Biodiversity of ectomycorrhizal fungi in surface mine spoil restoration stands in Poland – first time recorded, rare, and red-listed species

Results of mycological research conducted in the years 2001–2013 in the restoration stands growing on reclaimed mine spoils are presented. Four opencast lignite mine spoil heaps in Poland were examined: Pątnów-Jóźwin, Adamów, Mt Kamieńsk, and Turów. The paper focuses on 71 species of ectomycorrhizal fungi: recorded for the first time in the country (16 taxa), currently red-listed (23 taxa), known from few localities only (32 taxa). Notes on their ecology and habitats are provided, as well as their distribution in Europe and in Poland. Restoration tree stands, established as part of the reclamation process of mine spoils, form a unique habitat for many rare and interesting fungal taxa. Among them are pioneer species, species known mainly from Northern Europe or mountainous locations, highly specialized and narrow-niche taxa, and many threatened species. Afforested mine spoils contribute significantly to the fungal biodiversity, both at a local and at a larger scale.


Introduction
The research on biodiversity of macrofungi in Poland has been most frequently carried out in the areas, which represent more or less natural or developed vegetation or habitats valuable for plant diversity and conservation [1].Often, they are mature or old-growth forest communities in protected areas -nature reserves, national parks, that are usually expected also to show high fungal species richness and the presence of rare and valuable species.Young, dynamic vegetation, especially if developing on disturbed grounds, has rarely been an object of macrofungal biodiversity observations, although, it may be unexpectedly rich in that respect, as has been shown for spontaneously established young forest communities on abandoned fields [2] and isolated woodland patches in agricultural landscape [3].Also, the restoration tree stands, established as part of the reclamation process of post-industrial areas, have been hardly studied in the context of fungal biodiversity.Fungi, especially ectomycorrhizal (ECM) symbionts, are key organisms enabling trees to colonize open land, survive in a harsh environment, and form a healthy forest community [4].Although young reclamation stands planted on mine spoils are not believed to support diversified fungal communities valuable in biodiversity or conservation respect [5], it has been shown that older forests on spoil heaps may not differ very much in terms of ECM community richness and diversity from the surrounding woods [5,6] or other mature forests [7].The fungal species composition may not be equivalent, however, afforested mine spoil heaps may form a unique habitat for many rare and interesting fungal taxa.To outline the diversity of ECM fungi in young restoration forests on reclaimed mine spoils, extensive research has been conducted on the four largest lignite mine spoil heaps in Poland (Central Europe).Here, we present the species recorded for the first time in the country, species red-listed as threatened and rare, and interesting species known from just a few localities.These records are not only a contribution to the knowledge of the distribution of these species in Poland and Europe but also help our understanding of their specific ecological requirements.
The heaps Pątnów-Jóźwin and Adamów are situated in the area of Konin lignite region [8] in the eastern part of the Wielkopolska Province.According to Kondracki [9], the former is situated in the mesoregion of Pojezierze Gnieźnieńskie within the Pojezierze Wielkopolsko-Kujawskie macroregion (on younger deposits of the Vistulian glaciation moraine), and the latter in the mesoregion of Wysoczyzna Turecka within the Nizina Południowowielkopolska macroregion (on older deposits of the Riss glaciation).The hydroclimatic conditions within this area are rather poor [10].Mean annual temperature is 9.2°C and shows an increasing trend in the last 30 years (1980-2012).The mean annual precipitation is 517 mm and is lower than the mean in the Wielkopolska region and in Poland.
Pątnów-Jóźwin spoil heap (52.322574°N, 18.212444° E) was built in the period from 1962 to the beginning of the 1980's, west of Kamienica village (north of Konin town) as an outer dumping ground for the overburden from the Pątnów opencast pit.Its altitude reaches 70 m above the neighboring land (ca.165 m a.s.l.), it covers an area of ca.340 ha and has a cubature of 133.6 mln m 3 [11].Its strongly diverse relief includes long mounded ridges and very steep slopes prone to water erosion, with numerous landslides, precipices, cracks, deep troughs and alluvial fans, as well as leveled plateaus and small, periodic or permanent water bodies [12].Non-selective methods of overburden deposition have been applied, resulting in high spatial diversity of superficial physical and chemical features: on the heap surface, mainly grey boulder clays rich in CaCO 3 (Riss glaciation), Neogene clays of the Poznań series, and Quaternary sands and gravels of different origin have been tipped.These materials, most often of neutral or calcareous character, are thought to form a bedrock for better soils than fluvio-glacial or fluvial materials.However, there are also sites where Neogene acid sands and materials containing toxic sulphur compounds or mobile aluminum are present [10,13,14].After the heap formation, the reclamation measures were limited and rather made to the final content.If the certificate is missing or invalid it is recommended to verify the article on the journal website.ineffective [12].Most of the heap has been afforested.At present, the stands are managed by the State Forests National Forest Holding (Kazimierz Forest District, Konin Inspectorate).Clays, sandy clays, and sandy clay loams