New Record of Macrofungi for the Mycobiota of the Cieszyn Municipality (Polish Western Carpathians) Including New Species to Poland

In this paper, we present the results of mycological research carried out between 2015 and 2018 in the Cieszyn township, in the Silesian Foothills (Outer Western Carpathians). The list of 417 species of macrofungi from the Cieszyn area reported in our previous study, has been expanded further by the addition of 37 taxa found in the current study. Among these, the following deserve special attention: fungi that are new to Poland’s mycobiota (six species: Bryoscyphus dicrani, Discina martinii, Elaphomyces aculeatus, Tuber brumale, T. foetidum, and Russula cerea), taxa subject to legal protection (four species: Disciotis venosa, Grifola frondosa, Mitrophora semilibera, and Sparassis brevipes), as well as fungi that are rare in Poland, included in national or regional red lists, and in the registers of rare and endangered species (24 species including Amanita echinocephala, Arrhenia retiruga, A. spathulata, Catinella olivacea, Elaphomyces maculatus, Hygrophorus discoxanthus, Ophiocordyceps entomorrhiza, Pluteus diettrichii, Tuber aestivum, and T. fulgens). This paper presents the distribution and location of 32 species of fungi along with a short description and illustration of the macroand micromorphological features of select species and their habitats.


Introduction
Information about the various species of macrofungi that exist in the Cieszyn township should be considered relatively comprehensive. Between 2013 and 2015, the authors of this paper carried out extensive research in the area, which resulted in the inventarization of 417 species of fungi that grow in the administrative limits of the municipality (Chachuła et al., 2015). As a result of continued mycological exploration of the area in the following years, additional 37 species of fungi have been found. Among these are fungi that are protected and rare in Poland as well as species hitherto not reported in Poland. The article presents detailed description about their localities and ecology.
Microscopic slides were prepared from the dried fruiting bodies. For the new species of the Polish mycobiota, the measurements were made using 30 samples (n = 30) for each characteristic structure (including spores, spines, asci, and cystyds). Spores were measured in side view, and without ornamentation and hilar appendix. Melzer's reagent, Congo red solution (in ammonia), and cotton blue solution (in water) were used for staining for observation of the micromorphological characters (Clemençon, 2009). Identification of the species was made according to mycological keys (Hansen & Knudsen, 2000;Knudsen & Vesterholt, 2008;Montecchi & Sarasini, 2000). Nomenclature of the taxa followed the Index Fungorum (http://www.indexfungorum.org/). The specimen of Pluteus ephebeus was additionally identified using molecular methods at the Institute of Botany, Polish Academy of Sciences in Kraków. The species are presented in alphabetical order, according to the phyla Ascomycota and Basidiomycota. When a taxon is subject to species protection (Regulation of the Minister of Environment, 2014) or included in the red list of macrofungi endangered in Poland (Wojewoda & Ławrynowicz, 2006) and in Polish Carpathians (Wojewoda, 1991), a square bracket with appropriate labeling follows the species name. It should be noted that the information on the occurrence included in the GREJ register , literature database (Kujawa, 2018), and in the checklists (Chmiel, 2006;Wojewoda, 2003) were also considered.
The description of localities include information about the localization including ATPOL squares (Komsta, 2016), habitat conditions including substrate or potential ectomycorrhizal plant partners observed in their surroundings, the number of fruiting bodies found, the date of observation, and individuals that collected and identified the specimens. The herbarium materials were deposited at the Jagiellonian University Herbarium in Kraków (KRA), as well as in P. Chachuła's private collection (PCh). In the case of rare hypogeous fungi, the authors have chosen not to provide more detailed information about the site/location of the species to protect and conserve the species.
The municipality of Cieszyn is situated in the western part of the Silesian Voivodeship. It covers an area of ca. 2,861 ha. According to the physicogeographical division of Poland (Kondracki, 2002) the municipality of Cieszyn belongs to the Cieszyn Foothills, which is the westernmost part of the Silesian Foothills mezoregion, and in turn a part of the Western Beskidian Foothills in the Outer Western Carpathians.
