hindu Kush Mountain range between lat. 34°34'–35°55' N and long. 72°08'–72°50' E. Woody vegetation, dense forest, and humid climatic conditions of the forests favor the growth of many wood-rotting fungi. During regular surveys, polypore specimens were collected from the area. The ephemeral macroscopic characteristics of basidiocarps were carefully documented in a field notebook. Terminologies introduced by Petersen were utilized to describe colors[32]. All studied specimens were carefully dried at a temperature between 30 to 35 °C for 72 h until constant weight was obtained as suggested by Hu et al.[33] and deposited at the herbarium of the University of Malakand, Pakistan (BGH).

      The basidiocarps were subjected to microscopic examination using the methods and techniques outlined in previous studies[14,34]. The standard notations described by Ji et al. were used for documentation[34]. Freehand anatomical sections were prepared and analyzed under a microscope at magnifications up to 1,000×. The hyphal system, septal features, hymenial elements, and spore characteristics were studied in detail. Reagents such as 5% KOH, lactophenol cotton blue (CB), and Melzer's reagent (IKI) were employed to test for basidiospore staining. The resulting reactions were categorized as amyloid or Melzer's-positive (IKI+) if basidiospores showed staining, or inamyloid or Melzer's-negative (IKI−) for a negative reaction. The presence or absence of cyanophilic reaction was determined, and CB- denotes acyanophilous samples and CB+ for cyanophilous samples, following the classification by Banik et al.[35]. The mean spore length (L) and width (W) were calculated by determining the arithmetic average of all measured spores. The variation in the length-to-width ratios (L/W) among the specimens examined was represented as Q. The spore measured, denoted as 'n(a/b)', where 'a' represents the number of spores studied and 'b' the total number of specimens examined. Measurements of various structures were conducted using Image J software[36].

    • For gDNA extraction, a standardized CTAB method was followed with some modifications[3739]. Around 50 mg of a pore surface piece was homogenized in 400 µl of a 2% CTAB buffer containing 0.2% β-mercaptoethanol using a multi-beads shocker. The homogenate was then incubated at 65 °C for 1 h and added with 350 µl of chloroform: isoamyl alcohol (24:1) and then vortexed until a cloudy white mixture was obtained. Subsequently, the solution was centrifuged at 13,200 rpm at 4 °C for 20 min. The resulting supernatant, at the aqueous phase, was carefully transferred to a new autoclaved microtube. For the precipitation of DNA, 133 µl of ice-cold isopropanol was added to the supernatant, followed by centrifugation at 13,200 rpm at 4 °C for 20 min. The supernatant was discarded, and the resulting DNA pellet was washed twice by adding 500 µl of ice-cold ethanol. After each addition, the mixture was briefly vortexed and centrifuged for 3 min. The resulting pellet was air-dried and subsequently re-suspended in 100 μl of distilled water. The DNA extract was preserved at −20 °C until further use. The quantity and purity of the DNA extracts were assessed using the NanoDrop 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific, Wilmington, DE, USA).

    • The entire ITS and partial nrLSU regions were PCR-amplified on an Applied Biosystems Veriti thermal cycler using PuReTaq Ready-To-Go PCR Beads (GE Healthcare, Buckinghamshire, UK). The primer pairs used for ITS and nrLSU regions amplification were ITS1/ITS4 and LROR/LR5, respectively[3941]. The PCR reaction mixture (total vol. of 20 μl), consisted of 2 μl of genomic DNA (approx. 100 ng), 0.5 μl of each forward and reverse primer, 2.6 μl of Taq PCR buffer (10X), 11.4 μl of sterile deionized water (Fisher Scientific), 0.5 μl of Taq DNA polymerase from Takara BIO INC., 2.0 μl of MgCl2, and 0.5 μl of dNTPs. The following thermocycling parameters were used for the amplification: initial denaturation at 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension step performed at 72 °C for 8 min[42]. Negative controls were included in the PCR reactions to ensure the absence of contamination. The PCR products were then run on an ethidium bromide-stained 1% agarose gel (Fisher Scientific, Waltham, MA, USA). A 1 Kb DNA ladder from Promega was used as a size reference to estimate the size of the amplified bands. After purification of the PCR products using the MEGA quick-spinTM PLUS Total Fragment DNA Purification Kit from Thermo Fisher Scientific, Sanger sequencing was performed at the sequencing facility of Macrogen Inc. in Seoul, South Korea (https://dna.macrogen.com). The purified PCR products were sequenced using the BigDye Terminator v3.1 cycle sequencing method.

    • To confirm the identity of query nucleotide sequences, each sequence fragment was subjected to an individual Basic Local Alignment Search Tool (BLAST)[43]. An initial dataset was obtained by retrieving and examining all publicly available ITS sequences in GenBank of NCBI under the query genera[44]. To ensure the correct identification and retrieval of the right reference sequences, protocols recommended by Nilsson et al.[45] and Schoch et al.[46] were followed. Specimen names associated with valid herbarium codes were included in the final data set.

      Multiple alignments of the final data set were performed using an online version of MAFFT v. 7 software[47]. The alignment was manually adjusted by removing gaps and ambiguous sites using BioEdit 7.2.5 software[48], using methods from Shen et al.[49]. The outgroup taxa Perenniporia ochroleuca (Berk.) Ryvarden and Perenniporia medulla-panis (Jacq.) Donk were used, following Soares et al.[22]. Three phylogenetic analyses were performed on the combined ITS + nrLSU data set[34,50]. The maximum parsimony (MP) analysis encompassed 1,000 iterative heuristic search replicates, employing random addition of taxa searches alongside tree-bisection-reconnection (TBR) branch exchanges. A consensus tree following the 50% majority rule was constructed, and the tree topology was assessed by computing parameters such as tree length, consistency index (CI), homoplasy index (HI), and retention index (RI)[3].