prevailing on the heap surface, form the habitat for mesophilous (fresh) deciduous forest [15].The prevailing type of reclamation stands are poplar (Populus spp.) stands 28, 25, and 16 years old, at places mixed with willow (Salix spp.), black alder (Alnus glutinosa) and black locust (Robinia pseudoacacia), and pure black locust stands.Also, smaller fragments of pedunculate, sessile and northern red oak (Quercus robur, Q. petraea, and Q. rubra), beech (Fagus sylvatica), and European larch (Larix decidua) stands are present (trees 16-28 years old) as well as groups of sycamore maple (Acer pseudoplatanus), hornbeam (Carpinus betulus), common ash (Fraxinus excelsior), Scots pine (Pinus sylvestris), birch (Betula pendula), and lime trees (Tilia cordata).More humid places are covered with willow shrubs (Salix spp.).Adamów spoil heap (52.032322°N, 18.582870° E) was tipped in the period 1959-1970 north west of Warenka village (north east of Turek town) as an outer dumping ground of the Adamów opencast pit.It is a ca.38-m-high hill (ca.150 m a.s.l.) covering 318 ha, formed of 125 mln m 3 of the overburden [16,17].Non-selective methods of overburden deposition have been applied.The heap is formed of similar deposits to the Pątnów-Jóźwin heap, mainly Quaternary boulder clays, clayey sands, and other fluvio-glacial deposits as well as some Neogene Poznań clays [18].The slopes are intensively eroded and unstable, with numerous forms of landslide microrelief, including embankments, precipices, cracks, deep troughs, and alluvial fans.The landslides continue to happen today and are especially intensive in the areas devoid of trees.Most of the heap has been afforested.At present, the stands are managed by the State Forests National Forest Holding (Krwony Forest District, Turek Inspectorate).Potentially, the heap is a habitat of mesophilous (fresh) deciduous forest (on clays, sandy clays, and sandy clay loams) as well as of mesophilous (fresh) mixed deciduous forest (mainly on sandy loams) [15].The reclamation stands are variable and form a mosaic of different tree species, e.g., sessile oak (Quercus petraea), poplar (Populus spp.), birch (Betula pendula), black alder (Alnus glutinosa), black locust (Robinia pseudoacacia), Scots pine (Pinus sylvestris), European larch (Larix decidua), and spruce (Picea abies), with groups or single trees of beech (Fagus sylvatica), aspen (Populus tremula), willow (Salix spp.), Norway and sycamore maple (Acer platanoides and A. pseudoplatanus), common ash (Fraxinus excelsior), and lime (Tilia cordata) at different ages (16-45 years old).
Mt Kamieńsk spoil heap (51.215085°N, 19.428860° E) is situated west of Kamieńsk and south of Bełchatów town, in the Bełchatów lignite region [8], in the geographical mesoregion of Wysoczyzna Bełchatowska within Wzniesienia Południowomazowieckie macroregion [9].Interactions between polar maritime and continental air masses result in variable and transitional climatic features.The average annual temperature is 7.6°C and the average annual precipitation is 580-600 mm [19].Mt Kamieńsk was formed in the years 1977-1993 as an outer dumping ground of the Bełchatów opencast lignite mine.It is the second largest mine spoil heap in Poland and the highest hill in the central part of the country; its height ranges from 120 to 180 m (386 m a.s.l.) and it covers 1483 ha [19,20].According to some authors, the height of the heap reaches 195 m [11].The mine spoil comprises 1350 mln m 3 of various Quaternary and Tertiary overburden sediments covering the coal seam.Selective methods of overburden deposition have been applied, so that the surface spoil substrates are mainly of slightly alkaline sandy-clayey character (mixed sands, gravel, loams, silts, clays, boulder clays, sometimes with brown coal admixture).In some places, Neogene clays of Poznań series are present as well as phytotoxic acid sands, frequently with loads of geogenic carbon and sulphur compounds from Tertiary pyritic strata.In cases where such materials had not been isolated inside the heap, but were instead deposited near the spoil surface, extensive amounts of lime (lacustrine chalk from the open pit) and alkaline ash (from the nearby power plant) were applied to neutralize the high acidity.Top soil, humus, lignite, and charcoal were also used to enhance the soil substrate features.The reclamation procedure involved mineral fertilization (N and K -60 kg ha −1 , P -70 kg ha −1 ) and sowing mixed grass species and legumes (50-85 kg ha −1 ) [19][20][21][22][23][24][25].Reclamation works, aiming at forest restoration, started in the mid-eighties and were finished in 1994; 10 to 14 thousand tree seedlings per hectare were planted [25,26].Most of the reclaimed grounds have been handed over to the State Forests National Forest Holding (Adamów Forest District, Bełchatów Inspectorate).The most common tree species used for afforestation were Scots pine (Pinus sylvestris; 50%), birch (Betula pendula; 20%) and other deciduous species, e.g., black alder (Alnus glutinosa), black locust (Robinia pseudoacacia), pedunculate, sessile, and northern red oak (Quercus robur, Q. petraea, and Q. rubra), aspen (Populus tremula) and other poplar species (Populus spp.), different willow species (Salix spp.), Norway and sycamore maple (Acer platanoides and A. pseudoplatanus), common ash (Fraxinus excelsior), and European larch (Larix decidua) [20,27].The heap is a habitat of mesophilous (fresh) mixed coniferous forest [15].The reclamation stands are (10)15-30 years old, usually mixed in different proportions, composed mainly of clusters of Scots pine, birch, black locust, alder, larch, and poplar, with smaller patches of oak and single trees of various species.Accumulation of organic matter and formation of the soil horizons is very slow, even under the oldest stands [28,29].
