[ 55 ] Trans. Br. mycol. Soc. 88 (1), 55-61 (1987) Printed in Great Britain

ZEUS OLYMPIUS GEN. ET SP.NOV. AND NECTRIA GANYMEDE SP.NOV. FROM MOUNT OLYMPUS, GREECE

By D. W. MINTER, ROSALIND LOWEN CAB International Mycological Institute, Ferry Lane, Kew, Surrey, TW9 3AF, U.K.

AND S. DIAMANDIS Northern Forest Research Station, Loutra Thermi, Thessaloniki, Greece

Zeus olympius gen. et sp. nov. (Ascomycotina, , ) and Nectria ganymede sp. nov. (Ascomycotina, Hypocreales, Hypocreaceae) are described, illustrated and discussed, based on collections on dead branches of Pinus leucodermis on Mount Olympus in northern Greece. Zeus olympius appears to occur exclusively on Pinus leucodermis and Neetria ganymede exclusively on old ascomata of Zeus olympius.

During a recent visit by the senior author to Mount Ascomata apothecial, scattered or in small groups Olympus in northern Greece, two interesting fungi in rather pale dead twigs and small branches of were collected. They are described, illustrated and Pinus leucodermis, not closely associated with scars discussed in the following paragraphs. of short shoot needle bundles; when immature immersed below the bark and a black fungal Zeus Minter & Diamandis gen.nov. covering layer; when mature circular or rather Etym.: named after the king of gods in ancient angular, 0'5-2 mm diam; becoming erumpent, Greek mythology, who was believed to inhabit throwing back the bark and breaking the black Mount Olympus covering layer by several irregular radial splits to Hie ascophorus, ad ordinem Rhytismatalium et reveal the dark fawn-coloured hymenium (frag- familiam Rhytismatacearum pertinens, corticola, erum- ments of the bark often remaining attached to the pens, c1ypeo nigro ascomatum sane statu tectus, asco- black covering layer hiding its dark appearance), sporas habet elJipsoideas vaginis indutas mirabilibus et raising the substratum surface, but lacking a stalk, ascosnil in iodocoerulescentes. Habet ascomata Therryae and so not protruding beyond the level of the vel Coccomyceti aliquid similia, et ascos multarum dislodged bark covering-layers. Rhytismatacearum generibus similes, sed ascosporas In vertical transverse section: the hymenium adspectu mentem revocantes Ceratophacidium. arising from a yellow-brown subhymenium about typica : Zeus olympius sp.nov. 15-30 lIm thick composed of poorly defined textura intricata situated on a layer of colourless, poorly Zeus olympius Minter & Diamandis sp.nov. defined textura intricate embedded in mucus and (Fig. 1) about 100-200 pm thick; the blackened covering HaecspeciesincolatIovismontem, virgasPini leucodermis layer, approximately 30-80 lIm thick, composed et ramulos mortuos habitans. Habet ipsis e virgis for the most part of cells 5-12 lIm in diameter erumpentia ascomataquae corticem arboris reflectant ut forming a textura angularis or globulosa with a dark aperiantur. Ascomaquodque diametrum circa 0'5-2 mm brown pigment in the cell walls and between habet, et nigro a c1ypeo tegitur quod conpluribus in partibus statu maturo fissumest ut discum videatur. Asci individual cells; these cells gradually intergrading plus minusve cylindrici, circa 100-120 x 10-12'5,um via textura epidermoidea to become a textura oblita magnitudinis respectu, nil in apicibus crassiores, sed or intricata towards the torn edges of the covering saepiusaliquanto curvati, seriatimmaturant et ascosporas layer; heavily tanned tissue of the substratum octo quique ferunt, fissura apicali quique dehiscentes ut present below the ascoma, and on the outside of the ipsae liberentur. Ascosporae nee colore nee septis sese blackened covering layer. gaudentes, tenuitunicatae sunt et unicum habent parie- Asci developing from croziers, maturing in suc- tern. In quibusque adest vagina mucosa sane aliquid cession, with ascospores becoming visible a little eleganteret suaviter in apicibus fastigiata. Sporae autern, before elongation of the ascus is completed, sine vaginis circa 12-15 x 5-8 pm magnitudinis respectu metiuntur. uniformly thin-walled, with only one wall layer Emortuis in virgiset ramulis Pini leucodermidis Antoine visible at all stages of development, more or less ad altitudinem circa 1300m in Olympi monte collectusa cylindrical, slightly tapered towards the base, D. W. Minter & S. Diamandis, Graecia, 2 April 1986, rounded at the apex, sometimes also curved IMI 303384,holotypus. characteristically towards the apex, not turning New ascomycetes from Greece

