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The genus Pyrenomyxa and its affinities to other cleistocarpous Hypoxyloideae as inferred from morphological and chemical traits

     Naturwissenschaftlicher Verein Wuppertal, Mykologische Sektion, Pahlkestraße 17, D-42115 Wuppertal, Germany, and Bayer Health Care, Pharma Division, Natural Products Research, Wuppertal, Germany

Thomas Læssøe

     University of Copenhagen, Institute of Biology, Department of Microbiology, Øster Farimagsgade 2D, DK-1353 Copenhagen K, Denmark

Larissa Vasilyeva

     Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 

Types and authentic specimens of Hypoxylon piceum, Pulveria porrecta, and Pyrenomyxa invocans were studied for morphological traits and extrolite (= secondary metabolite) profiles generated by analytical HPLC with UV-visual and mass spectrometric detection. The orange stromatal pigments of P. invocans are rubiginosin A and mitorubrinol. It lacks three different types of extrolites (BNT, macrocarpone and hypomiltin) that are known from Hypoxylon taxa and occur in H. piceum and P. porrecta. In agreement with morphological traits, the latter two names are regarded as synonymous and transferred to Pyrenomyxa. Another species from Eastern Russia, Pyrenomyxa morganii sp. nov., is recognized. It contains yet unidentified azaphilones besides BNT and orsellinic acid, and its culture produces 5-methylmellein and a virgariella-like anamorph. These findings suggest a close relationship of Pyrenomyxa to Hypoxylon and emphasize the utility of chemotaxonomic traits for fungal taxonomy in general. Pyrenomyxa is accepted ad interim until the phylogenetic relationships among Hypoxylon have been further evaluated by means of chemotaxonomic, morphological and molecular methods.

Key words: Xylariales, Xylariaceae, chemotaxonomy, systematics, extrolite

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 
Several taxa are considered to belong to the Xylariaceae despite the fact that they lack the salient features of amyloid apical apparatus and forcible discharge of their ascospores. Their stromata are infrequently encountered in temperate North America and Eastern Russia on decorticated wood. Typically they develop beneath loose bark, although one sample was found in a hollow trunk. Their gross stromatal morphology suggests a Hypoxylon, and indeed the first of these ‘aberrant’ taxa was described by Ellis (1883) as Hypoxylon piceum Ellis from Iowa. He noted the conspicuous, laterally compressed ‘navicular’ ascospores but found no asci. Some years later, Morgan (1895) described Pyrenomyxa invocans Morgan, based on material collected from hickory (Carya sp.) in Ohio. He recognized the ascospores to be ‘lying edge to edge in oval to spherical clusters’. Interestingly, the generic name relates to its stromatal habit, which is reminiscent of the aethalial stage of a myxomycete (Morgan 1895). Lloyd (1912, 1924) synonymized P. invocans under Hypoxylon turbinatum Berk., i.e. Phylacia turbinata (Berk.) Dennis. Malloch and Rogerson (1977) erected Pulveria to accommodate Pulveria porrecta Malloch & C. T. Rogerson. They provided a detailed description of its teleomorph, including the development of globose, evanescent asci, which they recognized to be derived from the typical centrum structure of the Xylariaceae. They concluded that the cleistothecial features of Pulveria are adaptations to its habitat, reasoning that active discharge of ascospores becomes superfluous for fungi whose stromata develop under the bark of the host. Hence, they regarded Pulveria as a simplified rather than a primitive xylariaceous genus with affinities to the Hypoxyloideae.

However, Malloch and Rogerson did not mention similarities to the aforementioned taxa. Neither did Speer (1980), who found similar ascal structures to Pulveria in Phylacia Lév. and transferred both genera to the Phylaciaceae, a family which has not been generally accepted. While the cultures obtained by Malloch and Rogerson (1977) from ascospores of Pulveria did not sporulate, Rodrigues and Samuels (1989) found ‘Geniculosporium-like’ anamorphs in cultures of two species of Phylacia, providing evidence that the genus Phylacia belongs to the Xylariaceae. The anamorphs described by Rodrigues and Samuels (1989) for Phylacia species are more nodulisporium-like, and representative of the Hypoxyloideae, than geniculosporium-like and representative of the Xylarioideae. Ju and Rogers (1996) referred Phylacia to the Hypoxyloideae.