      The maximum likelihood (ML) analysis was performed using IQ-TREE version 1.6.12[51]. To compute the best-fit substitution model via the Akaike information criterion (AIC), jModelTest was used[52]. The final tree was inferred using 1,000 bootstrap values and setting all the base frequencies and substitution parameters as stated by Hoang et al.[53]. The Bayesian Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) inference was employed through MrBayes version 3.1[54]. For each partition, substitution models were defined as nst = 6, rates = invgamma (ITS), and nst = 6, rates = invgamma (nrLSU). Model parameters, such as base frequencies, substitution rates, gamma shape, and p.inv., were set for each partition accordingly. The analysis involved an operation of four chains throughout two million generations. Trees were sampled at an interval of 100 generations. The initial 5,000 trees (25% of the total) were excluded as burn-in, and the consensus tree was constructed from the remaining samples. The stop rule was set at stopval = 0.01[55]. The generated tree was visualized using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and subsequently subjected to editing. The ideal topologies derived from ML analysis were exhibited and verified using thresholds of ML-BS score (≥ 75), MP-BS score (≥ 50), and Bayesian posterior probabilities (BPPs) (≥ 0.95)[34].

    • The phylogenetic relationships of the species were analyzed using three methods: MP, ML, and Bayesian analysis. All inferred trees for each region were largely congruent, leading us to combine the matrices for further analysis. The results from the ML analysis are presented in Fig. 1, showing bootstrap proportion (MP), bootstrap (ML), and BPP values. The dataset consisted of 198 sequence variants, 99 ITS and 67 nrLSU sequences, including our subject sequences (Table 1). This added up to 37 species, with Perenniporia ochroleuca (Berk.) Ryvarden and Perenniporia medulla-panis (Jacq.) Donk serving as outgroup taxa. The concatenated data matrix, after alignment, consisted of 1,458 nucleotide characters, including gaps. Of these, 928 (86.3%) characters remained constant, 101 (4.91%) were variable or singleton sites considered parsimoniously uninformative, and 429 (8.8%) were parsimony informative characters, with a total of 552 distinct patterns. During the heuristic search of MP analysis, tree-bisection-reconnection (TBR) branch swapping was employed, resulting in 100 equally parsimonious trees on one island. From these trees, a 50% majority rule consensus parsimonious tree was constructed, with the following description: tree length = 2,138, consistency index = 0.394, retention index = 0.818, rescaled consistency index = 0.322, and homoplasy index = 0.606. A two-partition dataset (ITS + nrLSU) was used for the ML analysis, and the initial log-likelihood was −11,213.440. The best evolutionary models for the ITS and nrLSU regions were GTR+I+G, with estimated gamma shape parameters of 0.6590 and 0.4810, and p-invariance values of 0.2310 and 0.4950, respectively. The analysis converged after 20,000,000 generations, with a standard deviation of split frequencies of 0.009983. The results from both the Bayesian and likelihood analyses exhibited similar topologies and clade distributions.

      Figure 1. 

      ML consensus phylogenetic tree of species from brown-rotting genera including Antrodia, Daedalea, Fomitopsis, Neoantrodia, Rhodofomes, Rhodofomitopsis, and white-rotting Climacocystis species based on ITS and nrLSU sequences. Node support values are shown in the order of MP bootstrap/ML bootstrap/Bayesian posterior probabilities. Collection/voucher numbers are appended as tip labels and our specimens shown as underlined. Two new species R. flavomarginatus and C. temperata are indicated in bold and three previously unrecorded species in Pakistan are underlined.

      Table 1.  Names, collection numbers, and corresponding GenBank accessions of the taxa used in the phylogenetic analyses of this study.

