Hymenoscyphus subcarneus, a little known bryicolous discomycete found in the Białowieża National Park

B a r a l H . O . , K r i e g l s t e i n e r L .: Hymenoscyphus subcarneus, a little known bryicolous discomycete found in the Białowieża National Park. Acta Mycol. 41 (1): ..... 2006. The discomycete Hymenoscyphus subcarneus was found to grow parasitic on the liverwort Cephalozia catenulata in the Białowieża National Park (Poland), and is described and illustrated from the fresh collection. Two characters, the ascus apical ring structure of the Calycinatype and the contents of the living paraphyses (multiguttulate by low-refractive vacuolar bodies), have not been reported previously. In addition, a dried collection on Pohlia gracilis from Switzerland, Grimsel area, was studied. The relationship, taxonomy and infraspecific variation of the fungus are discussed. A new genus, Roseodiscus, is established to accomodate this bryicolous fungus together with two very similar equiseticolous species, Hymenoscyphus rhodoleucus and H. equisetinus. The three species are macroscopically characterized in the fresh state by a pale rosaceous-lilaceous hymenium and a mostly slender, concolorous or whitish stipe. Roseodiscus resembles Hymenoscyphus in various respects, but sharply deviates in the apical ring type which appears to indicate a more close relationship with genera like Calycina or Stamnaria. Rhizoscyphus ericae which forms a mycorrhiza with roots of Ericaceae, is compared with Roseodiscus. Based on vital observations of R. ericae this species is believed to be congeneric with the type of Pezoloma, P. griseum.


REPORTS OF HYMENOSCYPHUS SUBCARNEUS IN THE LITERATURE
A d e (1935: 24) reported under the name Phialea subcarnea two collections from Austria (Tirol, Vennatal and Roßkogel above Inzingeralpen, summer 1925 and1927).His description fits more or less to ours, except that he states the spores to be "elongate, often slightly curved, with narrow, bright mucilaginous sheath".The "sheath" might be a mistake for dead spores with detached plasma, perhaps observed from within the asci.Ade gave the spore size as "6-7 x 2-5" μm, but in view of the indicated ascus size (40-50 x 5-6 μm) and the irregularly biseriate spore arrangement, Ade´s spore width is probably an error for 2-2.5 μm.The rather narrow excipular cells ("upto 18.5 x 3.5 μm") forming a t.porrecta might be due to the immature collection.
W h i t e (1942, as Helotium desctructor) examined much over a dozen of collections from U.S.A. (New York, Michigan) and Canada (Alberta, New Hampshire, Ontario) including the type specimen (U.S.A., New York, Indian Lake, on Jungermannia sp., C. H. Peck 319).The apothecia are distinctly smaller than in our collection: 0.2-0.4(-0.5)mm diam., total height upto 1 mm, "scarcely visible to the unaided eye" (possibly measured in the dry state).Ascus (40-50 x 5-6 μm) and spore size (4.5-6x 2-2.5 μm) are a bit smaller than in our collection when comparing the dead state.The pyriform spore shape as well as the conical ascus apex and presence of croziers as depicted by White perfectly fit to our collection.D e n n i s (1964: 68, as Hymenoscyphus subcarneus) studied the excipular characters from the type.In concordance with our fungus, Dennis reported the cells to be rather broad, "about 20-30 x 10 μm, lying at a very low angle to the surface".
Tr i e b e l (1999: 10) reported a recent collection from Canada (ca. 75 km NNE of Québec, Forêt Montmorency).The fungus was identified by P. Döbbeler and said to grow on at least five different bryophytes.Except for the apothecial colour (distinctly pink) no further comments were published.
The here studied collection from Switzerland (on Pohlia gracilis) differs from all the above in distinctly longer asci (80-100 x 6-9 μm, living state) and ascospores (ellipsoid-oblong, *8.5-13.5 x 2.7-3.2μm).The question whether or not this represents a separate species must await further field work.Similar variability was observed by us in Hymenoscyphus rhodoleucus (Fr.: Fr.) W. Phillips) and H. equisetinus (Velen.)Dennis, two species which are obviously closely related to H. subcarneus (see below).Due to a rather high variation in spore size, the delimitation between these two equiseticolous taxa is still not fully settled.
A somewhat similar species with a similar name, Phaeohelotium subcarneum (Schumach.)Dennis, has also pinkish apothecia but lacks a stalk and was mainly reported from bark and wood.It was redescribed by H ö h n e l (1926: 99), G r e l e t (1948: 108), D e n n i s (1971: 355), B r e i t e n b a c h , K r ä n z l i n (1981, pl. 173), and B a r a l (1986: 17) from non-authentic material.The species was even combined in Hymenoscyphus Gray as H. subcarneus (Schumach.)J. Schröt., four years after O. Kuntze transferred Peziza subcarnea Cooke and Peck to that genus.D e n n i s ( 1971) is obviously in error when citing the species as Phaeohelotium subcarneum (Schumach.ex Sacc.)Dennis, because Peziza subcarnea Cooke and Peck is illegitimate, not the earlier Peziza subcarnea Schumach.