Læssøe (1994) suspected that Pulveria and Pyrenomyxa are congeneric from studies of type and authentic material of the latter, with Pulveria being a later name. He also reported that the stromatal pigments of P. invocans are red, suggesting affinities to Hypoxylon haematostroma Mont. and other Hypoxyloideae. Concurrently, Ju & Rogers (1996) noted the similar ascospore morphology and green pigments of P. porrecta and Hypoxylon piceum. However, since they found the holotype of H. piceum (NY ) to be depauperate, they refrained from drawing a taxonomic conclusion. Affinities between the aforementioned taxa and other Xylariaceae still remain to be established, since no culturable material has been available and morphological studies on the old herbarium specimens only provided limited information. Moreover, the discrepancies in previous reports on stromatal pigment colors (red in P. invocans and green in the other two taxa mentioned above) suggested the involvement of more than one taxon.

Recently, the utility of HPLC profiling was demonstrated to be of great value as a complementary chemotaxonomic tool in revealing infrageneric and intergeneric affinities in the Hypoxyloideae. Several new secondary metabolites (e.g. 1–7 in FIG. 1), which are referred to as extrolites, following Samson and Frisvad (2005), were identified from stromata and cultures by chromatographic and spectral methods. We have found that HPLC profiles are characteristic, reliable species-consistent features that may substantially facilitate the segregation of morphologically similar species (Stadler et al. 2001a, 2004a, Stadler et al, b, Mühlbauer et al 2002, Hellwig et al 2005, Quang et al 2004a, b, 2005). Highly sensitive, non-invasive HPLC profiling techniques were developed that are even applicable to herbarium specimens more than 200 years old (Stadler et al 2004a, Hellwig et al 2005, Quang et al 2005), because their characteristic extrolites may remain surprisingly stable. In these studies, various materials of Phylacia and one specimen of Pulveria porrecta were included. While the extract of Phylacia turbinata had only shown BNT (1) and other naphthalenes, Pulveria porrecta was found to contain no less than four different chemical types of extrolites: BNT (1), orsellinic acid (2), hypomiltin (5), and macrocarpon A (6). All of them are present in certain members of the Hypoxyloideae but apparently rare or absent in other fungi. This technique was therefore employed in an effort to obtain independently derived data that would contribute to resolution of the taxonomic position of these unusual xylariaceous fungi. Recently collected specimens from Eastern Russia were studied for comparison.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Extrolites of Pyrenomyxa species that also occur in stromata (1–6) or cultures (7) of other Hypoxyloideae. 1, BNT; 2, orsellinic acid; 3, mitorubrinol; 4, rubiginosin A; 5, hypomiltin; 6, macrocarpon A, 7, 5-methylmellein.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 
The results are divided in a taxonomic and a chemotaxonomic part.

	CHEMOTAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 
Aside from morphological studies, the stromata of all specimens examined and the culture of TL-9502 were also studied by analytical HPLC. The extrolite profiles of type materials of H. piceum and P. porrecta agreed well with that previously reported on by Stadler et al (2004a) and Hellwig et al (2005). Both contained BNT (1), orsellinic acid (2) and 5–6 (FIGS. 1, 2). In contrast P. invocans contained only 2 out of the former compounds. Instead of 1, 5 and 6, the azaphilones 3 and 4, both of which give an orange-red color in 10% KOH (see Stadler et al 2004b), as well as further congeners that are probably chemically related to them, were detected in minor quantities. The HPLC profile of P. invocans resembles that of H. rubiginosum (Pers. : Fr.) Fr., or even Entonaema cinnabarinum Cooke & Massee) Lloyd (cf. data in Quang et al 2004b, Stadler et al 2004a, b), despite the strongly differing morphological features of these fungi.

View larger version (21K): [in this window] [in a new window]   FIG. 2. HPLC-UV chromatograms (210 nm) of methanolic extracts of stromata of Pyrenomyxa species, showing characteristic UV/visual spectra of prevailing extrolites: a) P. invocans (ISC HOLOTYPE), b) P. picea (NY TYPE of Pulveria porrecta); (c) P. morganii (C HOLOTYPE). above: crude extract showing main peak with PM1 and BNT (1) overlaid at the same retention time; below: chromatograms of fractions containing purified BNT and PM1 after HPGPC. Correspondence of peaks with known chemical structures refers to FIG. 1. PM1: unknown azaphilone of P. morganii.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 
Pyrenomyxa is emended, and three species are recognized in this xylariaceous genus. An extended description including pigment colors, extrolite profiles and other features is provided for these species, and anamorphic and SEM data are presented.

