Boletoid fungi (Boletaceae, Basidiomycota) of the Bidoup – Nui Ba National Park (Vietnam)

Data on the diversity of boletoid fungi of the Bidoup – Nui Ba National Park are presented here and analyzed. An annotated list of 13 species are published for the first time for the National Park and 8 for the first time for Vietnam (Porphyrellus nigropurpureus, Phylloporus pachycystidiatus, Ph. rubiginosus, Pulveroboletus brunneopunctatus, Strobilomyces brunneolepidotus, S. calidus, Veloporphyrellus pseudovelatus, Xerocomus subparvus). Among plant communities of the National Park the mountain evergreen coniferous-broad-leaved forests dominated by Fagaceae, Lauraceae and Magnoliaceae and with the participation of representatives of Podocarpaceae and Pinaceae are characterized by the highest diversity of boletoid fungi. The species are illustrated with color photographs. The nucleotide sequences obtained during the study were deposited in NCBI GenBank.


Introduction
Boletoid fungi due to well-developed mycelium and more or less large basidiomata play a significant role in tropical ecosystems, forming mycorrhizal associations with forest-forming tree species of more than 10 families, including Dipterocarpaceae, Fabaceae, Fagaceae, and Pinaceae, improving their nutrition and growth, increasing resistance to adverse environmental influences, as well as participating in the decomposition of organic matter (Thoen, Bâ, 1989;Sato et al., 2007;Becerra, Zak 2011). Many of them are important as a source of nutrition for humans and as potential producers of biologically active substances for medical purposes, what is very actual in recent years. The Southeast Asia region is the center of diversity for this group.
The aim of the present work was to study the diversity of boletoid fungi in the Bidoup -Nui Ba National Park, located in the central part of the Dalat Plateau. Large tracts of mountain and foothill forests have been preserved here. The study of the fungal diversity of typical tropical forests complements the information on the biota of macromycetes in Vietnam and may be useful in the development of measures for the conservation of these valuable nature areas and species inhabiting them.

Bidoup -Nui Ba National Park
Bidoup -Nui Ba National Park (VQG Bidoup Núi Bà) is located in the northwest of Lam Dong province, Lac Doung District, in the Central Highlands of Vietnam (Tây Nguyên), in the central part of the Dalat plateau between 12°00'-12°19'N and 108°21'-108°44'E, occupying an area of 648 km 2 . The park relief is dominated by mountains with heights of more than 1400 m a. s. l., the highest peaks exceed 2000 m -Bidoup (2287 m), Lang Bian (2167 m) and Hon Giao (2062 m). The hydrographic network of the National Park belongs to Krong-No (Krong Nô) River basin (flowing to Mekong (Mê Kông)) and Da Nhim River (flowing to Dong Nai (Đồng Nai)). Climate is tropical monsoon. The average annual temperature is 18 °C, in winter in the mountains there can be short-term frosts down to -0.1 °C. Average annual rainfall is about 1800 mm (Eames, 1995;Sourcebook…, 2004;Brodribb, Field, 2008).

Materials and methods
Collections were made in different types of tropical forests in Bidoup -Nui Ba National Park during 2010-2014 in course of the investigation of mycobiota of the Central Highlands (Tay Nguyen). Boletoid fungi in the territory of the National Park were collected in the following main biotopes: Mountain evergreen coniferous-broadleaved forests dominated by Fagaceae, Lauraceae and Magnoliaceae and with the participation of representatives of the Podocarpaceae and Pinaceae (1500-1700 m a. s. l.).
The collected specimens were examined using light and electron microscopy, as well as by analyzing DNA sequences (ITS and tef1a sites). Macromorphological features have been studied in fresh collections and later through detailed photographs taken in the field. Microscopical characters were studied with a light Zeiss Axioscope A1 microscope with AxioCam 1Cc 3 camera and program tools AxioVisionRel.4.6. (Carl Zeiss, Germany). SEM photos were also used for the identification of material. They have been made by L. A. Kartseva on a scanning analytical electron microscope JEOL JSM-6390LA of Core Facility Centre 'Cell and Molecular Technologies in Plant Boletoid fungi of Bidoup -Nui Ba National Park Science' of Komarov Botanical Institute and by A. V. Alexandrova (Lomonosov State University) on scanning analytical electron microscopes JSM-6380LA and Camscan-S2 (Cambridge Instruments, 1990). Basidiospores, basidia, and hymenial cystidia were observed in squash preparations of small parts of the tubes in 5 % KOH. The pileipellis was examined on a radial section of the pileus, the stipitipellis -on longitudinal slice of the stipe in 5 % KOH. Basidiospore dimensions are based on 20 measurements, whereas cystidia and basidia dimensions are based on observing at least 10 structures per collection.
The molecular study is based on a phylogenetic species recognition (Taylor et al., 2000). To assess the taxonomic status of the identified phylogenetic lineages and put forward species hypotheses, we relied on the accepted boundaries of the possible variability of the ITS1-5.8S-ITS2 and tef1α nDNA regions within the species (Petersen et al., 2008;Hughes et al., 2009).
Collections studied are kept in the Herbarium of the Komarov Botanical Institute (LE).