Turów spoil heap (50.947965°N, 14.974363° E) is situated south of Zgorzelec town, between Działoszyn and Bogatynia, in the Kotlina Turoszowska basin, the southern part of the geographical mesoregion of Obniżenie Żytawsko-Zgorzeleckie within Pogórze Zachodniosudeckie macroregion [9].Kotlina Turoszowska is a tectonic depression filled with Tertiary (Miocene) lacustrine deposits with brown coal seams, which are part of the Western lignite region [8,9].According to data from the closest Polish (Zgorzelec) and German (Zittau-Hirschwelde) meteorological stations, mean annual temperature is 8.3 and 7.9°C, and mean annual precipitation reaches 706 and 716 mm, respectively.The climate is of mountain type [30].The spoil heap is an outer dumping ground of the Turów opencast lignite mine and it is the highest and largest object of this kind in Poland, 245 m high (465 m a.s.l.), with the surface of ca.2175 ha and cubature 1470 mln m 3 [30,31].Tertiary deposits comprise 90% of the overburden, with compact clays prevailing.Among them are kaolinitic clays with various proportions of sandy materials, mica and illite, and other sandy clays, sandy loams, sands and gravels, frequently carbonated and sulfurized with iron sulfides, e.g., pyrites, causing strong acidification.Quaternary deposits from surface strata consist of fluvio-glacial sands, gravels, and various loams as well as Holocene sands and gravels of river terraces.Also, slightly acidic, low-Ca ash from the Turów power station and the products of dry flue gas desulphurization were mixed with the overburden and deposited in the years 1972-1999 and 1994-1999, respectively [30].The heap has been continuously used for overburden deposition for over 60 years, which makes it the longest functioning outer spoil heap in the world.Its formation was eventually finished in 2006, although many landslides, faults and horizontal displacements of the hydrated clayey spoil occurred [31].The heap has been formed non-selectively.The surface materials are mainly clays (over 80%) with high proportion of fine fractions (up to 37%), and poor in N, P, K, and Ca [30].They are highly compact during drought and insufficiently aerated when wet [32].Most of them were strongly acidic (pH <3.5) before reclamation, sometimes showing phytotoxic effects due to sulphur compounds.Biological reclamation comprised surface neutralization of acid spoil (with a mixture of calcium oxide, dolomite and phosphate rock flour) followed by cultivation, then hydroseeding grasses and legumes (usually 35-65 kg ha −1 ) mixed with mineral or lignite-based mineral-organic fertilizer, and tree planting.Most frequently used species include birch (Betula pendula), black and grey alders (Alnus glutinosa and A. incana), European larch (Larix decidua), pedunculate and northern red oak (Quercus robur and Q. rubra), Norway and sycamore maple (Acer platanoides and A. pseudoplatanus), Scots and black pine (Pinus sylvestris and P. nigra), lime (Tilia cordata), hornbeam (Carpinus betulus), common ash (Fraxinus excelsior), and beech (Fagus sylvatica).Within the first years after outplanting, the stands were additionally fertilized with urea and potassium salts [30,33].The pH of the upper several centimeters of the spoil in the restoration stands of different ages varies between 3.8 and 6.2 in H 2 O, and between 3.4 and 5.6 in KCl and decreases towards deeper layers; the content of mobile aluminum is high, frequently exceeding 2.5 cmol kg −1 .Accumulation of organic matter is slow and becomes more visible in 12-16 years old stands [30].Afforestation of the heap, which had been carried out in older parts since the 1960s, was generally accomplished in 2008 [34].Ten thousand tree seedlings per hectare were usually outplanted [35].Most of the reclamation stands are currently managed by the State Forests National Forest Holding (Bogatynia Forest District, Pieńsk Inspectorate) or other State Treasure entities.The majority of the heap is a habitat of mixed deciduous upland forest [33].The reclamation stands are 4-50 years old, mostly consisting of alders and birch in different proportions with smaller clusters and single trees of other species.In some places, there are also some stands with prevailing black locust, Scots pine, larch, poplar or northern red oak, or with a higher proportion of aspen or willows [15].

Mycological surveys and methods of analysis
The observations were focused on sporophores of ectomycorrhizal (ECM) fungi forming symbiotic relationships with trees on the mine spoils examined.The species were regarded as ECM following Tedersoo and Smith [36] The surveys were conducted from spring (March) till late fall (November) including sporophore collections for documentation and identification purposes.Collected materials were studied with standard methods used in the taxonomy of macromycetes.They were macroscopically examined, dried and preserved as herbarium vouchers in the University of Łódź as part of the ILK collection (with letters IK and collection number).Analyses of microscopic characters and microscopic measurements were performed on preparations fixed in KOH or Melzer reagent under 400 or 1000 magnification using a light microscope.Species nomenclature follows Index Fungorum [37]; for some species frequently used synonyms are also given.