Geologically, the area is located within two unit -the Podśląska Nappe (in small part) and the Cieszyn Nappe. The latter consists mainly of the lower and upper Cieszyn schists divided by layers of Cieszyn limestone. In these layers, teschenites -intrusions of igneous rock -can also be found. In the hilly areas, the rock waste of Cieszyn Nappe form silty clay, roughing clay, sand, gravel, and loess. In the river valleys, coarse-grained gravels covered with alluvial mud soil, minerals, and organic mules may be found (Kasprowska & Ciborowski, 2010).
Upland and hilly landscape dominates the topography of Cieszyn, 80% of which is covered by synanthropic systems (urban areas, farmlands, wastelands, and green areas). Relatively small areas, occupying less than 12% of the township, are overgrown with dispersed forest complexes and other wooded areas. In terms of composition, the forests comprise of pockets of fertile carpathian beech forests (Figure 2A), oak-hornbeam forests, riverside riparian ash-alder forests, and piedmont riparian ash forests. Less than 8% of the township area is occupied by meadows and pastures.

Results
During the mycological research conducted in the Cieszyn area between 2015 and 2018, 38 species of macrofungi were found, of which 37 had not been reported previously from the area. One species -Calvatia gigantea -was mentioned in the literature (Kujawa & Gierczyk, 2011), but its presence was not confirmed during the comprehensive mycological research carried out previously in this area between 2013 and 2015 (Chachuła et al., 2015). Of the 38 fungal species, 15 belonged to the phylum Ascomycota and 23 to Basidiomycota. Of special interest are taxa not reported in Poland (six species), fungi subject to legal protection (four species), as well as rare taxa included in national or regional red lists and in the registers of rare and endangered species.
Among the six taxa found in Cieszyn, and not mentioned in any prior publication from Poland, five species belong to Ascomycota -Bryoscyphus dicrani, Discina martinii, Elaphomyces aculeatus, Tuber brumale and T. foetidum, and one species belongs to hypogeous Basidiomycota -Russula cerea.
In addition, owing to continued research on the mycobiota of Cieszyn, four more protected species were observed within the city limits, including Disciotis venosa (subject to strict protection) and Mitrophora semilibera, Grifola frondosa, and Sparassis brevipes (under partial protection) (Regulation of the Minister of Environment, 2014). Thus, the number of species subject to legal protection and occurring in the administrative limits of the township increased to 10, including the species already reported from the area: Clavariadelphus pistillaris, Fistulina hepatica, Ganoderma lucidum, Hericium coralloides, Holwaya mucida, Trichoglossum hirsutum (Chachuła et al., 2015).
In the following section, we present a list of the 31 most interesting fungal species not previously reported from Cieszyn (and Calvatia gigantea), with their localities and details about their morphology and distribution. It should be noted that, due to their relatively frequent occurrence in Poland, six species are not included in the list, although they also belong in the group of 37 new species of mycobiota of Cieszyn, and these are: Agrocybe erebia (Fr.) Kühner ex Singe, Helvella acetabulum (L.) Quél., Hemipholiota populnea (Pers.) Bon., Mycena polygramma (Bull.) Gray, Pluteus romellii (Britzelm.) Lapl., and Strobilurus tenacellus (Pers.) Singer.
Abbreviations: * -new taxon for Polish mycobiota; GREJ -a species included in the GREJ register; RLP -species listed on red list of macrofungi in Poland with appropriate the category of threat (Ex -extinct and probably extinct; Eendangered; V -vulnerable; R -rare; I -indeterminate); RLK -species included in the red list of the macrofungi of the Polish Carpathians with appropriate conservation status (R -rare); §-species protection (SP -strict protection; PPpartial protection); phot. Localities and Habitat. Cieszyn, landslide area near Błogocka Street, on the periphery of "Lasek Miejski nad Puńcówką" nat. res., DG00, few groups of ascomata on Bryum argenteum and B. caespiticium mosses, cocreating initial habitats on geotextile surfaces lined on exposed surfaces due to the mass movements, 2017-12-26, leg. RR and MF det. RR (PCh 1/26122017).
