Callistosporium pinicola ( Basidiomycota ) , a fungus species new to Poland

Basidiomata of Callistosporium pinicola were collected in years 2012–2013, on strongly decayed fallen trunks of Picea abies and Abies alba on two remote sites, localized in Northeastern and southern Poland. These localities represent the first records of this fungus in Poland. A full description and illustration of C. pinicola based on Polish specimens are provided and the distinguishing features and delimitation of the species are briefly discussed. The ecology of C. pinicola, its general distribution and threat are also presented. As all specimens of C. pinicola were collected only in recent years we suggest that this species is increasing its distribution in Poland. However, we cannot rule out the possibility that, because of its cryptic nature, C. pinicola has been much overlooked in the past.


Introduction
The genus Callistosporium Singer, which contains 13-16 species [1,2] belongs to the tricholomatoid clade and to the family Tricholomataceae within Basidiomycota [3,4].However, members of this genus may have a relationship with the entolomatoid clade, and analysis of only rRNA data place Callistosporium in the Entolomataceae [3,5].Members of the Callistosporium genus are generally characterized by the collybioid habit, the white spore deposit, smooth spores without iodine reactions, the presence of yellow pigment bodies inside the spores, and also often other elements of the hymenophore ("necropigments" colorless in fresh condition but visible in exsiccates), the absence of true cystidia and clamp-connections as well as the lilac, violet to purple staining of tissues in KOH and NH 3 •H 2 O [4,6].The fungi live as saprotrophs on humus, litter and various kinds of decaying wood, also on Sphagnum, mostly in forests [4,[6][7][8][9].The genus Callistosporium is completely unknown in Poland and appears poorly investigated in other European and non-European countries.The most of known Callistosporium species are probably under-collected due to their somewhat cryptically colored basidiomata and superficial similarity to Simocybe, Gymnopilus or Cortinarius (Dermocybe).
During field studies, both authors of this paper independently found an interesting small-spored Callistosporium basidiomata growing on strongly decayed wood of conifers.In comparison with recent literature we identified these records as C. pinicola Arnolds, a species new to the mycobiota of Poland.This wood-inhabiting basidiomycote was recognized for the first time by Bon [10], but at the species level it was described and validly published first by Arnolds [11] on the basis of Dutch material collected on strong rotten stump of conifer in mature beech forest (Lheederzand, Dwingeloo) [12].Callistosporium pinicola is generally known to prefer strongly decayed wood of conifers.It is reported from heterogeneous both deciduous and coniferous forests and from mixed stands.The known distribution of the species seems to be limited to the East-Central and Western Europe, and only exceptionally does it reach the southern and the northern part of the continent.This fungus is rather rare in all parts of its distribution [4,11,13].
The main aim of this paper is to describe morphologically the first collections of C. pinicola for Poland, and to compare their characters with published data.Furthermore, this paper aims to evaluate its ecology and distribution in Poland (Fig. 1) in regard to the previous records in Europe.

Material and methods
Material has been collected occasionally by the authors during several field trips in different regions of Poland in 2012 and 2013.Description of macroscopic features was based on fresh material comprising over 15 basidiomata in all stages of development, from three collections.Microcharacters were observed with a Nikon Eclipse E-400 light microscope equipped with a Nikon digital camera (DS-Fi1).For microscopic observations, dried materials were placed in 95% ethanol for about 1 min, and then transferred to 5% NH 3 •H 2 O solution until they became pliable.Free-hand sections of the rehydrated pieces of basidiomata were examined in 5% NH 3 •H 2 O and Congo red in ammonia.Image-grabbing and biometric analyses were done with NIS-Elements D 3.1 imaging software.Dimensions of microcharacters are given as (minimum) average ±standard deviation (maximum), and additionally in the form of the main data range (5-95 percentile values).Q value refers to the length/width ratio of basidiospores.For basidiospores size measurements, randomly selected mature spores were used, and measured without hilar appendix.Lengths of basidia were measured excluding sterigmata.Microphotographs were taken using Nikon DS-Fi1 digital camera.Statistical computations employed Statistica software (StatSoft).For morphological terminology see "Flora agaricina Neerlandica" [14,15].The fungus nomenclature and its synonyms follow Arnolds [11] and Antonín et al. [13].The nomenclature of forest communities follows Matuszkiewicz [16].Specimens have been deposited in the Herbarium of the Museum of Natural History at the Wrocław University (WRSL), Poland.

