A confusing duo: Calocybe cerina and Callistosporium pinicola (Agaricales)

The name Calocybe cerina has recently been applied for two different species of agarics. After studying collections from different European countries it is concluded that the true Calocybe cerina is very close to C. chrysenteron and best regarded as a variety. The new combination Calocybe chrysenteron var. cerina is proposed and a full description of that taxon is given. The majority of collections, identified as C. cerina, appear to belong to a fungus, provisionally described by Bon as Callistosporium luteoolivaceum fo. minor. It is described here as a new species: Callistosporium pinicola. Calocybe juncicola is another species that might be confused with the two species mentioned before and therefore a full description is provided. The status of the genera Rugosomyces and Calocybe is discussed. The new combinations Calocybe obscurata and Calocybe pudica are made.


INTRODUCTION
In The Netherlands in recent years several collections were made of a fairly small, white-spored agaric with an orange-brown pileus and crowded, orange-yellow lamellae, growing on stumps of coniferous trees.It was initially identified as Calocybe cerina (Pers.: Fr.) Donk (A r n o l d s , B e c k e r 1993).An important indication was the presence of granules in the basidia that were apparently siderophilous, staining dark violet in acetocarmine with addition of iron.However, some characters were deviating from most descriptions in literature, in particular the striking red to violet staining of all tissues in KOH and ammonia, the complete absence of clamp-connections and the lignicolous habitat, suggesting that it could be an undescribed taxon.
Recently I came across descriptions of Callistosporium luteoolivaceum var.minor M. Bon ined.(L u d w i g 2001; Wi l h e l m 2003) that showed striking resemblance with our material, e.g. the red staining in ammonia and KOH.In this paper I shall try to unravel the identity of the collected material and I will discuss the taxonomic position of Calocybe cerina (Pers.: Fr.) Donk and the related C. juncicola (Heim) Sing.

E. Arnolds
WHAT IS CALOCYBE CERINA?Agaricus cerinus was initially described by Pe r s o o n (1801: 321) as a fairly small agaric, growing in pine forests (substrate not explicitly mentioned) with dark waxyellow ('flavo-cerinus opacus'), fleshy, flattened to depressed pileus; crowded, narrow lamellae and a taste becoming bitter after a while.In his sanctioning description Fr i e s (1821:89) mainly copied Persoon's description, adding that the context and spore print are white.Most authors have interpreted the name as a species close to or identical with Calocybe chrysenteron (Bull.: Fr.) Sing.Pe r s o o n (1801) treated Agaricus chrysenterus Bull.immediately after his A. cerinus.The two taxa are microscopically characterized by e.g very small, ellipsoid spores, a pileipellis in the form of a cutis with transitions to a trichodermium, and presence of clamp-connections.
A different interpretation of Agaricus cerinus was published by A r n o l d s & B e c k e r (1993) and accepted by e.g.K a l a m e e s (1995,2004).The basidiocarps of that fungus occur on dead wood, are slightly smaller and thin-fleshed, their tissues turn reddish in alkaline solutions and clamp-connections are absent.
After comparing collections and modern descriptions of these taxa I believe that the first-mentioned interpretation is in better agreement with the original diagnosis, mainly in view of the larger size of the basidiocarps, the thicker context and the substrate on soil and litter.Therefore I present first a description of this little-known fungus.
Chemical reactions: No part of basidiocarp reddening with KOH or ammonia.Granules in basidia staining dark purple in acetocarmine with iron (siderophilous).
Habitat and distribution: Mainly on needle litter in coniferous forests (Picea, Abies), also reported under broad-leaved-trees (Quercus); on mesic to dry, calcareous soil.Probably widespread in Europe but much rarer than var.