      TaxaCollection no./voucherITSnrLSUOrigin
      Antrodia albidaCBS 308.82DQ491414NAKorea
      A. serpensX1508KC543167NAFrance
      A. serpensX1511KC543169NAFrance
      A. heteromorphaCBS 200.91DQ491415NAKorea
      A. heteromorpha0509-187KC543121KC543121USA
      A. heteromorphaX1474KC543150KC543150Norway
      A. heteromorphaX1841KC543177KC543177Finland
      A. mappaAm4132KC543130KC543130Canada
      A. mappaAm4239KC543113KC543113Finland
      Brunneoporus malicolaBCRC 35452DQ013299AY333837Taiwan
      B. malicolaMJL1167SPAY966449NATaiwan
      Climacocystis borealisDai 3703KJ566626KJ566636China
      C. borealisDai 11798KJ566632KJ566641China
      C. borealisDai 13208KJ566635KJ566642China
      C. borealisDai 4014KJ566627KJ566637China
      C. borealisFD-31KP135308KP135210USA
      C. borealisKHL13318 (GB)JQ031126NASweden
      C. montanaCui 10603KJ566634NAChina
      C. montanaCui 17122ON682359ON680811China
      C. montanaCui 17502MW377276MW377356China
      C. montanaCui 9610KJ566630NAChina
      C. montanaCui 17123ON682360ON680812China
      C. montanaCui 9607KJ566629KJ566639China
      C. montanaCui 9612KJ566631KJ566640China
      C. montanaDai 23003ON682358OL423570China
      C. montanaCui 9603KJ566628KJ566638China
      C. temperataMUSl21-41OR364522OR364606Pakistan
      Daedalea circularisCui10134JQ314352KP171221China
      D. circularisDai 13062KP171200KP171222China
      D. dickinsiiYuan 1090KR605790KR605729China
      D. dickinsiiYuan 2685KP171201KP171223China
      D. dickinsiiYuan 2707KP171202KP171224China
      D. dickinsiiMUBS40OM533594NAPakistan
      D. modestaCui 10151KP171205KP171227China
      D. modestaDai 10844KP171206KP171228China
      D. neotropicaDLC04-100FJ403218NABelize
      D. neotropicaDLC04-80FJ403217NAUSA
      D. quercinaDai 12659KP171208KP171230Finland
      D. quercinaDai 12697KP171209KP171231Czech Republic
      D. quercinaDai 2260KR605792KR605885China
      D. quercinaDai 12152KP171207KP171229China
      Fomitopsis betulinaCBS:377.51MH856908MH868430China
      F. betulinaCui 10756KR605797KR605736China
      F. canaCui6239JX435777JX435775China
      F. canaDai9611JX435776JX435774China
      F. meliae1P_1_1FJ372673FJ372695Thailand
      F. meliaeDai 10035KR605774NAChina
      F. palustrisCBS 283.65DQ491404MH870206Korea
      F. pinicolaCBS 221.39DQ491405NAKorea
      F. pinicolaH:HK-19330KF169655NARussia
      F. pinicolaPeruMyc1520MG820763NAItaly
      F. subtropicaCui 10578KR605787KR605726China
      F. subtropicaCui10140JQ067651NAChina
      Neoantrodia serialisCBS 306.82DQ491417NAKorea
      N. serialisMUJMk14OR364518NAPakistan
      N. serialisP213AJ344139NAGermany
      N. variiformisCBS 309.82DQ491418NAKorea
      N. variiformisFP90100SPAY966453NAChina
      Niveoporofomes spragueiCBS 365.34DQ491406NAKorea
      Perenniporia medulla-panisCui14515MW989399NAChina
      P. ochroleucaDai11486HQ654105JF706349China
      Rhodofomes cajanderiCBS 127.24DQ491407MH866275Korea
      R. cajanderiCBS 195.37DQ491399NAKorea
      R. cajanderiCui 9871KC507158KC507168.1China
      R. cajanderiCui 9888KC507156KC507166China
      R. cajanderiHOU 10773DQ491413NAKorea
      R. cajanderiLE-BIN 3546MG735350NARussia
      R. cajanderiV 0410/14a,b-J USAKR605768KR605707USA
      R. carneusLeif Ryvarden 10118KF999921KF999925China
      R. carneusO 15519KC507155KC507165China
      R. cystidiatusYuan 6304KR605769KR605708China
      R. flavomarginatusMU1EOR364739OR364741Pakistan
      R. incarnatusCui 10348KC844848KC844853China
      R. incarnatusHSJ-2006aDQ491411NAKorea
      R. incarnatusSNU m-05072501DQ491409NAKorea
      R. incarnatusYuan 2653KC844849KC844854China
      R. roseusCui 10633KR605782KR605721China
      R. roseusCui 10520KC507162AY333809China
      R. roseusCui 10551KC507163KC507173China
      R. roseusJV 1110/9KR605783KR605722Czech Republic
      R. roseusLE-BIN 3844MG734829NARussia
      R. roseusMUBS85OR364705OR364719Pakistan
      R. roseusRLG-6954KC585353KC585181USA
      R. subfeeiCui 9229KR605789KR605728China
      R. subfeeiDai 10430KR605788KR605727China
      Rhodofomitopsis africanaMUCL 43284DQ491422NACameroon
      R. cupreoroseaCBS 236.87DQ491400AY515325Costa Rica
      R. cupreoroseaNM692MF589757MF590128Brazil
      R. feeiCBS 424.84DQ491402NAKorea
      R. feeiJV 0610/K9KF999922KF999926Kout Mexico
      R. feeiOinonen 6011906KC844851KC844856Brazil
      R. feeiRyvarden 37603KC844850KC844855Venezuela
      R. feeiUotila 42928KF999924KF999928Australia
      R. lilacinogilvaSchigel 5193KR605773KR605712Australia
      Rubellofomes cystidiatusCui 5481KF937288KF937291China
      R. minutisporusRajchenberg 10661KR605777KR605716Argentina
      Subantrodia juniperinaFP97452TAY966454NATaiwan
      S. juniperinaCBS 117.40DQ491416MH867551USA
      Ungulidaedalea fragilisCui 10919KF937286NG_060408China
      * NA = Not available.

      Based on molecular and morphological analyses, two new species were confirmed: one in Climacocystis and the other in Rhodofomes. The sample sequence MUSl21-41 forms a distinct and independent lineage within Climacocystis, receiving strong support (100% MP, 100% ML, and 1.00 BPP). In addition, it differs significantly in microscopic characteristics from the two well-supported species within the genus, namely C. montana B.K. Cui & J. Song and C. borealis (Fr.) Kotl. & Pouzar. Similarly, the sample sequence MU1E exhibited robust clustering with significant support (71% MP, 97% ML, and 1.00 BPP) within the Rhodofomes clade. However, it is noteworthy that this sequence forms an independent lineage, distinctly separate from other known Rhodofomes species. Despite some morphological similarities with the known species within the genus, a few characteristics are unique to this new description.

      Additionally, through phylogenetic analysis, three polypores new to Pakistan were identified. MUBS40 corresponds to Daedalea dickinsii Yasuda. This assignment received substantial phylogenetic support (88% MP, 100% ML, and 1.00 BPP). Specimens examined under MUJMk14 are classified as Neoantrodia serialis (Fr.) Audet, with strong support values (98% MP, 100% ML, and 1.00 BPP). Finally, the sample MUBS85 groups together with Rhodofomes roseus (Alb. & Schwein.) Kotl. & Pouzar, showing significant support values (87% MP, 98% ML, and 1.00 BPP) (Fig. 1). The morphological characteristics of these three species from Pakistan corresponded to each of the descriptions of type specimens.

    • Climacocystis temperata S. Hussain, M. Nisar & Y.W. Lim sp. nov.

