Selected rare and protected macrofungi ( Agaricomycetes ) as bioindicators of communities of xerothermic vegetation in the Nida Basin

Results of mycological investigations conducted in xerothermic grasslands of the Nida Basin in the years 1984–2013 are presented. Our research brings up to date the current knowledge on ecological requirements of threatened species of macrofungi. Strong affiliations between macrofungi and specific phytocenoses of the alliances Festuco-Stipion and Seslerio-Festucion duriusculae were detected. A considerable influence of the occurrence of certain species of steppe fungi on the composition of the mycobiota in the Nida Basin was identified.


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association Tulostomato (brumali)-Gastrosporietum simplicis, which exhibits a strong relationship with habitats of xerothermophilic grasslands of the alliance Festuco-Stipion, is a good example of such a specialized mycoassociation.Species of the genera Tulostoma and Gastrosporium simplex are the main components of this mycoassociation [6][7][8].Due to high ecological specialization and a close relationship between fungi examined by us and specific plants of steppe origin, many of these species can be treated as important bioindicators of threatened and endangered habitat types of xerothermic vegetation in Poland [9,10].The occurrence of the species identified in our study indicates that such communities are persistent and have typically developed phytocoenoses.The bioindicator value of fungi in a variety of plant communities has been noted by, e.g., Kornaś [11], Bujakiewicz [12][13][14], Barkman [15], Kałucka [16].
Multidimensional methods were used in our study to assess changes in the species composition of fungi in selected xerothermic communities.Species defined in the literature as thermophilous, xerothermic and steppe fungi related to the communities of the class Festuco-Brometea [6,8] were chosen for statistical analyses.
Specific aims of the study are: (i) to analyze the occurrence of ten selected rare and protected species of fungi in chosen communities of xerothermic vegetation in the Nida Basin by correspondence analysis; (ii) to analyze in detail fruitbody productivity in xerothermic communities using statistical methods; (iii) to identify bioindicator species within fungi examined by us whose occurrence indicates an undisturbed type of communities of xerothermic vegetation.

Material and methods
The results presented in our study are based on investigations conducted in two stages.Six communities of xerothermic vegetation were examined: three of the alliance Cirsio-Brachypodion pinnati (Adonido-Brachypodietum pinnati, Inuletum ensifoliae, Seslerio-Scorzoneretum purpureae), two of the alliance Festuco-Stipion (Koelerio-Festucetum rupicolae, Sisymbrio-Stipetum capillatae) and one of the alliance Seslerio-Festucion duriusculae (Festucetum pallentis) [3].A total of 33 permanent research plots were established.Three research plots were established in the community Sisymbrio-Stipetum capillatae in the first stage of research, i.e., in the years 1984-2013.Thirty research plots, five in each of the six communities, were established in the years 2010-2013.Plot sizes were determined by the size of a homogenous vegetation patch and corresponded with the standards of an area of a phytosociological relevé used in these types of communities (from 30 m 2 to 150 m 2 ).Phytosociological relevés were performed with the commonly used Braun-Blanquet method at all permanent plots.Investigations were conducted regularly every two weeks after snow receded until a new snow cover appeared.In total, each plot was observed 15 times per year.All fruitbodies of selected gasteroid fungi were collected and counted each time during mycological observations.To avoid double counting of fruitbodies, all fruitbodies of fungi under consideration were collected.
Fungal fruitbodies were identified taxonomically in the laboratory.Mycological studies were used to determine the fungi: Pilát [17], Rudnicka-Jezierska [18], Sarasini [19], Wright [20].A light microscope, a scanning electron microscope and standard chemical reagents were used for analysis (10% KOH, IKI).Spores, basidia and capillitial threads whose size and shape are taxonomically important were measured with a light microscope.All microscopic characters were observed with an immersion lens.Examinations using a scanning electron microscope were performed in the Department of Environment Protection and Modelling, Jan Kochanowski University in Kielce, Poland.Sampled material (soil samples containing spores) was transferred on a special sticky aluminium disc and sputter coated with 24-carat gold.Spores were photographed at 3000, 5000, 10 000 and 12 000 × magnifications.