Pyrenomyxa Morgan emend. M. Stadler, Læssøe & Lar. N. Vassiljeva.

= Pulveria Malloch & C. T. Rogerson, Can J Bot 55:1505, 1977.

Etymology.— – Refers to its stromata being reminiscent of the aethalial stage of a myxomycete (Morgan 1895).

Anamorph.— – nodulisporium-like.

Type species.— – P. invocans Morgan.

Stromata effused-pulvinate, unipartite, developing on decorticated or under corticated wood of various angiosperms (known host genera: Acer, Carya, Fagus, Fraxinus, Populus) appearing erumpent (fide Malloch and Rogerson 1977) or superficial. Surface plane, lacking perithecial mounds and ostiolar openings, soft and felty when fresh but becoming melanised and wrinkled in age or upon drying. Reddish black or orange pigment granules beneath stromatal surface, yielding dense olivaceous, green, red, or orange pigments in 10% KOH. Ascomata monostichous, tubular, thin-walled, 800–1300 µm long x 300–400 µm diam. Asci globose or subglobose, mostly with a short stipe, lacking an apical ring or pore, evanescent. Ascospores unicellular, mostly phaseoliform, laterally compressed, 9–17 x 3–6 x 3–4.5 µm, olivaceous or brown to dark brown by LM, black in mass, with straight dorsal germ slit nearly spore length, perispore indehiscent in 10% KOH, smooth, epispore appearing smooth by light microscopy and SEM.

Commentary.— – For the history of the taxa included in Pyrenomyxa as understood here see INTRODUCTION. Its affinities are probably with Hypoxylon ss. Hsieh et al (2005) (i.e. Hypoxylon sect. Hypoxylon ss. Ju & Rogers 1996). We do not regard Phylacia Lév. as a closely allied genus, despite the report by Speer (1980) on a similar ascal ontogeny, since it differs from Pyrenomyxa in its strongly carbonized and highly melanized stromata, by the lack of germ slits in its ascospores, and by producing different anamorphs (see Rodrigues and Samuels 1989). In addition, Pyrenomyxa appears to be distributed in the Northern temperate America and Asia, while Phylacia is known only from tropical regions. For chemical characters and affinities of other Xylariaceae see further below (Discussion).

Pyrenomyxa invocans Morgan, J Cincinnati Soc Nat Hist 18:42. 1895. FIGS. 3a, 4a, d

View larger version (91K): [in this window] [in a new window]   FIG. 3. Stromata: a) Pyrenomyxa invocans K(M) 125652; b, c) P. picea (Hypoxylon piceum, ISOTYPE K); d) P. morganii (HOLOTYPE C), bar 1 cm.

View larger version (134K): [in this window] [in a new window]   FIG. 4. Ascospores of Pyrenomyxa species. FIGS. 4a, d: P. invocans (from ISC HOLOTYPE); FIGS. 4b, f: P. picea (K ISOTYPE of H. piceum); FIGS. 4c, f: P. morganii (C HOLOTYPE). FIGS. 4a–c: Phase contrast microscopy, bar 10 µm. FIGS. 4d–f: SEM (FIG. 4d, f: bar 5 µm; FIG. 4e: bar 2 µm).

Anamorph. – unknown.

Specimens examined. – USA. OHIO: Morgan, A. P. (ISC HOLOTYPE).—IOWA: Turkey Run State Park, on standing decorticated beech, 14 Oct 1956, collector unknown (K(M) 125652 ex IA).

Commentary.— – The type species of Pyrenomyxa is characterized by its relatively large ascospores, its reddish brown stromatal surface, the orange granules beneath the stromatal surface, and reddish orange pigments in 10% KOH corresponding with the same compounds that are also found in many Hypoxylon species that show similar orange pigment colors in KOH. Morgan (1895) gave the spore size as 12–15 x 5–7 µm but we found them slightly larger.

Pyrenomyxa picea (Ellis) M. Stadler, Læssøe & Lar. N. Vassiljeva, comb. nov. FIGS. 3b, 4b, e

Basionym: Hypoxylon piceum Ellis, Amer Nat 17:194. 1883.