Notes. Species thus far not reported from Poland. Occurs in Northern, Western, and Central Europe, but not common (Hansen & Knudsen, 2000;Kirk & Spooner, 1984;Knutsson, 2005). It is a parasite of moss. Its occurrence time is during the cold period of year, often from fall to spring.
Description of Examined Specimens. Apothecia brown, flat, round in outline wrinkled, 6 (7.5) cm in diameter, with short and veined stem. Outer layer clearly brighter from hymenium layer. Spores oval, 30-32 × 13-15 µm, usually with one central and two smaller oil drops on the sides and of the fingerlike 1.5 µm long appendages. Notes. In Poland, observed in small populations, mainly in the Polish Carpathian Mountains and their foothills, more rare in the Upper Silesia, Kuyavia, and Gdańsk Pomerania (Chmiel, 2006;Snowarski, 2018;Wojewoda, 2003). Notes. Species not reported before in Polish mycological literature (Chmiel, 2006;Kujawa, 2018;Ławrynowicz, 1988;Skirgiełło & Wosińska, 1963). For over a decade, it has been observed at another site in the Cieszyn Foothills -Tuł Mt area (RR -mat. unpb.). This hypogeous, ectomycorrhizal fungus is known from its few localities in western, central, and northern part of Europe, but some of the data is obsolete. It is considered as a relic species for deciduous and mixed forest with Corylus, Quercus, and Carpinus (Minter, 2013).
Description of Examined Specimens. Ascomata spherical, 1-4 cm in diameter, surrounded by a crust, consisting of closely interwoven hyphae, pink in color, strongly connected to the surface of the peridium. Peridium black and pink (if without crust), 1 mm thick, covered with acute pyramidal warts protruding above the surface of the crust. Gleba homogeneous powdery at maturity, blackbrown. Spores globose, 13.3-18 µm in diameter (average 15.6 µm), rusty brown, densely and evenly ornamented with small spines, 0.5-0.8 µm high. The macroand micromorphological features of examined specimens correspond to other descriptions published (Paz et al., 2017). Notes. Very rare species known from two sites in the country, not recorded for several years (Ławrynowicz, 1988;Schroeter, 1889;Skirgiełło & Wosińska, 1963).
Description of Examined Specimens. Ascomata spherical, 1-3 cm in diameter, sometimes deformed. Usually occurs in groups in the subsurface layer of soil. Ascomata are surrounded by loosely adherent mycelium, with a characteristic graygreen color. This fungus is characterized by black, suede peridium and relatively large spores (28-42 µm in diameter) (Paz et al., 2017). Notes. Apothecia round, flat or slightly concave, 1-3 mm in diameter, without stipe. Coloring of apothecia ranges from yellow to orange. Ascomata occure singly and grow directly from moss. Octospora leucoloma is a parasitic species, growing on Bryum mosses which are widespread in Poland (Chmiel, 2006). Notes. Relatively rare taxon in Poland from the Cordycipitaceae family, parasitizing mainly on beetles from the Carabidae family and also on the Hemiptera of the Nabidae family. Typical stromata develop on beetles located in the subsurface layer of soil, several cm layer of soil. Perithecia of O. entomorrhiza develop on stromata. Observed on atrophied beetles in conidial form, also known under the synonymous name Tilachlidiopsis nigra (Bałazy, 1982). In Cieszyn, the perfect stages and conidial forms were observed developing on larvae and imago beetles of the Carabus (mainly Carabus violaceus). Notes. Very rarely recorded fungus, until 2006 considered extinct (Wojewoda & Ławrynowicz, 2006). Few sites concentrated in the south Polish highlands belt (Ławrynowicz et al., 2008).
