Bull 655 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Suliaman, S. Q. Bull. Iraq nat. Hist. Mus. (2023) 17(4): 655-668. https://doi.org/10.26842/binhm.7.2023.17.4.0655 ORIGINAL ARTICLE NEW RECORDS OF TWO MACROFUNGI SPECIES BASED ON MORPHOLOGICAL AND MOLECULAR IDENTIFICATION IN IRAQ Sara Q. Suliaman Biology Department, College of Science, Tikrit University, Tikrit, Iraq. *Corresponding author E-mail: saraqahtan@tu.edu.iq Recived Date: 15 May 2023, Accepted Date 03 August 2023, Published Date:20 June 2023 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT This study was done in Al-Alam City, Salah Al-Din Province, to determine the diversity of the macrofungi in it. The results of the field study showed two species were recorded in Iraq for the first time, Inocutis tamaricis (Pat) Fiasson & Niemelä, 1984 (Basidiomycota, Hymenochaetales) and Melanoleuca castaneofusca Contu, 1998 (Basidiomycota, Agaricales). These species were diagnosed based on macroscopic and microscopic, DNA sequence analyses and environmental charactes. The study included the adoption of the ITS gene for molecular diagnosis, the results of which were confirmed for morphological and environmental diagnosis, and the specimens were registered in the NCBI Global GenBank under the international accession numbers OP153814.1 and MZ334407.1 for the species I. tamaricis and M. castaneofusca, respectively. Keywords: Basidiomycota, Inocutis tamaricis, Iraq, Macrofungi, Melanoleuca castaneofusca. INTRODUCTION There has been great interest in mapping the macrofungi of the main geographical regions to obtain their distribution records similar to those of flowering plants (Meuller et al., 2007). However, unlike plants, measuring macrofungal diversity depends on the collection of fruiting bodies, which in turn depends to a large extent on the availability of moisture, that is, on seasonal rains, so it abounds in the spring and autumn due to the high humidity and abundance of plants (flora) at these times (Sibounnavong et al., 2008). Iraq occupies a total area of 437,072 km2 (Al-Ansari, 2021) and is characterized by its different ecosystems and plant diversity. However, Iraqi macrofungi are overlooked and unexplored in many regions, despite their environmental and applied importance. There are very few and scattered studies in this regard, including the study of Aziz and Toma (2012), Toma et al. (2013), Al- Qaissi )2013), Al Anbagi (2014), Suliaman et al. (2017), Owaid et al. (2018), Al- Khesraji et al. (2019, 2020, 2022), Al Anbagi et al. (2021), Al- Khesraji et al. (2021), and Al Anbagi and Al- Khesraji (2022) reflecting an increase in the macrofungal species recorded with most of the documented species belonging to the phylum BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Iraq Natural History Research Center & Museum, University of Baghdad https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799-Print ISSN: 1017-8678 https://doi.org/10.26842/binhm.7.2023.17.4.0655 https://orcid.org/0000-0002-7590-7702 mailto:saraqahtan@tu.edu.iq https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 656 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM New records of two macrofungi Basidiomycota and the remaining species belonging to the phylum Ascomycota. That indicates the rich diversity of macrofungi in Iraq. During the field trips to the Al-Alam area, the two species were collected and identified. Therefore, the present study is a new addition to the macrofungal record in Iraq. MATERIAL AND METHODS Collecting, identification and preserving macrofungi specimens: Macrofungi were collected from the orchards of Al-Alam City (34°38′41″N43°42′0″ /elevation 96 m), Salah Al-Din Province, in December-January 2022. Information about the habitat, habit, substrate, the host, and the nature of growth was recorded if it was solitary, overlapped, or