      Mycobank no.: MB852033

      Holotype: Climacocystis temperata, voucher no. MUSl21-41, PAKISTAN, KP PROVINCE, Sailand, Swat District, (lat. 34º59'09'' N and long. 72º10'55'' E, 2,748 m a.s.l.) in mixed coniferous forest on dead tree inside the hollow stumps (6 feet) of Abies pindrow (Royle ex D. Don) Royle, July 29, 2021, MUSl21-41(BGH F000501), Mycology section of Botanical Garden Herbarium, University of Malakand (BGH). nrRNA gene sequences holotype: ITS (OR364522), nrLSU (OR364606).

      Diagnosis: Distinguished from other Climocystis spp. by having larger (up to 19 cm wide) and leathery basidiocarp, with distinclty zonate pileal surface, pores 1–3 (usually 2)/mm, with entire to serrate margin, possessing varied shaped hymenial cystidia, elongated clavate basidia up to 47 µm long, notably larger ellipsoid basidiospores (6.7–10.7 × 3.9–6.1 μm), showing Q = 1.40–1.51, and associated with A. pindrow.

      Etymology: 'temperata' is a Latin word characterizing the distribution of C. borealis and C. montana in the temperate forests, used in analogy with the other species of the genus.

      Description: Basidiocarps are large sized, annual, imbricate, connate at the attachment, dimidiate to applanate, laterally stipitate, substipitate or almost sessile, thick fleshy and leathery textured, watery, becoming lightweight and hard woody when dry, having a distinct pungent odor and acidic flavor. Pilei growing up to 5–12 cm long, 10–19 cm wide, and 1.5–3.5 cm thick at the attachment point, hairy, showing distinct margins up to 2.5 mm of white colored; upper surface plano-concave, whitish or pale to cream-colored, distinctly zonate in mature basidiocarp, furrowed or striated along the radii, tomentose or somewhat velutinate, becoming dull yellowish brown when dry. Margin whitish, sterile, smoothly blunt, and entire to serrate in mature basidiocarp, distinct on both surfaces up to 2.5 mm wide. Stipe almost absent or with short robust stipe. Pore surface pale yellow when fresh, bruises brown to clay buff when dry, rounded to radially elongated (reaching up to 1.8 mm long), or elliptic and irregularly distributed. Pore lining first rounded then more or less elliptical angular, number of pores 1–3 (2) mm−1; dissepiment 0.2–1.12 (0.49) mm in thickness, and usually lacerate. The tube layer is detachable, yellow to pale-colored, and up to 3 mm at the widest point. Context whitish showing prominent radial ridges when the tube layer is peeled up, becoming spongy when dry, context up to 1.5 cm thick at the widest point, distinctly colored from the tube layer (Fig. 2).

      Figure 2. 

      Basidiocarps of Climacocystis temperata (a), (b), (c) from its natural habitat; (d) close-up view of the pileal surface (fresh); (e) pore surface; (f) cross sectional view of fresh specimen showing yellow tube layer and white context. Scale bars: (a), (d) = 1 cm; (b), (c) = 2 cm; (e) = 4 mm; (f) = 5 mm.

      Hyphal system monomitic; generative hyphae in the tube and context layer are IKI–, CB– unaffected by KOH. The contextual hyphae are light yellow or rarely hyaline, usually branched, occasionally septate, frequently clamped (compound clamps) showing interwoven, subparallel arrangement, 5.2–8.1 (6.5) μm in diam. Hymenial cystidia, 22.8–58.5(39.8) × 3.8–9.4(6.1) μm, variable in shape, often capitated, ventricose, rarely hyphoid, basally septate, smooth, thinwalled, usually yellowish colored, lacking encrustation. Basidia long, clavate, granular yellowish content, with four short sterigmata and basal clamp, 21.5–47.6(33.8) × 5.4–8.2(6.6) μm (n = 35/3). Basidiospores are ellipsoid shaped, thin, smooth, uniguttalate with hyaline lining, non-dixtrinoid, acyanophilous, varied size, 6.7–10.7 × 3.9–6.1 μm, L = 7.72 ± 0.35 μm, W = 5.34 ± 0.51 μm, Q = 1.40–1.51 (n = 90/3) (Fig. 3).

      Figure 3. 

      Drawing from the microscopic examination of holotype specimen of Climacocystis temperata. (a) Contextual generative hyphae showing branches and clamps; (b) tramal monomitic hyphal construct showing generative hyphae; (c) simple and compound clamp connections; (d) hymenial cystidia of different shapes; (e) basidia; (f) basidioles; (g) basidiospores showing different aspect view. All scale bars are 10 µm.

      Additional specimens cited: Vouchers including MUSl23-40 (lat. 34º59'27'' N and long. 72º10'52'' E, 2,935 m a.s.l., on stump of A. pindrow), MUSl23-27 (lat. 34º59'36'' N and long. 72º10'54'' E, 2,895 m a.s.l., on stump of A. pindrow) were collected from the type locality and examined for the morpho-anatomical characterization.

      Rhodofomes flavomarginatus S. Hussain, M. Nisar & Y.W. Lim sp. nov.

      Mycobank no.: MB852034

      Holotype: Rhodofomes flavomarginatus, voucher No. MU1E, PAKISTAN, KP PROVINCE, Malam Jabba, district Swat, (lat. 34º47'37'' N and long. 72º34'38'' E, 2,680 m a.s.l.) in the mixed coniferous forest on dead tree stumps (7 feet) of Abies pindrow (Royle ex D.Don) Royle, 1st September, 2016, MU1E (BGH F000502), Mycology section of Botanical Garden Herbarium, University of Malakand (BGH), nrRNA gene sequences holotype: ITS (OR364739), nrLSU (OR364741). Rarely distributed in the type locality.