= Pulveria porrecta Malloch & C. T. Rogerson, Can J Bot 55:1505. 1977.

A detailed description of teleomorphic features was published by Malloch & Rogerson (1977). New data reported here allow for a better discrimination against the other species: Stromata effused-pulvinate, with surface Chestnut (40) or Sepia (63), blackening with age to some extent, partly covered with an Olivaceous (48) to Fawn (87) pruina, with dull blackish red granules immediately beneath surface, with KOH-extractable pigments Greenish Olivaceous (90), Dull Green (70), or Yellow-Green (71). Ostioles absent. Ascocarps located in the upper part of the stroma, ellipsoid to fusiform, 800–1100 µm long, thin-walled. Asci globose to subglobose, 100–180 µm diam, with a short stalk (15–25 µm), containing eight spores. Ascospores light brown to brown, unicellular, phaseoliform, ellipsoidinequilateral, laterally compressed, with broadly to narrowly rounded ends, 9–12(–14) x 4–6 x 3–3.5 µm, with straight dorsal germ slit spore-length; perispore indehiscent in 10% KOH; perispore and epispore smooth by light microscopy and SEM (> 10 000x).

Anamorph.— – unknown.

Specimens examined. – CANADA. ONTARIO: Carleton Co., 2.4 km NW of Bell’s Corners, 27 Sep 1974, Malloch, D., wood of Acer saccharum (NY ex TRTC 47274 ISOTYPE of P. porrecta); loc. cit., 14 Sep 1974, Malloch, D. (NY ex TRTC 47273 PARATYPE of P. porrecta).

USA. CONNETICUT: Litchfield Co., Camp Jewell YMCA Camp, Colebrock, on cut end of decorticated log, 24 Sep 1983, Rogerson, C. T. (NY ). IOWA: Decorah, 10.1882, Holway, E.W. 289, ex herb Ellis 1361, decorticated wood, (NY HOLOTYPE, K(M) 123177 ISOTYPE of H. piceum). NEW YORK: Ulster Co., Ashokan Campus, State University of New York, south of Ashokan reservoir, New Paltz, on standing decorticated trunk of tree, 9 Sep 1972, Rogerson, C.T. 72–226 (NY PARATYPE of P. porrecta); loc. cit., on standing decorticated trunk of Fraxinus, 9 Sep 1972, Malloch, D. (NY ex TRTC 47271 PARATYPE of P. porrecta). OHIO: Hocking Co., Cedar Falls State Park, Acer saccharum, 30 Sep 1979, Cooke, W. B. &.V. G. 56012 (NY and duplicate in personal herbarium of J. D. Rogers).

Illustrations.— – Malloch and Rogerson (1977), FIGS. 1–6 (as P. porrecta); Ju and Rogers (1996), FIG. 15 J (as H. piceum, ascospores).

Commentary.— – The teleomorph, as well as the ascal development and further details of the biology of this species were described by Malloch and Rogerson (1977) as P. porrecta. An isotype of H. piceum was located at K (FIG. 3b). Unlike the holotype studied by Ju and Rogers (1996), it still contained stromata with intact ascomata that allowed us to establish its identity with P. porrecta. The species has the smallest ascospores of all Pyrenomyxa species. Although they are also strongly laterally compressed, they appear rather stout in lateral view when compared to those of P. morganii (FIG. 4b, e). The etymology of H. piceum remains unclear because Ellis (1883) did not explain his choice of epithet, but it probably refers to ‘pix’ (Latin for pitch) rather than to the gymnosperm genus Picea. There is no indication that this fungus has ever been found from non-angiospermous hosts, but, indeed, its old, overmature stromata may eventually turn pitch black.

Pyrenomyxa morganii M. Stadler, Læssøe & Lar. N. Vassiljeva, sp. nov. FIGS. 3c, 4c, f, 5

View larger version (70K): [in this window] [in a new window]   FIG. 5. Cultures and anamorph of Pyrenomyxa morganii (ex-type) on Difco OA after 2 wk. a) Culture on agar plate (9 cm diam). CR, conidiogenous regions, EX, exudates; b), c) micrographs (water mounts, phase contrast, 1000x, bars 10 µm). b) conidiogenous cells of virgariella-like anamorph; c) conidia.

