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Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (3): 314-326 Research Article Kastamonu Univ., Journal of Forestry Faculty Doi:10.17475/kastorman.499076

Phylogeny of Some (Fungi: ) in Turkey and Identification of Melanoleuca angelesiana A.H. Sm. As a First Record

Aysenur KALMER1, İsmail ACAR2, Ayten DIZKIRICI TEKPINAR1*

1Department of Molecular Biology and Genetics, Van Yüzüncü Yıl University, 65080, Van, Turkey 2Department of Organic Agriculture, Başkale Vocational High School, Van Yüzüncü Yıl University, 65080, Van, Turkey *Corresponding author: [email protected]

Received Date: 28.05.2018 Accepted Date: 08.11.2018 Abstract Aim of Study: The purposes of the present study are to describe and identify all collected Melanoleuca samples using morphological characters, determine phylogenetic relationships among species using DNA sequences of ITS and LSU regions and check the number of subgenera of Melanoleuca . Area of Study: Samples were collected from different parts of Turkey and the study was conducted at the Department of Molecular Biology and Genetics in Van Yüzüncü Yıl University. Material and Methods: Twenty-two samples, representatives of fourteen species, were used. Structures of , , lamellae and basidia, cystidia, spores were used as macroscopic and microscopic features, respectively. The nuclear ribosomal internal transcribed spacer region (nrITS) and largest subunit (LSU) were amplified for molecular analyses. Main results: Taxonomic positions of some Melanoleuca species from Turkey were evaluated based on both morphological characters and sequences of two different nuclear DNA regions. nrITS region was more informative (112 variable sites in ITS1, 1 in 5.8S and 95 in ITS2 subregions) compared to LSU (46 variable sites). Analyses of ITS region revealed that the genus Melanoleuca was monophyletic and divided into two subgenera as Melanoleuca and Urticocystis. Research highlights: Within studied species, Melanoleuca angelesiana was firstly reported for mycobiota of Turkey Keywords: ITS, LSU, Melanoleuca, Fungal , New record Türkiye’deki Bazı Melanoleuca türlerinin (Fungi: Basidiomycota) Filogenisi ve Yeni bir Kayıt Olarak Melanoleuca angelesiana A.H. Sm. Türünün Tanımlanması Öz Çalışmanın Amacı: Bu çalışmanın amacı toplanan Melanoleuca örneklerinin morfolojik karakterlerle tanımlanması, ITS ve LSU DNA sekanslarını kullanarak türlerin filogenetik ilişkilerinin belirlenmesi ve Melanoleuca cinsinin alt cins sayısının teyit edilmesidir. Çalışma Alanı: Örnekler Türkiye'nin farklı bölgelerinden toplanmış ve çalışma Van Yüzüncü Yıl Üniversitesi Moleküler Biyoloji ve Genetik Bölümü'nde gerçekleştirilmiştir. Material ve Yöntemler: On dört türü temsil eden yirmi iki örnek kullanılmışır. Şapka, sap, lamel yapıları ve bazidya, sistidya, spor yapıları sırasıyla makroskopik ve mikroskopik özellikler olarak kullanılmıştır. Nükleer ribozomal iç aralayıcı bölge (nrITS) ve ribosomal en büyük alt birim (LSU) moleküler analizler için amplifiye edilmiştir. Ana sonuçlar: Bu çalışmada, Türkiye'den bazı Melanoleuca türlerinin taksonomik konumları hem morfolojik karakterleri hemde iki farklı nükleer DNA bölgesinin sekansları ile değerlendirilmiştir. nrITS bölgesi (ITS1’de 112, 5,8S’de 1, ve ITS2’de 95), LSU bölgesine (46 değişken alan) kıyasla daha bilgilendirici olmuştur. ITS bölgesinin analizleri Melanoluca cinsinin monofletik olduğunu ve Melanoleuca ve Urticocystis olarak iki alt cinse ayrıldığını göstermiştir. Araştırma konuları: İncelenen türler içerisinde Melanoleuca angelesiana Türkiye mikobiyotası için ilk defa rapor edilmiştir. Anahtar kelimeler: ITS, LSU, Melanoleuca, Fungal taksonomi, Yeni kayıt

Citation (Atıf): Kalmer, A., Acar, İ. & Tekpınar Dizkırıcı, A. This work is licensed under a Creative Commons (2018). Phylogeny of some Melanoleuca species (Fungi: Attribution-NonCommercial 4.0 International License. Basidiomycota) in Turkey and identification of Melanoleuca 314 angelesiana A.H. Sm. as a first record. Kastamonu University Journal of Forestry Faculty, 18 (3), 314-326. Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (3): 314-326 Kalmer et al. Kastamonu Univ., Journal of Forestry Faculty

