Reassessment of the bondarzewiaceae (aphyllophorales)

Trans. Br. mycol. Soc. 58 (1),49-58 (1972) Printed in Great Britain

REASSESSMENT OF THE BONDARZEWIACEAE

(APHYLLOPHORALES) By D. N. PEGLER

Royal Botanic Gardens, Kew

T. W. K. YOUNG Botany Department, Chelsea College, Hortensia Road, London and Birkbeck College, London AND

(With Plates 6-9 and

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Hyphal systems of the basidiocarps of Bondarzeuiia montana, B. berkeleyi, Amylaria himalayensis, Hericium erinaceus and Meripilus talpae have been investigated and the surface structure of the basidiospores determined by light, transmission and scanning electron microscopy. It is concluded that the Bondarzewiaceae, as constituted at present, should be limited to the single genus Bondarzeuna.

The Bondarzewiaceae is a small family accommodating polyporoid, clavarioid and hydnoid members of the Aphyllophorales in which the component species are apparently related largely on the basis of similarities shown in the structure of their spores. The advisability of erecting a family on the characters selected has been questioned on more than one occasion, Singer (1940) proposed a new genus, Bondareetoia, for the European polyporoid fungus Polyporous montanus (Quel.) Ferry, distinguished from other genera of the Polyporaceae by the ornamented, amyloid basidiospores. Bondarzew & Singer (1941) subsequently added P. talpae Cke and P. berkeleyi Fr. to Bondarzetoia, placing the genus in the Scutigeraceae. Finally, a fourth species, B. perniciosa Singer, was described from Argentina by Singer (1953). Bondarreioia remained in a somewhat isolated position until Corner (1955) described a new clavarioid genus, Amylaria, based on collections from the Himalayan areas of Bhutan and Nepal. Amylaria himalayensis Corner also produces small basidiospores with an amyloid, ridged ornamentation and reference was made to the similarity of the spores of Amylaria to those of Bondarreioia, and to the dimitic nature of their hyphal systems. However, Corner (1955) has suggested Amylaria to be more closely related to Hericium, which produces large, fleshy hydnoid basidiocarps, than to Bondarzetoia. The amyloidity of the spores is envisaged as a phyletic link between the erect, flabellately branched fruit-bodies of Amylaria and the horizontal decurved ones of Hericium. Kotlaba & Pouzar (1957) erected the Bondarzewiaceae to accommodate Bondarzetoia, Amylaria and Hericium and gave the following diagnosis - 'Fungi lignicoli; molliter carnosi, annui, carposomatibus polyporoideis, hydnoideis vel clavarioideis. 4

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Sporis hyalinis, verrucosis, asperulatis vel laevibus, fortiter amyloideis.' Clearly, the family was based only on the fleshy consistency of the context and the amyloidity of the spores. Donk (1962) regarded these characters, even in combination, as apparently insufficient to indicate a natural family relationship. Later Donk (1964) excluded Hericium when he designated it the type genus of a new family, the Hericiaceae, owing to the presence of an extensive system of gloeo-vessels in the context terminating in hymenial gloeocystidia. Amylaria was only retained in the Bondarzewiaceae provisionally, because detailed analyses of the hyphal structure were necessary before any definite relationship with Bondarreuna could be confirmed. In this paper, hyphal systems of the genera in the Bondarzewiaceae and surface structure of the spores are compared, and the systematic position of Bondarzeuna evaluated. MATERIALS AND METHODS

For light microscopy, spores and hyphae were mounted and examined in 10 % potassium hydroxide, cotton blue in lactic acid, and aqueous cresyl blue. Photographs were taken with a Nikon microscope. Fine surface structure of spores was determined by transmission and scanning electron microscopy. Carbon replicas of spores were prepared according to Pegler & Young (1971) and examined and photographed using a Zeiss E.M. 9A electron microscope. Samples for scanning electron microscopy were prepared by attaching a piece of undeveloped film, emulsion surface uppermost, with double-sided adhesive tape to a specimen stub. A drop of distilled water was pipetted onto the emulsion and hymenial fragments were placed in the water and gently agitated with a needle point to release spores. The preparation was allowed to dry in air. Specimens were coated with gold/palladium alloy on a rotating turntable in an Edwards high vacuum coating unit and were examined in a Cambridge Instrument Company Stereoscan 2 A electron microscope. RESULTS

Basidiospores BONDARZEWIA MONTANA (Fr.) Sing., Rev. Mycol. 5, 4 (1940).