A Pyrenomyxae piceae differt ascosporibus maiores, 11–14(–15) x 4–5 x 3–4 µm, granulis stromatisque KOH dissolutis olivaceofuscis. Status anamorphosis Virgariellam similis. Cellulae conidiogenae 14–26 x 2.5–3 µm, conidiae 4–6 x 2.5–4 µm.

Stromata effused-pulvinate, up to 7 cm long x 2–5.5 cm broad x 4–11 mm thick, plane, surface Sienna (8) to Brick (59) when fresh, becoming Bay (6) to Sepia (63) when dry, blackish red granules immediately beneath surface, with KOH-extractable pigments Citrine (13), Hazel (88) or Isabelline (48), sometimes turning purplish after 5–10 min of incubation, the tissue below ascocarp layer ca 200–700 µm thick, dark brown, woody. Ascocarps located in the upper part of the stroma, ellipsoid to fusiform, 400–450 µm diam x 800–1100 µm high. Ostioles absent. Asci globose to subglobose, short stipitate, 140–200 µm diam, stipe 20–30 µm. Ascospores light brown to olivaceous, unicellular, phaseoliform, laterally compressed with narrowly rounded ends, 11–14(–15) x 4–5 x 3–3.5 µm, with straight dorsal germ-slit spore length or nearly so; perispore indehiscent in 10% KOH, smooth by light microscopy and SEM (> 10 000x).

Anamorph.— – Colonies on OA reaching the edge of Petri dish in 10–13 d, at first whitish, velvety, with diffuse margins, later becoming Saffron (10), Salmon (41), Honey (64) or Hazel (88), then frequently with brownish exudates. Reverse initially becoming Umber (9), but blackening after 3–4 wk. Sporulating regions appearing after 10 d in the center of colony, later scattered over entire surface of colony in Dark Brick (60) to Sepia (63) patches. Conidiogenous structure with virgariella-like branching pattern as defined in Ju and Rogers (1996). Conidiophores hyaline to pale brown, finely roughened, up to 140 µm long, unbranched or dichotomously branched (and then usually with additional branches arising from the first level of conidiogenous regions), 2.5–3 µm diam, with 1–2 conidiogenous cells arising from each terminus. Conidiogenous cells terminal or rarely intercalary, cylindrical, hyaline, smooth or finely roughened, 14–26 x 2.5–3 µm, bearing one to several poroid conidial secession scars on apical region. Conidia produced holoblastically in sympodial sequence, hyaline, smooth, ellipsoid, 4–6 x 2.5–4 µm, sometimes with flattened base. Extrolites in liquid YMG culture: 5-methylmellein (7) and further derivatives of this type of dihydroisocoumarins.

Specimens examined. – RUSSIA. KHABAROVSK TERRITORY: Mt. Matai, cross-way southwest of mountain, on hard, naked wood inside a corticated Populus-branch, 21 Aug. 1998, Læssøe, T., TL-5205 (C-61362, culture CBS 116990). Petropavlovka Lake, on huge decorticated trunk of cf. Fraxinus, associated with Nemania and Hypoxylon, 14 Aug. 1998, Læssøe, T., TL-5134 (C-61291, culture in CBS, HOLOTYPE). Petropavloka Lake, on hard dicot wood, 23 Aug 1998, Læssøe, T., TL-5222 (C-61380). PRIMORSKY TERRITORY: Ussuriysky district, vicinity of Gornotayezhnoye, 18 Sept. 2001, Vasilyeva, L. (VLA)