Introduction the genus have always been unclear (Yu et Melanoleuca Pat. ( R. al., 2014) since some morphological and Heim ex Pouzar) is a mostly edible ecological features can be changed due to saprotrophic fungal genus growing on soil geographic distribution. Interpretations of in , evergreen forests and sand morphological characters often varied among dunes (Boekhout, 1988; Phillips, 2006). mycologists and resulted in disagreements Edible fungi have economic and ecologic (Hyde et al., 2013). The molecular data may importance (Yıldız, Gürgen & Can, 2017). provide invaluable information to identify The database of Mycobank lists 333 macrofungus correctly (Undan et al., 2016). Melanoleuca species worldwide Therefore, researchers have begun to use (http://www.mycobank.org, accessed; molecular and morphological data together. 25.02.2018) and only 23 of them have been Vizzini et al. (2011) used sequence of nrDNA identified up to now in Turkey (Sesli and ITS region to check the traditional Denchev, 2014; Solak, Işıloğlu, Erbil & Allı, Melanoleuca classification. Their results 2015; Acar, Dizkirici Tekpınar, Kalmer & indicated that Melanoleuca is a monophyletic Uzun, 2017). genus including only two subgenera, The genus is characterized by: collybioid Urticocystis and Melanoleuca. to tricholomatoid basidiomata; emarginate to In the present study, specimens were adnate to subdecurrent lamellae; a white to collected from different locations of Turkey pale yellowish ; hyaline spores and identified by using morphological with ellipsoid, mostly amyloid characters. DNA sequences of ITS (Internal ornamentation; absent or present Transcribed Spacer) and LSU (Large Subunit cheilocystidia of two types; pleurocystidia of rRNA) regions were analyzed to similar to cheilocystidia; pileipellis a cutis to understand phylogenetic relationships among a trichoderm and an absence of clamp Melanoleuca species. These two regions connections (Singer, 1986; Bon, 1991; were selected for the study because rDNA Boekhout, 1999). Especially, structure of the gene has been conserved during evolutionary cheilocystidia is a valuable character to process and usefulness of ITS and LSU identify taxonomic position of the studied regions has been proved by several studies species within the genus. Furthermore, the for the Melanoleuca genus (Hinrikson, Hurst, size, amount and ornamentation of spores are Lott, Warnock & Morrison, 2005; Brown, important features for delimitation of species Rigdon-Huss, & Jumpponen, 2014). (Kibby, 2016). The genus Melanoleuca can Especially, ITS region is accepted as a be distinguished from other genera with valuable DNA marker for fungal taxonomy specific shapes of cystidia (thin walled and (Schoch et al. 2012). fusiform to lageniform). Even though these The purposes of the present study are to i) mentioned characters are useful to identify a describe and identify all collected samples sample at the genus level, lots of problems using morphological characters ii) determine are encountered during identification of a phylogenetic relationships among species sample at the species level because of using DNA sequences of ITS and LSU morphological variabilities and regions iii) check the number of subgenera. macroscopic/microscopic similarities (Vizzini, Para, Fontenla, Ghignone & Ercole, Materials and methods 2011; Duriska, Antonin, Para, Tomsovsky & Taxon sampling and morphological Jancovicova, 2017). studies Some researchers divided Melanoleuca The macrofungus samples were collected genus into three subgenera as Acystis, from different locations of Turkey (Table 1). Urticocystis and Melanoleuca, using Twenty-two samples, representatives of 14 traditional classification which is based on species, were collected and used for presence and shape of cystidia (Bon 1991). morphological and molecular studies. All Acystis is characterized by missing cystidia, collected samples were deposited in the Urticocystis with urticiform cystidia and Fungarium of Van Yüzüncü Yıl University Melanoleuca with macrocystidia (Bon, 1991; (VANF). Specimens were photographed in Bas, Kuyper, Noordeloos & Vellinga, 1999). situ, using with a Canon (EOS 60D) camera However, the infrageneric classifications of equipped with Tokina 100 mm macro lens

315 Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (3): 314-326 Kalmer et al. Kastamonu Univ., Journal of Forestry Faculty during field work. Macroscopic characters program and described based on different were recorded using fresh materials. Pilea, studies (Murrill, 1913; Bresinsky and Stangl, cheilocystidia and basidia were observed in 1977; Bon, 1991; Breitenbach and Kränzlin, distilled water under a Leica EZ4 stereo 1991; Dähncke, 2004; Jordan, 2004; Gerault, microscope and sections were examined 2005; Clémençon 2009; Vizzini et al., 2011; under a Leica DM500 research microscope. Buczacki, 2012; Garcia, Blanco & Matheney Microscopic characters were measured with 2013; Kuo and Methven, 2014; Desjardin, the Leica Application Suite (version 3.2.0) Wood & Stevens, 2015).

Table 1. List of studied Melanoleuca species, their localities, collected date, subgenera and NCBI accession numbers. Subgenera were decided using study of Vizzini et al. (2011). Species Locality Collected Subgenera Accession no. date ITS LSU 1. M. brevipes (Bull.) Pat. Bingöl 2009 Urticocystis MG989678 MG989689 Tatvan-Bitlis 2010-2011 Hani- 2009- 2. M. exscissa (Fr.) Singer Diyarbakır 2010 Urticocystis MG989683 MG989694 Erzincan 2006 Bingöl 2009 Artvin 2009 3. M. graminicola (Velen.) Baykan-Siirt 2012 Urticocystis MG989684 MG989695 Kühner & Maire 4. M. grammopodia (Bull.) Hani- 2010 Urticocystis MG989681 MG989692 Murrill Diyarbakır 5. M. paedida (Fr.) Kühner & Bingöl 2009 Maire Tatvan-Bitlis 2010 Urticocystis MG989679 MG989690 6. M. substrictipes Kühner Selim-Kars 2013 Urticocystis MG989680 MG989691 Tatvan-Bitlis 2011 7. M. dryophila Murrill Şemdinli- 2014 Urticocystis KX507366 KX507363 Hakkari 8. M. microcephala (P. Karst.) Hakkari 2014 Urticocystis MG989677 MG989688 Singer 9. M. angelesiana A.H. Sm. Soğanlı-Kars 2011 Urticocystis MG989682 MG989693 10. M. arcuata (Bull.) Singer Siirt 2016 Urticocystis MG989686 MG989697 11. M. (Pers.) Erzincan 2006 Murrill Bingöl 2008 Melanoleuca MG989685 MG989696 12. M. polioleuca (Fr.) Hani- 2009 Kühner & Maire Diyarbakır Melanoleuca MG989687 MG989698 Hakkari 2014 13. M. heterocystidiosa Yüksekova - 2014 Melanoleuca KX507368 KX507365 (Beller & Bon) Bon Hakkari 14. M. communis M. Sánchez- Hakkari 2015 Melanoleuca KX507367 KX507364 García&Cifuentes