Description of Examined Specimens. Ascomata irregularly globose, often with hollow base or slightly deformed, with diameter 2-10 cm, recessed in the subsurface layer of soil. The outer surface of asomata is covered with conical, black, polygonal, furrowed warts 3-5 mm in diameter. Gleba brownish with a white-veined labyrinth. Mature ascomata have intense odor. Spores elliptic, surrounded by openwork regular reticulum are usually gathered in ascus (two-six spores). Notes. Not reported from Poland yet. Tuber brumale ist a fairly common in the Europe (Montecchi & Sarasini, 2000). The red-brown color in the grooves between the verrucae distinguishes T. brumale from the other black Tuber found in Poland.
Description of Examined Specimens. Ascomata irregularely oval, 1-2 cm in diameter. Surfce black, covered with four-six angled, distinct warts, between which reddish colored fragments of the peridium were visible. Gleba light to dark brown, depending on the maturity of ascomata with white veins. Odor fruity, intense at maturity. Spores ellipsoid, 23.3-34 × 15.3-23.3 µm, Q = 1.5 on average, covered with long-spiny ornamentation, 2-5 µm long (up to 6 um long in the case of spores formed singly in ascus). Notes. Species of so-called white truffle, relatively rare reported from Europe (Montecchi & Sarasini, 2000), not previously reported from Poland, but observed in the Stołowe Mountains National Park (PC -mat. unpb.). This species among the other truffles from the Tuber puberulum group distinguished by the morphology of peridium surface -brown plates on a yellowish background, and by pseudoparenchymatus peridium structure consisting of round cells (Montecchi & Sarasini, 2000;Orczán et al., 2010). There is a light gleba in young ascomata, which becomes darker as it matures, becoming russet-brown with white veins. Fungus gives off an unpleasant odor, these traits also distinguish T. foetidum from another similar Tuber.

Auricularia mesenterica (Dicks.) Pers. [RLP-R, RLK-R, GREJ]
Localities and Habitat. Cieszyn, "Lasek Miejski nad Puńcówką" nat. res., DG00, on a cut stump of Ulmus sp., aggregation of many, overgrown basidiomata, 2017-04-21, leg. AD and MF, det. PC. Notes. Species moderately widespread in Poland, but relatively rare reported in the Carpathians. Found once in Cieszyn (Kujawa & Gierczyk, 2011), but not confirmed during the mycological research carried out in this area in 2013-2015 (Chachuła et al., 2015). Notes. Spring fungus, reported from Świętokrzyskie Mts and from the Kurnik Arboretum (Łuszczyński, 2007;Rudawska & Leski, 2013). Notes. Species not often observed in the country. Of the other Hebeloma, it is distinguished by a elongated root-like stipe base, occurence of ring on stipe and a very strong odor, resembling marzipan or bitter almonds (rarely described like a smell of fennel or prussic acid) (Halama et al., 2017). Documented are a few cases of the correlation between the presence of the fungus with the presence of underground nests of mice, moles, voles, or badgers (Sagara et al., 2006(Sagara et al., , 2008. Notes. The species was recorded so far only in the Tatra National Park, Bieszczady Mts, and Przemyśl Foothills (Gierczyk et al., , 2019Ronikier, 2012). Notes. Species previously reported from Poland only from Bieszczady Mts (Gierczyk et al., 2019), although in view of taxonomic revision published in 2016 (Paz et al., 2016), it is justified to verify specimens collected in the country, identified as Octaviania asterosperma Vittad, because in Poland occur only O. mutabilis and O. lutea. The basic differences between the two species concern on color of peridium: O. lutea -peridium usually thick, colored grayish white or yellowish, with faint spots bluish or greenish that, with manipulation, hardly redden Finally it is yellow ochraceous covered with blackish-gray fibrils. Peridium meat that does not redden to the cut; O. mutabilis -peridium generally thin, initially pure white with light bluish or greenish spots, which oxidizes quickly with vinous red manipulation and, more slowly, to blackish-gray. Peridium meat from pink color to the cut (Paz et al., 2016).