As most of the collections described in this study are difficult to identify morphologically and represent rare species, part of the material examined was subject to molecular analysis using the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes (nrDNA).ITS sequence data were generated based on PCR amplification with primer pair ITS1f or ITS5 and ITS4 [38,39] and direct sequencing of both strands from total DNA extracts of dried specimens.The extracts were prepared with GeneMATRIX Plant & Fungi DNA Purification Kit (EURx Ltd., Gdańsk, Poland) according to the manufacturer's instructions using small hymenophore portions.Further procedure followed Leski and Rudawska [40].The sequences obtained were manually edited and aligned using BioEdit Sequence Alignment Editor (7.2, 2013) [41].Selected sequences have been deposited in GenBank (Tab.S1).

Soil analyses
Thirty-two soil samples of the surface mineral layer (0-15 cm) from each of the heaps Pątnów-Jóźwin, Adamów, and Turów were collected in 2013, following fungal collection routes (96 samples).Similar soil samples from Mt Kamieńsk were collected in 2015, five from each of the 22 permanent plots mentioned under (B) above (115 samples).The soil pH in H 2 O (potentiometric method, according to the PN-ISO 10390:1997 standard) and the content of CaCO 3 (Scheibler method) were measured in each sample.Also, the soil texture in plots (B) was examined.Soil parameters of the plots mentioned under (A) above were published by Świtoniak et al. [28,29].

Results and comments
On the mine spoils examined, over 180 species of ectomycorrhizal fungi were identified (Kałucka, unpublished).Here, we present 71 species that we consider to be the most interesting.Among them are 16 species whose sporophores were recorded for the first time in Poland (marked with an asterisk " " in the list below), 23 species which are currently red-listed (#) [42], among which are two species that are protected by law in the country ( §) [43], and the other 32 species that are currently known from not more than 10 contemporary (recorded after 1970) localities in Poland and may be worth considering for conservation reasons, e.g., for red-listing.The numbers of localities of the species in Poland were estimated based mainly, but not exclusively, on the records listed by Wojewoda [44], and according to the data accessible at the Grzyby Polski (Fungi of Poland) website, namely, the records published after the year 2000 [45] and records included in the register of protected and threatened species (GREJ) [46].If a particular record is specified in the text, full citation is provided.The data on the occurrence of the species in Europe were retrieved and compiled from Knudsen and Vesterholt [47], Krieglsteiner [48,49], Legon et al. [50], Global Biodiversity Information Facility (GBIF) [51], and the UNITE database [52,53].
In the checklist below, up to three collections from each spoil heap examined are quoted.If not otherwise indicated, all the specimens mentioned were collected and determined by the first author (ILK).Color photographs of selected species collected in the study sites are provided (Fig. 2-Fig.6).
Soil characteristics are given whenever the specimens were collected in the sample plots or near the sites of soil sampling.The soil acidity was expressed using the terms described in the U.S. Department of Agriculture Soil Survey Manual [54].The results of soil analyses in the mine spoils examined are summarized in Tab. 1.

Ascomycota
Geopora arenosa (Fuckel) S. Ahmad (Fig. 5a) Commentary.The species is known from the loess ravine in the Kazimierz Landscape Park [55], Kampinos National Park [56], a few records on Mt Kamieńsk and a few records near the gravel pit in Ładzice commune [57].The present study showed that Geopora arenosa appeared in Mt Kamieńsk in thousands of ascomata.It was especially abundant in open sites on bare ground or among sparse vegetation, usually near pines and/or birches, and in young pine plantations until canopy closure (e.g., in 1000 m 2 of 5-year-old pine plantation on strongly alkaline calcareous sand, ca.14 400 ascomata were recorded over three seasons).Later, the species gradually disappeared and in stands older than 15 years could be found only occasionally (e.g., in 1000 m 2 of 14-year-old pine plantation on moderately alkaline calcareous loamy sand, 22 ascomata were found over three seasons, while no ascomata were found in older pine stands).It prefers alkaline (calcareous) soil and seems much less abundant on the acid substrates.The presence of mycorrhizas of Geopora arenosa on the roots of Scots pine was confirmed (Mleczko and Kałucka, unpublished).
Commentary.R. The species is widespread across Europe.

Commentary. R (as Helvella queletii).
The species is widespread across Europe.
Commentary.The species is known mainly from Northern Europe.Four records of this species were reported in Poland; one in the Karkonosze Mts (the Sudetes), two near Świebodzin (southwestern Poland) [58], and one near Krynki (northeastern Poland) [59].
Cortinarius diasemospermus Lamoure (Fig. 2d) Commentary.The species is common in Fennoscandia, single records from mountainous areas are known from France, Germany, and Slovakia.In Poland, the species was recorded in Kampinos National Park [56].It was found also as mycorrhiza on poplar roots near Kórnik (based on ITS) [60].On Mt Kamieńsk, it was found more than 10 times, usually as groups of sporophores.Commentary.The species is widespread across Europe.It was reported from Kampinos National Park [56].In our study, it was recorded fairly frequently in pine stands of different age.Commentary.The species was described for the first time in 2014 from Finland, and a locality in Canada was also confirmed [61].On Mt Kamieńsk, it was recorded several times in different stands, most often under birch.