Molecular studies DNA amplification of each studied region DNA isolation, PCR amplification and was performed in a 25 µl volume mixture DNA sequencing containing genomic DNA (10 ng/µl), 10X Genomic DNA was isolated from fungal PCR Buffer, MgCl2 (25 mM), dNTP mixture material using the CTAB (10 mM), selected primer pair (10 µM), Taq (Cetyltrimethylammonium bromide, polymerase (5u/µl) and sterile water. PubChem ID 5974) method (Doyle and To amplify ITS (ITS1-5.8S-ITS2) and Doyle, 1987). The purity and quantity of LSU regions, primer pairs N-nc18S10 extracted DNA were determined with 5'AGGAGAAGTCGTAACAAG3'/C26A NanoDrop2000c UV–Vis Spectrophotometer 5'GTTTCTTTTCCTCCGCT3' (Wen et al. (Thermo Scientific) and 0.8% agarose gel 1996) and LROR 5’ACCCGCTGAACT electrophoresis. Isolated stock DNA was TAAGC3’/LR5 5’TCCTGAGGGAAACTT stored at -20°C prior to PCR amplification. CG3’ (Vilgalys & Hester 1990) were used,

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Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (3): 314-326 Kalmer et al. Kastamonu Univ., Journal of Forestry Faculty respectively. ITS region was amplified under ML tree. The tree with the highest log a program consisting of a hot start at 95 ºC likelihood was shown. Initial tree(s) for the for 2 min, followed by 30 cycles of heuristic search were obtained automatically denaturation at 94 °C for 1 min, annealing at by applying Neighbor-Join and BioNJ 54° C for 1 min, extension at 72 °C for 2 algorithms to a matrix of pairwise distances min, and a final 72 ºC step for 10 min. Same estimated using the Maximum Composite temperature and time conditions were used Likelihood (MCL) approach, and then, the for amplification of LSU region except topology with superior log likelihood value annealing (52 °C). PCR products were was selected. The Tree-Bisection- checked in 1% TAE agarose gels staining Reconnection (TBR) search method was with Gelred dye, and positive reactions were employed with 100 random addition sequenced with forward and reverse PCR replications to construct the MP trees and the primers. Chromatograms were checked for consensus tree inferred from 10 most putative reading errors and corrected. The parsimonious trees was used. All positions sequences of species were deposited in NCBI containing gaps and missing data were database and their accession numbers were eliminated. given in Table 1. Results Sequence alignment and phylogenetic Taxonomy analysis Macroscopic and microscopic characters BLAST (Altschul, Gish, Miller, Myers & of studied Melanoleuca species were given in Lipman,1997) was used to select the most Table 2. Within the studied specimens, closely related ITS and LSU rDNA Melanoleuca angelesiana A.H. Sm. was sequences from NCBI database (Appendix 1) reported firstly for Turkey and its and all sequences were aligned with the aid characterization was done by using of the program ClustalW (Thompson, morphological characters as follow. Higgins & Gibson, 1994). Alignments were Specimen examined: Turkey, Kars, Soğanlı, checked and manually adjusted where it was under , 40°24'053" K, 42°29'117"D, necessary. Totally 40 sequences (26 of them 2525 m, 03.10.2011, Acar, I., 971, VANF. retrieved from NCBI) of the genus were NCBI numbers: MG989682 (ITS region), included in the phylogenetic analysis of ITS MG989693 (LSU region). regions. LSU region was less studied compared to ITS so 10 LSU sequences could Description of Melanoleuca angelesiana be downloaded from NCBI and analyzed A.H. Sm. with our samples. cervinus (Schaeff.) Pileus: 60-100 mm across; convex to young, P. Kumm. was used as outgroup for testing then flatted, often with a superficial or the monophyly of Melanoleuca (ITS: evident central lump, smooth, damp when JX857448, LSU: HM562221, Garcia et al., fresh, dark brown, discoloration to 2013). Borders of regions were decided tan. Lamellae: notched, attached to the stipe, considering sequences downloaded [ITS close to cramped, at first greyish-tan, grayish (JX429148, Garcia et al., 2013) and LSU to whitish, sometimes growing lightly (JX429179, Garcia et al., 2013)]. pinkish color. Stipe: 30-60 × 5-15 mm, more Phylogenetic trees were constructed using or less equal, sometimes lightly bulb at the both Maximum Likelihood (ML) and base, fairly smooth, surface fibrillose-striate Maximum Parsimony method with aid of when maturity, whitish at the apex but brown Molecular Evolutionary Genetics Analysis like the pileus below, basal mycelium white software (MEGA 6; Tamura, Stecher, and absent. Basidia: 23.5-37.2 × Peterson, Filipski & Kumar, 2013). Tamura- 6.8-10.7 µm. Spores: 7-10 × 5-6.5 µm, more Nei model (Tamura and Nei, 1993) and or less elliptical, amyloid, finely warts. bootstrap analysis with 1000 replications Pleurocystidia and cheilocystidia absent (Felsenstein, 1985) were selected to construct (Figure 1).