E. Arnolds
The studied collections differ from typical Calocybe chrysenteron morphologically only in the duller, more brownish colours of pileus and stipe and the paler context.In var.chrysenteron all parts of the basidiocarps are bright yellow to orange-yellow (e.g.L u d w i g 2000).In addition var.cerina seems to be almost confined to coniferous forests, whereas var.chrysenteron is mostly (but not always) found in deciduous forests.Both taxa are calciphilous.Some modern authors synonymize C. chrysenteron and C. cerina (e.g.L u d w i g 2001), others treat them as separate species (e.g.B o n 1999; H o r a k 2005).The differences are small but seem to be rather constant and warrant in my opinion a distinction in the rank of variety.
Many modern authors regard Tricholoma pseudoflammula J. Lange as a synonym of Calocybe chrysenteron (e.g.L u d w i g 2001; K a l a m e e s 2004), but Lange's plate (24B) is typical of var.cerina in view of the orange-brown pileus, already in young basidiocarps, and the pale context.
Chemical reactions: all parts of the fresh basidiocarp turning immediately dark red-brown to violet-brown with KOH 5% and 10% ammonia (macroscopically); preparations of all tissues turning immediately violet in KOH 5% and reddish in 10% ammonia, also staining surrounding liquid; granules and clots becoming vinaceous red or dark violet.Granules staining dark purple in acetocarmine with iron (seemingly siderophilous).
Habitat and distribution: Saprotrophic, solitary or in small groups, on strongly decayed stumps or dead trunks of coniferous trees, mainly recorded from Pinus sylvestris, also on Pinus pinea and Picea abies, in coniferous and mixed stands, mainly on acidic, sandy and loamy soils.July-Nov.Callistosporium pinicola does not belong to the genus Calocybe in view of the presence of small granules and larger clots of brown necropigment in the basidia, spores trama and pileipellis, as well as by the complete absence of clamp-connections.However, the necropigment can only be recognized as such in preparations in water (also of exsiccata), because it is immediately turning red to violet in ammonia and KOH.Since exsiccata are usually revived in these solutions the brown pigment is easily overlooked in dried specimens.Moreover the necropigment is strongly staining purple with acetocarmine, thus mimicking the siderophilous granules of Calocybe species.Therefore it is not surprising that the majority of herbarium collections, identified as Calocybe cerina, appears to belong to Callistosporium pinicola.In this context it is interesting to note that M a i r e (1937) described Callistosporium luteoolivaceum under the name Tricholoma chrysenteron (= Calocybe chrysenteron) var.olivascens Maire (Re d h e a d 1982).Apparently confusion between the two genera has occurred before.
The presence of necropigment, the red staining of tissues in KOH and ammonia and the absence of clamp-connections are all characters of the genus Callistosporium Sing.The present species belongs to subgenus Callistosporium in view of the crowded lamellae, small, ellipsoid spores, short basidia and the pigment not turning blue in ammonia (Bon, 1991).Taxonomy and nomenclature of this group are still rather controversial.Bon (1991)  Callistosporium pinicola differs from all species mentioned above in the first place by the very small spores.Ludwig ( 2001) described the spores of C. luteoolivaceum as 5-5.5(-6.5)x 3.5-4 μm and B o n (1991, as C. xanthophyllum) as (5.0-)5.5-6.5(-7)x (3-)4-4.5(-5)μm.Spores in the type collection of C. elaeodes measured 6.5-7.5 x 3.5-4.4(-5.0)μm (B o n 1976).In addition it is striking that the basidiocarps of C. pinicola are generally lacking green colours, so characteristic for other species of Callistosporium.Only in one of the studied collections a weak olivaceous tone was observed in the centre of the pileus.The habitat on stumps and trunks of coniferous trees is also characteristic for C. pinicola.Other species are usually growing on deciduous wood or litter.In view of these characteristics the identity of the collection, depicted by L u d w i g (2000) in Pl. 6.2 B as C. luteoolivaceum var.minor, is in my opinion doubtful since the pileus is distinctly green and the spores are described as larger: 4-5 x 3-4 μm.
In the field Callistosporium pinicola may also be mistaken for a species of Simocybe or Gymnopilus.