Description of Examined Specimens. Basidiomata irregularly spherical, up to 1.8 cm in diameter, initially white, browning as they mature. Hymenophore loculate, with white-cream color, brown in mature basidiomata. Spores subglobose, yellowish at light microscope, 9.3-11.3 µm in diameter, ornamentation of slender, conical, and cylindrical amyloid spines of lenght 1.3-2.3 µm. Notes. Species closely related with a similar species of the genus -S. spathulata. In a narrow view only individuals growing in Europe are included to the species (Petersen et al., 2015;Wang et al., 2004).

Discussion
Continued research on the mycobiota of the Cieszyn has contributed significantly to enhancement of knowledge and information about the regional mycological biodiversity, including rare or previously unreported taxa in the country. The discovery of 37 more fungal species in the Cieszyn area has increased the total number of mycobiota species studied area to 454 taxa (Chachuła et al., 2015).   Among them, 99 species belong to the phylum Ascomycota, 350 to Basidiomycota, and five to Mucoromycota.
The number of valuable species in the supraregional categories (protected, rare, and endangered) increased to 127 taxa. Among them are 13 species of fungi whose first national reports come from Cieszyn. Finding additional four protected fungal species has increased the total number of legally protected taxa to 10 (one with strict protection and nine with partial) (Regulation of the Minister of Environment, 2014). As a result of the comprehensive research of mycobiota of Cieszyn, the number of macrofungi taxa that figure in the red list of macrofungi in Poland (Wojewoda & Ławrynowicz, 2006) that were observed in the township increased by 14 taxa and currently includes 82 such fungal species in Cieszyn (Ex -1, E -13, V -21, R -44, I -3).
Another interesting aspect of the study that merits discussion is the relatively large number of hypogeous fungi that were found despite the small size of the forest area and the fact they were found accidentally -15 hypogeous fungi were found within the administrative limits of Cieszyn, including four reported for the first time in Poland: Elaphomyces aculeatus, Tuber brumale, T. foetidum, and Russula cerea.
Despite the small size of the total area of the township analyzed (ca. 2,861 ha) and the relatively limited area that could be considered good for the development of mycobiota (nonurbanized) there were suitable microclimate, soil, and phytocoenosis conditions (mild-climate with high precipitation sum and higher average annual temperature, presence of limestone soil covered with deciduous forests) that supported the growth of such interesting and rarely recorded taxa in Europe, including: Amanita echinocephala, Elaphomyces aculeatus, E. maculatus, or T. brumale. These discoveries provide a reason of intensification of further mycological research in the remaining areas of the Cieszyn Foothills, which are abundant in various age-old complexes of beech, oak-hornbeam, and riparian forest growing in limestone soil. No such comprehensive mycological research has been conducted in the Cieszyn Foothills area, although several causative character publications have appeared in the recent times (Beczała, 2016;Chachuła et al., 2015Chachuła et al., , 2018Krotoski, 2009;Rutkowski et al., 2014). Previous observations and reports clearly indicate that the Cieszyn Foothills are one of the national centers of mycological biodiversity.
Also worth mentioning is the fact that majority of the rare and protected macrofungal species found in the Cieszyn township were found in different types of nature protection areas including "Lasek Miejski w Błogocicach" nature and landscape complex, "Cieszyńskie Pogórze" protected landscape area, "Kopce, " "Lasek Miejski nad Olza, " and "Lasek Miejski nad Puńcówką" natural reserve. The occurence of the protected fungal species increases the natural value of the nature protection areas, and also indicates a properly functioning system of nature protection in the Cieszyn township.