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Figure 1. Melanoleuca angelesiana, a) b) Spores (distilled water) c) Basidia d) Basidioles e) Hyphae

Table 2. Considerable morphologic characters of studied Melanoleuca species Pileus Lamellae Stipe Basidia Cystidia Spores References M. brevipes (Bull.) Pat. 50-90 mm, Light cream, later 30-50 × 8-15 30-40 × Cheilocystidia Elliptic, convex when with gray to mm, usually 8-9 µm, of the stinging irregularly Boekhout, young, then lilactints, slightly with a clavate clavate hair type verrucose, 1988; depressed notched to base and urticifom, hyaline Breitenbach & center subdecurent widened apex, Caulocystidia with drops, Kränzlin,1991 somewhat surface with at the stipe 6.9-9.7 × umbonate, ocherish on a apex 4.6-7.0 µm radially brown fibrillose, background, beige to gray- covered with brown, grading white to to black-brown ocherish toward the powder center, M. exscissa (Fr.) Singer 40-70 mm, White to cream, 40-60 × 5-8 22-25 × Cheilo- and Elliptic, convex when edges slightly mm, 6.5-8 µm, pleurocystidia verrucose, Breitenbach & young, later undulating to somewhat cylindric fusiform to hyaline, Kränzlin,1991 plane and crenate enlarged to lageniform 7.1-8.8 × sometimes toward the cylindric 4.2-5.5 µm with a apex, surface clavate depressed fibrillose, center, apex white- obtusely pruinose, umbonate in dark-brown the center toward the base

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Table 2. (continued) M. graminicola (Velen.) Kühner & Maire 20-30 mm, White, later 40-50 × 3-4 23-35 × Cystidia Elliptic, convex- cream-whitish, mm, slightly 7-8.5 µm absent verrucose, Breitenbach & campanulate sometimes also clavate toward clavate, hyaline, Kränzlin,1991 when young, with a warm pink the base, with drops, with a distinct tint surface cream 5.6-7.9 × papilla, later to pink- 4.6-6.0 µm plan to slightly whitish, with depressed, white fibrils dark-brown to dark-gray brown, center darker M. grammopodia (Bull.) Murrill 70-150 mm, Crowded sinuate, 50-120 × 10- 30-40 × Cheilocystidia Elliptical, bell-shaped rather thick, 15 mm, 8- 13 µm, with swollen minutely Boekhout, then expanded, triangular- swollen at the clavate and long, warted, 1988; Phillips, often ventricose, pale base; grey- narrow neck amyloid, 2006 becoming cream-beige brown, 8.5-9.5 × 5- centrally longitudinally 6 µm depressed; fibrillose light to dark gray-brown M. melaleuca (Pers.) Murrill 30-80 mm, White, 55- 90 × 5- 8 25- 40 × Cystidia Hyaline, dark-brown, emarginated, mm, 7- 10 µm, absent warty, hygrophanous, broad, crowded cylindrical, clavate ellipsoid, Boekhout, drying buff; at slightly amyloid, 1988; Jordan, first flattened broader droplets, 7- 2004 convex, towards base, 8.5 × 5-5.5 becoming sometimes µm umbonate- becoming pale depressed beige to brown-grey longitudinally fibrillose with pruinose apex M. paedida (Fr.) Kühner & Maire 35-50 mm, Gray-whitish to 30-50 × 7-10 27-30 × Cheilocystidia 8.1-9.6 × hemispherical gray, with mm, cylindric, 6-8 µm, 40-50 × 5-8 5.5-6.3 µm Breitenbach & when young, yellowish-brown some with a cylindric µm, stinging- elliptic, Kränzlin,1991 later convex to edges, bulbous base to hair shaped, finely plane, gray- or enlarged cylindric- sometimes verrucose, brown with a apex, surface clavate sparse to hyaline silver tinge longitudinally absent when dry, fibrillose, gray-beige cream to gray- when wet brown M. polioleuca (Fr.) Kühner & Maire 25-70 mm, at Crowded, adnate 35-85 × 3-8 15- 40 × Cheilocystidia 6.3-9 × 4-5 first convex, to sinuose, mm, 6-10 µm, 45-75 × 8-15 µm finally with mostly with cylindrical clavate µm, fusiform ellipsoid to Boekhout,1988 somewhat subdecurrent clavate base, to lageniform, elongate, depressed toothlet, stuffed, at first at apex rather centre, mostly ventricose to whitish, encrusted with ornamented with low broad triangular, becoming pale crystals umbo, when whitish to sordid grey- moist brown yellowish brown to dark grey-brown