      Diagnosis: This species differs from other Rhodofomes species by having large, thick, woody perennial, ungulate basidiocarps with clearly broad, obtuse, yellow-colored margins, possessing dimitic hyphal construct, short, broadly clavate basidia, 6.3–16.7(11.7) × 3.5–5.7(4.8) µm, rare occurrence of cystidioles and slightly small sized basidiospore (5.5–6.0 × 2.9–3.2 µm).

      Etymology: 'flavomarginatus' is a Latin word combining 'flavus' and 'marginatus', signifying the yellow bordered basidiocarps of the species.

      Description: Basidiocarps pileate, perennial, sessile to effused reflexed, solitary, ungulate, dimidiate or semicircular on the underside, hard, woody textured, becoming lightweight when dry, indistinct odor and taste. Pileus 14 cm long, 10 cm wide, 20 cm thick at the base; upper surface dark grayish brown, becoming dull brown when dry, glabrous, azonate surface with 1–2 deep grooves or fissures behind the margin. Margin rounded, smooth, sterile, blunt, undulating, forming over-grown rim beyond the pileal and hymenophore surface, yellow color, fading to light ochraceous when dry, 3–4 cm wide. Stipe absent, broadly attached to the wood but easily separable from the substrate. Pore surface pinkish white, tuberculate, producing amber to red colored exudates, pores minutes (0.05–0.19 mm in diam.), mostly rounded, irregular in distribution, pore-lining entire, number of pores 5–6(5) mm–1; dissepiment uniform in thickness 0.16–0.31 mm. Tube layer tightly affixed to the context, striated or multilayered, dark brown colored, each layer 2–3 mm thick. Context brownish, fibrous, or cottony, up to 5 cm in thickness (Fig. 4).

      Figure 4. 

      Basidiocarps of Rhodofomes flavomarginatus (a), (c) from its natural habitat showing pileal surface; (b), (d) close-up view of pores surface (fresh) showing exudates. Scale bars: (a), (b), (d) = 1 cm; (c) = 5 cm.

      Hyphal structure dimitic both in context and hymenophore of the basidiocarp; skeletal hyphae predominate in the context, 3.3–7.4(5.4) μm in diam (n = 40/1), rarely branched, non-septate, yellowish to dark brown in KOH, solid to semisolid, interwoven; generative hyphae found both in trama and context, usually dominating in the trama, branched, clamped, septate, light yellow to hyaline, thin-walled, ranging 2.6–3.9(3.2) μm in diam (n = 43/1), interwoven arrangement. Basidia small sized, broadly clavate, with short 4 sterigmata and a basal clamp, 6.3–16.7(11.7) × 3.5–5.7(4.8) µm (n = 28/1). Cystidioles club shaped, 23.2–30.6(27.3) × 3.4–4.2(3.9) µm, rarely observed. Basidiospores cylindric to ellipsoid, straight, hyaline, thin, smooth, non-dixtrinoid, acyanophilous, 5.1–6.0 × 2.6–3.1 µm, L = 5.54, W = 2.97 μm, Q = 1.86 (n = 32/1) (Fig. 5).

      Figure 5. 

      Microscopic structures drawn from holotype specimen of Rhodofomes flavomarginatus. (a) Context showing generative and skeletal hyphae; (b) dimitic hyphal construct from trama; (c) cystidioles; (d) basidioles; (e) basidiospores; (f) basidia. Scale bars are 10 µm.

      Daedalea dickinsii Yasuda, Bot. Mag., Tokyo 36: (128) (1922)

      Mycobank no.: MB481905

      Vouchers: PAKISTAN, KP PROVINCE: (i) Daedalea dickinsii voucher No. MBS40, Sailand, district Swat, (34º59'48'' N and 72º11'07'' E, 2,888 m a.s.l.), mixed coniferous forest on living trees and dead tree stumps of Quercus semecarpifolia Sm., August, 2020, MBS40 (BGH F000503), Mycology section of Botanical Garden Herbarium, University of Malakand (BGH).

      Description: Basidiocarps are small, perennial, effused-reflexed, imbricate, dimidiate, ungulate, broadly sessile thick corky textured or coriaceous, becoming woody when dry, having a distinct pungent odor and acidic flavor. Pilei 3–4 cm long, 5–6 cm wide, 1–2 cm thick at the broader base; upper surface velutinate, brown or buff brown, becoming greyish brown or dull brown when dry, distinctly zonate, sulcate, concentric zonation at growing margin distinctly yellow. Margin whitish or pale, sterile, smoothly rounded, and blunt or obtuse up to 1 cm wide, fading to light brown when dry. The stipe are almost absent but broadly attached to the exposed wood. Hymenophoral surface ochraceous or light brown or cream when fresh, becoming brown when dry; pores mostly rounded or angular and irregularly distributed, labyrinthine or daedaleoid, elongated and deep towards the base and shallow to the growing margin, number of pores 1–2(2) mm–1; dissepiment 0.3–0.9 mm in thickness, and usually entire. The tube layer is tightly affixed to the context, whitish brown colored in the young layer and light brown in the older layers, about 1–1.5 cm thick, almost concolorous with pore surface. Context multilayered or zonate showing thin cuticle layer; tubes light brown, coriaceous, spongy on drying, 0.3–0.8 cm at the widest point near the attachment. Context to tube layer ratio is 1–2.5:1 Indistinct taste and order (Fig. 6).

      Rot type: brown-rotting

      Figure 6. 

      Basidiocarps of Daedalea dickinsii (a) from its natural substrate; (b) pileal surface, also showing margin; (c) pore surface (dried); (d) cross section showing context and tube layers; (e) close-up view of pores; (f) microscopy of context showing skeletal and generative hyphae; (g) skeletal and generative hyphae from trama; (h) cystidioles; (i) basidiospores; (j) basidioles and basidia. Scale bars: (a)–(d) = 1 cm; (e) = 5 mm; (f)–(j) = 10 µm.