A B 250 /l m

50 /lm

Fig. 1. Zeus olympius . (A) Habit view of ascornata on dead twig. (B) Detail of single ascoma as seen with the dissecting microscope . (C) Ascoma in vertical transverse section as seen at low power with a compound microscope. (D) Detail of part of ascoma in vertical transv erse section as seen at a higher power with a compound microscope (random stippling indicates fungal pigments, linear stippling indicates tanned deposits in the substratum. (E) Asci, ascospores and paraphyses. D. W. Minter, Rosalind Lowen and S. Diamandis 57 blue in iodine, with no discernible apical structures, been provided by Darker (1967), Korf (1973), containing eight ascospores arranged in a single Sherwood (1980) and Cannon & Minter (1986). file, opening by a single apical crack which may In making the following assessment, an extensive extend some way down the sides, becoming checklist of the families, genera, species and collapsed and bearing longitudinal creases and sub specific taxa of the Rhytismatales was consulted latitudinal striations after ascospore discharge, (M int er, unpubl.), and the recent monograph of approximately 110-120 x 10-12'5 pm in size when the other similar family, Phacidiaceae Fries, by mature and before ascospore discharge. DiCosmo, Nag Raj & Kendrick (1984) was also Ascospores colourless, thin-walled, smooth, more scanned. Evidence relating to mucous sheaths or less ellipsoidal, aseptate (perhaps sometimes around the ascospores and paraphyses was becoming r-septate around or after the time of particularly carefully checked because such sheaths discharge) bearing many guttules and surrounded often take on different appearances when different by an inconspicuous but broad mucous sheath stain s are used in slide preparations (Punithalingam which is flared out and markedly wider beyond each & Woodhams, 1984). end of the ascospore and constricted around its In external appearances the ascomata of Zeus equator, thus appearing to be two separate sheaths, olympius resemble those of Wallroth, approximately 12-15 x 5-8 pm excluding sheath Saccardo or bark-inhabiting species of when mature; sheaths appearing similar in eryth- de Notaris and, to a lesser extent, rosin in ammonia, cotton-blue and Meltzer's species of Massee and Ocotomyces Evans & reagent (without pretreatment in KOH). Minter. This general similarity with such taxa Paraphyses with unpigmented walls and yellow- continues when the ascomata are examined in ish globular contents, thin-walled, smooth, sparse- vertical transverse section, although on close ly septate, covered in a mucous sheath visible in inspection numerous small differences may be erythrosin in ammonia, cotton-blue and Meltzer's observed in tissue types and their extent and reagent (without pretreatment in KOH), filiform, distribution. None of these genera, however, apparently unbranched, slightly swollen at the apex contains species with ellipsoid ascospores and , which often appears ruptured in slide preparations, since ascospore shape remains an important sometimes anastomosing near the base, arising (like criterion at generic level within the Rhytis- the croziers) from the sub-hymenium. mataceae, the present species is clearly not congeneric with the se taxa. Sp ecimens examined : on dead twigs and small branche s Ellipsoid or nearly ellipsoid ascospores are of Pinus leucodermis at an altitude of about 1300 m, above known from a rather limited number of genera Prionia by the path to the mountain refuge on Mount within the Rhytisrnataceae, notably Cerato- Olympus, Greece, D . W. Minter & S. Diamandis.z April phacidium Reid & Pirozynski, Myriophacidium 1986, IMI 3°3384, holotype, Isotype in the herbarium of Sherwood, Darker and Vladracula the Pathology Division of the Northern Forest Research Station, Loutra Thermi, Thessaloniki, Gree ce. On dead Cannon, Minter & Kamal. Of these, Ploioderma branches of Pinus leucodermis at an altitude of about contains strong needle pathogens, mainly of north 1300 m, above Prionia, on Mount Olympu s, Greece, American pines, while species of both Myrio- S. Diamandis, late April 1986, IMI 3°3844. phacidium and Vladracula tend to be leaf-inhabiting and , in any case, lack the bulky nature of ascomata of the present species . Ceratophacidium is interest- ing because its only species also has ascospores Discussion of Z. olympius with a mucous sheath drawn out at both ends . Asci Many features of this fungus suggest it should be of this species were, however, reported by Reid & placed in the order Rhytismatales and family Pirozynski (1966) to have each a minute apical ring Rhytismataceae Chevallier (syn. Hypodermataceae turning blue in iodine (not noticed by Bonar (1942) Rehm): in particular the paler appearance of the when the type was first described), and the twigs ; the immersed apothecial ascomata, which ascomata are much smaller, less pigmented, and open by splits in the blackened covering layer- less bulky than those of the present species. swelling to reveal their discs in wet conditions and Furthermore, on careful inspection of Reid & shutting again when dry; the iodine-negative asci Pirozynski's (1966) illustration and of a slide made with no apical modification, which open with from part of the type collection of the type species irregular apical cracks (a particularly important of Ceratophacidium (IMI 120921 ), the tapered feature (M int er & Cannon, 1984)); the probable mucous sheaths around ascospores in Cerato- violent discharge and the colourless asco- phacidium (which Reid & Pirozynski stated were with large mucous sheaths. All of such examined in an iodine mountant) seem rather features of this family have been reviewed by different from the flared and divided sheaths in Cannon & Minter (1986), and keys to genera have Zeus. New ascomycetes from Greece

