Species of Odontia and Tomentella (Thelephorales, Basidiomycota) new to Dagestan, Russia

Aim. To obtain new data on the species diversity, phylogenetic structure, and ecological characteristics of thelephoroid fungi (Thelephorales, Basidiomycota) in the Republic of Dagestan. Material and Methods. Both micromorphological and molecular analyses were used for studying of the fungal specimens collected by the authors in 2018 ‐ 2019 in the Gunibsky and Magaramkentsky Districts of Dagestan. Additional speci ‐ mens from the Mycological Herbarium of the Komarov Botanical Institute of the Russian Academy of Sciences (LE) were studied. The ITS region of nrDNA was amplified with two pairs of primers, ITS1F/ITS4 and ITS5/ITS4. Results. Sixteen ITS sequences belonging to eight species were obtained from the studied material. Of them, 14 sequences clustered in the Tomentella clade and two sequences nested within the Odontia clade. Four species – Odontia duemmeri , Tomentella lapida , T. radiosa , T. terrestris – were registered for the first time for Dagestan. Detailed information on the specimens studied is pre ‐ sented. Species identification of Odontia fibrosa , Tomentella badia , T . ferruginea , and T . stuposa was confirmed by ITS nrDNA analysis. Conclusion. Data on the species richness of the genera Odontia and Tomentella in Dagestan is updated, and the species T. lilacinogrisea is excluded from the regional funga. To date the genera Odontia and Tomentella in the Republic of Dagestan are represented by three and fifteen species, respectively.


INTRODUCTION
Despite rather detailed scientific research on the flora and fauna of Dagestan, mycological studies have not been given due attention; they have been episodic and unsystematic in nature. In general, the data on the diversity of aphyllophoroid fungi are available for the protected natural territories [1][2][3][4]. This publication continues the series of works devoted to the inventory of species diversity and ecological characteristics of aphyllophoroid fungi in Dagestan [2][3][4][5][6][7][8], in particular of the genera Odontia and Tomentella [9].
Taking into account the widespread use of molecular techniques in mycology both to describe new taxa and to study the modern species composition of regional mycobiotas, we carried out a comparative study of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) from the specimens of the genera Odontia and Tomentella, which allowed us to identify species new to Dagestan.
The aim of the study was to obtain new data on the species diversity, phylogenetic structure, and ecological characteristics of thelephoroid fungi (Thelephorales, Basidiomycota) in Dagestan.

MATERIAL AND METHODS
Specimens of basidiomycetes were collected during a routine survey of forest ecosystems in the Gunibsky and Magaramkentsky Districts of the Republic of Dagestan within the protected areas: the Upper Gunib Nature Park and the Samursky National Park in May and September-October 2018-2019. Additionally, specimens stored in the Mycological Herbarium of the Komarov Botanical Institute of the Russian Academy of Sciences (LE) were studied. Microscopy-based identification of fungi as well as reexamination of herbarium specimens was done at magnifications up to ×1000 using LOMO Mikmed-6 optical microscope, Carl Zeiss AxioImager A1 microscope and a standard set of reagents (5% potassium hydroxide solution, Melzer's reagent).
DNA was extracted from small pieces of dried basidiocarps using the FitoSORB DNA extraction kit (Syntol, Russia) according to the manufacturer's instructions. PCR reactions were performed in 25 μL of reaction mixtures containing 5 μL of Fidelity Buffer (5X), 0.5 μL of KAPA HiFi HotStart DNA Polymerase, 0.75 μL of dNTPs, 0.5 μL of each PCR primer, 12.75 μL of deionized H 2 O, and 5 μL of template DNA. The ribosomal ITS1-5.8S-ITS2 region was amplified with two pairs of the primers: ITS1F and ITS4 or ITS5 and ITS4 [11; 12]. PCR products were visualized using agarose gel electrophoresis and GelRed staining, and subsequently purified with the Fermentas Genomic DNA Purification Kit (Thermo Fisher Scientific, Lithuania). Purified PCR products were sequenced on an ABI model 3130 Genetic Analyzer (Applied Biosystems, CA, USA). Raw data were edited and assembled in MEGA 6 [13]. Newly generated sequences were deposited in the GenBank. Additionally, 46 ITS sequences were retrieved from GenBank [14] and UNITE [15] ( Table 1). Sequences were aligned with the MAFFT version 7 web tool [16; 17] using the E-INS-1 option. Maximum Likelihood (ML) analysis was performed in the IQ-TREE Web Server [18] with 1000 ultrafast bootstrap replicates.