Cortinarius murinascens
Cortinarius pseudofallax Carteret (Fig. 2f) Commentary.The species was described for the first time in 2004 from France [62].Its occurrence in Spain, Finland, Sweden, and Estonia was also confirmed [63].On the Turów spoil heap, the species was found a few times, but seems rather rare.
#Cortinarius saniosus (Fr.)Fr.Commentary.The species occurs mainly in Northern Europe.Only two records of this species were reported in Poland in Szklarska Poręba (the Sudetes) [58] and in Kampinos National Park [56].According to the species hypothesis (SH) concept [53], the specimens found on the Adamów spoil heap belong to the Cortinarius balaustinus complex (with at least 98.5% similarity) [64].However, the collections' characteristics, especially microscopic ones, match the description of C. subbalaustinus Rob. Henry and not C. balaustinus Fr. [47].Moreover, collections of C. subbalaustinus from Finland (JX407335) and Germany (AY669692) are included in this SH complex, as well.Taxonomical interpretation of C. subbalaustinus is uncertain and needs further studies.
Commentary.The species is widespread across Europe.In Poland, it was listed from Kampinos National Park [56], however, it was synonymized with C. erythrinus.According to the present knowledge, these are two different species [37,47], so the record should be verified.
Commentary.The species is one of the most common and widely distributed Hebeloma spp. in Europe and it was found also in a number of localities in Poland [56,65,69].It was recorded (based on the ITS sequence) also as mycorrhizas on the roots of beech seedlings from a nursery in Prudnik [70].
Commentary.The species is widespread across Europe and often locally common, especially in sandy habitats, under Pinus [65].It was reported in Poland from a few localities at the Baltic coast; Słowiński National Park [65,73], Białogóra reserve [65], and Wejherowo region [74].On Mt Kamieńsk, six collections of the species were recorded.One of them, IK-H0050 (HJB13069), was published by Beker et al. [65].
Commentary.The species is widespread in Europe, although it seems to be rare.Its presence in France, Denmark, Finland, Sweden, and Poland was confirmed by Beker et al. [65].The Polish collection cited in this monograph (IK-H0205, HJB14170) comes from Mt Kamieńsk and was collected in the same oak plantation as the specimen mentioned above.As the only record of H. danicum had been listed by Kaufmann [75] from the vicinity of Elbląg (north Poland), the species was considered extinct [76].More recently, Łuszczynski [77]  Commentary.The species is fairly common in Europe, it occurs in woodlands with Salicaceae, also in slag heaps [65].In Poland, it was reported from the Tatra Mts [78], Kampinos National Park [56], Mt Kamieńsk [65], and near Białystok (northeastern Poland) [79].Hebeloma xerophilum Rudn.-Jez.(published as H. xerophila [80]) described from the Kampinos National Park turned out to be H. dunense [65].In the Pątnów-Jóźwin and Mt Kamieńsk spoil heaps, the species was relatively frequent.
Hebeloma eburneum Malençon (Fig. 3c) Commentary.The species is widely distributed in Europe.However, it is very similar to the more common H. pseudofragilipes, both morphologically and molecularly (the ITS does not separate these two species), so at least part of the known collections of H. fragilipes are misidentified [81].In Poland, the species was reported from Jelonka reserve (northeastern Poland) [65].Also, the collection reported from the Jodły Łaskie reserve (central Poland) as H. testaceum by Kałucka [82]  Commentary.Macroscopically, the species may be confused with H. leucosarx and H. velutipes.Also, it is not always possible to distinguish between H. incarnatulum and H. velutipes based on ITS [71].The species seems widespread in Northern Europe, especially around the Baltic area, it occurs also in more southerly locations in subalpine areas [65].In Poland, it was reported from Słowiński National Park (Czerwona Szopa neighborhood) [65,71], further record from Sarbska Spit reserve is shown on the distribution map of H. incarnatulum European database collections (p.371 in [65]).
Commentary.The species is of rather Northern European distribution, with most eastern known localities in Poland [65].In Poland, it was reported from Mt Kamieńsk [65] from the same tree stand as the records published in the present paper Commentary.The species is widely distributed across Europe and locally very common in calcareous areas [65].Contemporary localities of this species in Poland include the Świętokrzyskie Mts [77], Cieszyn (the Silesian Foothills) [83], and Mt Kamieńsk [65].
Commentary.The species appears to be widespread across Europe, however, it must be rare and/or overlooked as only three records of this taxon have been confirmed so far, namely from England, France, and Greece [65,81].However, many other Hebeloma species had been wrongly originally determined as H. populinum.In Poland, this species has not been reported, so far.The record verified as probable H. populinum mentioned by Vesterholt [84] and later cited by Wojewoda [44], which was a part of the mixed collection described by Rudnicka-Jezierska [80]  Commentary.Although the species has been described only recently [81], it appears to be widespread across Europe and even more frequent than the very similar H. fragilipes.The Polish records shown on the distribution map of H. pseudofragilipes European database collections (p.306 in [65]) include the collections from Hel Spit (near Jastarnia, Baltic coast), Ojców National Park (two collections), and Jurassic Landscape Parks (two collections from Góra Zborów and Parkowe reserves).Commentary.The species seems to be widespread across Europe, although all the so-far known records originating from latitudes lower than 48° N. are alpine.It occurs almost always with Salicaceae in various, mostly Western European woodlands [65].Although it is different morphologically from H. cavipes, it cannot be separated from this species by ITS [65,81].The only two published Polish records come from the Świętokrzyskie Mts [77] and Kampinos National Park [56].All three collections found on the Turów spoil heap were recorded with Betula pendula, although the presence of a small, overlooked Salix or Populus in the vicinity cannot be excluded.Commentary.The species is known to form ectomycorrhiza with Larix and, in Europe, it occurs together with its host.It is known from less than 15 localities in Poland (always with larch), most of them represent mountainous habitats, nearly all are located in the southern part of the country.On the Turów spoil heap, it was recorded fairly frequently in relatively older larch stands.