Specimen examined. Czechoslovakia, Dolroc Virgin Forest, 24 Sept. 1965, D. A. Reid. Under the light microscope the spores measure 5'6-8 x 5-7 (6'7 x 6'0) Ilm without ornamentation; the ornamentation ranges from 0'3 to 0'75 pm high, Q = r-r , Spores are subglobose to broadly ellipsoid, with an applanate suprahilar region, cyanophilous in cotton blue, non-metachromatic with cresyl blue, and the ornamentation is strongly amyloid with Melzer's reagent. In micrographs using carbon replicas and stereoscan techniques, the individual ridges of the ornamentation are approximately rod-shaped in plan view, and rectangular in side elevation, slightly broader towards their bases, with the slightly swollen apices gently undulate or centrally depressed. The surface on the sides of the ridges is either smooth

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or shows several faint vertical ridges. The spore surface between the ridges is smooth. The length of each ridge is about twice that of the width. The ridges are arranged in approximately 11-15 subparallel rows with a longitudinal orientation following the strongly curved distal abaxial surface. The total number of visible ridges varies from 30 to 56 (average 39), although in light micrographs this number is reduced to 11-18 because only those ridges with perisporial fragments are observed on staining. The adaxial surface bears much lower ridges, more crowded, with little or no obvious orientation (PI. 6, figs. 2, 6; PI. 7, figs. 9, 10; PI. 8, figs. 13, 14; PI. 9, fig. 23; Text-fig. IA).

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F G Text-fig. I. A-E. Camera lucida drawings of basidiospores as observed in Melzer's Reagent (x 1000). A, Bondarzeuna montana; B, B. berkeleyi (type); C, Amylariahimalayensis (type); D, Hericium erinaceus; E, Meripilus talpae (type). F, G. Diagrams comparing the arrangement of the exosporial ridges and the longitudinal axis of the mature spore. F, Russula laurocerasi with a delimited suprahilar plage on the adaxial surface; G, Amylaria himalayensis with the entire adaxial surface confined to the suprahilar region.

BONDARZEWIA BERKELEYI (Fr.) Bond. & Sing., Annls Mycol. Berl. 39, 47 (194 1 ) . Specimen examined. U.S.A., North Carolina, 547, Herb. Berkeley (K, holotype). Under the light microscope the spores measure 6-8'3 x 6-7'5 (7'2 x 6'5) p,m without ornamentation; the ornamentation ranges from 0'4 to 1'2 p,m high, Q = 1'1. Spores are subglobose to broadly ellipsoid, with a slightly applanate suprahilar region, cyanophilous, non-metachromatic, and the ornamentation is strongly amyloid. Ultrastructural observations reveal a similar pattern of ridges to those found in B. montana. The total number of ridges varies from 30 to 33, whilst in light micrographs the apparent number is reduced to 11-16. The individual ridges are slightly higher than those of B. montana (PI. 6, fig. I; PI. 8, fig. 15; PI. 9, figs. 25, 26; Text-fig. IB).