Statistical method
The indicator species analysis was conducted with the indicator value index (IndVal) [21,22].The IndVal is the product of two components called A and B. Quantity A is defined as the mean abundance of the species in the target site group divided by the sum of the mean abundance values over all groups.Quantity B is defined as the relative frequency of the occurrence (presence-absence) of the species inside the target site group.
Statistical significance of the relationship between the species and the site group was tested using a permutation test where the P-value <0.05 was considered significant.
The relationship between site groups and fungi species was also studied by correspondence analysis.
All computations were performed with package R, version 3.1.2(R Core Team; http://www.R-project.org) The nomenclature of fungi was accepted after Wojewoda [23] while that of plants after Mirek et al. [24].
Detailed data regarding the number of fruitbodies of individual species of fungi in selected plant communities is given in Tab. 1. Phytosociological features and a list of collected fruitbodies of the fungi examined in the study based on the community Sisymbrio-Stipetum capillatae are presented in Tab. 2. Tab. 3 contains the IndVal and its components.
Exiccata are deposited in the Fungarium (KTC) of the Faculty of Mathematics and Natural Sciences, Jan Kochanowski University in Kielce, Poland.

Xerothermic communities and fruitbody productivity: an analysis
The most favorable growth conditions for fruitbodies of fungi examined by us were noted in the community Festucetum pallentis.A total of 837 fruitbodies of all species Tab. 1 The number of fruitbodies of selected species of gasteroid fungi in communities of xerothermic vegetation.The diversity of fruitbody production of macrofungi is plotted against xerothermic communities in figures below (Fig. 1a-f).As the box plots show (Fig. 1a,b,f), G. simplex has a narrow range of ecological requirements.Its fruitbodies produce white, long (ca.50-200 mm) mycelial cords, rhizomorphs, which they use to entwine grass roots.Gastrosporium simplex is an obligatory parasite of grass roots, mostly of the genus Stipa but it has also been noted on roots of Brachypodium, Bromus, Festuca, Koeleria and Sesleria [17,25].Many authors: Šmarda [26], Bujakiewicz [27], Łuszczyński and Łuszczyńska [28], Stasińska [29,30], stress its strong affiliation with communities of the alliance Festuco-Stipion but they also show that the species is exceptionally thermophilous, associated with dry, warm and sun-exposed sites where soil temperature often exceeds 39°C.Gastrosporium simplex was also recorded most frequently in the communities Sisymbrio-Stipetum capillatae and Festucetum pallentis (Fig. 1a,b).The most numerous production of fruitbodies was recorded on roots of grasses of the genus Stipa, which also confirms its obligatory relationship with the host.
The mycobiota of extremely dry and warm habitats of xerothermic and psammophilous grasslands is differentiated by species of the genus Tulostoma: T. brumale, T. kotlabae, T. melanocyclum and T. squamosum.Species of this genus develop the mycelium and fruitbodies among tall herb vegetation, in southfacing sites.They prefer dry, sandy soils  rich in calcium carbonate [17,18].In our study, fruitbodies of fungi belonging to the genus Tulostoma were collected on south-facing slopes, in summit parts, strongly insolated, where mean soil temperatures often exceeded 35°C in summer months.Fruitbodies of these fungi were most frequently recorded on soils such as shallow clay rendzina.The closest biocoenotic affiliation with phytocoenoses of the associations Festucetum pallentis, Sisymbrio-Stipetum capillatae and Koelerio-Festucetum rupicolae was recorded for Tulostoma squamosum and T. melanocyclum (Fig. 1a-c).Tulostoma brumale can also be a characteristic species of the above-mentioned grasslands as fruitbody abundance is considerably higher in them.
Two species of the genus Disciseda: Disciseda bovista and D. candida, associated with Pontic-steppe vegetation, are appropriate species of the associations Festucetum pallentis and Sisymbrio-Stipetum capillatae [6,19,[31][32][33][34].Localities noted in our study confirm their affiliation with grasslands of the alliances Festuco-Stipion and Seslerio-Festucion duriusculae.Our results confirm the occurrence of the mycocoenosis of the association of fungi Tulostomato (brumali)-Gastrosporietum simplicis in the study area.Species of the genus Geastrum also characterize well thermophilous, easily warming areas of steppe grasslands.A strong affinity between Geastrum campestre, G. minimum and G. schmidelii and Stipa grasslands containing Stipa capillata and S. joannis as well as Festuca grasslands dominated by Festuca pallens and F. rupicola (Fig. 1b) was recorded in our study.
Several extreme data points, relating to Gastrosporium simplex, Geastrum schmidelii, G. campestre, Tulostoma kotlabae and T. squamosum, are noticeable in some diagrams (Fig. 1d-f).These values suggest that these species can also be identified with phytocoenoses belonging to the alliance Cirsio-Brachypodion pinnati, which would indicate a broader ecological scale of these taxa.However, local lithological-pedological conditions in the Nida Basin make the microhabitats in this seemingly homogenous type of communities highly mosaic.Without specialized pedological examinations, the richness of the microhabitats is not reflected in the floristic diversity of the communities, especially in the alliance Cirsio-Brachypodion pinnati.Small plots are often distinguished in floristically homogenous patches due to outcrops beneath upper soil layers.This local diversity can cause the development of fungi associated with xerothermophilic communities of the alliance Festuco-Stipion and not those associated with mesoxerothermic phytocoenoses of the alliance Cirsio-Brachypodion pinnati.
The results of mycological observations conducted in the phytocoenoses of Sisymbrio-Stipetum capillatae, successively burnt out since 1984, are very different.Not only is an impoverished species composition observed at these plots but also a total disappearance of the biota of macrofungi is noted (Tab.2).Therefore, fungi examined by us are an important component of the biocoenosis.Their occurrence indicates its mature character and complex structure while its type remains undisturbed by burning.

