Fungi of the Wolin National Park – New Data on Macromycetes

The paper contains results of mycological examinations conducted in the Wolin National Park fromMay to October 2017, and data previously unpublished. Exploration was carried out using the route method in the whole Park, with particular emphasis on its western part. The paper includes 205 taxa (25 from Ascomycota and 180 from Basidiomycota), including 32 new ones for the Wolin National Park. Among the identified taxa, 17 were threatened. The endangered species (Category E) were represented by Aleurodiscus disciformis, Calcipostia guttulata, and Geastrum triplex, the vulnerable species (V) included Inocybe grammata, Inocutis rheades, and Xylobolus frustulatus, and the rare species (R) consisted of 10 taxa, including Helvella lacunosa, Gloeoporus taxicola,Mycena crocata, Plicaturopsis crispa, and Pseudomerulius aureus. Some species are known only from a few sites in Poland, e.g., Hohenbuehelia auriscalpium and C. guttulata. Currently, the number of macromycetes species known from the Wolin National Park is 508.


Introduction
Large protected areas, such as national parks, have become particular points of interest in the mycological explorations conducted in Poland in recent years. They are characterized by a great richness of mycobiota (Gierczyk et al., 2009;Halama & Romański, 2010;Karasiński et al., 2015;Kujawa et al., 2012Kujawa et al., , 2015Ławrynowicz, 2012;Ruszkiewicz-Michalska et al., 2015;Wojewoda et al., 2016). The specificity in the appearance of fruiting bodies means that subsequent years of research will still provide new data on macromycetes of the studied area (e.g., Gierczyk et al., 2017, Gierczyk, Szczepkowski, et al., 2019a, 2019bGrzesiak et al., 2017). This is also reflected in the case of the Wolin National Park (WNP). The first mention of macromycetes of the current area of the WNP dates back to the 1930s (Stier, 1939;Ulbrich, 1932). They consisted of several macrofungi species. A comprehensive study on macromycetes of the WNP was conducted by Lisiewska (1966), presenting the share of macroscopic fungi in plant communities -dunes (Elymo-Ammophiletum and Helichryso-Jasionetum), coastal pine forest (Empetro nigri-Pinetum), and mixed forests (Querco roboris-Pinetum), as well as beech forests (Galio odorati-Fagetum and Fago-Quercetum petraeae). At that time, a total of 283 species and 11 varieties and forms of macrofungi were found. It was only after 50 years that another study was published on the biota of macrofungi occurring in the forest communities of the Park (Stasińska & Sotek, 2016). The study mainly covered the areas of strict protection, within which well-developed patches of Cephalanthero rubrae-Fagetum, Galio odorati-Fagetum, Luzulo pilosae-Fagetum, and Fago-Quercetum petraeae complexes have been preserved. It constitutes a significant contribution to the knowledge on mycobiota of the WNP, since the study caused the number of fungi taxa recorded in this area to increase to 476. However, they did not fully exhaust the existing richness of mycobiota in this area. This is evidenced by continued research, which has resulted in the discovery of many species that were not previously recorded. This study is treated as the second part of the article "New data to the knowledge of macrofungi of Wolin National Park, " and presents new data supplementing the knowledge on the diversity of macrofungi in the Park.