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BONDARZEWIA TALPAE (Cke)Bond. & Sing., Annls Mycol. Berl. 39, 47 (194 1). Specimen examined. Brazil, Minas Geraes, on stumps, Glaziou 16679 (K, holotype of B. talpae); Sierra Leone, Njala, 12 June 1952, F. C. Deighton M4732 (P. mesotalpae). Under the light microscope the spores measure 5'7-7'5 x 4'2-6 (6'3 x 5'3) /lm, Q. = 1'19. Spores are subglobose to broadly ellipsoid lacking any applanation or depression in the suprahilar region, with wall surfaces appearing smooth in potassium hydroxide, lactophenol and Melzer's reagent. They are cyanophilous, non-metachromatic, and completely inamyloid even after hydrolysis with ammonium hydroxide. From the evidence of carbon replicas and scanning electron micrographs the wall surface is smooth ultrastructurally. A similar investigation of Polyporus mesotalpae Lloyd confirms the spores as smooth, and the two fungi are considered conspecific (PI. 9, figs. 21, 22). The absence of both surface ornamentation and an amyloid perisporium casts doubt on the retention of this species in Bondarretoia. Reid (1963) transferred the species to Meripilus Karst. on the basis ofbasidiocarp habit, hyphal analysis and spore characters, and this view was subsequently supported by Fidalgo & Fidalgo (1967)' AMYLARIA HIMALAYENSIS Corner, Bull. Br. Mus. (Nat. Hist.) I, 197 (1955)· Specimen examined. Bhutan, Trashiyangsi, Rocha Chu Valley, alt. 3000 m, 21 Sept. 1934, Ludlow and Sherriff 974 (K, holotype). Under the light microscope the spores measure 5-6'5 x 4'5-5'2 (6 x 5'7) /lm without ornamentation and the ornamentation is 0'3-0'75 /lm high, Q. = I '05. Spores are subglobose to broadly ellipsoid with a slight suprahilar depression, cyanophilous, non-metachromatic, and the ornamentation of elongate, orientated ridges is very strongly amyloid. In micrographs, using carbon replicas and scanning microscope techniques, the ridges or crests of the ornament are an elongate rod-shape in plan view, and crescent shape in side elevation, with the length three to eight times that of the width. Each ridge is slightly broader towards its base, although not lobed at the upper surface which may, however, be irregular in outline. The surface of the ridges is smooth yet uneven. They show a strongly subparallel orientation on the abaxial and lateral surfaces of the spore, radiating from the small and restricted adaxial surface. These are densely crowded, with very little of the intervening spore surface visible, and arranged in approximately 10-14 rows. The individual ridges or crests extend from one quarter to one half of the total length of the spore and the number varies from 19 to 40 (average 29). In light micrographs, however, this number is reduced to 7-1 lowing to the restricted distribution of the fragmented perisporium. On the adaxial surface the exosporium outgrowths are confined to densely crowded, hemispherical verrucae, lacking any orientation. The surface structure of the spores of Amylaria is similar to that of Bondar-

