KSÜ Tarım ve Doğa Derg 23 (3): 600-605, 2020 KSU J. Agric Nat 23 (3): 600-605, 2020 DOI:10.18016/ksutarimdoga.vi.671275 Morphological and Molecular Phylogeny of Cortinarius rufo-olivaceus (Pers.) Fr. (subgenus Phlegmacium sect. Calochroi) Collected from Tokat Region Meryem Şenay ŞENGÜL DEMİRAK1, Hakan IŞIK2, İbrahim TÜRKEKUL3 1Tokat Gaziosmanpasa University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, 60200, Tokat, 2Tokat M.Emin Saraç Anatolian Religious High School, 60030, Tokat, 3Tokat Gaziosmanpaşa University, Faculty of Arts and Sciences, Department of Biology, 60200, Tokat, Turkey 1https://orcid.org/0000-0003-4879-1908, 2https://orcid.org/0000-0001-8241-0078, , 3https://orcid.org/0000-0002-1036-9835 : [email protected] ABSTRACT Research Article Cortinarius (Pers.) Gray samples were collected in the oak forests from Tokat province. Macro and micromorphological features as well Article History as molecular phylogenetic analyses according to the DNA sequences Received : 06.01.2020 corresponding to internal transcribed spacer region (ITS) and the Accepted : 20.02.2020 large subunit (LSU) of nuclear ribosomal RNA gene regions indicated that the studied specimen is a Cortinarius rufo-olivaceus Keywords (Pers.) Fr. This study determined the first microscopic and Cortinarius macroscopic morphological description and molecular phylogenetic Macrofungi New locality analysis of the Cortinarius rufo-olivaceus (Pers.) Fr. in Turkey. ITS LSU Tokat Yöresinden Toplanan Cortinarius rufo-olivaceus (Pers.) Fr. (subgenus Phlegmacium sect. Calochroi) Türünün Morfolojik ve Moleküler Filogenisi ÖZET Araştırma Makalesi Cortinarius (Pers.) Gray örnekleri Tokat ilinden meşe ormanlarında toplanmıştır. Makro ve mikromorfolojik özellikler ile nükleer Makale Tarihçesi ribozomal iç aralayıcı bölge (ITS) ve ribozomal en büyük alt birim Geliş Tarihi : 06.01.2020 (LSU) gen bölgelerine karşılık gelen DNA dizileri ile moleküler Kabul Tarihi : 20.02.2020 filogenetik analizler, çalışılan numunenin Cortinarius rufo-olivaceus (Pers.) Fr. türü olduğunu göstermiştir. Bu çalışma, Türkiye’de C. Anahtar Kelimeler rufo-olivaceus’un mikroskobik ve makroskobik morfolojik tanımı ve Cortinarius Makrofungus moleküler filogenetik analizini belirleyen ilk çalışmadır. Yeni lokalite ITS LSU To Cite : Şengül Demirak MŞ, Işık H, Türkekul İ 2020. Morphological and Molecular Phylogeny of Cortinarius rufo-olivaceus (Pers.) Fr. (subgenus Phlegmacium sect. Calochroi) collected from Tokat region. KSU J. Agric Nat 23 (3): 600-605. DOI: 10.18016/ksutarimdoga.vi.671275. INTRODUCTION has been possible to measure the taxonomic value of The genus Cortinarius belongs to the order Agaricales the morphological characters used in this genus and is one of the most diverse group of fungi. (Orton, 1955; Breitenbach and Kränzlin, 2000; Ortega Members of this genus establish ectomycorrhizal et al., 2008; Stensrud et al., 2014; Itoo et al., 2015). associations with members of the genus Quercus and Worldwide, the genus Cortinarius is represented by Pinus. They can be distinguished by the cortina more than 5000 records (Kirk, 2011). Approximate100 between the cap and the stem especially when young, records of this genus have been reported from our rusty brown lamellae and spore print and bulbous country and more records are being added gradually stipe. The subgenus Phlegmacium has characteristic with the support of molecular data (Sesli and features such as viscid cap especially when young, dry Denchev, 2014; Akata et al., 2015; Sesli et al., 2015; stem, arachnoid cortina and rust-yellow to rust brown Sesli et al., 2016; Sesli and Liimatainen, 2018; Sesli, spore print. In addition to morphological studies, 2018; Kalmer et al., 2019) molecular systematic analysis of the genus Cortinarius rufo-olivaceus has been previously Cortinarius has provided clarification of the reported in Turkey from Osmaniye province (Isıloglu phylogenetic relationships among its species, and it and Öder, 1995). However, no morphological or KSÜ Tarım ve Doğa Derg 23 (3): 600-605, 2020 Araştırma Makalesi KSU J. Agric Nat 23 (3): 600-605, 2020 Research Article molecular data have been provided for this species. A a programme 3 min initial denaturation at 95ºC detailed morphological interpretation is needed for followed by 40 cycles of denaturation at 95ºC for 30 the correct identification of this species. Moreover, sec, annealing at 48ºC for 30 sec and extension at molecular data will provide invaluable information to 72ºC for 1 min and a final extension for 10 min. PCR support morphological data and support the products were checked in a 1 % agarose gel identification. Thus, DNA sequences corresponding to electrophoresis and positive PCR products were gel internal transcribed spacer region (ITS) and the large purified by using Wizard SV Gel and PCR Clean-up subunit (LSU) of nuclear ribosomal RNA genes were System (Promega). Purified PCR products were analyzed to understand the phylogenetic relationship sequenced in both directions using forward and of this species in Cortinarius genus. Although, the reverse primers (BM Labosis Inc., Ankara). existence of C. rufo-olivaceus has been reported previously, this study contributes the first Sequence and Phylogenetic analysis documentation of full description of the species and Chromatograms for forward and reverse primer the new locality supported by both morphological and sequencing were checked for any nucleotide errors. phylogenetic data. All assembled rDNA sequences were examined using Basic Local Alignment Search Tool (BLAST) MATERIALS and METHODS programme using the National Center for Morphological studies Biotechnology Information (NCBI) nucleotide Mushroom samples were detected in the oak forest database. For phylogenetic analysis, representative during a field trip in Akbelen village of Tokat in ITS and LSU sequences of Cortinarius species were autumn 2019. Color photographs of basidiocarps were retrieved from GenBank. The multiple sequence taken and brought to the laboratory in wrapped paper alignments and phylogenetic trees for each genomic for microscopic studies. Spore print was obtained region were done using Molecular Evolutionary from a mature basidiocarp and the samples were Genetics Analysis software (MEGA 7.0; Kumar et al., dried using a heater. The samples were placed into 2016). Phylogenetic trees were constructed using the polyethylene bags and kept in the fungarium of Tokat maximum likelihood (ML) and maximum parsimony Gaziosmanpasa University, Department of Biology, (MP) methods. ML method was based on Tamura-Nei for later studies. Microscopic studies were performed model (Tamura and Nei, 1993) with bootstrap support on dry samples using some chemicals (such as of 1000 replicates and default settings. Initial tree(s) distillate water, KOH, Congo red, etc.). Comparing for the heuristic search were automatically obtained the microscopic, macroscopic and ecological features by using Neighbor-Joining and BioNJ algorithms to a the sample were identified according to the literature matrix of pairwise distances estimated using the such as Bon, 1987; Breitenbach and Kränzlin, 2000; Maximum Composite Likelihood (MCL) approach and Phillips, 1981; Moser, 1983. topology with superior log likelihood value was selected. MP trees were constructed using the Tree- Molecular studies Bisection-Reconnection (TBR) search method with 100 random addition replications. The bootstrap DNA extraction, PCR amplification and Sequencing support values > 50% were marked on the branches of The genomic DNA (gDNA) was extracted form dry the tree. samples using GeneMATRIX Plant & Fungi DNA purification kit (EURx, Poland) following RESULTS and DISCUSSION manufacturer’s protocol. For DNA amplifications, Taxonomy primer pairs ITS4-ITS5 (White et al., 1990) and LROR-LR5 (Vilgalys and Hester, 1990) was used to Description of macrofungi, photographs showing the amplify ITS1-5.8S-ITS2 and 28S LSU rRNA gene morphological features of basidiocarps and regions, respectively. Each polymerase chain reaction microphotographs of basidia and basidiospores, (PCR) was performed in 30 µl volume mixture locality and collection number are given below. The containing 3 µl 10X buffer, 3 µl dNTP mix, 3 µl systematics of the macrofungi are in accordance with degenerate primer pair (final concentration of 1 µM Index Fungorum (http://www.indexfungorum.org: each), 0.3 µl Dream Taq DNA polymerase (Thermo), accessed 14 December 2019). 10 µl gDNA and 7.7 µl sterile ddH2O. A negative PCR Fungi control reaction was prepared in the absence of Basidiomycota gDNA, which included sterile ddH2O instead. ITS Cortinariaceae region was amplified using a programme containing 5 min initial denaturation at 95ºC followed by 40 cycles Cortinarius rufo-olivaceus (Pers.) Fr., Epicr. Syst. of denaturation at 95ºC for 30 sec, annealing at 53ºC Mycol. 268 (1838). for 30 sec and extension at 72ºC for 1 min and a final Pileus 5−10(13) cm across, hemispherical at first, extension for 10 min. LSU region was amplified using then convex to plane, sometimes depressed, slightly 601 KSÜ Tarım ve Doğa Derg 23 (3): 600-605, 2020 Araştırma Makalesi KSU J. Agric Nat 23 (3): 600-605, 2020 Research Article indented, pink- to purple-red or reddish-copper with molecular phylogeny of this species. paler margin. Flesh thick, whitish, light violet under In this study, an approximately 688 bp long region for cap cuticle. Stipe 4−9(10) × 1.2−2.0 cm, cylindrical, ITS1-5.8S-ITS2 and 948 bp long region for the 28S solid,