Commentary. – Pyrenomyxa morganii differs from P. picea in having orange brown tones on its stromatal surface, and in having different KOH-extractable pigments. Furthermore, it has larger asci, and its ascospores are longer and more slender than in the former species. It is the only species reported from Eastern Russia, while the other two species of the genus were collected from Eastern temperate North America. Pyrenomyxa morganii has different surface colors, orange or red KOH-extractable pigments, and slightly larger ascospores. Pyrenomyxa picea shows quite similar KOH-extractable pigments as the current species but contains hypomiltin (5) and macrocarpone A (6). The prevailing stromatal pigment of P. morganii appears to be an unidentified azaphilone (PM1 in FIG. 2). Color changes from greenish to purplish tones occurred after 10 min in some of the stromatal samples that were treated with 10% KOH to determine the pigment colors. We assume that those are due to the presence of the naphthalene, BNT (1), which has a purple color in KOH (Stadler et al 2001a) and was also detected in all collections studied. In contrast to azaphilones, BNT is rather stable and may be retained in the extract after decay of the unstable major pigment has occurred. Orsellinic acid (2), which is not a pigment, was also present in minor quantities. Interestingly, the anamorph of P. morganii is similar to that of H. macrocarpum Pouzar (see for comparison Ju and Rogers 1996), which also contains BNT, orsellinic acid and macrocarpon A (1, 2, 6; Mühlbauer et al 2002) but no hypomiltin (Hellwig et al 2005). Some hypomiltin (5) containing taxa treated in the preceding study, such as H. trugodes Berk. & Broome, and H. perforatum (Schwein. : Fr.) Fr. also have a virgariella-like anamorph. However, while H. hypomiltum Mont., the species from which 5 was originally isolated shows a nodulisporium-like anamorph and the conidiogenous structure of H. intermedium (Schwein. : Fr.) Y.-M. Ju & J. D. Rogers, another representative of this chemotype, is highly reduced. It only occurs on media with high contents of free sugars (Greenhalgh and Chesters 1968, Stadler et al 2004b) and is referable to the Sporothrix type sensu Ju and Rogers (1996).

	KEY TO PYRENOMYXA SPECIES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 

1. KOH-extractable pigments dense Orange (7), Sienna (8), Umber (9), or Luteous (12) and remaining so. Ascospores (12–)14–17 x 4.5–7.5 x 3–4.5 µm P. invocans 1. KOH-extractable pigments greenish or olivaceous, sometimes with color changes into purple after some minutes, and ascospores smaller 2 2. Stromatal surface lacking reddish or orange brown tones. KOH-extractable pigments Greenish Olivaceous (90), Dull Green (70), or Yellow-Green (71). Ascospores 9–13(–14) x 4–6 x 3–4 µm P. picea 2. Stromatal surface with reddish to orange brown tones. KOH-extractable pigments Citrine (13), Hazel (88) or Isabelline (48), sometimes turning purplish after 5–10 min of incubation. Ascospores longer and more slender than in the former species, 11–14(–15) x 4–5 x 3–3.5 µm P. morganii

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 
While this study reinforced the view that the taxonomic affinities of Pyrenomyxa (and its synonym as understood here) are with the Hypoxyloideae, its affinities to other xylariaceous genera that show similar reductions of ascal and ascospore structures remain unclear. Notably, Phylacia spp. also contain similar stromatal metabolites as the Hypoxyloideae (Stadler et al 2004a), but these are different from the specific azaphilone and macrocarpone polyketides of Hypoxylon sect. Hypoxylon that are present in Pyrenomyxa. The only metabolite found to occur in both Phylacia and Pyrenomyxa is the naphthalene BNT (1), whose biogenesis from the ubiquitous 1,8-dihydroxynaphthalene pathway of fungal melanin biosynthesis is easy to conceive. BNT may therefore have arisen convergently and/or constitute an evolutionary rather old molecule (see discussion in Quang et al 2005). Accordingly, BNT is omnipresent in Hypoxylon sect. Annulata sensu Ju and Rogers (1996) (= Annulohypoxylon gen. nov. fide Hsieh et al [2005]), Daldinia Ces. & de Not., and widespread in Hypoxylon sect. Hypoxylon. It is also typical of another genus of Xylariaceae that shows tendencies to attain a cleistocarpous habit: Rhopalostroma D. Hawksw., whose nodulisporium-like anamorph suggest its affinities to the Hypoxyloideae (Hawksworth and Whalley 1985), despite the fact that it contains rather similar metabolites as Phylacia in its stromata, and even has the same metabolites as Daldinia in its cultures (Stadler et al 2004a). Pyrenomyxa morganii cultures yielded 5-methylmellein (7), which was identified previously as a common extrolite of various taxa that were at times included in Hypoxylon s. lat. (sensu Miller 1961) but are now distributed over Biscogniauxia, Camillea, Hypoxylon, and Nemania (Anderson et al 1983). Interestingly, 7 was lacking in H. multiforme (Fr. : Fr.) Fr. and H. (= Annulohypoxylon) cohaerens (Pers. : Fr.) Fr., as well as in Euepixylon udum (Pers. : Fr.) Laessøe & Spooner, H. fragiforme (Pers. : Fr.) J. Kickx fil., and H. howeianum Peck, cultures of which contained other mellein type dihydroisocoumarins. Among the Hypoxylon spp. reported to contain the compound (7) were ‘H. rubiginosum’ (although it is not clear which segregate of this species in the broad concept of Miller [1961] the material examined by Anderson et al 1983 actually belonged to) and H. argillaceum (Pers.) Nitschke, i.e., H. intermedium (Schwein. : Fr.) Y.-M. Ju & J. D. Rogers fide Ju and Rogers (1996).