History of the studies on of boletoid fungi in the Bidoup -Nui Ba National Park
Information on the history of the investigation of macromycetes in the Central Highlands of Vietnam, including the Bidoup -Nui Ba National Park, is presented in the works of Morozova et al. (2012) and Pham et al. (2018).
The table below (Table 1) summarizes the published data on Boletaceae in Bidoup -Nui Ba National Park, including species described as new to science from this area at different times.
A series of works on the diversity of macromycetes of the Central Highlands, containing information on 24 species of boletoid fungi, belongs to Lê Bá Dũng (Le, 2001(Le, , 2003. However, these works do not indicate the exact locations of the fungi, so we cannot consider these findings as collected in the territory of the Bidoup -Nui Ba National Park. It should be noted that previously species were identified on the basis of morphological and ecological characters only, and often for identification of paleotropic species European and North American keys were used. Species described from Madagascar also present in the list. In light of recent data on the geographical distribution of fungi, such determinations are questionable and require confirmation by the molecular methods. In this regard, we take into account only the species, which collections were confirmed by molecular data (including those determined only to the genus) and / or described directly from the territory of the National Park. Attempts to isolate DNA from old type specimens, unfortunately failed.

Results and discussion
Thirteen species not previously recorded for the Bidoup -Nui Ba National Park are listed below, including eight species published for the first time for Vietnam (marked with "!").     In addition, thanks to molecular genetic methods, 6 genotypes were revealed, which could not be attributed to any known species either by molecular or morphological data. There are: Austroboletus sp., Boletellus sp., Leccinellum sp., Phylloporus sp., and two species of Strobilomyces.
Thus, we can say about the presence of 25 species of Boletaceae family in the studied area, which were confirmed by the molecular data or were taken into account as species with a type locality in a National Park ( Table 2). The species are distributed between 14 genera, belonging to five subfamilies and the Pulveroboletus group.
Among plant communities of the National Park the mountain evergreen coniferous-broadleaved forests dominated by Fagaceae, Lauraceae and Magnoliaceae and with the participation of representatives of Podocarpaceae and Pinaceae are characterized by the highest diversity of boletoid fungi. The most favorable conditions for their growth have developed in these types of forest: the largest number of possible symbionts grows; the temperature and humidity indicators are optimal. In addition, the largest number of observations was carried out in these biotopes. In secondary pine and high mountain foggy and crooked forests the diversity of the boletoid fungi was found to be distinctly lower.
The available information on the nutritional properties of tropical boletes is very limited. We can only assume them, based on knowledge of the phylogenetic relationships in this group of fungi. Boletellus annamiticus and Crocinoboletus rufoaureus are most likely edible, as the very closely related species Boletellus emodensis and Crocinoboletus laetissimus are eaten (Laessøe et al., 2018;Wu et al., 2019). Zangia roseola considered edible (Wu et al., 2019). Aureoboletus viscosus and some species of the genus Strobilomyces regarded as potentially edible (Laessøe et al., 2018). Therefore, the related Aureoboletus longicollis and other species of the genus Strobilomyces can also be considered as potentially edible. It is known that Pulveroboletus brunneopunctatus, as well as other species of this genus, are poisonous (Chen et al., 2014, Laessøe et al., 2018. However, this question requires further study.