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:::;ewia in the production of subparallel, longitudinally orientated, exoepisporial ridges or crests, the strongly expanded abaxial surface and correspondingly unexpanded adaxial surface, the absence of a defined plage characteristic ofthe Russulaceae, and the retention of a very strongly amyloid perisporium. Spores of the two genera differ in size, form of the exo-episporial outgrowths, and the degree of crowding of the outgrowths (PI. 6, figs. 4, 5; PI. 7, fig. II; PI. 8, fig. 16; PI. 9, fig. 24; Text-fig. I C). HERICIUM ERINACEUS (Bull. ex Fr.) Pers., Mycol. Europ. 2, 153 (1825). Specimen examined. England, Berkshire, Bagley Wood, 15 Sept. 1969, D. N. Pegler. Under the light microscope the spores measure 5-6'2 x 4-5'3 (5.6 x 4'5) ftm, Q. = 1'24, with the wall surface smooth to finely asperulate but the ornament not measurable. Spores are subglobose to broadly ovoid, lacking any suprahilar depression or applanation, cyanophilous, non-metachromatic and both the smooth and the ornamented spores are uniformly amyloid. From carbon replicas and scanning micrographs the spore surface ranges from smooth to weakly rugose, or fairly strongly ornamented with numerous low, verrucose to elongate rod-like exo-episporial outgrowths. There is no indication of any regular orientation and no pattern of distribution comparable to Bondarzetoia or Amylaria. The small hilar appendix bears an open-pore hilum (PI. 6, figs. 3, 8; PI. 8, fig. 12; PI. 9, figs. 17-20; Text-fig. I D). Hyphal analysis BONDARZEWIA MONTANA AND B. BERKELEYI In both species the hyphal system forming the mid-context is dimitic with an abundance of both generative and skeletal hyphae. The generative hyphae are loosely interwoven, frequently branched, hyaline with very thin walls, and regularly septate but lacking clamp connexions. The generative hyphae of B. montana measure 2-5 (-7) ftm diam, and of B. berkeleyi 2-6 ftm diam. The skeletal hyphae in both fungi show an essentially radially parallel arrangement, and are hyaline to pale melleous, with a thickened wall (0'5-3'0 ftm thick) which often obliterates an otherwise continuous lumen. They are non-septate, generally sinuous and unbranched, although in both species some irregular branching has been observed to give rise to a condition approaching arboriform skeletal or binding hyphae. In B. montana the skeletal hyphae measure (1'5-) 3-7 ftm diam, and in B. berkeleyi 1'5-6'0 ftm diam. Neither gloeo-vessels nor conducting elements have been observed in either species (Text-fig. 2 A). AMYLARIA HIMALAYENSIS The hyphal system is at first monomitic but finally becomes dimitic with generative and skeletal hyphae. The generative hyphae are the dominant type, measuring 1'5-4 ftm diam, and are hyaline, very thin-walled, only rarely branched, and septate bearing large and conspicuous clamp connexions. The skeletal hyphae or unbranched binding hyphae (according to

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Fig.

2.

For legend see facing page.

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Corner) develop secondarily and are not so frequent as the generative hyphae. They are mostly unbranched, although not always so, hyaline with a thickened wall (-1·5 pm diam) yet always retaining a continuous lumen, and measure 1.5-4.0 pm diam. The skeletal hyphae ramify through the system binding together the essentially vertically parallel generative hyphae. There is no conducting system present (Text-fig. 2B).

MERlPILUS TALPAE The hyphal system is monomitic with generative hyphae but these may be modified to form a discontinuous conducting system. The generative hyphae are loosely and irregularly interwoven without any orientation, 2-10 (-14) pm diam, hyaline or pale brownish, with a thin to slightly thickened wall (-1·5 pm), scarcely to frequently branched, septate and often constricted at the septa, lacking clamp connexions. The conducting elements are modified generative hyphae with abundant granular, yellowish brown contents, sometimes extending into the hymenium by not forming typical gloeocystidia. The basidiocarps of M. talpae clearly differ from those of Bondarzetoia in the hyphal system. In some ways, the hyphal system is also atypical of Meripilus as exemplified by M. giganteus (Pers. ex Fr.) Karst. Fidalgo & Fidalgo (1967) showed that the context of M. giganteus is fibrous in texture, consisting of radially orientated hyphae, whilst that of M. talpae is spongy to crumbly, with the hyphae lacking any true orientation. These observations are confirmed in the present study. HERlClUM ERlNACEUS The hyphal system of the context is monomitic and lacks a conducting system, although generative hyphae in the trama of the spines become modified and filled with oleaginous contents to form an extensive conducting system which terminates in the hymenium as abundant and conspicuous gloeocystidia (PI. 9, fig. 27). The generative hyphae are loosely interwoven, 2.5-6.0 pm diarn, with hyaline, thickened walls (0.5-3.0 pm thick) and a very narrow continuous lumen, frequently branched, septate with large conspicuous clamp-connexions (Text-fig. 2 C). DISCUSSION