clustered. Other information related to the date and place of collection, the color of the fruiting bodies with macrofungi in different parts, and the names of the plants prevalent in the area have also been documented. The specimens were placed in plastic storage containers and transferred to the laboratory for macroscopic and microscopic examinations. Later, species were identified according to Ghobad-Nejhad and Kotiranta (2008), Sharma et al. (2013), Chinan et al. (2015), Kibby (2016), Sicoli and Mannarino (2017), and Antonín et al. (2021). Classification, synonyms, and basionyms were provided according to the GBIF Secretariat (2022). Some of the specimens were preserved in a preservation solution, ethanol alcohol 70%, with adhesive paper placed on the box. Information of the fungus, date and place of collection was written down, while the rest of the fruiting bodies were cut to small parts and dried on sterile paper by exposing them to indirect sunlight. The fruiting bodies were grinded by electric mill. The powder of the specimens was kept in a plastic storage container with a tight lid until use for genetic analysis. The identified fungi were deposited in the Department of Biology, College of Science, Tikrit University, Iraq. Molecular studies: The DNA extraction was conducted using the MG Tissue Genomic DNA Extraction SV kit (Doctor protein INC, South Korea, Cat. no. MD014). The DNA amplification was completed using Dr. MAX DNA Polymerase (Doctor protein, cat. no.: DR00302). The primers for the amplification of the internal transcribed spacer region (ITS) were ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). The PCR conditions were a denaturation at 95o C for 5 min, followed by 35 cycles for a secondary denaturation at 95o C for 30 sec; annealing at 55o C for 30 sec; and an elongation at 72o C for 1 min with a final extension step of 72o C for 10 min. The PCR products were stored at 4o C. Later, the PCR products were purified using Multiscreen filter plate, merck millipore. The amplified DNA was sequenced by Applied Biosystems ABI 3730XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit, merck millipore Macrogen / Korea. The sequences were aligned using NCBI’s Basic Local Alignment Search Tool (BLAST). The phylogenetic tree analysis was performed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 7 (Tamura et al., 2013). 657 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Suliaman, S. Q. RESULT AND DISCUSSION Morphological identification In this study, two species belonging to the class Agaricomycetes were reported as new records for Iraq. Macroscopic and Microscopic descriptions and photographs were provided. The classification of them is as follow: Kingdom: Fungi Phylum: Basidiomycota Class: Agaricomycetes (1) Order: Hymenochaetales Family: Hymenochaetaceae Genus: Inocutis Fiasson & Niemelä, 1984 Speceis: I. tamaricis (Pat.) Fiasson & Niemelä (1984) (2) Order: Agaricales Family: Pluteaceae Genus: Melanoleuca Patouillard, 1897 Species: M. castaneofusca Contu, 1998 Inocutis tamaricis (Pat.) Fiasson & Niemelä, 1984 (Pl. 1) Basionym: Xanthochrous tamaricis Pat., 1984 Synonyms: Inonotus tamaricis (Pat.) Bondarzew & Singer Inonotus tamaricis (Pat.) Maire Inonotus tamaricis f. corneus Bondartseva Polyporus tamaricis (Pat.) Sacc. & D.Sacc. Xanthochrous rheades subsp. tamaricis (Pat.) Bourdot & Galzin Basidiocarp: Sessile, hemispherical, rough texture (woody), 5.5-7.0 cm wide, 3-4 cm thick, creamy to rusty-brown with a lighter at the margin. Flesh: woody texture, dark brown at the center, mixed with pale yellowish and white mycelium. Hymenial layer: tubular, creamy to pale brown become dark brown at the edges ends with irregular pores. Basidia: 4-spored, hyaline in H2O. Basidiospores: 5.5-7.5x5.0-6.0µ, oval to elliptical, yellowish to pale brown, thick-walled, smooth. Habit