Two New Additions to Turkish Tulostoma

The purpose of the present work is to identify Tulostoma samples collected from Ankara and Kırıkkale provinces (Turkey). Both traditional methods and ITS rDNA-based molecular phylogeny were implemented to identify the specimens. When the high sequence similarities were taken into account, the collected specimens ANK Akata & Altuntaş 647 and ANK Akata & Altuntaş 675 were identified as T. simulans and T. subsquamosum respectively; and the morphological data further supported these findings. Short descriptions of the species are given together with their macro-and micromorphology and spore images taken by a scanning electron microscope (SEM). Research Article Article History Received : 12.12.2020 Accepted : 11.02.2021


INTRODUCTION
Tulostoma Pers., commonly known as stalked puffballs, is a large gasteroid genus containing approximately one hundred and seventy currently existing species (www.indexfungorum.org; accessed 20 November 2020). Members of the genus are characterized by globose and stalked spore-sac opened by an apical mouth, double peridium, pulverulant gleba, globose to ovoid, smooth or ornamented basidiospores, simple or branched capillitium with septa. Members of the genus are cosmopolitan, which prefer primarily sandy and calcareous soil in temperate and tropical regions (Pegler et al.,1995;Calonge, 1998).
Four species T. fimbriatum, T. laceratum (syn. Schizostoma lacerata), T. tortuosum, and viz. T. mammosum (including T. squamosum) were described by Fries (1829). However, Fries (1829) subsequently renamed Tulostoma with Tulasnodea, for the honor of the Tulasne brothers, who were his mycologist colleagues from France. However, considering the nomenclature rules, "Tulostoma" coined by Persoon should be regarded as the legitimate name for this typical genus (Persoon, 1801 Wright (1987), included 139 species. Wright's species concept mostly relied on studies of type specimens and various herbarium samples. With the advancements in scanning electron microscopy (SEM), fine spore morphology could be employed as a guide for the taxonomic revisions and appendance of new species.
The inclusion of the molecular analysis resulted in fundamental changes in the fungus systematics and taxonomy. The order Agaricales was established to include basidiomycetous gasteroid fungi including the puffball genus Tulostoma by Hibbett et al. (1997) Although a considerable amount of morphological data concerning the genus Tulostoma have been accumulated, relatively less molecular phylogenetic studies related to this genus have been reported. These published studies mostly focus on a low number of species and they are geographically restricted (Jeppson et al., 2017).

Determination of the ITS rDNA Sequences
For the genomic DNA extraction from ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675, CTAB method was implemented as described previously (Rogers and Bendich, 1994). For the quality and quantity measurements, the isolated genomic DNA was spectrophotometrically (Nanodrop Lite Thermo Scientific) analyzed, and later it was utilized as the template in order to amplify the Internal Transcribed Spacer (ITS) rDNA regions via the polymerase chain reaction (PCR) method. By using the ITS1 forward and ITS4 reverse universal oligonucleotides, PCR amplification of the ITS rDNA regions was implemented as described elsewhere (Stielow et al., 2015). The presence of the amplification products was electrophoretically verified on an agarose gel and then they were purified with Expin Gel, PCR, and CleanUp SV Kit (GeneAll) and sequenced with Sanger dideoxy sequencing method. For the sequencing PCR conducted using the BigDye™ Direct Cycle Sequencing Kit (Thermo Fisher Scientific), the same ITS1 and ITS4 oligonucleotides were employed and the fragment analyses were carried out by using ABI Prism 3130 Genetic Analyzer. Agarose gel electrophoresis and the Sanger sequencing were conducted as described previously (Chen et al., 2014).

Molecular Phylogeny Study
DNAMAN Version 10 sequence assembly software (Lynnon Corporation) was used to assemble the sanger reads obtained from ITS1 and ITS4 primers were assembled and BLASTn search was conducted with the assembled sequence for the identity index analysis. Based on the results of the BLAST search, the in-group and the out-group sequences were retrieved from NCBI GenBank and used in the phylogenetic analysis (Table 1) (Pawlik et al., 2015;Jeppson et al., 2017). The ClustalW algorithm of MEGAX software was used to align the assembled sequences and the nucleotide sequences of the retrieved in-group and out-group members (Kumar et al., 2018). The phylogenetic tree exhibiting the evolutionary history of ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675 was predicted using the Maximum Likelihood method and GTR+G+I nucleotide substitution model (Nei and Kumar, 2000). The bootstrap method was selected for improving the accuracy of the estimation using 1000 bootstrap replicates (Felsenstein, 1985). Remarks: Although it is difficult to distinguish T. simulans and T. brumale in detail by conventional methods, the exoperidial characteristic, spore dimensions, and the presence of crystals may help to separate these species (Jeppson et al., 2017;Rusevska et al., 2019).
Distribution: Asia, Europe, North, and South America (Jeppson et al., 2017). Remarks: T. subsquamosum can be distinguished from other Tulostoma members by its verrucose-echinate spores (under LM) and hyphal exoperidium with scattered sphaerocyst-like cells (Rusevska et al., 2019) The nuclear ITS rDNA sequences of ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675 obtained from Sanger dideoxy sequencing were deposited into NCBI GenBank with the accession numbers MT798590.1 and MT798591.1 respectively. In phylogenetic analyses of ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675, 13 different nuclear ITS rDNA sequences belonging to 11 different Tulostoma species were retrieved from NCBI GenBank database and used as in-group sequences in the phylogenetic analysis. As the out-group sequence, nuclear ITS rDNA sequences of Coprinus comatus (O.F. Müll.) Pers. was selected.  As a result of the phylogenetic analysis, the specimens ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675 were clustered with species of T. simulans and T. subsquamosum respectively. On the other side, Coprinus comatus fell into a distinct branch separate from the Tulostoma species and formed an out-group as expected. The BLAST analysis implemented with the nuclear ITS rDNA sequence of ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675 revealed as high as 100% similarity rates between these specimens and different isolates of T. simulans and T. subsquamosum. The phylogenetic analyses conducted herein further solidified the close identity relationship of the specimens ANK Akata and KSU J. Agric Nat 24 (5) For the reliable identification of fungal taxa, conventional methods employing morphological data may not always sufficient per se. Hence, conserved regions of genomic DNA including nrITS, nrSSU, and nrLSU as well as sequences of protein-coding genes are benefited for molecular taxonomic studies for decades (Raja et al., 2017). Furthermore, ITS is the most generally used DNA barcoding marker for fungi and this reason endows valuable information for molecular phylogenetic studies. Thus, we benefited from nuclear ITS rDNA sequences for the molecular identification of the specimens ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675. Figure 3. The Maximum Likelihood tree demonstrating the phylogenetic relationships of 39 fungi predicted from the nuclear ITS rDNA region. Percentage bootstrap values that are more than 50 were stated for each branch. All of the sequences included in the phylogenetic analysis were retrieved from GenBank except for specimens ANK Akata and Altuntaş 647 and ANK Akata and Altuntaş 675. Nuclear ITS rDNA sequences of Coprinus comatus was included as the outgroup sequence in the phylogenetic analysis.
GenBank accession numbers are also stated. The scale bar at the lower left shows a genetic distance of 0.02.