From a chemotaxonomic standpoint, Pyrenomyxa therefore appears more closely related to the group of Hypoxylon (sect. Hypoxylon sensu Ju and Rogers 1996) with olivaceous pigments and sporothrix- or virgariella-like anamorphs comprising, e.g. H. macrocarpum and H. intermedium, while Phylacia appears more closely related to Rhopalostroma, Thamnomyces Ehrenb., and Daldinia. Further work, including molecular studies, may eventually reveal whether or not the cleistocarpous lineages of Xylariaceae have arisen independently from one another, but current evidence, including the bio-geographical patterns of these taxa, strongly suggest this is the case.

Orsellinic acid (2), which is not found in Phylacia or Rhopalostroma (Stadler et al 2004a), is the only extrolite detected in all three species of Pyrenomyxa. Conjugates of this compound with azaphilone pigment systems (3–5; PM1), as well as the non-azaphilone pigment macrocarpone A (6), which was so far only found in P. picea and H. macrocarpum, are held responsible for the colors of their stromatal granules, and their pigments in KOH. In general, such chemical traits should not be underestimated in their importance. Even though it is not clear whether the biogenetic prerequisites for these characteristic major constituents in stromata of Hypoxyloideae have arisen convergently several times or whether they were originally omnipresent in certain ancestral taxa and have been lost in some of their descendants through evolution, their occurrence can be as characteristic as, e.g. a particular ascospore size range or anamorphic structure, at least in particular species and species groups.

Specific stromatal extrolites that can serve as chemotaxonomic markers because they remain in old herbarium specimens for decades are a valuable tool to link type material to recently collected culturable specimens. When not only one, but three or four classes of chemical compounds can be combined to a characteristic HPLC profile as demonstrated here for H. piceum and P. porrecta, such results become even more consequential. Notably, the structures of the major azaphilones of all Pyrenomyxa species differ only slightly from each other and the deviations can be explained easily by merely one or two alterations in their biogenesis. For instance, P. picea may be the closest relative of H. macrocarpum and the other two species derived from the same lineage but which eventually lost their ability to produce macrocarpone (6) and changed their biosynthetic pattern of azaphilones. Then again, P. morganii may as well be derived from common ancestors along with particular species of the H. rubiginosum complex that also contain rubiginosin A as prevailing metabolite, while the other two Pyrenomyxa spp. constitute members of a distinct evolutionary lineage. Such chemotaxonomic traits, despite their importance in resolution of species complexes, can only facilitate the interpretation of morphological and/or molecular data. In order to test the monophyly of Pyrenomyxa and its position in relation to Hypoxylon, further data, including identification of the unknown dominant metabolite PM1, anamorphic features and sequence analyses, must be obtained. Pyrenomyxa is therefore accepted ad interim, until such work has been accomplished.

	ACKNOWLEDGMENTS

 
We are grateful to the curators of the herbaria K (Begoña Aguirre-Hudson), IAS (Deborah Harris), NY (Ellen Bloch) and their colleagues, and to our colleague Jack D. Rogers (Pullman, Wa.) who kindly sent us specimens. Furthermore, we thank Stephan Seip (BHC, Wuppertal) for recording HPLC-MS, Klaus Ide (Bayer Technology Services, Leverkusen, Germany) for his help with SEM, and Dang Ngoc Quang and Yoshinori Asakawa (Tokushima Bunri University, Tokushima, Japan) for providing standard compounds and for confirming the identity of 5-methylmellein. The foundations controlled by the Danish Botanical Society are thanked for financially aiding the fieldwork by TL, and so is Victor Muhkin.

	FOOTNOTES

 
Accepted for publication July 6, 2005.

¹ Corresponding author. E-mail: marc.stadler{at}t-online.de

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CHEMOTAXONOMY TAXONOMY KEY TO PYRENOMYXA SPECIES DISCUSSION LITERATURE CITED

 
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