      Hyphal system dimitic; skeletal hyphae dominant in the context layer, IKI–, CB–, range 3.1–6.3(5.0) μm (n = 35/1) characterized by hyaline, rarely branched, non-septate, interwoven; generative hyphae 2.6–4.0(3.2) rarely observed in the context. Basidia, clavate, with short sterigmata, 12.9–28.8(20.8) × 3.2–5.4(4.4) μm (n = 40/1). Cystidoles are fusoid or tubular, thin-walled, hyaline, septated at the base 13.2–24.9(18.2), rarely observed. Basidiospores narrowly cylindrical, thin-walled, non-dixtrinoid, acyanophilous, 6.6–11.3 × 2.7–3.9 μm, L = 9.71 μm, W = 3.48 μm, Q = 2.79 (n = 32/1) (Fig. 6).

      Neoantrodia serialis (Fr.) Audet, Mushrooms nomenclatural novelties 6: [2] (2017)

      Mycobank no.: MB552864

      Vouchers: PAKISTAN, KP PROVINCE: (i) Neoantrodia serialis voucher No. MUJMk14, Jabba Mankyal, district Swat, (35º19'26'' N and 72º39'26'' E, 2,650 m a.s.l.), mixed coniferous forest on dwoned and dead tree logs of Abies pindrow (Royle ex D. Don) Royle, July, 2019; (ii) Neoantrodia serialis voucher No. MUJMk14b, Jabba Mankyal, district Swat, (35º20'53'' N and 72º40'50'' E, 2,563 m a.s.l.), mixed coniferous forest on sloping tree logs of Picea smithiana (Wall.) Boiss., July, 2020, MUJMk14 (BGH F000504), Mycology section of Botanical Garden Herbarium, University of Malakand (BGH).

      Description: Basidiocarps large sized, annual to perennial, usually completely resupinate, evenly flat, covering up to 80 × 40 cm of the substrate surface, very rarely reflexed, closely affixed or adnate to the downed logs or partly detaching at the senescent portion, about 3–4 mm thick, margin compact or slightly upwardly curved, wavy margin up to 0.5 mm thick and indistinct at other parts; pore surface regular, white or cream, becoming pale brown when dry, pores regular, rounded, 3–5(3) mm1, soft leathery textured becoming woody and lightweight when dry; tubes layer concolorous with pore surface, 2–3.5 mm thick, non-stratified; dissepiment thin and entire; odor unrecorded, taste bitter (Fig. 7). Context white, 0.1–0.3 mm.

      Hyphal system dimitic: skeletal hyphae were prominent, solid or semisolid (with capillary to fairly wide lumen), rarely branched and accidentally septated, 2.6–5.1(3.5) μm in diam. (n = 38/2), thick-walled, interwoven; generative hyphae abundant, yellowish, thin-walled, branched, rarely septate and clamped, occasionally inflated and tortuous, 2.4–4.9(3.9) μm in diam. (n = 42/2). Cystidia frequenty occurring, deeply located skeletocystidia possessing clavate apices and thick-walled, 10.6–18.7 × 3–5.5 μm with hyphoid cystidioles. Basidia broadly clavate, agglutinated, with short 4-sterigmate and a basal clamp, 10.2–15.8(13.6) × 4.5–7.2(5.4) μm. Basidiospores are narrowly cylindrical to oblong, thin-walled, non-dixtrinoid, acyanophilous, tapering towards the distal end, 5.6–8.7 × 2.2–3.2 μm, L = 6.52 ± 0.34 μm, W = 2.81 ± 0.55 μm, Q = 2.16–2.45 (n = 65/2). Large sized diffused crystals were also found in the tramal tissue (Fig. 7).

      Rot type: brown-rotting

      Figure 7. 

      Basidiocarps from Neoantrodia serialis. (a) Pore surface showing close-up view of pores; (b) resupinate basidiocarp adnate to the substrate; (c) skeletal and generative hyphae of context showing branches; (d) generative hyphae from the trama showing sepatation and branching; (e) basidiospores; (f) basidia and hyphoid cystidia. Scale bars: (a) = 5 mm; (b) = 10 cm; (c)–(f) = 10 µm.

      Rhodofomes roseus (Alb. & Schwein.) Kotl. & Pouzar, Česká Mykol. 44 (4): 235 (1990)

      Mycobank no.: MB127496

      Vouchers: PAKISTAN, KP PROVINCE: Rhodofomes roseus voucher No. MUBS85, Sailand, Swat District, (34º59'22'' N and 72º10'56'' E, 2,833 m a.s.l.), mixed coniferous forest on living trees and dead tree stumps of Abies pindrow (Royle ex D. Don) Royle, August, 2020, MUBS85 (BGH F000505), Mycology section of Botanical Garden Herbarium, University of Malakand (BGH).

      Description: Basidiocarps, annual, perennial, effused reflexed, solitary, pileate dimidiate to applanate, semicircular, sessile to broadly attached, hard, woody, tough textured, becoming lightweight when dry, having indistinct odor and taste. Pilei 2–4 cm long, 5–8 cm wide, and 1 cm thick at the base, upper surface dark brownish, light pinkish brown towards the margin, showing blackish stains, glabrous, azonate surface no groove or fissure observed, Margin acute to rounded, smooth, sterile, blunt, undulating, indistinct on both surfaces, light pink brownish or clay pink. Stipe absent, broadly attached to the woods. The pore surface is pinkish white or rosaceous, producing cream colored exudates, pores minutes mostly rounded to elliptical and irregular in size (0.04–0.36 mm in diam.) and distribution. Pore lining entire, number of pores 5–6(4) mm1; dissepiment non-uniform in thickness 0.1–0.3 mm. Tube layer tightly affixed to the context, striated or multilayered dark brown colored, 4–4.5 mm thick. Context brownish, fibrous, pinkish white to light brown color, up to 7–9 mm thick at the attachment with distinct cuticle of pileal surface. Section exhibits distinctive zonation and layers representing seasonal growth variation (Fig. 8).

