First report of Rutstroemia elatina (Ascomycota) from Turkey

The purpose of this research was to identify Rustroemia samples from Bolu province (Turkey) on June 22, 2019. The samples were identified based on both conventional and molecular methods (ITS region of the rDNA). By considering the high sequence similarity of the collected samples (Akata 7020) with Rutstroemia elatina (Alb. & Schwein.) Rehm, the relevant specimen was considered to be R. elatina and the morphological data supported this finding. This species was firstly reported from Turkey. The results of the molecular analysis and a short description of the newly reported species along with its colored images associated with macroscopic and microscopic structures were conferred. Research Article Article History Received : 29.09.2019 Accepted : 29.11.2019


Rutstroemia elatina Ascomycota
. The genus comprises roughly 75 widely distributed species particularly in temperate regions and its members are mainly characterized by brownish to black, cup to funnel or goblet shaped apothecia with short stipe, eight-spored, uni to biseriate, amyloid and cylindric asci, branched to unbranched, cylindrical to filiform paraphyses sometimes thickenings toward to tips, ellipsoid to allantoid, hyaline, smooth, mostly uni to biseptate, more rarely multiseptate spores (Hansen and Knudsen, 2000;Kirk et al., 2008). R. elatina is an uncommon species growing on the fallen branchs, twigs or needles of Abies alba (silver fir). Although it is considered to be specific to silver fir, it has also been reported on Picea abies (Palmer et al., 1994). According to the literature (Işık and Türkekul, 2018;Sesli and Denchev, 2008;Öztürk et al., 2010), R. conformata (P. Karst.) Nannf. and R. firma (Pers.) P.
Karst. have hitherto been registered from Turkey but there was no report of R. elatina (Alb. & Schwein.) Rehm for Turkish Rutstroemia. The purpose of this study was to make a contribution to the larger Ascomycota of Tukey.

MATERIALS and METHODS
Rutstroemia specimens were collected from Bolu province during the field study in 2019. Macroscopic characteristics of the fresh specimens were noted and their photographs were taken at the growth sites. Microscopic features such as asci, paraphyses and spores were examined with Leica DM 1000" bright field light microscope and some chemicals (Congo red, Melzer's reagent, 5% potasium hydroxide, 10% ammonium hydroxide etc.) were utilized for this purpose. We benefited from the relevant literature (Breitenbach and Kränzlin, 1984;Hansen and Knudsen, 2000) for identification. The identified voucher samples were deposited to the Herbarium of Ankara University (ANK).

Molecular study DNA Isolation
The genomic DNA was isolated from the sporophores of the specimen, according to the modified CTAB method (Aras et al., 2013). NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermofisher) was used to calculate the concentration and purity of the extracted DNA.

PCR Amplification and Sequencing
The ITS1, 5.8s rRNA gene and ITS2 regions were amplified with PCR by using the universal ITS1 and ITS4 oligonucleotides (White et al., 1990). PCR was conducted in a reaction volume of 25µl. The final concentrations of the PCR ingredients were adjusted as follows: 1× Taq DNA polymerase buffer, 2 units of Taq DNA polymerase (Fermentas), 0,4 mM dNTPs, 3 mM MgCl2, and 15 pmol of both ITS 1 and ITS4 primers. PCR was carried out in a Thermal Cycler (Applied Biosystems MiniAmp Plus) with the following thermal cycling protocol: first denaturation step of 95°C for 4 min, persued by 35 cycles of 95°C for 30s, 56°C for 15s, and 72 °C for 40s, and a last elongation step of 7 min at 72°C. The PCR amplicons were electrophoretically analyzed in 1.2% agarose gel containing ethidium bromide, and the amplicon sizes were defined by the aid of a DNA size marker (GeneRuler 100 bp Plus DNA Ladder, Thermofisher). The sequences of the amplicons were determined with Sanger dideoxy chain termination method at the laboratory of Macrogen Europe in Amsterdam, The Netherlands using the same oligonucleotide primers.

Sequence Analysis
The ITS gene sequences of some relevant fungal species were obtained from GenBank and used for the phylogenetic analysis of the 'Akata 7020. While the ITS sequences of the genera Rutstroemia and Lanzia, two of the most well-known genera of the Rutstroemiaceae family, were used as ingroup sequences, the ITS sequences of Gyromitra esculenta and Morchella angusticeps were used as the outgroup sequences. The sequences were assembled by 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 aligned using the CLUSTALW and molecular phylogenetic analyses were conducted by using the neighbor joining method based on the Kimura 2-parameter substitution model via MEGAX software with using 1000 bootstrap replicates (Felsenstein, 1985;Kumar et al., 2018).    (Breitenbach and Kränzlin, 1984;Palmer et al., 1994).

DISCUSSION
R. elatina is a sabrobe species and its most common host species is considered to be Abies alba. Moreover, its samples have also been collected on Picea abies (Palmer et al., 1994). R. elatina is characterized by dark brown to black, stipitate, goblet to cup shaped apothecia up to 5mm in width, smooth hymenium, uniseriate, amyloid and cylindric asci containing eightspores, septate, unbranched and filiform paraphyses slightly thickening at the tips, smooth, hyaline, ellipsoid to allantoid spores sometimes with a single central septum (Hansen and Knudsen, 2000).
R. elatina may be confused with Rutstroemia bulgarioides (P. Karst.) P. Karst. because of their similar color and macroscopic appearance. Though both species have dark olive brown to black, goblet to cup shaped and stipitate apothecia, the latter is separated from the former by its shorter asci (up to 100 μm long) and spores (up to 9 μm long). The most important characteristics of R. bulgarioides are to grow on damp spruce (Picea Link) cone lying on the ground between february and may, especially after the snow melts (Breitenbach and Kränzlin, 1984).
As the morphological data is not sufficient per se for the accurate identification of fungal species, the use of sequence data from the conserved DNA regions such as ITS is utilized as a useful tool in taxonomic studies for the last three decades (Baldwin, 1992;Raja et al., 2017). Additionally, ITS is one of the most common DNA barcoding markers and therefore serves as an important source of information for the researchers to make comparisons of data. Hence, we used ITS region for the molecular identification of the Akata 7020. The BLAST and phylogenetic analyses carried out based on the ITS regions revealed 100% genetic similarity between the Rutstroemia elatina and the new record (GenBank ID: MN263048) (Figure 3).
In this present study, R. elatina was firstly recorded from Turkey and species numbers of Turkish Rutstroemia increased to total of three. Additionally, after Abies alba and Picea abies, Abies nordmanniana subsp. equi-trojani was also reported as a new host species for R. elatina. Figure 3. The neighbor joining tree demonstrating the phylogenetic relationships of 11 fungi inferred from the nuclear ITS region. Bootstrap values from 1000 bootstrap replicates were given next to the branches.
All sequences were obtained from GenBank except for Akata 7020. Gyromitra esculenta and Morchella angusticeps were used as the outgroup samples. Accession numbers are given in parentheses. The scale bar shown at the lower left indicates a genetic distance of 0.05.