and habitat: solitary; fruiting on live and dead trees of Tamarix spp. Edibility: locally and globally unknown. Distribution: Greece (Piatek, 2001), Southern Europe, Northern Africa, Southern Asia, China (Ryvarden, 2005), Iran (Ghobad-Nejhad and Kotiranta, 2008), India (Sharma et al., 2013), Romania (Chinan et al., 2015), Italy (Sicoli and Mannarino, 2017; Girometta et al., 2020). Note: The current results were consistent with the aforementioned sources, which also confirmed that the presence of I. tamaricis on Tamarix trees is a distinctive feature in determining its identity. Melanoleuca castaneofusca Contu, 1998 (Pl. 2) Basidiocarp: Cap: 4.8-10.0 cm broad, smooth, depressed, slightly reflexed towards margin, laccate, dark gray to brown with a silver appearance, and darker spots around the cap margin, and entire cap margin. Flesh: white, fragile, the smell is similar to that of mushroom. Gills: white, subdeccurent, crowded, smooth. Stipe: 3.0-7.0x3.5-4.0 cm, white changed to brown 658 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM New records of two macrofungi after harvest, central, equal, solid, fibrillose, longitudinally striate. Volva and ring and absent. Basidia: 4-spored, hyaline in H2O, 12.5-15.0 x75.0µ. Basidiospore: 6.25-7.5 x 5.0-7.5µ, elliptical with central oil droplets and an ornamented wall. Spore print: is white to light yellow. Cheilocystidia: 35.0x7.5-10.0µ, lageniform with crystals at the apex. Pleurocystidia present and have a similar shape to cheilocystidia. Habit and habitat: solitary, collecting from the soil of barley fields. Edibility: locally unknown, globally edible (Singer, 1986). Distribution: It has been collected from several countries such as Italy, Czech Republic, Slovakia, France, Britain, and Sweden (Kibby, 2016; Antonín et al., 2021). Molecular identification and phylogenetic analysis The analyzed portions of ITS rRNA sequencing for both presented species were between 660 and 689 base pairs. The blast search of the sequence similarities was identified the first and second species sequences as M. castaneofusca and I. tamaricis, respactively. The two identified sequences were submitted to the NCBI GenBank under the accession numbers OP153814.1 and MZ334407.1 for species, I.tamaricis and M. castaneofusca, respectively. The pairwise sequence alignment of I. tamaricis appeared transversion and transition in the 152-158 and 282 nucleotide positions, respectively, when being compared with the refrance isolate with accession number GQ253453.1 form the Mediterranean Sea (Diag.1). On the other hand, the sequence alignments of M. castaneofusca exhibited transition at the 312 alignment position once the Iraqi isolates paired with Italyi isolates with the acession number MW491323.1(Diag.2) (Chinan et al., 2015; Zhuo et al., 2016; Wu et al., 2019). 659 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Suliaman, S. Q. Plate (1): I. tamaricis; (A, B, C, D) Fruiting bodies on Tamarix tree, (E) Basidium and basidiospores, (F) Basidiospores. (40x). Plate (2): M. castaneofusca; (A) Fruiting body in lab, (B) Gills, (C) Long section of fruiting body, (D) Basidia and basidiospores, (E) Basidiospores, (F) cystidia. (40x). 660 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM New records of two macrofungi Diagram (1): The pairwise sequence alignments of ITS region for both isolates I. tamaricis from Iraq (the query) and the Mediterranean Sea (the subject) with the accession number GQ253453.1. The alignment starts at the first base in the query sequence and progresses upwards to base 60 in the first alignment line; however, for the subject sequence, the alignment starts at base 57 and progresses downwards to base 745. 