The Bondarzewiaceae was erected on the basis of the texture of the basidiocarp context and the structure of the spores. Context texture is directly related to the nature of the hyphal systems involved, thus it could be expected that closely related genera would show basic similarities in their hyphal systems. Indeed, hyphal analysis has been shown to be of Text-fig. Il. Hyphal systems taken from the mid-context of the basidiocarp, A, Bondarzewia montana, dimitic, with the generative-hyphae lacking clamp-connexions ; B, Amylaria himalayensis, dimitic, with abundant clamp-connexions on the generative-hyphae; C, Hericium erinaceus, monomitic, with the thick walled generative hyphae bearing clampconnexions: D, Meripilus talpae, monomitic, with the generative-hyphae lacking clampconnexions. (All x 1000.)

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primary taxonomic importance throughout the families of the Hymenomycetes, particularly within the Aphyllophorales. A comparative study of the hyphal systems found in the genera assigned to the Bondarzewiaceae has revealed fundamental differences and little or no correlation with respect to the characters considered. A dimitic hyphal system is present in Bondarzetoia and Amylaria, but secondary hyphae are not developed in either Hericium or Meripilus. Clamp-connexions are found in Amylaria and Hericium but are lacking from Bondarreuna and Meripilus. A gloeo-system is strongly developed in Hericium particularly towards the hymenium but is absent from Bondarzeioia and Amylaria. Similarly, a comparative study of the spores brings into question the concept of the family envisaged by Kotlaba & Pouzar (1957). Although Bondareetoia and Amylaria produce strongly amyloid, verrucose spores, these characters are also evident in members of the Russulaceae (Agaricales) and many genera in the Astrogastraceous series. The latter differ only in the lack of any parallel orientation of the ornamentation. Orientation of crests and ridges has been developed in Boletellus Murr. of the Strobilomycetaceae, although no amyloidity has been observed in this family. The spores of B. montana, as observed under the light microscope, have always been compared with those of the Russulaceae, with a surface ornamentation of short isolated crested ridges which stain violaceous-black with Melzer's reagent. Besson (1970) published micrographs showing ultrathin sections of the spores of B. montana which demonstrated the similarity of the wall layers to those of the Russulaceae. A thin, inner endosporium is surrounded by a thick episporium which in turn is coated by a continuous exosporium giving rise to the large crests and ridges forming the ornamentation. Finally, the exosporial outgrowths are partly covered by a discontinuous or fragmenting perisporium. It is the perisporial remains which react with iodine and stain deeply in mounts with Melzer's reagent. The final ornamentation is therefore the result of two overlapping processes, the production of the exo-episporium and disintegration of the perisporium. However, the pattern of ornamentation is very different in Bondareetoia in which the ridges are basically orientated longitudinally, whereas in Russula the pattern is irregular without definite orientation (PI. 6, fig. 7). In Bondarzetoia, approximate rows of ridges radiate from the spore base, i.e. the hilar appendix, appearing subparallel over the broad, strongly inflated abaxial and lateral surfaces. The flattened adaxial surface is covered by densely crowded, more irregular outgrowths, probably through lack of expansion in this region during sporogenesis, no separation or orientation having occurred. In this way the adaxial suprahilar area is formed by the entire unexpanded adaxial surface, and not by a specialized and well-developed plage as in the Russulaceae (Text-fig. If-g). The hilar appendix is well developed although not as strongly as in the Russulaceae and the hilum is of the nodulose type in both. The inclusion ofpolyporoid, clavarioid and hydnoid genera in the same family is not altogether unusual, for several examples may be found in such well-established families as the Hymenochaetaceae and the Thelephoraceae although in the Bondarzewiaceae a comparative analysis of the macroscopic and microscopic characters argues against family relationship