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Table 2 (continued) M. substrictipes Kühner 25-70 mm, White, later 40-70 × 3-8 33-38 × Cheilocystidia 8.1-10 × 5- convex when cream colored mm, 7-9 µm, fusiform to 6.3 µm, young, later with a pink tint cyclindric, clavate, subulate, elliptic, Breitenbach & plane to base bulbous almost like finely Kränzlin, 1991 irregularly and up to 10 stinging hair verrucose, undulating, mm thick, hyaline, with an sometimes with drops umbonate twisted, center, satiny, surface white when smooth, young, later longitudinally cream-whitish fibrillos, white when young later yellow- brownish M. dryophila Murrill 30-150 mm, Deeply sinuate to 60-130 × 10- Not seen 5-7 × 3.5- convex, adnexed, close, 45 mm, 4.5 µm, gibbous, viscid narrow, plane, stuffed at globose, Acar et al., when flesh, white maturity, smooth, 2017 subshining, equal to hyaline whitish, enlarged at the stained with base, fibrillose rusty-brown, brown over a margin paler, pallid context white, background with below farinaceous M. microcephala (P. Karst.) Singer 10-30 mm, low white or tinged Cheilocystidia 6-8 × 4-5 convex to pale buff 40-60 × 6-10 μm, Vesterholt and plane with a μm, urticoid ellipsoid, Knudsen, 2008 small umbo, septate, often verrucose smooth, yellow with crystals at brown to dark apex brown M. heterocystidiosa (Beller & Bon) Bon 50-70 mm., Somewhat 50-80 × 4-7 Cheilocystidia 7-9 × 4-5 convex to fragile, closed, mm, slightly 35-90 × 8-20 µm, smooth, white pruinose, µm, abundant elliptical, Acar et al., slightly grey-sepia, and variable in warty 2017 pruinose, gray- pale greyish, size and shape, dark brown downwards mostly with olive in striate, at the vertical- the center, then base dark shaped greyish ochre M. communis M. Sánchez-García&Cifuentes 30-150 mm, Sinuate, 40-170 × 4-16 20-40 × Cheilocystidia 6-9 × 4-6 plane-convex uncinateoradnate, mm, 5-10μm, 50-80 × 8-18 μm, to plane, close to crowded, cylindrical to clavate μm, fusiform, ellipsoid, Acar et al., sometimes white to slightly usually with ornamented 2017 umbonate, yellowish with attenuated crystals with margin entire edges towards the amyloid decurved, base, white to brown, greyish pale yellow, brown, slightly with yellowish- yellowish brown towards longitudinal the margin striates

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Table 2 (continued) M. arcuata (Bull.) Singer 50-100 mm, Emarginate, 40-80 × 7-14 Cystidia 40-60 9-10 × 5-6 Carleton, 1922 convex, then deccurrent with a mm, × 10-12 μm, μm, plane, pale long tooth, fibrillose, base lanseolate punctate brownish, yellowish thickened brownish clay or coffee and milk

Molecular phylogeny Melanoleuca) and B (subgenera ITS data matrix comprised a total of 40 Urticocystis). Clade A and B were well sequences including 26 from NCBI. The supported with 99 and 98% bootstrap, amplified DNA fragment of the region was respectively. Clade A consisted of four of approximately 650 bp long encompassing studied species; M. communis, M. complete ITS1, 5.8S and ITS2 subregions. heterocystidiosa, M. arcuata and M. The data set of the region included a total of polioleuca. Melanoleuca arcuata from these 731 positions, of which 508 were conserved, species was placed unexpected clade. Clade and 208 were variable (112 variable sites in B consisted of studied M. brevipes, M. ITS1, 1 in 5.8S and 95 in ITS2 subregions). dryophila, M. substrictipes, M. paedida, M. Number of variations was higher in ITS1 grammopodia, M. exscissa, M. angelesiana, (112) compared to ITS2 (95). M. graminicola, M. microcephala and M. The second region, LSU, comprised 24 melaleuca. In this clade, Melanoleuca sequences (10 from NCBI) and yielded total melaleuca was placed unexpected clade. lengths of 885 nucleotides with 21 nucleotide There was no discrimination at the section variations. In the study, we mainly used the level in the tree. results taken from ITS region that was more LSU tree was not given due to less informative than LSU. The topologies of the informativeness to understand phylogeny and MP and ML phylogenetic trees had no low bootstrap values. The species could not considerable differences, so only one tree be separated at the subgenus level (ML) was given to indicate phylogenetic (Melanoleuca and Urticocystis) and they relationships. Phylogenetic tree constructed scattered in the tree so taxonomic relations based on ITS region (Figure 2) composed could not be resolved. Only M. paedida, M. from two major which were named substrictipes, M. grammopodia and M. according to the study of Vizzini et al. (2011) exscissa evolutionarily separated from other and called as clade A (subgenera species and caused a small cluster which was also observed in ITS tree.

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Figure 2. Maximum Likelihood phylogeny of from the studied species were marked with nrITS sequences of Melanoleuca taxa. white square. Clade A; subgenus was used as outgroup taxon. Melanoleuca. Clade B; subgenus Urticocytis. Studied species were marked with black square in Clade A and black circles in Clade B. Species whose position was incorrect