Material and Methods
The location and physiographic and floristic characteristics of the WNP were presented in the article by Stasińska and Sotek (2016), which is the first part of the research on macromycetes in this area. Currently, the list of macrofungi species contains data from field studies conducted from May to October 2017 and earlier data that were not included in the first article. The list includes only new, unpublished locations of fungi species. The study was conducted using the route method in the whole Park, with particular emphasis on its western part.
Due to the significant mutual similarity of Russula sardonia and R. xerampelina basidiomes and the high variability of their morphological structure, molecular methods were used to identify these species. DNA was extracted using the GeneMATRIX Plant & Fungi DNA Purification Kit (EURx, Poland). Each sample consisted of a dry fragment of basidiocarp. DNA samples were analyzed based on PCR amplification (with primers ITS-1F and ITS-4 and a Type-it Microsatellite PCR Kit; Qiagen, Germany) and sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) (Gardes & Bruns, 1993;White et al., 1990). Amplification was confirmed using gel electrophoresis. The PCR products obtained were sequenced using an ABI Prism 3130XL Analyzer (Applied Biosystems, USA) sequencer with ITS-1F/ITS-4 primers, in the Laboratory of Molecular Biology at the Adam Mickiewicz University in Poznań (Poland). The consensus sequence was created and unclear readings were corrected manually using BioEdit 7.2. The sequences were compared to the GenBank and UNITE databases using BLAST search (Altschul et al., 1990).
The specimens were identified by examining macroscopic and microscopic features, using standard methods of studying macrofungi, and monographs by Aronsen and Laessøe (2016), Bernicchia and Gorjón (2010), Breitenbach and Kräzlin (1984, 1986, 1991, 1995, 2000, Knudsen and Vesterholt (2012), Kränzlin (2005), Romagnesi (1996), and Stangl (1989). The fungal nomenclature and the synonyms were given according to the Index Fungorum database (http://www.indexfungorum.org/). The names of vascular plants in the present paper follows the description by Mirek et al. (2002), and the names of the plant communities follows the description by Matuszkiewicz (2006). The identified specimens were deposited in the Herbarium of the University of Szczecin (SZUB-F), Poland.
More than half of the macromycetes included in this study were recorded outside well-developed patches of plant communities, while 89 species were found in Luzulo pilosae-Fagetum, and only one species in Galio odorati-Fagetum and Fago-Quercetum petraeae. Taking into account the substrate on which the fungi grew, the group of saprotrophic wood decay fungi was the most abundant, as it was represented by 95 species. Among this group, macrofungi on deciduous trees dominated, i.e., 27 species were recorded on Fagus and 18 on Quercus. The share of macrofungi growing on coniferous wood was also noticeable, i.e., 22 species were found on Pinus. Thirty-two species of fungi were found on wood, whose taxonomic affiliations were difficult to determine. Terrestrial and litter saprotrophic fungi were not numerous groups, 25 and 19 species, respectively. On the other hand, mycorrhizal fungi were numerous -58 taxa, which constituted almost 1/3 of the species included in this study. Parasitic fungi were represented by nine species.
The macromycetes new to the Park include taxa that were rarely recorded in Poland, most often in well-preserved old stands under protection. In this group of fungi, Aleurodiscus disciformis and Calcipostia guttulata attracted special attention, and were included in the endangered category (E) of the "Red list of the macrofungi in Poland" (Wojewoda & Ławrynowicz, 2006). Aleurodiscus disciformis was reported from only a few localities, including nature reserves "Trębaczew" and "Parkowe, " the Białowieża Primeval Forest (Wojewoda, 2003), and the Kashubian Landscape Park (Karasiński, 2016). This species is critically endangered (CR) in the Czech Republic (Holec & Beran, 2006) and is known mainly from the southern part of this country (Zíbarová, 2015). According to Bernicchia and Gorjón (2010), it has a wide range in Europe, including Denmark, France, Germany, Italy, Portugal, Slovakia, Sweden, and Ukraine. In Poland, C. guttulata is rarely found as the previous species, e.g., in the Beech Forest near Szczecin , the Białowieża Primeval Forest Szczepkowski et al., 2008), and the Kaczawskie Mts (Gierczyk et al., 2018). In neighboring Germany, on the other hand, it has been reported from a number of localities (Dämmrich et al., 2019). In Finland, until recently, it was red-listed in the category: near threatened (NT) and has now been downgraded to the category: least concern (LC) (Kotiranta et al., 2019). Other species of fungi newly recorded in the Park are also known from single or a few sites in Poland, such as Hohenbuehelia auriscalpium -from the Wigry National Park (Halama & Romański, 2010), the "Ostrzycki Las" reserve (Kujawa & Gierczyk, 2016), and the Bieszczady Mts , and Hohenbuehelia grisea -from Częstochowa Upland (Adamczyk, 2011), the Kampinos National Park (Karasiński et al., 2015), and the Bieszczady Mts .
A large number of the saprotrophic wood decay fungi (95 species) resulted from a significant accumulation of substrate in the form of decaying logs and trunks, which were left to naturally decay, and have become suitable habitats for the development of this group of organisms. The share of mycorrhizal fungi (58 taxa; 28.3%) in the studied western part of the Park indicates the good health condition of the stand. Moreover, few parasitic fungi (nine species; 4.4%), of which Phellinus pini was the most frequent (it grew on old pines), do not pose a threat to it. The significant species diversity of mycobiota of the WNP shown in the current and previous studies (Czubiński & Urbański, 1951;Dominik, 1957;Friedrich, 2011;Lisiewska, 1966;Ławrynowicz, 1983Ronikier, 2005;Skirgiełło, 1970;Stasińska & Sotek, 2016;Stier, 1939;Ulbrich, 1932;Wojewoda, 2002;Wojewoda et al., 2002), confirms the very high natural value of this area. The number of macrofungi (508 species) found in the WNP is almost 1/5 higher than the number of species recorded in two other national parks in Pomerania, the Słowiński National Park (429) (Bujakiewicz & Lisiewska, 1983) and the Drawa National Park (379) (Stefaniak, 2013, as cited in Karasiński et al., 2015). In terms of the number of identified taxa, the WNP is only slightly inferior to the Bory Tucholskie National Park (517) (Grzesiak et al., 2017). National parks located in other regions of Poland are much richer in species of fungi, e.g., the Białowieża National Park (1585) (Karasiński et al., 2010, as cited in Karasiński et al., 2015, and the Kampinos National Park (1,611) (Gierczyk, Szczepkowski, et al., 2019a). Differences in the number of species observed between the WNP and other national parks are related, inter alia, to the duration and intensity of mycological research, as well as to the size of objects, the diversity of ecosystems, and the plant communities.
The presented data only, to some extent, supplement the knowledge about macromycetes in the Park. Due to unfavorable weather conditions for macromycetes occurrence in recent years, their biology, and the lack of systematic mycological observations, the list of fungi species illustrating the richness of the WNP biota is still open.