Determination of indicator species: statistical analysis
In our data, six out of ten of the species analyzed were significantly related to site groups.Tulostoma squamosum is an indicator of Festucetum pallentis, Tulostoma kotlabae is an indicator of Koelerio-Festucetum rupicolae and Geastrum minimum, Gastrosporium simplex, Disciseda bovista and Tulostoma melanocyclum are indicators of Sisymbrio-Stipetum capillatae (Tab.3).
The highest values of IndVal were observed for Geastrum minimum and Gastrosporium simplex, which were significantly associated with Sisymbrio-Stipetum capillatae.The species of fungi Gastrosporium simplex and Geastrum minimum are mainly restricted to Sisymbrio-Stipetum capillatae (A > 0.7) and each of them occurs at all plots of Sisymbrio-Stipetum capillatae.
In the correspondence analysis based on all 30 plots, the first two dimensions accounted for only 49.7% of inertia (Fig. 2) and no clear clusters were found.When we restricted our analysis to those six species that were significantly related to site groups by IndVal analysis, the first two dimensions accounted for 71.2% of inertia (Fig. 3) but clear clusters were still not observed.

Fig. 1
Fig.1 The number of fruitbody production of Basidiomycetes in communities of xerothermic vegetation in the Nida Basin in the years 2010-2013.

Fig. 2
Fig.2The correspondence analysis based on all 30 plots in the years 2010-2013 (abbreviations are as in Tab. 1).
Phytosociological features and a list of collected fruitbodies of fungi examined by us: Sisymbrio-Stipetum capillatae (phytosociological data according to Braun-Blanquet method).Tulostoma squamosum, which accounted for 48% of fruitbodies of all species of fungi examined by us, was the dominant species at the Festucetum pallentis plots.The number of fruitbodies of individual fungal species collected in the patches of Seslerio-Scorzoneretum purpureae phytoceoneses ranges quite considerably; a single dominant species, however, was not recorded.Very good conditions for the growth of fruitbodies of fungi investigated by us were also noted in the community Sisymbrio-Stipetum capillatae.A total of 770 fruitbodies were noted in the plots of this phytocoenosis, Abbreviations: Fp -Festucetum pallentis; Sc -Sisymbrio-Stipetum capillatae; Sp -Seslerio-Scorzoneretum purpureae; K -Koelerio-Festucetum rupicolae; I -Inuletum ensifoliae; A -Adonido-Brachypodietum pinnati; T. sq.-Tulostoma squamosum; T. mel.-Tulostoma melanocyclum; T. kot.-Tulostoma kotlabae; T. bru.-Tulostoma brumale; G. schm.-Geastrumschmidelii; G. min.-Geastrumminimum; G. cam.-Geastrum campestre; G. sim.-Gastrosporium simplex; D. can.-Disciseda candida; D. bov.-Disciseda bovista.Tab. 2 with Gastrosporium simplex as a dominant species.A large number of fruitbodies was also produced by Geastrum minimum in Sisymbrio-Stipetum capillatae, where 153 of its fruitbodies were collected.Neither G. simplex nor Geastrum minimum developed fruitbodies in the communities Koelerio-Festucetum rupicolae and Inuletum ensifoliae.The least favorable conditions for the development of fruitbodies of fungi examined by us were noted in the community Inuletum ensifoliae.Fruitbodies of only one species, Geastrum campestre, totaling 19 fruitbodies, were collected at the plots comprising phytocoenoses of this community.Detailed data regarding the number of fruitbodies of individual species in plant communities is given in Tab. 1.