      Hyphal system dimitic both in the context and hymenophore; skeletal hyphae indeterminate, rarely branched, non-septate, hyaline, thick-walled, interwoven, 2.9–6.1 (4.6) μm in diam; generative hyphae predominant in the trama, branched, rarely clamped, septate, light yellow, thin-walled, ranging 2.7–5.4(3.8) μm in diam, interwoven arrangement. Basidia clavate to broadly cylindrical, with short sterigmata and a basal clamp, 7.2–16.2(11.9) × 3.7–5.9(4.8) µm. Basidioles frequently small sized, pyriform to globose shaped 9.2–12.7(11) × 3.3–5.6(4.8) μm. Cystidial elements were not found. Basidiospores oblong to cylindric, hyaline, thin-walled, non-dixtrinoid, acyanophilous, varied in size, 5.5–7.7 × 2.3–3.6 µm, L = 5.84 μm, W = 2.72 μm, Q = 2.15 (n = 32/1) (Fig. 8).

      Rot type: brown-rotting

      Figure 8. 

      Basidiocarps of Rhodofomes roseus (a) from its natural habitat showing pore surfaces and margin; (b) view of pileal surface; (c) cross section view of fresh specimen depicting zonation; (d) close-up view of pores; (e) dimitic construct of context showing dominant skeletal hyphae and generative hyphae; (f) skeletal and generative hyphae from trama; (g) basidioles; (h) basidia; (i) basidiospores. Scale bars: (a), (b) = 1 cm; (c) = 5 mm; (d) = 2 mm; (e)–(i) = 10 µm.

    • The analyses of both morphoanatomical characteristics and phylogenetic data of the collected specimens revealed noteworthy findings, contributing three new records to Pakistan and two novel species. The species examined in this study are distributed across distinct clades within two families, Fomitopsidacae and Climacocystaceae, located within the Polyporales[4,28]. The inferred phylogeny of the species highlights their interrelationship in the broader context (Fig. 1). Phylogenetically, our findings additionally corroborate the clustering of brown-rotting genera, as indicated in previous studies[20,21,28,56,57]. Nevertheless, certain branches in the heterogeneous brown-rotting cluster exhibited limited resolution[4,8,58]. These species may be distinguished morphologically (Table 2).

      Table 2.  Morphoanatomical comparisons of the new Climacocystis and Rhodofomes species and their relatives.

      GenusRhodofomesClimacocystis
      SpeciesR. flavomar ginatus1R. roseus2R. incarnates3R. subfeei4R. cajanderi5C. montana6C. borealis7C. temperata1
      Basidiocarp shapeUngulateDimidiateDimidiateApplanate to triquetrousApplanate, broadly convexapplanate to dimidiateApplanate to dimidiateApplanate to dimidiate
      Basidiocarp dim. (L × W × T) cm.14 × 10 × 20nil × 5–10 × 3–813 × 6 × 74 × 10.6 × 2.5nil × 3–10 × 107 × 14 × 211 × 12 × 312 × 19 × 3.5
      MarginObtuseAcute to roundedAcuteObtuse to acuteAcuteNAacuteEntire to serrate
      Growth habitSolitaryimbricateNASingle or imbricateImbricateImbricateImbricateimbricate
      Pileal surface colorDark grayish brownBrownish blackBrownish gray to grayish blackcinnamon brown to fawn brownPink to grey, brown, or blackWhite to pale creamWhite to creamWhitish or pale
      Surface textureGlabrousNAGlabrous, finely velvetyGlabrous, rough,finely velvety or hairyTomentose to hirsuteTomentose to hirsuteTomentose
      Marking or zonationAzonate, sulcateZonate, sulcateSulcatesulcateFaintly zonateAzonateAzonateZonate
      Pore surface colorPinkish whitePinkPinkish whitePink to brownish vinaceousRosy pinkWhite to creamWhite to creamPale yellow
      No. pores (mm−1)5–6 (5)3–56–84–63/4–51–31–31–3(2)
      Hyphal structureDimiticTrimiticTrimiticTrimiticTrimiticMonomiticMonomiticMonomitic
      Cont. GH (µm)2.6–3.9NA2.3–32–3NA5–83–74.7–11.4
      Tramal GH (µm)2.3–3.6NANANANA3–42.5–43.9–7.2
      CystidiaAbsentNANANANAVentricose, without crystals,
      35-60 µm
      Ventricose, with apical crystals,
      30-50 µm
      Varied in shape,
      22.8–58 µm
      CystidiolesClub shapedAbsentSubulateFusoidSubfusiform or irregularAbsentAbsentAbsent
      Basidial sterigma4–sterigmate4–sterigmate2–sterigmate4–sterigmate4–sterigmate4–sterigmate4–sterigmate4–sterigmate
      Basidia dim. (µm)6.2–13.8 × 3.5–5.710–18 × 4–615–19 × 4–6.39–18 × 4–610–12 × 3–420–27 × 6–725–30 × 6–821.5–47.6 × 5.4–8.2
      Basdiospore shapeCylindric to ellipsoidSubcylindrical to slightly ovoidEllipsoid, frequently curvedCylindrical to oblong-ellipsoidAllantoidEllipsoid to subcylindricalEllipsoid to subcylindricalEllipsoid to subcylindrical
      Basidiospores length (µm)5.1–6.0 × 2.7–3.95.5–7.5 × 2–2.54.5–6.3 × 2.2–2.94–5 × 1.9–2.45–7 × 2–36–8.8 × 3–4.25–6.8 × 3.2–46.7–10.7 × 3.9–6.1
      Q range1.7–2.0NANA2.05–2.1NA1.85–1.891.6–1.671.40–1.51
      Host plantAbies pindrowPicea abiesFraxinus mandshurica, Pinus sp.Cunninghamia lanceolataPinus spp.Picea spp.Pinus spp., Picea spp.Abies pindrow
      Species references: 1. This study; 2. Carranza-Morse & Gilbertson[64]; Gilbertson & Ryvarden[17]; Han et al.[5]; Justo et al.[8]; 3. Kim et al.[59]; 4. Han & Cui[21]; 5. Justo et al.[8]; 6. Song et al.[11]; 7. Gilbertson & Ryvarden[17]; Núñez & Ryvarden[13]; Dai[14]; Song et al.[11]. * NA = Not available.