RESULTS AND DISCUSSION
The ITS dataset includes 16 newly generated sequences and 46 sequences of 19 species downloaded from public databases (GenBank, UNITE). The genus Odontia was used as an outgroup. The final ITS alignment contained 858 positions (including gaps). The ML tree is shown in Fig. 1. Our 16 sequenced specimens appeared in eight separate wellsupported clades, which correspond to different species. Among them, 14 sequences clustered in the clade comprised of Tomentella species and two sequences nested within the Odontia clade. The ITS nrDNA analysis confirmed the microscopybased taxonomic assignment of the specimens from the Republic of Dagestan belonging to three species from the genus Tomentella (T. badia, T. ferruginea, T. stuposa) and one species from the genus Odontia (O. fibrosa) [7][8][9][10].
The first finding of Tomentella badia from Dagestan was recorded on a fallen trunk of Juniperus oblonga from the Gunib Plateau (Gunibsky District) as a result of the special study of juniper-associated aphyllophoroid fungi [7]. This fungus was also collected from fallen trunks of Betula sp. in the same area by Sergey Volobuev and Aziz Ismailov in October 2018 and these specimens (LE 314772, LE 314775) were sequenced now ( Table 1).
The species Tomentella ferruginea was registered for Dagestan based on four specimens mentioned by U. Kõljalg [10], one specimen (LE F-332319) from the Samursky National Park (Magaramkentsky District) [8], and the specimen from the Gunib Plateau (Gunibsky District) collected from a fallen trunk of Betula sp. by Yuliya Ivanushenko in May 2019 (LE 314778). The last two specimens mentioned were sequenced in this study.
T. stuposa, one of the most common species of the genus Tomentella, was reported for Dagestan [10], but the specimens from the Gunib Plateau (Gunibsky District) collected from fallen trunks of Betula sp. by Sergey Volobuev and Aziz Ismailov in October 2018 (LE 314774, LE 314779) were sequenced and included in the phylogenetic analysis for the first time (Table 1, Fig. 1). The newly generated ITS sequence of Odontia fibrosa was obtained (Table 1) for the specimen from the Samursky National Park (Magaramkentsky District) [8], which is the second finding of the species besides the record in the Upper Gunib Nature Park (Gunibsky District) [9]. At the same time, four other species are new to Dagestan -Odontia duemmeri, Tomentella lapida, T. radiosa, and T. terrestris. Detailed annotations for specimens of these species and some taxonomic and distributional remarks are presented below. The second species of the Odontia genus, followed by O. fibrosa [9], which is reported for Dagestan. As stable isotope analyses showed, all representatives of Odontia possess a non-ectomycorrhizal lifestyle, but their nutrition differs from typical xylotrophic basidiomycetous fungi [19]. Micromorphology of O. duemmeri is carefully described and illustrated by E. Martini [20]. This species occurs both on deciduous (Quercus robur) and coniferous (Juniperus communis) trees, but it was not previously collected from the wood of birch. Our finding (Fig. 2) is the first one of the species on the Caucasus. This is a common species with a worldwide distribution. T. lapida is close to T. stuposa (Fig. 1), but it differs distinctly from the latter in its encrusted subicular hyphae and smaller basidiospores. At the same time, it was noted previously [10] that T. lapida is also close to T. lilacinogrisea. Apparently, the similarities in the incrustation of thick-walled and brown subicular hyphae as well as shape of basidiospores, which can be slightly globose in frontal and lateral face in both species, were taken into account. The main differences between T. lapida and T. lilacinogrisea are in the size of spores (6-7 μm in T. lilacinogrisea and 7.5-9.5 μm in T. lapida) and the diameter of subicular hyphae. Our specimen (LE F-332369) has a smaller size of spores (6.5-7(7.5) μm). In a previous microscopic study [8], the specimen was incorrectly identified as T. lilacinogrisea. Based on the molecular analysis and additional examination of the micromorphology, the specimen was redetermined as T. lapida. The species was known previously in the Caucasus from Russia (Krasnodar Territory) and Armenia [10]. A widespread species in the Caucasus, in particular, in its north-western part (Karachay-Cherkessia Republic, Krasnodar Territory) and Transcaucasia (Armenia, Azerbaijan, Georgia) [10]. This species grouped together with sequences of Thelephora terrestris Ehrh. in our phylogenetic tree (Fig. 1) that supports the nomenclature combination of Thelephora terrestris f. radiosa (P. Karst.) Zmitr. [21] for this taxon. This is a remarkable species in the genus Tomentella due to the size of its basidia, which are up to 15-20 μm in diameter. This species is widely distributed in the Caucasus and is known from Russia (Karachay-Cherkessia Republic, Krasnodar Territory) and Azerbaijan [10]. The basidiocarps of T. terrestris were found during this study not only on well-decayed wood but also on soil.

CONCLUSION
Based on morphological and molecular evidence, four species of thelephoroid basidiomycetes -Odontia duemmeri, Tomentella lapida, T. radiosa, T. terrestris -were recorded for the first time to Dagestan. The data on the species richness of the genera Odontia and Tomentella in this region are updated and the species T. lilacinogrisea is excluded from the regional funga. Currently, the genera Odontia and Tomentella in the Republic of Dagestan are represented by three and fifteen species, respectively.