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suggesting, on the present evidence, the family to be confined to the single genus Bondarzetoia, as proposed by Donk (1962). Hericium is the type genus of the Hericiaceae, which includes a natural group of genera possessing a gloeo-system and amyloid spores, whilst Amylaria might be retained in the Clavariaceae, being possibly linked phyletically with the amyloid-spored species of Muctonella Fr. The authors are grateful to the Science Research Council for supporting this study; to Mr A. D. Greenwood (Botany Department, Imperial College, London) for use of the scanning electron microscope; to Miss J. Fillery for technical assistance and advice on preparation of specimens; and to the Cambridge Instruments Co. Ltd. for permission to publish PI. 6, fig. 7. REFERENCES

BESSON, M. (1970). Ultrastructure de la paroi sporique amyloide et ornee de quelques Hymenomycetes. Compte rendu hebdomadaire des seances de l'Academie dessciences, ser. D,

271 , 964-967.

BONDARZEW, A. & SINGER, R. (1941). Zur Systematik der Polyporaceen. Annalesmycologici 39,43-65· CORNER, E.]. H. APUD BALFOUR-BROWNE, F. L. (1955). Some Himalayan Fungi. Bulletin of the British Museum (Natural History) I, 187-218. DONK, M. A. (1962). Notes on resupinate Hymenomycetes 6. Persoonia 2, 217-238. DONK, M. A. (1964)' A conspectus of the families of Aphyllophorales. Persoonia 3, 1993 24. FIDALGO, O. & FIDALGO, M. E. P. K. (1967)' Polyporaceae from Trinidad and Tobago 2. Mycologia 59, 833-869. KOTLABA, F. & POUZAR, Z. (1957). Notes on classification of European pore fungi. Ceska mycologie II, 152-17°. PEGLER, D. N. & YOUNG, T. W.K. (1971). Basidiospore morphology in the Agaricales. Cramer Press. REID, D. A. (1963). New or interesting records of Australasian Basidiomycetes. Kew Bulletin 17, 267-308. SINGER, R. (1940). Notes sur quelques Basidiomycetes. 6e serie. RevuedeMycologic, 5, 3-13. SINGER, R. (1953). Four years of mycological work in South America. Mycologia 45, 86 5- 89 1. EXPLANATION OF PLATES

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Figs. 1-4. Light micrographs of basidiospores stained to show the amyloid ornamentation. I, Bondarzewia berkeleyi (type). x 2000; 2, B. montana. x 2000; 3, Hericium erinaceus. x 2000; 4, Amylaria himalayensis (type). x 2000. Figs. 5--8.Scanning micrographs. 5, Amylaria himalayensis, abaxial surface. x 10200; 6, Bondarreuna montana, lateral view. x 10000; 7, Russula laurocerasi, abaxial surface, x 4800; 8, Hericium erinaceus, single spore and basidium, x 5800. PLATE

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Figs. 9-1 I. Replicas showing surface ornamentation. 9, Bondarzetoia montana, exosporial ridges in profile, x 31500; 10, B. montana, ridges in surface view; I I, Amylaria himalayensis, ridges in surface view, x 4075°. PLATE 8 Figs. 12-15. Carbon replicas. 12, Hericium erinaceus, detail of surface ornamentation, x 17500; 13, Bondarzsuna montana, x 8600; 14, B. montana, x 19600; 15, B. berkeleyi, x 7980. Fig. 16. Scanning micrograph. Amylaria himalayensis, x 10200.

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Figs. [7-23 . Carbon replicas. 17-20, Hericiumerinaceus, showing rang e of sur face ornamentation, all x 7000; 21, Meripilus talpae (type), lateral view, x 7000 ; 2 2, Polyporus mesotalpae, adaxial surface, x 7000; 23, Bondarzeuiia montana, adaxial surface , x 8000 . Fig. 24. Amylariahimalayensis (type ), la teral view, scanning micrograph, x 10 200 . Figs. 25, 26. Bondarzeuna berkeleyi (type ), repli cas showing surfac e ornamentati on, x 7000. Fig. 27. Hericium erinaceus, gloeocystidiurn an d basidia , scanning micrograph, x 580 0.

(Acceptedfor publication 6 July 197 I)

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