Commentary.The species was described for the first time in 2002 from France [90].Until present, apart from the type specimen, only single collections of the species have been recorded from the Netherlands [91] and Croatia [92].
Commentary.The species was described for the first time in 2014 from Spain [93].Three collections were made in pine forests consisting of Pinus radiata, P. sylvestris, and P. pinaster, on acid soil and humus.One record of the species is known from Estonia, submitted as Inocybe sp.P156 to GenBank [94].The collection from Pątnów-Jóźwin spoil heap is the fifth known record of this species.
Commentary.The species seems to occur occasionally in Europe.It was reported from two localities in Poland; Lublin (east Poland) [95] and Kampinos National Park [56].On Mt Kamieńsk, it occurs frequently and was recorded in dozens of sporophores in most of the Scots pine stands examined.
Commentary.The species was described for the first time in 2011 from Finland and Sweden [96], where it was found on calcareous soil, in localities often close to limestone processing plants (chalk dust emission), in association with birch.Other records come from Svalbard and Estonia [97].Similarly to the populations found in Fennoscandia, those found in the spoil heaps examined were growing in abundant groups of sporophores.On Mt Kamieńsk, they were recorded fairly frequently, especially in birch and Scots pine stands.
Commentary.According to Stangl [98] and Index Fungorum [37], I. ochracea and I. alnea are two different species.However, Vauras and Kokkonen [99] claim that microscopical features of the type specimens of both species proved to be nearly identical, and the DNA analysis supported their conspecificity.Indeed, the sequence KX602264 (Tab.S1) representing the collection IK-00287 differs in one gap and one base pair from the holotype of I. ochracea (J755803) and differs in one gap from the two sequences from the holotype of I. alnea (FJ755801 and FJ755802).Both I. ochracea and I. alnea seem to be rare in Europe.Inocybe alnea was reported from Poland from different alder forests in the Babia Góra massif (the Western Carpathians) [100][101][102].
Commentary.The species is widespread and rather common in Europe.It was reported from six localities in Poland; in the Babia Góra massif [102], Białowieża Forest (two records [103,104]), Kampinos National Park [56], Biebrza National Park [87], and in Radomsko district [87].In the spoil heaps examined, it seems not to be rare.
Commentary.E (as I. jacobi).The species is fairly widespread in Europe, especially in the northern part, but occurs occasionally.It was reported from five contemporary localities in Poland, always in open or disturbed sites (e.g., meadows, coal mine dumps, road sides); Kampinos National Park (two records [56,105]), near Gliwice [106] and in Katowice [107] (the Upper Silesia), and near Krasny Las (northeastern Poland) [108].On Mt Kamieńsk, it was, fairly frequently, observed under pines.
Commentary.So far, few basidiomata of the species have been collected in Europe, only in Estonia and Germany [109], and in Finland (as I. auricoma) [110].In Poland, I. semifulva was also found as mycorrhiza on poplar roots (based on ITS sequence, as I. hirtella) [60].

Inocybe subporospora Kuyper
Specimens examined.MtK, in young Scots pine stands (strongly alkaline sand and moderately alkaline clay loam, 3.5% CaCO 3 ), soil profiles 5 and 7 in Świtoniak et al.Commentary.The species is widespread but occasional across Europe.It was recorded only from one locality in Poland in Kampinos National Park [56].According to GBIF [111], 40 occurrences of the species are known from Norway, Sweden, Denmark, Netherlands, Germany, Spain, and Portugal.However, only one ITS sequence from an identified sporophore is available (AM882931 [112]).On Mt Kamieńsk, fairly numerous sporophores were observed a number of times, always in young, semi-open pine stands.
Commentary.E. The species is widespread across Europe.It is not common in Poland, however, it seems to be not very rare either, having more than 15 known localities in the country.The species was found also on the Adamów spoil heap (Ada), in poplar stands (slightly alkaline to neutral soil, 0.25-7.75%CaCO 3 ), among deep litter and in the site of humid organic matter accumulation (groups of sporophores, 2012-10-09, 2013-09-12, 2013-09-22; no voucher collection).Commentary.The species is known mainly from Northern Europe.It has been recently recorded from two localities in Poland; Białowieża Forest [104] and Kampinos National Park [56].It is very similar to L. obscuratus (Lasch) Fr., which is not rare in Poland.The collections presented here show the features highlighted by Heilmann-Clausen et al. [120]; L. cyathuliformis forms relatively larger, more robust basidiomata which are usually tinted olivaceous, and has slightly larger spores than L. obscuratus.