B . - _. -=----

Fig. 2 . Nectria ganymede (IM I 305041), mountants (A) Lactophenol, (B-1) lactophenol and cotton-blue. (A) Ascoma in vertical transverse section with immature ascoma and cottony mycelium. (B) Detail of wall of ascoma. (C) Cells of ascoma in surface view. (D) Ascus and ascospores. (E) Thickened hyphal cells. (F) conidiophore. (G) ' Macroconidia ' from SNA after 1 month, (H) ' Macroconidia ' from PSA after 1 month. (I) Distorted old conidium. D. W. Minter, Rosalind Lowen and S. Diamandis 59 It is therefore concluded that the present species uniformly thin-walled, with only one wall layer represents a new of the Rhytismataceae. visible at all stages of development, clavate, Almost nothing is known of its biology: it fruits as rounded at both apex and base, not turning blue in a saprobe, producing asci and ascospores in spring iodine, with no apical structures visible, attaining and releasing them in wet conditions; it appears to full size, 90-100 x 10-14 psn, before ascospore be restricted to pine twigs; no anamorph has been discharge, 8-spored; the ascospores arranged observed and the mode of colonization of substrata unevenly within, uniseriate near the base and apex, is not known. It may inhabit living twigs as but irregularly biseriate above the mid-point. an endobiont, waiting until the death of the Ascospores 8-23 (-26) x 6-8 pm, tending to before fruiting, or its whole life cycle may be deviate from ellipsoid, one side curved more than saprobic: ascomata were observed at the same stage the other, often wider in the top half, colourless of development on branches and twigs of different and r-septate, becoming light brown, 3-septate ages in the type collection, but that provides little and spinulose (visible with oil immersion) when further evidence for either an endobiotic or a mature; usually containing 1-2 guttules per cell, saprobic existence. with a thickened ring visible at the edges of the Nectria ganymede Lowen & Minter sp.nov. central septum which is occasionally slightly (Fig. 2) constricted; germinating from all four cells. Paraphyses not observed. Etym.: named after Ganymede, 'a beautiful youth of Phrygia who was taken up to heaven by Characteristics in pure culture: very slow growing, Zeus to become cup-bearer to the gods and to sit attaining 3 mm diam on PCA+, OA + and on Zeus' lap' (Lernpriere, 1788) CMA after 7 days and on SNA after 19 days; slimy, with sparse aerial mycelium; colour no. 35, Hie fungus ad genus Nectriae et subgenus episphaeriae strong red orange (Kelly, 1965), plate 7, 8B pertinens, habet ascornata perithecialia, rubra, pyri- (Kornerup & Wanscher, 1978), becoming colour formia, sparsa, circa 30011m diam, lateraliter collapsa. Asci adsunt clavati, apice et in fundamento rotundati, no. 38, dark red orange (Kelly, 1965), plate 7, 6C, 90-100x circa 14flm, octospori, irregulariter biseriati. brownish orange (Kornerup & Wanscher, 1978), Ascosporae sunt 8-23 x 6-8 flm, ellipsoideae, primo sin with a raised central area which is darker, and a colore et uniseptatae, deinde leviter brunneae et trisep- reverse which is slightly lighter; producing tatae. Paraphyses non visaesunt. a Fusarium 'macroconidial' anamorph, but no Hymenioin Zenos olympi derelictaad altitudinem circa 'microconidial' anamorph, after 7 days. 1300m in Olympi monte collectus a S. Diamandis, Mycelium colourless, thin-walled, smooth, sep- Graecia,April 1986,1M1 305041, holotypus tate, branched at acute angles; producing thick- Ascomata perithecial, translucent, colour no. 13, walled, enlarged, globose to ellipsoid or almost deep red (Kelly, 1965), plate 10, 8C (Kornerup & rectangular cells, 6-22 x 4-6 flm, in chains. Coni- Wanscher, 1978), 290-300 pm diam, pyriform, diogenous cells colourless, thin-walled, smooth, often collapsing laterally, scattered or in groups of terminal and intercalary, produced directly or on up to 10, immersed at the base in the hymenial short branches, sometimes in whorls on undifferen- remains of ascomata of Zeus olympius, surrounded tiated hyphae, 12-16 x 2'5-3 flm, more or less by a white, sparse cottony mycelium; ascomatal cylindrical, but tapered slightly at the base and walls turning darker red in KOH then yellow in strongly towards the apex, with apical wall- lactophenol (KOH+), composed of cells approxi- thickening sometimes visible. Conidia colourless, mately 6-10 flm diam forming a textura angularis falcate, thin-walled, smooth, aseptate or r-septate to epidermoidea in surface view. (occasionally 3- or more septate or swollen and In vertical transverse section: ascomatal walls distorted in cultures 6 weeks old), strongly curved, 