THE TAXONOMIC POSITION OF CALOCYBE JUNCICOLA
Next to Calocybe cerina sensu Arnolds & Becker, there is a second European species of Calocybe in which red staining of tissues in alkaline solutions has been reported, viz.Calocybe juncicola (R. Heim) Sing.(M o r e n o 1995).Since this reaction is considered a characteristic feature of the genus Callistosporium, I have examined two collections of that species in order to re-evaluate its taxonomic position.
Chemical reactions: Preparations of all tissues turning immediately reddish to violet in KOH 5% and ammonia 10%; macroscopic reactions on fresh basidiocarps unknown.Granules in basidia staining dark purple in acetocarmine with iron (siderophilous).
The reddish staining of tissues in alkaline solutions was only reported by M o r e n o et al. (1995), who mentioned a violet discoloration of the solution when making a preparation of the lamellae in KOH.The reaction was not mentioned in other descriptions (H a u s k n e c h t , Z u c c h e r e l l i 1994; C e t t o 1989; L u d w i g 2001) or in the descriptive notes accompanying the collections.The reddening in ammonia and KOH is very similar to that of Callistosporium pinicola, although slightly weaker.Nevertheless, Calocybe juncicola seems not to be related to the latter species in view of the absence of brown necropigment, the abundance of clamp-connections and the presence of truly siderophilous granules in the basidia, although these granules seem to be less numerous and striking than in other Calocybe species.Otherwise, C. juncicola differs from C. chrysenteron (incl.var.cerina) mainly in duller colours, striate stipe, larger and more elongate spores and specific habitat.
A second European species of Calocybe with violaceous red discoloration of tissues in KOH is Rugososmyces pudicus M. Bon & Contu, collected in moist forest of Populus on Sardegna (C o n t u , B o n 2000).It has small basidiocarps with a pinkish brown to yellow-brown pileus up to 20 mm, differing mainly from C juncicola in immediate reddening of the context on exposure to the air and broader spores (5.0-6.0 x 3.5-4.5 μm) that are slightly rugulose, as was also demonstrated with SEM microscopy (C o n t u , O r t e g a 2001).

CALOCYBE OR RUGOSOMYCES?
The genus Calocybe was erected (but invalidly published) by K ü h n e r (1938) in order to accommodate tricholomoid, white-spored agarics with basidia containing siderophilous granules in combination with vividly coloured basidiocarps.Donk (1962) validated this name and selected Calocybe georgii (L.) Kühner (= C. gambosa (Fr.: Fr.) Donk) as type species.Several authors have discussed the possible heterogeneity of this genus since.K a l a m e e s (1992) has proposed the genus Tricholomella for Calocybe constricta (Fr.: Fr.) Sing., deviating from other species in presence of a partial veil, verrucose spores, large basidia and ecological preference for substrates with high ammonia content.This distinction was later confirmed by phylogenetic studies using molecular characters (H o f f s t e t t e r et al.The genus Gerhardtia was created by B o n (1994) for Calocybe borealis A. Riva (= Lyophyllum incarnatobrunneum Ew.Gerhardt), a species deviating in minutely verruculose spores and absence of clamp-connections.Since verruculose spores have also been observed in Rugosomyces (Calocybe) pudicus (C o n t u and O r t e g a 2001), the status of this genus seems questionable.The phylogenetic relationships with the rest of Calocybe (and Lyophyllum) has not yet been studied.
Earlier Ra i t h e l h u b e r (1979) erected the genus Rugosomyces for the two species of Calocybe with a cellular pileipellis (hymeniderm) instead of a pileipellis of slender hyphae (cutis): the type species Rugosomyces onychinus (Fr.)Raithelh.and R. fallax (Sacc.)M. Bon.This concept was adopted by e.g.H o r a k ( 2005), but Bo n (1991) extended Rugosomyces to all small species with collybioid habit, a pileipellis with more or less free ending hyphal tips, often with inflated cells, and mixed parietal and intracellular pigments.Calocybe was restricted by B o n (l.c.) to a few large, tricholomoid species with intracellular pigments.This concept was accepted by e.g.K a l a m e e s (2004).However, molecular studies of the Lyophylleae have demonstrated that Calocybe (with the exception of C. constricta, possibly also C. borealis; see above) forms a single clade and is probably of monophyletic origin (H o f f s t e tt e r et al. 2002; M o n c a l v o et al. 2002).Therefore there seems to be no good reason to recognise Rugosomyces as a separate genus.

CALLISTOSPORIUM AND CALOCYBE
The genus Callistosporium is assigned to the Tricholomatoideae and not to the Lyophylloideae in view of the absence of siderophilous granules in the basidia.Also in phylogenetic analysis the genera seem not to be closely related to each other (M o n c a l v o et al. 2002).However, it might be interesting to study the relationships between these groups more closely in view of the remarkable convergence between some species.Also the chemical composition of the necropigment in Callistosporium deserves attention.
distinguished two species, C. xanthophyllum (Malenc.& Bert.)M. Bon and C. elaeodes (Romagn.ex) M. Bon, but Re d h e a d (1982) and N o o r d e l o o s (1995) united these taxa in one species and synonymised it with the North-American C. luteoolivaceum (Berk.& Curtis) Sing, which name has priority in that case.L u d w i g (2001) described C. foetens E. Ludwig as additional new species in this subgenus, characterized by a strong unpleasant smell.
2002; M o n c a l v o et al. 2002).