      The combined data from both nrRNA genes (ITS + nrLSU) showed two large groups of clades, each consisting of a single new species reported in this study. The group of Antrodia s.l.-Daedalea s.l.-Fomitopsis s.l. all comprised brown-rotting genera (Fig. 1), and this includes the recently established Rhodofomes, occupying a distinct and prominent clade[4,20,59,60]. Rhodofomes clade is closely affiliated with Brunneoporus malicola (Berk. & M.A. Curtis) Audet., but exhibits minimal similarity, both morphologically and phylogenetically. Compared to other known species, distinguishing features of R. flavomarginatus sp. nov. include dimidiate, solitary, larger, and thicker basidiocarp possessing wide obtuse margin, with capitate basidioles, short basidia (6.2–13.8 × 3.5–5.7 µm) cylindric to ellipsoid basidiospore (5.1–6.0 × 2.7–3.9 µm) (Table 2). The second group features sequences from Climacocystis, forming a highly cohesive cluster of white-rotting species. Each of the tree species formed a well-defined clade with high supports. Climacocystis temperata sp. nov. is different from the other congeneric species by its larger basidiocarp (up to 19 cm wide) with abundant narrow, multiform cystidia, with zonate, white upper surface and yellowish pore surface, wide-pored 1–3 (usually 2)/mm, with distinctly larger ellipsoid basidiospores (6.7–10.7 × 3.9–6.1 μm). These morphological and phylogenetic differences provide the basis for proposing a new species.

      Daedalea dickinsii, Neoantrodia serialis, and Rhodofomes roseus were new records added to the country's polypore inventory. After the species identifications based on phylogenetic assessments, morphological characters were examined to confirm the identities. The examined Daedalea dickinsii morphologically corresponds to the original description by Yasuda[60]. Further, our collected specimen of R. roseus exhibited morphological and phylogenetic similarities with the description provided by Gilbertson & Ryvarden[17], Han et al.[61], and Justo et al.[8], corroborated the initial proposal made by Kotlaba & Pouzar[62]. Similarly, Neoantrodia serialis in Pakistan demonstrates noteworthy morphological similarity to available species descriptions. Neoantrodia serialis was originally identified as Antrodia serialis (Fr.) by Donk in 1966[63]. Later, phylogenetic reconfiguration of Antrodia s.l. has resulted in the establishment of multiple novel genera[6]. Among them, there is Neoantrodia Audet, designated for A. searilis and its related taxa (approx. 13 species)[22], which includes the studied species N. serialis.

    • Wood-rot fungal genera are well-studied in various countries, mainly from North America, Europe, and East Asia. However, despite these extensive efforts, significant unaccounted diversity still exists, particularly in several Asian countries, including Pakistan. Consequently, there is a critical need to identify species within these underreported genera, employing diverse taxonomic approaches incorporating molecular, ecological, and morphological evidence. The current study, therefore, aimed to rigorously examine collections based on morphological characteristics and phylogenetic evaluations. In conclusion, the rich biodiversity in the Hindu Kush region offers a favorable habitat for wood-inhabiting fungal species. The numerous collections from the Northern area of Pakistan deserve comprehensive morphoanatomical analysis and molecular investigation. Utilizing such taxonomic data is primarily essential for conservation efforts, resource management, and scientific research not only within the country but also on a global scale. Furthermore, the presence of wood-decaying fungi showcases the region's remarkable ecological and economic significance and contributes to our understanding of global fungal biodiversity.

    • The authors confirm contribution to the paper as follows: study conception and design: Hussain S, Lim YW, Nisar M; data collection: Hussain S, Ahmad W, Nisar M; analysis and interpretation of results: Hussain S, Cho Y, Ahmad W, Nisar M; draft manuscript preparation: Hussain S, Sher H, Lim YW, Jan T. All authors reviewed the results and approved the final version of the manuscript.

    • The authors of the manuscript confirm that data supporting our study findings are available in the article. Data regarding species/specimen DNA sequences are publically available on the accession provided in Table 1, in the GenBank data base of NCBI.

    • We gratefully acknowledge the support of the laboratories: Molecular Ecophylogeny, School of Biological Sciences, and Institute of Microbiology, Seoul National University, South Korea, and the Department of Botany, University of Malakand, KP, Pakistan. Their assistance was vital in conducting this study.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (8)  Table (2) References (64)
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    Hussain S, Nisar M, Lim YW, Cho Y, Sher H, et al. 2024. Morphology and multigene phylogeny reveal two novel species and three new records of Polypores in Swat, Pakistan. Studies in Fungi 9: e004 doi: 10.48130/sif-0024-0005
    Hussain S, Nisar M, Lim YW, Cho Y, Sher H, et al. 2024. Morphology and multigene phylogeny reveal two novel species and three new records of Polypores in Swat, Pakistan. Studies in Fungi 9: e004 doi: 10.48130/sif-0024-0005

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