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Discussions whitish lamellae and mainly lageniform Identification of Melanoleuca species is cystidia or rarely without cystidia (Bon, controversial due to similarities of 1991; Boekhout, 1988; 1999; Vizzini et al., morphological characters. Vizzini et al. 2011). Sample determined as M. arcuata (2011) indicates that environmental factors placed in this clade with M. arcuata affect these characters so molecular analyses (JX429187) and other species of subgenus are required for delimitation of the species. Melanoleuca. Melanoleuca arcuata differs For molecular analysis, one of the most mainly by a very dark pileus and yellow- important steps is to select useful region that ochre lamellae only at maturity (Boekhout, changes according to studied genus. 1988; Vizzini et al., 2011) so discrimination Therefore, two regions were used in the of M. arcuata is expected to be accurate. M. present study to compare and select the most heterocystidia clustered with representative useful one. Results indicated that ITS region NCBI sample (JN052140) with 79% was more useful compared to LSU to figure bootstrap value. The studied M. polioleuca out phylogenetic relationships among and its representative retrieved from NCBI Melanoleuca species. Genetic variation located distantly. Similar relations were seen observed in the LSU was lower than that of for other species such as M. communis, M. ITS and insufficient to figure out phylogeny. paedida, M. grammopodia and M. In our previous study, Melanoleuca species graminicola. This type relationship can be were separated at both subgenus and section originated from environmental differences. level (Acar et al., 2017) but separation was M. communis were given as new records in occurred at only subgenus level in the current our previous study and detailed information study. The fact that number of species is (spores, basidia, cystidia etc.) can be seen increased, analysis of taxonomy is (Acar et al., 2017). complicated. Clade B (subgenus Urticocystis) consisted Two major clades (Clade A and B) with of studied M. brevipes, M. dryophila, M. high bootstrap values were observed in the substrictipes, M. paedida, M. grammopodia, constructed ITS tree. Clade A mainly M. exscissa, M. angelesiana, M. graminicola, included species Melanoleuca subgenus and M. microcephala, M. melaleuca and their Clade B generally composed from species representatives. This subgenus characterized Urticocystis and Acystis subgenera. Bon by mainly with urticocystidia but also with (1991) claimed that three subgenera are macrocystidia and brightly colored pilei or found in the genus and one of them is called lacking cystidia and absent or very rare as Acystis. In the present study, this subgenus pleurocystidia (Bon, 1991; Vizzini et al., was represented by only two species, M. 2011). M. angelesiana and M. graminicola graminicola and M. angelesiana, and they species placed in this clade with M. paedida located distantly to each other. Therefore, our (JN616453), M. electropoda (JN616430) and outcome supported the results of Vizzini and M. rasilis (JF908355). These studied species his colleagues (2011) where Acystis was previously were accepted to be members of rejected to be a subgenus of Melanoleuca subgenus Acystis (Bon, 1991) however and number of the subgenus was decreased according to Vizzini et al. (2011) Acystis is a from three to two as Melanoleuca and polyphletic taxa and nests with Urticocystis. Urticocystis. Melanoleuca angelesiana was firstly Clade A (subgenus Melanoleuca) reported in Turkey with current study. It is consisted of species downloaded from NCBI distinguished from other species by greenish- and four of studied species; M. communis, M. brown stipe, amyloid warts, grey lamellae heterocystidiosa, M. arcuata and M. and central umbo pileus (Table 3) (Smith, polioleuca. This subgenus characterized by a 1944; Desjardin et al., 2015; Vizzini et al., dark-brown blackish, slate-grey pileus, 2011).

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Table 3. Comparison of Turkish specimens of Melanoleuca angelesiana and data of another authors. Pileus Lamellae Stipe Basidia Spores Referenc es 60-100 mm convex, attached to the 30-60 × 5-15 mm 23.5-37.2 × 7-10 × 5-6.5 This study smooth, central stipe, grayish smooth, fibrillose, 6.8-10.7 µm µm, elliptical, umbo, dark brown partial veil absent amyloid, finely warts 50-70 mm, broad, adnate, grayish 50-60 × 10-12 Four spored 7-9 × 4.5-6 µm, Smith, 1944 central umbo, mm, fibrillose elliptical, brown amyloid warts 20-110 mm, adnate, grayish 15-75 × 3-15 mm, 20-50 × 6-13 7.5-10 × 5-6 Bessette et convex, broad, fibrillose µm µm, elliptical, al. 1995 sometimes umbo, verrucose, dark brown amyloid warts 30-90 mm convex, adnate, grayish 30-50 × 5-15 mm - 8-10.5 × 5-6.5 Desjardin et low umbo, dark fibrillose, partial µm, elliptical, al., 2015 brown, veil absent amyloid, finely warts

Although M. graminicola clustered with cystidia (Kuo and Methven, 2014). However, M. rasilis (JF908355) with 100% bootstrap morphologic characters may not enough due value morphologically they are different. to homoplasy and may cause incorrect Melanoleuca graminicola has not cystidia identification. while M. rasilis has an urticoform cystidia As a conclusion, morphological (Boekhout, 1999). M. rasilis (JN616461) and characters of Melanoleuca species may show M. brevipes clustered together with 99% high level of similarities due to convergent bootstrap value in the tree. M. brevipes is evolution. Therefore, only morphology may differentiated from M. rasilis with its not be enough to study taxonomy of this fusiform to lageniform cystidia and small genus. Molecular techniques are also needed spores covered by coarse warts. M. exscissa, to resolve some problems and selecting M. grammopodia, M. paedida and M. useful region is important. ITS region may be substrictipes clustered together in both ITS a proper marker to solve some taxonomic and LSU (not shown) trees and problems but it is only enough to solve morphologically they share resemble problems at the subgenus level. So, characters. They have fibrillose-smooth stipe additional region should be used to figure out and urticoform cystidia. However, M. phylogenetic relations at the species level. grammopodia is characterized and separated This study is valuable because Melaleuca from others by a large pileus and a long stipe specimens from Turkey have never been (Antonin, Duriska, Jancovicova & studied by using morphological and Tomsovsky, 2015). So, it is expected that the molecular characters. Moreover, diagnosis is correct. M. dryophila found in Melanoleuca angelesiana was firstly Urticocystis subgenus was given as new reported for mycobiota of Turkey and record in our previous study and detailed detailed information about this species was information (spores, basidia, cystidia etc.) also given in the paper. can be seen Acar et al. (2017). is belonging to subgenus Melanoleuca but placed in Acknowledgments subgenus Urticocystis (clade B) with M. The authors are grateful to Van Yüzüncü melaleuca (HM622269) in the tree. Yıl University (BAP Project No. 2017-FBY- Interestingly, one of M. melaleuca sample 5560) for its financial support during the retrieved from NCBI (JN616448) placed in study. clade A while the other sequence (HM622269) placed in clade B with our sample M. melaleuca. This species is characterized by grey-brown or dark-brown pileus, amyloid warts spores and lacking