The few known reports of Phaeohelotium subcarneum are possibly not homogeneous, at least the brief descriptions do not allow such a conclusion.That P. subcarneum is a synonym of Hymenoscyphus imberbis (Bull.: Fr.) Dennis was erroneously cited by Ve r k l e y (1995: 180).In fact, Baral (B a r a l , K r i e g l s t e i n e r , 1985: 129) stated that P. subcarneum in the sense of Breitenbach and Kränzlin seems to belong to H. imberbis or a closely related species.P. subcarneum s.Baral (1986) has a Calycina-type of apical ring and is therefore unrelated to Hymenoscyphus (incl.Phaeohelotium Kanouse).The ectal excipulum of t. globulosa without a covering layer appears to separate it sharply at generic level from Hymenoscyphus subcarneus (Sacc.)O. Kuntze.Furthermore, the apothecia are fresh greyish-white and turn only pinkish with age or when dried.The generic position of this species remains unclear upto now.

GENERIC POSITION OF HYMENOSCYPHUS SUBCARNEUS (SACC.)
O. KUNTZE D e n n i s (1964: 43) and L i z o ň (1992) believed that the bryicolous species is a normal Hymenoscyphus.However, the rather wide generic concepts of the two authors included in Hymenoscyphus also species of Calycina (Nees) Gray, due to the frequent neglection of apical ring types in light-microscopical studies.The apical ring of H. subcarneus is of the Calycina-type: rather thick in optical section, widest in its upper part which extends to the very apex, here most strongly reactive (B a r a l 1987, fig.26; Ve r k l e y 1995: figs.4.74-76, 186, "type VIII: Chlorociboria-Pezizella-Calycina"), and the ascus apex is strongly conical.This type of ascus apex is very much unlike that of typical members of Hymenoscyphus with a truncate, broadly conical apex and a narrow ring that is usually only present in the lower half of the apical thickening as two very thin amyloid lines which are not widened apically (B a r a l 1986, figs.10-12).The guttulate paraphyses and the presence of a covering layer of narrow hyphae over the excipulum would, however, fit very well in that genus.
The genera Bryoscyphus Spooner and Muscicola (Velen.)Svrček likewise differ from our fungus in ascus structure (Hymenoscyphus-type; B a r a l , M a r s o n 2005), the latter also in the presence of distinct, strongly uncinate hairs.
Two taxa growing on stems of Equisetum, Hymenoscyphus rhodoleucus and H. equisetinus, strongly resemble our species in their pinkish apothecia, but also in their microscopical characters.Like H. subcarneus, both have apical rings of the Calycina-type (B a r a l , M a r s o n 2005) and are therefore clearly misplaced in the genus Hymenoscyphus.Svrček (1959) already compared one of the two (as Phialea rhodoleuca) with our species (as Phialea subcarnea).Placement of H. rhodoleucus in Phialea (Fr.) Gillet (= Cyathicula de Not.) was favoured by N a n n f e l d t (1932) while D e n n i s (1956,1978) preferred to assign the two equiseticolous species to Hymenoscyphus.
Macroscopically, our bryicolous species indeed somewhat resembles a Cyathicula.However, members of that genus have a strongly gelatinized excipulum and often also crystals on the exterior, both absent in our species.The ascus structure varies strongly among the accepted species (Tr i e b e l , B a r a l 1996 as Crocicreas Fr.), comprising both the Hymenoscyphus-and the Calycina-type.However, the latter occurs only in the type species of Crocicreas which should better be distinguished at the generic level.The guttulate paraphyses and the covering layer in our species would fit to Cyathicula.
Species of Calycina differ from our fungus in having a more gelatinized ectal excipulum of much shorter cells which often lie at a higher angle to the surface, and in constantly lacking an external covering hyphal layer.The hairs of Calycina emerge directly from the prismatic excipular cells.Furthermore, spores of Calycina are usually homopolar.Last but not least, the living paraphyses of Calycina species are generally strongly elongate, not multiguttulate.The genus Calycina was transferred form the Helotiaceae to the Hyaloscyphaceae by the first author (B a r a l , K r i e g l s t e i n e r 1985).
The genus Stamnaria Fuckel is characterized by a gelatinized layer outside the ectal excipulum, and the presence of carotenoids in both excipulum and paraphyses.Otherwise it shows some similarities with our species, including the apical ring which is of the Calycina-type in S. equiseti (Hoffm.)Sacc.(K ü n k e l e et al. 2005).
The genus Rhizoscyphus W. Y. Zhuang and Korf was described in Z h a n g , Z h u a n g (2004) to accomodate R. ericae (D.J. Read) W. Y. Zhuang and Korf (≡ Hymenoscyphus ericae (D.J. Read) Korf and Kernan), a species with a mycorrhizal connection to roots of Ericaceae.The erection of a new genus was mainly based on molecular data, but also on the symbiontic relationship with the host plants.Huhtinen (H a m b l e t o n et al. 1999) redescribed the species in detail including vital characters.Following his report, the living paraphyses are without any refractive vacuoles and the apical ring is of the Calycina-type.Characteristic of the species is the hyphoid margin which gives the apothecia a hairy appearance, also the sometimes furcate apices of paraphyses and the simple-septate ascus bases with often a downward protuberance.Clearly, the apical ring excludes Hymenoscyphus s.str.The microscopical data are indeed similar to H. subcarneus, but the subsessile hairy apothecia more resemble a Hyaloscyphaceae.