10-20 flm thick, forming a single layer several cells with a Fusarium-like foot cell sometimes visible, deep, composed of long thin cells 6-10 x 2-4 flm <)-18 x 2-2'5 flm on SNA, 16-28 x 1'5-2'5 flm on with thickened and fused walls about t :5 pm thick, PSA after 1 month. Conidial development by a and towards the base of the ascoma of more angular replacement wall-building apex system with per- cells 6-10 x 7-8 flm with thinner walls; inner current, enteroblastic proliferation and schizolytic surface of ascomatal walls lined with colourless secession, without elongation of the conidiogenous angular cells; papilla truncate at the apex, cell. 100-150 11m wide, the ostiole lined with slender hyphal cells which also extend sometimes down- Specimen examined: on the hymenial remains of Zeus olympius on dead twigs and small branches of Pinus wards from the upper colourless angular cells; leucodermis at an altitude ofabout 1300m, abovePrionia, centrum expanding in water, causing sections to on Mount Olympus, Greece, S. Diamandis, late April curl and break. 1986, 1M! 305041, holotype;isotypeNY. Livingcultures Asci arising mostly from the base of the ascoma, deposited in 1M1 305041, ATCC, NY (CTR). 60 New ascomycetes from Greece olympiusand its pine substratum. This fits well with Discussion of N. ganymede what is known of several other fungal associates of Nectria (Fr.) Fr. contains fungi with brightly the Rhytismatales (Darker, 1932; Mitchell et al., coloured, fleshy, perithecial ascomata which may 1976; Reid & Minter, 1979). or may not be on or in a stroma, and with asci having a single thin wall. Saccardo (1878, 1883) restricted Nectria to species with r-septate asco- CONCLUSIONS spores, and described several generic and sub- At first sight it is remarkable that such a generic segregates based on single characters. conspicuous fungus as Z eus, on such a well- Following Saccardo (1883), Ne ctria ganymede collected substratum, in Europe, should appear to conforms to the subgenus Hyphonectria Sacc., be both a new species and a new genus. It is, comprising species with ascomata on a cottony however, merely further evidence that the fungi of mycelial base. It might equally be placed in Greece are still very poorly known : this is, indeed , Calonectria de Not. because of its multiseptate the second large and conspicuous ascomycete of ascospores with a length-width ratio of less than this family to be described from pines from Greece 20 :1 (Rogerson, 1970). within a decade, the other being It has become clear in recent years, however, that torres-juanii Diamandis &Minter(1979).Thegenus such divisions of Nectria based on single characters Nectria is also usualIy considered to have been wel1 have led to the placement of closely related species studied in Europe, and the discovery of a new in diverse genera within the Hypocreales. Recent species from this continent is also rather surprising. researchers have accordingly attempted to divide The implication is clear: with further detailed Nectria into more natural groups of species based studies of the Greek forests and National Parks, on the correlation of many anamorphic and many beautiful and interesting new fungal species teleomorphic characteristics, with an emphasi s on remain to be discovered . the structure of the ascomatal wall and the form of the anamorph (Booth, 1959, 1978; Rossman , 1983; Drs C. T. Rogerson and G. J. Samuels are Samuels, 1976, 1978; Samuels & Rossman , 1979). thanked for their advice on the of Ne ctria Following these authors, N. ganymede is best species associated with other fungi. Dr E. Puni- placed in Nectria, within the episphaeria group, thalingam and Mr s J. E. Woodhams are thanked because of its ascomatal characteristics (simple wall for advice and technical assistance concerning ways structure, lateral collapse of walls, KOH + wall- of examining mucous sheaths of ascospores. colour reaction), its light brown spinulose asco- spores, its slow-growing Fusarium anamorph and REFERENCES its ecological association with other fungi. Nectria ganymede may be distinguished from N. BONAR, L. (1942). Studies on some California fung i. episphaeria (T ode:Fr.) Fr. by its larger, 3-septate Mycologia 34, 180-192. BOOTH, C. (1959). Studies of Pyrenomycete s : IV . ascospores, its larger asci and its unusual habitat. N ectria (Part 1). My cological Papers (CM f) 73, 1-115. The Fusarium anamorph is close to F . aquae- BOOTH, C. (1978). Presidential Address. Do you believe ductuum Lagerheim, with its characteristic 0- to in genera? 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(Received for publication 14July 1986)