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Appendix 1 Breitenbach, J. & Kränzlin, F. (1991). Fungi of Accession numbers of species Switzerland. Verlag Mykologia, 3rd ed. downloaded from NCBI database. Lucerne, Switzerland. Bresinsky, V.A. & Stangl, J. (1977). Beiträgezur ITS region: revision m. britzelmayrs hymenomycetenaus Melanoleuca angelesiana (JN616420), M. südbayern. Zeitsehr.f.Pilzkunde, (43) ,145- brevipes (JX429188), M. humilis 173. M. cinereifolia Brown, P.S., Rigdon-Huss, A.R. & Jumpponen, (JX987317), (JN052138, A. (2014). Analyses of ITS and LSU gene KU575243), M. graminicola (JN616438), M. regions provide congruent results on fungal melaleuca (HM622269, JN616448), M. community responses. Fungal Ecology, (9), grammopodia (KT279047, KP192269), M. 65-68. polioleuca (JF908358, JX429196), M. Buczacki, S. (2012). Collins fungi guide: the most communis (LT716069, JX429204, complete field guide to the and JX429208), M. heterocystidiosa (JN052140), toadstools of Britain & Ireland. London, UK: M. rasilis (JN616461, JF908355), M. friesii Harper Collins Publishers. (JN616437), M. exscissa (JX429121, Carleton, R. (1922). British basidiomycetae, JN616431), M. substrictipes (JN616474), M. Cambridge University Press, UK. Methods for working with microcephala M.arcuata Clémençon, H. (2009). (JF908348), macrofungi: laboratory cultivation and (JX429187), M. electropoda (JN616430), M. preparation of larger fungi for light paedida (JN616453), Pluteus cervinus microscopy. Berchtesgaden: Berchtesgadener (JF908614) Anzeiger. Dähncke, M. R. (2004). 1200 Pilze in farbfotos. References Switzerland: ATVerlag Aarau. Acar, I., Dizkirici Tekpınar, A., Kalmer, A. & Desjardin, D. E., Wood, M. G. & Stevens, F. A. Uzun, Y. (2017). Phylogenetic relationships (2015). California mushrooms: the and taxonomical positions of two new records comprehensive identification guide. Portland, Melanoleuca species from Hakkari province, OR.: Timber Press. 560. Turkey. Biodicon 10(3), 85-93. Doyle, J. J. & Doyle, J. L. (1987). A rapid DNA Altschul, S. F., Gish, W., Miller, W., Myers, E. isolation procedure for small quantities of W. & Lipman, D.J. (1997). Basic local fresh leaf tissue. Phytochemical Bulletin, (19), alignment search tool. Journal of Molecular 11-15. Biology, 215, 403-410. Duriska, O., Antonin, V., Para, R., Tomsovsky, Antonin, V., Duriska, O., Jancovicova, S. & M. & Jancovicova, S. (2017). Taxonomy, Tomsovsky, M. (2015). Identity of ecology and distribution of melanoleuca brevipes bull. (Melanoleuca brevipes, strictipes (basidiomycota, ) in tricholomataceae, basidiomycota). . Czech Mycology, 69(1), 15–30. Mycological Progress 14, 107. Felsenstein, J. (1985). Confidence limits on Bas, C., Kuyper, Th. W., Noordeloos, M. E. & phylogenies: An approach using the bootstrap. Vellinga, E.C. (1999). Flora Agaricina Evolution, (39), 783-791. Neerlandica. 4th ed. CRC Press. Rotterdam, Garcia, M. S., Blanco, J. C. & Matheny, P. B. Netherlands. (2013). Taxonomic revision of the genus Bessette, A. E., Miller, O. K., Bessette A. R. & Melanoleuca in Mexico and description of Miller H. H. (1995). Mushrooms of North new species. Revista Mexicana de America in color: A field guide companion to Biodiversidad, 111-127. seldom-illustrated fungi. China: Syracuse UP. Gerault, A. (2005). Florule evolutive des 172 pp. basidiomycotina du finistere Boekhout, T. (1988). Notulae ad floram heterobasidiomycetes tricholomatales. agaricinam neerlandicam, XVI – New taxa, Version 2.1. France. new combinations in Melanoleuca Pat. and Hinrikson, H. P., Hurst, S. F., Lott, T. J., notes on rare species in the Netherlands. Warnock, D. W. & Morrison, C. J. (2005). Persoonia, 13(4), 397–431. Assessment of ribosomal large-subunit D1- Boekhout, T. (1999). Melanoleuca, in: C Bas et D2, internal transcribed spacer 1, and internal al. (eds). Flora Agaricina Neerlandica 4.A.A. transcribed spacer 2 regions as targets for Balkema, Rotterdam. 153–165. molecular identification of medically Bon, M. (1991). Flore mycologiqued’Europe, 2 – important aspergillus species. Journal of Les tricholomesetressemblants. Revue de Clinical Microbiology, (43), 2092-2103. Mycologie Mémóire Hors-Série, 2, 163. Hyde, K. D., Udayanga, D., Manamgoda, D. S., Tedersoo, L., Larsson, E., Abarenkov, K.,