Commentary.E. The species is widespread in Europe, although more frequent in its northern part.It has less than 10 known localities in Poland.

Lactarius mammosus
Fr. (Fig. 5f) Commentary.The species is widespread across Europe.It was reported from two localities in Poland, in young Scots pine stand on post-arable land near Białowieża [2] and in Roztocze region (southeastern Poland) [121].
Commentary.The species seems to be infrequent outside Fennoscandia.It was reported from less than 10 contemporary localities in Poland (also as L. roseofractum).

Commentary. R (as P. arhizos).
The species is widespread and not rare across Europe, its recent distribution and ecology was presented by Fraiture and Otto [124].On Mt Kamieńsk, the species was recorded several times, usually as groups of basidiomata, always in places that were rather acidic.A few records of this species from Mt Kamieńsk were also reported to GREJ by another collector [125].Pisolithus arhizus was also found on the Adamów spoil heap (Ada) under Quercus robur and Populus tremula in oak/birch stand, not far away from a Scots pine stand (2012-08-21; no voucher collection).Commentary.The species is widespread across Europe.It was reported from three contemporary localities in Poland (as R. depallens); Lasy Łochowskie n.Wyszków [128], on zinc wastes near Olkusz [129], and in the Świętokrzyskie Mts [77].On all the spoil heaps examined in the present study, the species was frequently recorded, especially on Mt Kamieńsk.Commentary.V.The species is widespread across Europe and, in some regions, rather common.§#Tricholoma aurantium (Schaeff.)Ricken (Fig. 6g) Specimens examined.MtK, in Scots pine stand (very strongly acid to strongly acid loamy sand), among pine litter, IK-00604 (2012-09-28).Ada, in mixed birch/spruce/ larch stand (moderately alkaline soil, 4.88% CaCO 3 ), among abundant litter, IK-00620 (2012-10-09).

Russula medullata
Commentary.P, R. The species is widespread in Europe.It was reported from four contemporary localities in Poland; Tatra Mts National Park [131], Jasieniowa Mt in the Cieszyn Foothils [132], Pieniny Mts National Park [133], and Kampinos National Park [56].Tricholoma aurantium is a species found usually on calcareous soil.On Mt Kamieńsk, the species was found in the plot where all five soil samples were acid, however, solitary calcareous spots cannot be excluded.
#Tricholoma equestre (L.) P. Kumm.Commentary.The species is known also as Alnicola umbrina and mainly under this name is known to form ectomycorrhizas on Alnus spp.roots [135].The species seems to be overlooked but widespread in Europe.

Conclusions
Spoil heaps are piles built of loosened, mixed, and aerated masses of overburden and other waste rock materials brought to the earth's surface from the mine pits.They are not contaminated with any chemical waste but usually show poor physical and chemical features.For reinstatement of utility and natural functions, they are reclaimed by means of technical and biological recultivation, and frequently, the final stage of this process is achieved by afforestation [4].The restoration forests planted on mine spoils are thought to be important especially for their soil protective functions, landscape improvement, and recreational goals.However, their ecological functions associated with increasing local and general biodiversity seem equally significant.
Early successional habitats typical of opencast mine spoils are generally infrequent in the European landscape and they provide unique sites for pioneer ECM fungal species, species associated with transitional, short-lived open and semi-open early stages of forest development, on mineral soil with low organic matter accumulation, or adapted to disturbed soil conditions, like Geopora arenosa, Cortinarius helobius, Hebeloma dunense, Inocybe rufoalba, I. subporospora, I. tjallingiorum, and Pisolithus arhizus.
Tab. 1 Acidity and CaCO3 content of the superficial soil on the reclaimed lignite mine spoil heaps Pątnów-Jóźwin, Adamów, Turów, and Mt Kamieńsk (in four stand types).Mine spoils are isolated hills of waste rock material and raw overburden that is not present on the surrounding earth surface for hundreds of kilometers.They may serve as a refuge for many rare fungi known from distant, especially northern or mountainous locations, from oligotrophic or severe habitats.Such species are represented, e.g., by Cortinarius diasemospermus, C. murinascens, Inocybe griseotarda, I. lanatopurpurea, I. myriadophylla, I. semifulva, Laccaria macrocystidiata, Lactarius mammosus, and Sistotrema confluens.

Spoil heap
As a result of mixed overburden deposition and using different amelioration measures and amendments (like chalk, ash, mineral fertilizers, or geogenic carbon), the mine spoil surface is composed of various materials, patchily distributed, exhibiting different physical and chemical properties.Usually, as in the case of spoil heaps examined in this study, an afforestation process takes years and restoration stands are of different age and also are composed of different tree species.This, in turn, results in exceptional diversification and heterogeneity of habitats and site conditions on a small scale, and in a high diversity of ECM fungi.In the spoil heaps examined, a large group of species associated with rich and/or calcareous soils can be clearly distinguished, like Hebeloma odoratissimum, H. laterinum, Inocybe myriadophylla, Russula exalbicans, Tricholoma aurantium, T. populinum.However, species typical of poor and sandy soils are also present, like Cortinarius depressus, Hebeloma cylindrosporum, Inocybe subporospora, Suillus cavipes, as well as species occurring on mostly acidic soils, like Gomphidius glutinosus, G. roseus, Hebeloma cavipes, Tricholoma focale.It is also possible that in accordance with the successional model of ECM fungi proposed by Jumpponen and Egerton-Warburton [136], strong environmental filters, e.g., extreme soil pH, very low or high nutrient level, infrequent soil texture and physical conditions, harsh microclimatic conditions, and their unusual combinations, may eliminate great part of the locally and regionally available competitive ECM species pool and this, in turn, enables successful colonization and existence of vast range of rare, less competitive, highly specialized and narrow-niche fungal taxa.
