New localities of Chamonixia caespitosa (hypogeous Boletaceae) in Central Europe

Chamonixia caespitosa rolland, has been recently found in Poland for the first time after 1945. The basidiocarps, partially exposed from the humus layer, were found in two localities: in the spruce forest in the Polish tatra Mts., at the elevation of 1540 m a.s.l., and in the mixed forest with spruce and fir in the Beskid niski Mts. at the elevation of app. 400 m a.s.l. the description of the Polish specimens generally agrees with descriptions of the specimens found in other central european countries. the roundish to tuberculate basidiocarps were characterized by the presence of highly reduced stipe, whitish colour of the peridium changing rapidly to blue after exposure to air, small, complete or incomplete columella and brown, spongy gleba. typically 4-spored basidia were present which produced ellipsoid, brown spores with the ornamentation in the form of rough, interconnected ridges. taxonomic position, ecology and chorology of the species, the ontogeny of basidiocarps and description of ectomycorrhizae are summarized in the paper.

Soehner (Soehner 1949). Several studies, however, strongly supported Chamonixia as a separate genus and established its position within Boletales (see Discussion). Since its discovery Chamonixia caespitosa rolland has been found in several european countries and also outside europe.
The first Polish locality of Chamonixia was discovered in 1923 by Buchs in Karkonosze range (German name: riesengebirge), Sudety Mountains, above Karpacz Górny town (German name: Brückenberg), in that time within Germany (Soehner 1949).
recently after 85 years the species has been found in Poland in two localities. These are the first contemporary collections of this species, and the first records in the Polish carpathians. in the article the localities of Chamonixia caespitosa with description of macroscopic and microscopic features of the basidiocarps are presented. the authors discuss taxonomic position, ecology and chorology of this species, the ontogeny of its basidiocarps and description of its ectomycorrhizae.

METhoDS
Description of the basidiocarps was based on fresh and dry material. the measure-the measurements of microscopical features were taken on dry material mounted in 3% koH. the information on the dimensions of spores and other structures are based on 25-30 measurements. Measurements of spores include spore ornamentation but excluded apiculus.

Chamonixia caespitosa
Basidiocarps mostly single (not caespitose), in one case two young adhering basidiocarps with single stipe were found (on second locality), roundish and slightly flattened to irrergularely tuberculate, with well defined base formed as a short, tapered stalk, with some small rhizomorphs attached. Fruitbody surface slightly woolly, felty, cracked in places and exposing inner layers of the peridium, whitish to creme or ochraceous, when bruised rapidly changing colour becoming blue ( Fig.  1), greenish to olive on drying (Fig. 2). Peridium (Fig. 3) in cross section 0.1-0.2 mm thick, separating from gleba. whitish columella present, connected with a basal stalk, with context also turning blue after cutting. columella either complete (basidiocarps from second locality) or triangle in section and not percurrent, but than it was found to continue as a thin layer of hyphae oriented to the site rather than to the top of the gleba (basidiocarps from the first locality). Gleba spongy, labyrinthine, with Figs 17-18. Section through inner peridium layer (arrows indicate the direction from inner to outer layer of the peridium). Fig. 19. Peridium in section: outer, pigmented layer built by highly septated hyphae with inflated hyphal cells. Fig. 20. Peridium in section: the part exposed by outer peridium cracking, with hyphal ends arranged in parallel. Scale bars: 10 μm.
Spores elongated, ellipsoid to broadly ellipsoid, tapering towards the base, 16-23 (mean: 19.6) μm long and 12.5-16 (mean: 14.0) μm in diameter (including ornamentation), with an apiculus (hilar appendage) centrally positioned, straight and truncate; spore surface rough and grainy , covered with conspicuous, interconnected, rarely single, ridges up to 5 μm thick, which make the spores irregularly star-like in optical section; usually 4-6 (7) complete or partial, longitudinal ridges visible in side view, sometimes the ridges may be difficult to discern (see Fig.  15). The spore wall 1.5-3.5 μm thick (including ornamentation). The ornamentation of the top of the spore typically star-like, consisting of short lobes continuous with the ridges or independent of them (Figs 14,16). colour of mature spores yellow-to reddish-brown; young, immature spores hyaline then yellowish to yellowish-brown. the wall of young, immature spores slightly dextrinoid and strongly cyanophilous.
LocaLities. The fruitbodies were found in two localities in Southern Poland. 1. august 2008, the western Tatra Mts., in the upper part of the Tomanowa valley (49 o 13' 23''n, 19 o 53' 43''e) exposed to the west, at the elevation of 1540 m a.s.l. the collection site is situated in the part of the tatra Mts. with the limestone bedrock. Single basidiocarp was found in the montane old-growth spruce forest (association: Polysticho-Piceetum), on a gentle slope by the forest path, app. 0.5 m above the path's level. it was growing among spruce roots on the ground scarcely covered with mosses. the basidiocarp was partially exposed from the soil.