Notes
Though the holotype of C. pinicola from the Netherlands was not studied in this case, we consider the macro-and microcharacters exhibited by a number of Polish specimens to be consistent in principle with those described by Arnolds [11] and supplemented by others (e.g.[13]).The new findings presented below are characterized by rather variable shape of basidiospores.Although analyzed spores are ellipsoid in majority, they are also nearly amygdaliform (with adaxial side less convex than abaxial one) and subglobose in side view.In the protologue C. pinicola is described as having only four-spored basidia.However, the Polish material of the species revealed also a small proportion of two-spored cells.The presence of two-spored basidia in C. pinicola was also reported by Antonín et al. [13].Moreover, the dominant four-spored basidia are smaller in Polish material in accordance with those described in the protolog.
Arnolds [11] provides very interesting information on the affinities of C. pinicola with Calocybe cerina (Pers.: Fr.) Donk [the taxon is currently treated as Calocybe chrysenteron var.cerina (Pers.: Fr.) Arnolds or Rugosomyces chrysenteron (Bull.: Pers.)Bon].He noticed that C. pinicola was sometimes misidentified as C. cerina in the past and are occasionally considered under this name in the literature and herbaria.Although similar at first glance, mainly because their comparable size and color at early stages, C. cerina is unlikely to be mistaken for C. pinicola when studied in more detail, mainly in view of the thicker, sulphur-yellow context, a taste becoming bitter after a while, and the substrate on soil and litter (rarely on very rotten wood).Its additional distinguishing characters are a pileipellis in the form of a cutis with transitions to a trichodermium, presence of clamp-connections, and lack of reddish discoloration of their tissues in alkaline solutions [11,25,26].We checked the collections of the WRSL herbarium where few specimens of C. cerina are stored (as Tricholoma cerinum and Agaricus cerinus).However, all of them represent another taxon, namely Rugosomyces naucoria (Murrill) Boffelli (syn.R. fallax Bon, Calocybe fallax (Sacc.)Redhead & Singer invalid name, ICBN Art.35.1).
Antonín et al. [13] have discussed the ecological preferences of C. pinicola in detail.They have tentatively concluded that one should take into account a strongly rotten wood (of mostly conifers) and humid climate as well, when considering the environmental requirements of the species.In spite of these factors, various relations between the occurrence of C. pinicola and kind of habitat have been also noticed.The typical habitats of the species in the Czech Republic and the Ukraine are old-growth forests (include a wide range of forests with natural occurrence of Abies alba and/or Picea abies), in rather cold and humid stands (montane or submontane forests and stream valleys or gorges in highlands), most frequent between 200-500 and 800-1100 m altitude.However, according to Antonín et al. [13] C. pinicola was also found in a man-influenced stand and cannot be considered a species confined exclusively to natural and virgin forests in the area.In The Netherlands C. pinicola grows in first-generation coniferous (Pinus sylvestris, Picea abies) and mixed forest plantations [11][12][13].In France this species is reported from coniferous and mixed stands, with Quercus, Carpinus, Fagus and Pinus, in the colline belt [13,19].In Poland, C. pinicola has been found in highly natural forest communities (oak-hornbeam forest and fir-beech forest), possessing a natural tree species composition and multi-aged structure, and situated in planar and montane belt respectively.Thus, Polish findings seem to confirm a strong linkage of C. pinicola to old-growth forests in Central Europe.The humid meso-and microclimate of these forests and their rich supply of various decaying woody substrates (as measured by a diversity of diameters and decay classes) creates suitable conditions for this species.All specimens of C. pinicola in Poland were collected only in recent years (in the period 2012-2013), which may suggest that this species is increasing its distribution for unknown reasons.However, we cannot rule out the possibility that, because of its cryptic nature, C. pinicola has been much overlooked in the past.As an uncommon fungus restricted to threatened natural habitats (protected as nature reserve and the area under strict protection), C. pinicola should certainly be classified at least as a rare and included into the new edition of the red list of fungi of Poland.