All the seventy-one species of ECM fungi collected on the lignite mine spoil heaps examined, presented in this paper, are the species recorded for the first time in Poland or currently red-listed, or extremely rarely recorded.The number is unexpectedly high.It is even more impressive, as it comprises more than one third of all ECM taxa identified during this study.These findings reveal very high potential of young restoration forests for hosting a diversity of ECM symbionts.Such habitats should not be neglected or ignored in fungal biodiversity studies or in conservation considerations.New data on the currently red-listed species [42] may suggest, at least in the case of some taxa, the need for re-evaluation of the threat category.Examples include Helvella lacunosa (R), Pisolithus arhizus (R), Thelephora caryophyllea (V), Tricholoma equestre (I), and T. populinum (V), which seem not to be rare or threatened at present.Also, high threat category (E) may be no longer adequate in the case of Inocybe glabripes, I. rufoalba, Lactarius controversus, and Tricholoma focale.At the same time, some species may be worth considering for including into the red list, like the group of northern taxa (Cortinarius murinascens, C. depressus, C. diasemospermus, C. subbalaustinus, Hebeloma incarnatulum, Inocybe myriadophylla, I. vulpinella, Lactarius cyathuliformis, L. mammosus, Leccinum melaneum), and taxa which seem to be rare and scattered in Europe (Cortinarius pseudofallax, Hebeloma danicum, H. odoratissimum, H. populinum, Inocybe griseotarda, I. lanatopurpurea, I. ochracea, I. semifulva, I. subporospora, Laccaria macrocystidiata, Rhizopogon mohelnensis).
The results of the present study show the importance of detailed and long-term mycological field observations and taxonomical studies, which, especially if combined with molecular analyses, can provide reliable and credible data for further studies on fungal ecology and distribution.The importance of field observations and morphological analysis is particularly important in the context of numerous studies based exclusively on molecularly identified OTUs and environmental samples, which sometimes automatically or in indiscriminate manner use the sequence data available in public databases and duplicate misidentification errors (compare [137,138]).
The species is widespread across Europe.It was reported from less than 15 localities in Poland.Both on Mt Kamieńsk and Turów spoil heap, it was recorded fairly frequently, usually as groups of sporophores.
[65] Eberh.&BekerSpecimensexamined.Ada, in pedunculate oak/birch stand, among grass and mosses, IK-H0360 (2013-09-17).MtK, in alder stand with Betula pendula nearby (neutral to slightly alkaline clay with 3.84-11.83%CaCO3),amonggrassandlitter, IK-H0245 (2012-09-27).The species was first described in 2016[71]with many localities widespread across Europe, usually on calcareous soil and under a variety of trees, most commonly Fagus and Quercus.It belongs to a complex of three very similar species (not distinguishable by ITS) with H. quercetorum and H. erebium.In Poland, it was reported from Białowieża National Park[65], with further localities shown on the distribution map of H. celatum European database collections (p.360 in[65]): Biebrza National Park (two records), and Lisi Jar gorge near Jastrzębia Góra (the Baltic coast).
[65,66]ns examined.P-J, in oak plantation with some birch, among mosses and grass, IK-H0324 (2012-10-20; det.I.L. Kałucka, H. J. Beker & U. Eberhardt); in beech plantation, with Salix sp. in vicinity (slightly alkaline soil, with ca.11% CaCO 3 ), on ± bare soil, IK-H0325 (2012-10-20; det.I.L. Kałucka, H. J. Beker & U. Eberhardt).Commentary.As pointed out by Vesterholt et al.[72], the name H. crustuliniforme was used at various times for several different medium to large taxa most often from the sections Denudata and Velutipes, at best it represented a crustuliniforme-complex of four closely related species.Finally, it has been tied to one of them and epitypified, excluding the three remaining species: H. aanenii, H. eburneum, and H. geminatum[65,66], with a recommendation that this taxon be referred to as H. crustuliniforme (Bull.)Quél.emend.Beker, Vesterh.& U. Eberh.The species is ubiquitous and widespread in Europe, although so-far known records seem to be more confined to Western and Southern Europe.The two collections found on Pątnów-Jóźwin spoil heap are the first records of this species in Poland and in Central Europe.
[65]entary.The species seems to be widespread although relatively uncommon in Europe[65].The first records of this species in Poland were signaled by Tadeusz Twardy in 2010 and 2012 (unpublished, personal communication, 2012), who found numerous basidiomata in two localities in the Ustronie Morskie Commune (Baltic coast), in shallow wet grassy depressions with thickets of different willow species, birch and alder.The localities in Poland are the most eastern ones already known in Europe.