DiScUSSion
according to label (2006) australian species of Chamonixia may represent a different taxon and perhaps deserve a new generic name.
Chamonixia caespitosa is characterized by the presence of (in most cases incomplete) columella, the spores with (6) 8 or more (up to 16) ribs on their surface, and by (generally) 4-spored basidia. However, the differences between the original description by rolland and descriptions by other authors were realized (2-spored versus 4-spored basidia, caespitose versus mostly single basidiocarps, e.g. kotlaba 1971) and it was suggested that perhaps rollands species might represent a different taxon than the one described later by Soehner (1922) under the name Hymenogaster caerulescens. the problem has not been solved until today as the rolland's type collection of Chamonixia caespitosa was not found (capellano 1967).
The features of the specimens from the Polish Tatra Mts. and Beskid niski Mts. generally agree with the descriptions given by other authors (e.g., Soehner 1922;Lange, hawker 1951;capellano 1967;Kotlaba 1971;hagara 1985;Kers 1985;Breitenbach, Kränzlin 1986;haeggström 1987;Montecchi, Sarasini 2000). our fruitbodies were mostly single and not consisting of several basidiocarps joined by common stalk, as described for the type collection by rolland. However, more complex basidiocarps were also found. hagara (1985), Kers (1985) and cheype (1990) reported fruitbodies consisting of two even or uneven parts growing from a common base (stalk). the dimensions of spores only slightly vary in different descriptions; more variable dimensions were detected among basidia (tab. 1). the description of trama elements as well as the structure of the peridium and dimensions of it's hyphae given by kotlaba (1971) for czech specimens, one of the closest localities to the Polish one (no such data for Slovak specimens) correspond well with our observations. However, kotlaba did not mention a double-layered structure of the peridium, which in our specimen was evident not only in hyphal structure but also in colour (yellowish outer layer and hyaline inner one). this double-structure was, in turn, described in detail by e.g., capellano (1967) who, however, found no trichodermal structure in the outermost layer described for the american tramal hyphae were described by lange and Hawker (1951), and american authors, and this was also found in case of the Polish specimens.
in europe Chamonixia caespitosa is a mycorrhizal symbiont of spruce. its mycorrhizae with Picea abies were described and illustrated by raidl (1999,2008). They were also detected during studies of ectomycorrhiza in spruce forests by, e.g., Peter et al. (2001) in Switzerland. in western USa the species was also reported to be connected to other Pinaceae: Abies amabilis, Tsuga spp., Pseudotsuga menziesii (castellano et al. 1999), but probably this applies to the taxon different than european Chamonixia caespitosa (see above). in central and western Europe Chamonixia fruitbodies were found in spruce forests mostly in mountainous regions (alps, apennines, Sudety Mts., carpathians), and the distribution of this species was described by Groß et al. (1980) as montane-subalpine. however, in Scandinavia they occur at lower altitudes in regions with climatic conditions similar to those of spruce montane forests of central Europe (Bohlin, Jeppson 1983). The distribution of the species can thus be described as montane-subalpine-subboreal. although growing mostly in the sites with rather high lime contents, Chamonixia was also found in forests developed on the neutral or even slightly acid bedrock in Sweden and austria (Bohlin, Jeppson 1983;Kers 1985). Bohlin and Jeppson (1983) summarized ecological requirements of this species and stated that Chamonixia caespitosa occurs in regions with either high annual rainfall or at least high air humidity. Kers (1985) analyzed Scandinavian localities of Chamonixia and characterized the ecological requirements of the species precisely. He stated that the species occurs in old-growth spruce forests, mostly on slopes with northern exposition or in ravines. the localities are characterized by constant water supply, either from small streams or underground moving water, and usually by high accumulation of woody debris -decaying wood was also mentioned as a substrate of Chamonixia by, e.g., Groß (1974) and Bohlin & Jeppson (1983). Frequently, forests, where Chamonixia occurs, are of high value also as habitats of rare and threatened plant species. the observations of Kers have been recently confirmed during inventory of Chamonixia localities in Sweden made by Bohlin (2005Bohlin ( , 2006 and Vesslén and Bengtson (2008). the inventories of all known and potential localities of this species were undertaken in the frame of the national project aiming for protection of Chamonixia and its habitats -both are threatened mostly due to timber harvesting and forest practices (mostly drainage). the projects also aimed to assess the status of Chamonixia populations and evaluate the condition of habitats (Bohlin 2005;Vesslén, Bengtson 2008) and should eventually lead to developing suitable preservation measures. Very detailed descriptions of Chamonixia localities (including accompanying fungal and plant taxa) from outside Sweden can be found in, e.g., Groß (1974Groß ( ), haeggström (1987Groß ( ), and zureck et al. (1999. The basidiocarps of Chamonixia develop in soil, mostly under extensive moss or spruce needle cover, but they are frequently partially exposed when mature. For this reason the species was rated among semihypogeic fungi (Bohlin, Jeppson 1983).
the ontogeny of the fruitbodies of Chamonixia caespitosa has recently been investigated in detail by clémençon (2005). he showed that in the early stages of development the later gasterocarpic fruitobodies resemble those of some other Boletales: the tubular hymenophore is formed on the primordial stem, which is then covered by the hyphae of pileus margin and stem surface. in the later stages, those hyphae (the amphicleistoblema) cover completely the hymenophore and entire fruitbody. the author concluded that development of the fruitody is exocarpic, claustropileate and amphicleistoblemate, and found similarities of some stages of this development with development of fruitbodies of Suillus and Boletinus. clémençon pointed out that these similarities strongly support the taxonomical position of Chamonixia within Boletales.
the taxonomic position of Chamonixia was controversial for a long time. certain similarities (mostly spore morphology) made some authors place this species in the genus Hymenogaster or in close relationship to it (e.g., Soehner 1949;Lange 1956;Szemere 1965). another hypothesis placed Chamonixia close to Gautieria (e.g., zeller 1948;cribb 1958;Smith 1973), however, differences in spore print color and gasterocarp structure between both genera were soon realized (lange, Hawker 1951;capellano 1967). Smith and Singer (1959), found some similarities of peridium structure and spore morphology of the american species to some members of Boletales. they also realized that Chamonixia basidiocarps were parasitized by Sepedonium chrysospermum s.l., a typical boletophilous fungus. Similar color reaction of bruised basidiocarps and mycelium of Chamonixia and Gyroporus cyanescens was also evident. Thus, Moser (1978) and Jülich (1981) in their monographs placed Chamonixia in the family Boletaceae. The close affinity of Chamonixia caespitosa to Gyroporus cyanescens was suggested again by Bresinsky & wittmann-Bresinsky (1995) and Bresinsky (1996) due to the presence of pigments: gyrocyanin, chamonixin and gyroporin; the first two being responsible for blueing of the fruitbodies (Steglich et al. 1977;Bresinsky, Besl 1979). This, however, was not supported by molecular studies: Kretzer and Bruns (1999) found this species to be more related to Boletus, Chalciporus and Phylloporus than to Gyroporus, basing on the analysis of combined atp6 and mt LSU rDna gene sequences. The molecular analysis by Binder & Bresinsky (2002) and Binder & Hibbet (2006) revealed, however, that the closest relative of Chamonixia is genus Leccinum (genus Leccinum was not included in the study by kretzer & Bruns 1999).
the morphology and anatomy of Chamonixia caespitosa ectomycorrhizae with Picea abies have been studied in detail by raidl (1999,2008). They were characterized by a ring-like hyphal arrangement of the mantle hyphae and highly differentiated rhizomorphs of type F (agerer 1995), characteristic features of many members of Boletaceae. clamp connections were absent. the bluish reaction of the mantle and rhizomorphs was encountered after bruising or treatment with FEa. The ectomycorrhizae of Chamonixia differed from the ectomycorrhizae of Gyroporus cyanescens, which was suggested as a close relative of Chamonixia. in Gyroporus ectomycorrhizae the reaction was not caused by simple bruising but treatment with concentrated sulfuric acid. additionally, the hyphae of Gyroporus form clamps and the ring-like arrangement of hyphae in the mantle is absent. the bluish reaction has also been found in ectomycorrhizae of other members of Boletales, e.g., Leccinum (Müller, agerer 1990) and Alpova diplophloeus (Miller et al. 1988;wiedmer et al. 2001). close anatomical similarities of Chamonixia ectomycorrhizae with Leccinum ectomycorrhizae are especially interesting as molecular studies indicate that these genera are closely related (see above). Studies of ectomycorrhizae indicate that hypogeous structures may be regarded as a reliable source of taxonomic and phylogenetic information (e.g., agerer 1999, 2003, 2004eberhardt 2002;Martin, raidl 2002;Beenken 2004;Mleczko, ronikier 2007