325

Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (3): 314-326 Kalmer et al. Kastamonu Univ., Journal of Forestry Faculty

Bertrand, Y. J. K., Oxelman, B., Hartmann, humans and chimpanzees. Molecular Biology M., Kauserud, H., Ryberg, M., Kristiansson, and Evolution, (10), 512-526. E. & Nilsson, R. H. (2013). Incorporating Tamura, K., Stecher, G., Peterson, D., Filipski, molecular data in fungal systematics: a guide A., M. & Kumar, S. (2013). MEGA 6: for aspiring researchers. Current Research in Molecular evolutionary genetics analysis Environmental & Applied Mycology, 3(1), 1- Version 6.0. Molecular Biology Evolution, 32. 30(12), 2725–2729. Jordan, M. (2004). The Encyclopedia of fungi of Thompson, J. D., Higgins, D. G. & Gibson, T. J. Britain and Europe. UK.: Frances Lincoln (1994). Clustal W improving the sensitivity of Publishers P. 51. progressive multiple sequence alignment Kibby, G. (2016). Melanoleuca castaneofusca through sequence weighting, position-specific new to Britain. Field Mycology, 17(3):95-97. gap penalties and weight matrix choice. Kuo, M. & Methven, S. A. (2014). Mushrooms of Nucleic Acids Research, (22), 4673–4680. the Midwest. Urbana, Chicago and Undan, J. Q., Alfonso, D. O., Dulay, R. M., Springfield: University of Illinois Press. Leon, A. M., Kalaw, S. P. Undan J. R. & Murrill, W. A. (1913). The of the Reyes, R. G. (2016). Molecular identification Pacific Coast - IV. New species of and phylogeny of different macrofungi in Mt. and Melanoleuca. Mycologia, (5), 206-223. Bangkay, Cuyapo, Nueva Ecija, Philippines Mycobank database. (2018). Melanoleuca. based on ITS nrDNA region. Advances in http://www.mycobank.org/BioloMICS.aspx?T Environmental Biology, 10(12), 35-42 able=Mycobank&Rec=56901&Fields=All, Vesterholt, J. & Knudsen, H. (2008). Funga Westerdijk Fungal Biodiversity Institue, Nordica-Agaricoid, boletoid and cyphelloid München, Germany. (Accessed: 25.02.2018) genera. Nordsvamp, Copenhagen. Phillips, R. (2006). Mushrooms. Published Vilgalys, R. & Hester, M. (1990). Rapid genetic August, (18th ed.) by Pan MacMillan. identification and mapping of enzymatically Schoch, C. L., Seifert, K. A., Huhndorf, S., amplified ribosomal DNA from several Robert, V., Spouge, J. L., Levesque, C. A. & Cryptococcus species. Journal of Bacteriology Chen, W. (2012). Fungal Barcoding 172, 4239-4246. Consortium Nuclear ribosomal internal Vizzini, A., Para, R., Fontenla, R., Ghignone, S. transcribed spacer (ITS) region as a universal & Ercole, E. (2011). A preliminary ITS DNA barcode marker for Fungi. Proceedings phylogeny of Melanoleuca (Agaricales) with of the National Academy of Sciences, 109, special reference to European taxa. 6241-6246. Mycotaxon, 118, 361-381. Sesli, E. & Denchev, C.M. (2014). Checklists of Wen, J. & Zimmer, E. A. (1996). Phylogeny and the myxomycetes, larger ascomycetes, and Biogeography of Panax L. (the Ginseng larger basidiomycetes in Turkey. Mycotaxon Genus, Araliaceae): Inferences from ITS Checklists Online, 1-136. Sequences of Nuclear Ribosomal DNA. Singer R. (1986). The Agaricales in Modern Molecular Phylogenetics and Evolution 6, Taxonomy. (4th ed.) Koenigstein Königstein 167-177. im Taunus, Germany: Koeltz Scientific Yıldız, S., Gürgen, A. & Can, Z. (2017). In vitro Books. bioactive properties of some wild mushrooms Smith, A. H. (1944). New North American collected from Kastamonu province. Agarics. Mycologia, 36, 242-262. Kastamonu University Journal of Forestry Solak, M.H., Işıloğlu, M., Erbil, K. & Allı, H. Faculty, 17(3), 523-530. (2015). Macrofungi of Turkey checklist. (2nd Yu, X. D., Lv, S. X., Ma, D., Li, F. F., Lin, Y. & ed.) İzmir: Üniversiteliler Ofset. Zhang, L. (2014). Two new species of Tamura, K. & Nei, M. (1993). Estimation of the Melanoleuca (Agaricales, Basidiomycota) number of nucleotide substitutions in the from northeastern China, supported by control region of mitochondrial DNA in morphological and molecular data. Mycoscience, 55, 456-461.

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