661 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Suliaman, S. Q. Diagram (2): The pairwise sequence alignments of ITS region for both isolates M. castaneofusca from Iraq (the query) and Italy (the subject) with the accession number GQ253453.1. The alignment starts at the first base in the query sequence and progresses upwards to base 60 in the first alignment line; also, for the subject sequence, the alignment starts at base 60 and progresses downwards to base 660. The results of the ITS sequence region showed that the percentage similarities of the isolets I. tamaricis from Iraq were 99% with the Mediterranean Sea, Romania, and Greece, while they were matched as 98%, 97%, 96%, and 95% with South Korea, France, Italy, and China, respectively as shown in Table (1). Table (1): Similarity ITS gene for the Iraqi isolate of I. tamaricis with Gene Bank isolates. No. Accession number Country Source Similarity (%) 1 GQ253453.1 The Mediterranean Sea Inocutis tamaricis 99 2 KJ755854.1 Romania I. tamaricis 99 3 KX881614.1 Greece I. tamaricis 99 4 AY558604.1 South Korea I. tamaricis 98 5 MH855326.1 France I. tamaricis 97 6 GU111920.1 Italy I. tamaricis 96 7 HM050416.1 China I. tamaricis 95 8 JN169789.1 China I. subdryophila 89 9 KY907684.1 USA: Arizona I. jamaicensis 83 10 MN498104.1 USA: Arizona I. dryophila 94 11 MK422156.1 Tunisia Inonotus levis 79 https://www.ncbi.nlm.nih.gov/nucleotide/KJ755854.1?report=genbank&log$=nuclalign&blast_rank=2&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/KX881614.1?report=genbank&log$=nuclalign&blast_rank=3&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/AY558604.1?report=genbank&log$=nuclalign&blast_rank=4&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/MH855326.1?report=genbank&log$=nuclalign&blast_rank=5&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/GU111920.1?report=genbank&log$=nuclalign&blast_rank=6&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/HM050416.1?report=genbank&log$=nuclalign&blast_rank=7&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/JN169789.1?report=genbank&log$=nuclalign&blast_rank=11&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/KY907684.1?report=genbank&log$=nuclalign&blast_rank=13&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/MN498104.1?report=genbank&log$=nuclalign&blast_rank=12&RID=89EZ9MGZ01R https://www.ncbi.nlm.nih.gov/nucleotide/MK422156.1?report=genbank&log$=nuclalign&blast_rank=16&RID=89EZ9MGZ01R 662 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM New records of two macrofungi Further, the ITS sequencing results for Iraqi isolate of M. castaneofusca appeared the Similarity 99% with Italy , Czech Republic, Italy, Slovakia, and France , whereas compatible 98% with United Kingdom as in Table(2). Table (2): Similarity ITS gene for the Iraqi isolate of M. castaneofusca with GenBank isolates. The results of the phylogenetic tree analysis of I. tamaricis showed that the current isolate was close to the Romanian isolate (Diag. 3). The p-distances between the previous species were 0.0013 and between the isolates from Iraq and the Mediterranean Sea were 0.0020. Moreover, variable distances 0.013-7.8 were observed between the Iraqi sequence and other GenBank sequences. The current results were comparable with other reported mean sequence divergences for fungi of 0.004-0.036 (Chinan et al., 2015) and 0.025 (Schoch et al., 2012). The Iraqi isolate had a high bootstrap value (>79%) compared to other species in the GenBank isolates. Another study by Chinan et al. (2015) examined the phylogenetic evolution of I. tamaricis from Romania. The phylogenetic bootstrap value of the investigated species was high >65% compared with other sequences in GenBank. The phylogenetic tree analysis was generated for the Iraqi collected specimen of M. castaneofusca (Diag. 4). The sequences of the Iraqi isolate were close to other sequences from Italy, France, and Slovakia. Their p-distances between them were 0.0008. The distances were varied from 0.0008 