A New Lichenicolous Fungus Record From The Turkey , Tremella macrobasidiata ( Basidiomycota , Tremellales )

Despite the recent increase in studies on lichenicolous fungi in Turkey, there is still no checklist. A key to lichenicolous fungus prepared by Halici (2008) were published 118 taxa from the Turkey. So far, the numbers of lichenicol fungi known in Turkey is 193. Generally, lichenicol Ascomycota and mitosporic fungi records are reported, whereas lichenicol Basidiomycota recording is rare. Tremella macrobasidiata is reported on Lecanora chlarotera from Turkey for the first time. The morphological, anatomical and ecological characteristics of the species are given. In addition, a phylogenetic tree was constructed by comparing the sequence data of the ITS region of closely related species.


Introduction
The genus Tremella Pers.includes mostly mycoparasitic species.Many of these are host specific and limited to a single genus or fungal species.More than 50 lichenicolous species have been identified that grow up in lichenized fungi until now [1][2][3][4][5][6][7][8].In the Turkey, only one Tremella lichenicolous species have been reported so far [9].Lichenicolous Tremella species that development in the hymenium of the host inconspicuous at first but then the formation of waxy to gelatinous galls is observed on the thallus or apothecium [2,6].The phylogenetic position of the genus has been determined by Millanes [10].In this study, phylogenetic, morphological and ecological features of the species are given.

Material and Method
Morphological and anatomical examinations were all performed under stereomicroscopes (Olympus SZX10) and light microscope (BX53).Sections for the anatomical examinations were taken directly under the stereo microscope using manual razors and examined 10% KOH, Congo-red, and water.DNA isolation was performed with DNeasy Plant Mini Kit (Qiagen).PCR-amplification was carried out in 50 μL reaction volumes using 3 μl of 10 x reaction buffer, 3 μl MgCl2 (50 mM), 0.5 μl each primer (ITS1F and ITS4), 1 μl dNTP (10 mM), 0.1 μl Taq DNA polymerase, 3 μl of genomic DNA and 38.9 μl dH2O on a thermal cycler equipped with a heated lid.[11] and ITS1-F [12] were used to amplify the ITS sequence.Polymerase chain reaction (PCR) cycling parameters included an initial hold at 94°C for 5 min, then denaturating at 94°C for 1 min, anneling at 56°C for 1 min., decreasing 1°C per cycle fort he first 6 of the 39 cycles, and extension at 72°C for 3 min.PCR products obtained from lichen samples were sent to BMLabosis for sequence analysis.ITS sequence results were analyzed manually using samples from Genbank using the Clustal W option in the BioEdit program.For phylogenetic tree, MEGA 7 (Molecular Evolutionary Genetics Analysis) program was used.
Phylogenetic tree was constructed by Maximum Likelihood analysis using Kimura 2-parameter model.Pairwise deletion was performed for deleting data gaps and checking.The tree reliability was tested with 1000 bootstrap replications.Phaeotremella pseudofoliacea JN053502 was used as the outer group.The specimens are deposited in Bozok University Herbarium, Yozgat, Turkey, (DK 0.079, CMP 0.007).

Tremella macrobasidiata
A detailed description is provided by Zamora et al. [6].
Because lichenicolous Tremella species are inconspicuous species, they are overlooked by both micologists and lichenologists [7].The species not observed on Lecanora chlarotera Nyl., during our morphological studies was noticed as a result of molecular studies (Figure 1).Subsequent detailed anatomical examinations showed that L. chlarotera developed in apothecia.According to the literature, the species grows in the apothecia of epiphytic lichen L. chlarotera, first formed hymenium and later formed gall and is apparently common [6].

Ecology and distribution
Tremella macrobasidiata is known from Spain and appears to be host-specific in L. chlarotera.The distribution is mainly related to a cosmopolitan epiphytic lichen with a wide ecological diversity [13,14].For this reason, it is stated that new records are expected [6].Spanish specimens were collected at altitudes between 590 and 1340 m, extends from the meso-ordination of the Mediterranean Region by Zamora [6].The Turkish sample was collected at a height of 1020 m from the Black Sea Region.

Molecular results
Sequence results were checked in GenBank by blastn similarity search (Table 1).The ITS sequences of T. macrobasidiata collected from the Turkey were blasted with sequences of species from Spain and Portugal.It is seen that these species are matched in phylogenetic tree (Figure 2).

Figure 2 .
Figure 2. Maximum Likelihood (ML) analysis of the ITS region of T. macrobasidiata and related species

Table 1 .
Sequences used for analysis were downloaded from the gene bank, the new product is indicated by bold .