Recently the first author had the opportunity to study a fresh specimen of R. ericae detected by P. Zinth.The apothecia were formed on the rootlets of a Rhododendron sp. that grows in an semi-sterilized artificial medium in a pot culture in his house in Egelsbach near Frankfurt (Hessen).The specimen fully matches Huhtinen´s analysis.In addition, the mature living asci each contained one globose, refractive drop (4-4.5 μm diam.)below the pars sporifera similar as in the genus Psilachnum Höhn., which disappears in dead asci.This feature was also seen in fresh finds of Pezoloma ciliifera (P.Karst.)Korf and P. marchantiae (Sommerf.)Benkert (B a r a l , M a r s o n 2005), two species which share some further characteristics of R. ericae like the subsessile apothecia, the type of apical ring and the eguttulate, sometimes apically furcate paraphyses.The two species differ in an external gel layer and in marginal teeth composed of narrow agglutinated hyphae.These teeth are a striking feature of the typical species of Pezoloma Clem., but are absent in many other taxa assigned to that genus.The hyphae of the teeth indeed resemble the hair-like protrusions of R. ericae.Similar differences between dentate and adentate apothecia are accepted within the genus Cyathicula.However, the genus Pezoloma in the current circumscription (e.g., G a r c i a , Va n Vo o r e n 2005) is heterogenous and includes also taxa with a typical Hymenoscyphus-type of apical ring.The presence of an external gel layer which is used to define the genus is most probably a polyphyletic feature whereas differences in the apical ring type appear to have much more been conserved during evolution.
Based on the above data it seems wise to restrict the genus Pezoloma to species with a Calycina-type of apical ring.The morphology of R. ericae appears to be quite close to P. ciliifera and P. marchantiae.These in turn are surely very closely related to the type species of Pezoloma, P. griseum Clem., which has also a toothed margin.While the former two species are partly found growing on mosses (Sphagnum, Marchantia), P. griseum was reported by C l e m e n t s (1911) to grow on rootlets of Betula, suggesting a mycorrhizal life style similar as in R. ericae.Moreover, R. ericae was two times isolated as endophytic mycelium in a liverwort (Cephaloziella, Chambers et al. 1999).It is therefore believed that the type species of Rhizoscyphus and Pezoloma belong to the same genus.The following combination is therefore proposed.Molecular research on species of Pezoloma and a comparison with the sequence of R. ericae would highly be appreciated.
Pezoloma ericae (D.J. Read) Baral comb.nov.Basionym: Pezizella ericae D. J. Read, Trans. Brit. Mycol. Soc. 63: 381 (1974) The genus Moserella Pöder and Scheuer (1994), with the single species M. radicicola Pöder and Scheuer, was described as having very minute, strongly convex (capitate), white (to yellowish) apothecia with long and very slender stalks growing hypogeus on ectomycorrhizal rootlets of Picea abies.The ascus apex is very precisely illustrated, clearly showing the Calycina-type of apical ring.The microscopical features are not unlike our fungus, except for the spores which are said to be finely warted.Possibly Moserella is also closely related to Pezoloma.
There appears to exist no genus to which the three species with rose apothecia can safely be assigned.A new genus is therefore proposed.
The new genus is obviously more related to Stamnaria, Pezoloma and Calycina than to Hymenoscyphus.This is confirmed in the phylogenetic analysis of Z h a n g and Z h u a n g (2004) where H. rhodoleucus is found far away from the main part of Hymenoscyphus, but clustering together with Calycina herbarum (Pers.)Gray (Hyaloscyphaceae), whereas Pezoloma ericae is found in a separate clade quite far away from these two.

ECOLOGY AND PARASITISM
R. subcarneus appears to be a montaneous to subalpine and probably also boreal species, at least in Europe.A d e ´s (1935) collections were reported to have grown in the alps between 1500 and 1800 m on north-exposed slopes, the one of S v r č e k (1959) in c. 1200 m.In North America the species was collected, e.g., in the Adirondack Mountains (eastern U.S.A.) and in Canada.W h i t e (1942) assumed that the species is not uncommon in that area.The N-American collections were made in (August-)September, except for Peck´s type (July), while those from Europe were made during June-August.
R. subcarneus appears to be a parasitic species which has already been stated by Peck and H. S. Jackson (W h i t e 1942: 165), A d e (1935), and S v r č e k (1959).H. S. Jackson stated that "the apothecia are best located by first searching out the brown areas in green patches of mosses where the plants have evidently been killed by the fungus".Also Ade described a parasitic growth in his collections.The fungus is said to kill the tissue and thereby changes the colour of the moss population by forming circular yellow-brown lesions (Ade).More or less distinct lesions could also be observed in our specimen from Poland.