to 0.0025 between the sequences of the investigated Iraqi species and others in GenBank. The Iraqi isolate had a high bootstrap (>98%) with the isolates in GenBank. However, other studies showed two major clades: subgenera Melanoleuca (clade A) and subgenera Urticocystis (clade B) when phylogenetic analysis was conducted depending on the ITS region. These clades had high bootstraps of 99 and 98% respectively, (Kalmer et al., 2018). Another study conducted the phylogenetic analysis for other species of No. Accession number Country Source Similarity (%) 1 MW491323.1 Italy Melanoleuca castaneofusca 99 2 MW491321.1 United Kingdom M. castaneofusca 98 3 MW491320.1 M. castaneofusca 99 4 MW491325.1 Czech Republic M. castaneofusca 99 5 MW491324.1 M. castaneofusca 99 6 MW491322.1 Italy M. castaneofusca 99 https://www.ncbi.nlm.nih.gov/nucleotide/MW491323.1?report=genbank&log$=nuclalign&blast_rank=1&RID=ES015WE6013 https://www.ncbi.nlm.nih.gov/nucleotide/MW491321.1?report=genbank&log$=nuclalign&blast_rank=2&RID=ES015WE6013 https://www.ncbi.nlm.nih.gov/nucleotide/MW491320.1?report=genbank&log$=nuclalign&blast_rank=3&RID=ES015WE6013 https://www.ncbi.nlm.nih.gov/nucleotide/MW491325.1?report=genbank&log$=nuclalign&blast_rank=4&RID=ES015WE6013 https://www.ncbi.nlm.nih.gov/nucleotide/MW491324.1?report=genbank&log$=nuclalign&blast_rank=5&RID=ES015WE6013 https://www.ncbi.nlm.nih.gov/nucleotide/MW491322.1?report=genbank&log$=nuclalign&blast_rank=6&RID=ES015WE6013 663 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Suliaman, S. Q. Melanoleuca, including M. angelesiana, M. castaneofusca, M. luteolosperma, M. pseudopaedida, and M. robertiana and the bootstrap results was >75% (Antonín et al., 2021). Diagram (3): Maximum likelihood tree depended on ITS sequence. Bootstrap values > 79% based on 1000 replications. (The sequence of I. tamaricis of Iraq is pink circle). Diagram (4): Maximum likelihood tree depended on ITS sequence. Bootstrap values > 98% based on 1000 replications. The sequence of M.castaneofusca of Iraq is red triangle. CONCLUSIONS The present study reported the first I. tamaricis and M. castaneofusca collected from Tamarix tree soil, respectively. The study also indicates the possibility of the presence of several macrofungi that were not recorded in Iraq. The molecular analysis confirmed the phenotypic analysis. Also, the phylogenetic analysis of the Iraqi isolates for I. tamaricis and 664 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM New records of two macrofungi M. castaneofusca were appeared close to the isolates in GenBank. This shows the evidence of compatibility between phenotypic analysis and phylogenetic analysis. CONFLICT OF INTEREST STATEMENT The authors whose names are listed immediately below certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent- licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript. LITERATURE CITED Al-Anbagi R. A. 2014. 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(2023) 17(4): 655-668. استنادا الى Macrofungiتسجيل جديد في العراق لنوعين من الفطريات الكبيرة التشخيص املظهري والجزيئي سارا قحطان سليمان ، تكريت، العراققسم علوم الحياة، كلية العلوم، جامعة تكريت 20/12/2023تأريخ النشر: ، 3/8/2023القبول: ، تأريخ 15/5/2023تأريخ االستالم: الخالصة محافظة صالح الدين لتحديد تنوع الفطريات / أجريت هذه الدراسة بمدينة العلم Inocutis tamaricisالكبيرة فيها. أظهرت نتائج الدراسة امليدانية تسجيل جديد للنوعين (Pat) Fiasson & Niemelä, 1984(Basidiomycota Hymenochaetales,) و Melanoleuca castaneofusca Contu, 1998 (Basidiomycota, Agaricales) ألول مرة تم تشخيصهما استنادا الى الصفات املظهرية والجزيئية والبيئية. هذا اذ في العراق . والذي جاءت نتائجه مؤكدة لنتائج كل ITSالتشخيص الجزيئي اعتماد الجين وتضمن بيئية، فضال عن تسجيل العينات في بنك الجينات العالميمن الفحوصات املظهرية وال NCBI ضمن الرقمين الدوليةOP153814.1 وMZ334407.1 للنوعينI.tamaricis و M.castaneofusca .على التوالي