Callistosporium SINGER, A NEW GENUS RECORD FOR TURKISH MYCOBIOTA

This study aims to describe and introduce a new record for the Turkish mycobiota. Based on the similar macro- and micromorphology, and high nuclear ribosomal large subunit sequence similarity, the mushroom was identified as Callistosporium olivascens (Boud.) Bon. According to the literature research, we found out that this finding is the first record of the genus Callistosporium in Turkey.

I. Akata et al. Conca et al. 2017). The identified samples are deposited in Ankara University herbarium (ANK).

DNA Isolation
The genomic DNA of the specimens ANK Akata & Altuntas was isolated from the fruit bodies according to the CTAB method (Doyle & Doyle 1987). NanoDrop One© Microvolume UV-Vis Spectrophotometer (Thermofisher) was used to measure DNA concentration and purity.

PCR Amplification and Sequencing
The nuclear ribosomal large subunit (nrLSU) region of the rDNA was PCR amplified using the universal LR0R and LR5 oligonucleotide primers (Stielow et al. 2015). PCR was conducted in a reaction volume of 25µmL. The final concentrations of the PCR ingredients were adjusted as follows: 1 × Taq DNA polymerase buffer, 1 unit of Taq DNA polymerase (Fermentas), 0.4 mM dNTPs, 2.5 mM MgCl2, and 10 pmol of both LR0R and LR5 primers. PCR was carried out in a Thermal Cycler (Applied Biosystems MiniAmp Plus) with the following thermal cycling conditions: initial denaturation step of 95°C for 5 min, followed by 35 cycles of 95°C for 30s, 55°C for 30 s, and 72°C for 90 s, and a final extension step of 7 min at 72°C. The PCR amplicon was electrophoretically separated in 1% agarose gel containing the intercalating dye ethidium bromide, and the amplicon size was determined using a DNA marker (GeneRuler 100 bp Plus DNA Ladder, Thermofisher). The amplicon was sequenced bidirectionally using the LR0R and LR5 primers and the standard Sanger dideoxy chain termination method at the laboratory of Macrogen Europe (Amsterdam, The Netherlands).

Sequence Analysis
The nrLSU gene sequences of some relevant fungal species were obtained from GenBank and used for the phylogenetic analysis of the specimen Ank Akata & Altuntas 172. While the nrLSU sequences of the genera Callistosporium Singer, Singerocybe Harmaja, Tricholoma (Fr.) Staude and Lepista (Fr.) W.G. Sm. as some of the well-known genera of Tricholomataceae R. Heim ex Pouzar were selected as in-group sequences, the nrLSU sequences of Agaricus campestris L. and Marasmius oreades (Bolton) Fr. were selected as the out-group sequences. The sequences were assembled using Geneious Prime 2019.1.3 software (Biomatters Ltd) and used for the sequence identity analysis with Basic Local Alignment Search Tool (BLAST). The DNA sequences were then aligned using the CLUSTALW. Molecular phylogenetic analysis was conducted using the neighbor-joining method based on the K2 + G substitution model. MEGAX software was used for constructing the phylogenetic tree by applying one thousand bootstrap replicates (Kumar et al. 2018, Felsenstein 1985.

Macroscopic and microscopic features
Basidiomata clustered to solitary. Pileus 20-50 mm, hemispherical when young, later campanulate, convex to almost plane or funnel-shaped depending on weather conditions, with a wide umbo. Margin straight to wavy, entire, translucently striate; surface smooth, velutinous to apparently glabrous; mixture of brown, yellow and green pigments and hygrophanous. Lamellae sparse, L = 25-35, l = 1-3, emarginate, pale yellowish to beige when young, then rusty yellow or olive-yellow. Stipe 20-50 × 2-4 mm, generally central, cylindric, fused into a cluster, mostly curved, longitudinally compressed, fistulose, brown, yellowish-brown and olive-brown, sometimes minutely floccose or finely longitudinally fibrillose. Context 1.5-2 mm thick, olive-brown or yellowish. Taste mild, smell like beeswax. Basidiospores 7.5-9.5 × 5-6.5 µm, ellipsoid with small but distinct hilar appendage, hyaline, smooth, thinwalled with various content. Basidia 35-40 × 6-7 µm, clavate to cylindrical, 4-spored, clavate, thin-walled, hyaline with globose droplets. Hymenial cells are clavate, narrowly utriform and cylindrical with obtuse apex. Cheilocystidia and pleurocystidia not seen. Pileipellis a cutis, about 30-70 mm deep, made up of cylindrical, smooth or incrusted, thin-to thick-walled, 3-8 mm wide hyphae; Terminal cells cylindrical, narrowly cylindrical, clavate, narrowly clavate or narrowly lageniform. Stipitipellis a cutis of cylindrical, smooth or slightly incrusted, thin-to slightly thick-walled, up to 9 mm wide hyphae. Clamp-connections absent in all tissues.  Tricholoma and Lepista, respectively. On the other hand, A. campestris and M. oreades were branched far from the rest of the fungi species and formed an out-group as anticipated. The BLAST analysis carried out with the nrLSU sequences of Ank Akata & Altuntas 172 provided evidence for the 99.89% similarity of this new record with the two separately collected C. olivascens specimens. The phylogenetic analyses conducted based on the nrLSU sequences of this specimen further supported the close identity relationship of the specimen with C. olivascens with a percent bootstrap value of 98 (Fig. 3).

Discussion
Although the genus Callistosporium includes sixteen confirmed species, the most known European members are C. olivascens (Boud.) Bon, C. luteo-olivaceum (Berk. & M. A. Curtis) Singer and C. pinicola Arnolds (Jančovičová et al. 2016). These species may be confused in the field due to their collybioid habit but C. olivascens can be distinguished from the latter species by its different morphology and ecology. Callistosporium olivascens was described in this study with 20-50 mm, hemispherical, campanulate, convex to almost plane or funnel-shaped pileus; emarginate, pale yellowish to beige, rusty yellow or olive-yellow lamellae, 7.5-9.5 × 5-6 µm and ellipsoid basidiospores. Since the morphological data is not always adequate for the precise identification of fungal species, the sequence data from the conserved DNA regions such as ITS, nrSSU and nrLSU has been employed as a convenient tool in taxonomic studies in the last three decades (Raja et al. 2017). Furthermore, nrLSU is one of the most common DNA barcoding markers and thus confers important information for molecular phylogenetic studies. Therefore, we used nrLSU region for the molecular identification of the specimens Ank Akata & Altuntas 172. The phylogenetic analysis conducted based on the nrLSU regions revealed almost 100% genetic similarity between the C. olivascens (GenBank ID: MK277665) and the new record (GenBank ID: MN486509 for Ank Akata & Altuntas 172) (Fig. 3).
The molecular phylogeny of C. olivascens demonstrated herein, points out its significant distinction from the other species of the genus Callistosporium as C. olivascens clusters in a separate branch within the clade 1 (Fig. 3). Based on this finding, it is plausible to state that the taxonomic revision of this species is likely to be addressed in the future.