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Molecular evolution and systematics

Cryptadelphia (Trichosphaeriales), a new genus for holomorphs with Brachysporium anamorphs and clarification of the taxonomic status of Wallrothiella

     Department of Plant Taxonomy and Biosystematics, Institute of Botany, Academy of Sciences, CZ-252 43 Prhonice, Czech Republic

Keith A. Seifert

     Biodiversity (Mycology and Botany), Agriculture and Agri-Food Canada, Central Experimental Farm, Ottawa, Ontario, K1A 0C6, Canada

	ABSTRACT

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The genus Cryptadelphia (Trichosphaeriales) is described for the presumed teleomorphs of six species of the dematiaceous hyphomycete genus Brachysporium. Cryptadelphia is characterized by the production of dark, leathery perithecia with two-layered walls, cylindrical, long-stipitate asci with eight hyaline, initially aseptate but ultimately 1-septate as-cospores. Zignoëlla groenendalensis is transferred to Cryptadelphia with Wallrothiella melanostigmoides as a synonym. Five new species, i.e., Cryptadelphia abietis, C. brevior, C. obovata, C. pendulispora and C. polyseptata, with their respective Brachysporium anamorphs, are described and illustrated. Phylogenetic analysis of partial large subunit rDNA sequences of two species supports the monophyly of Cryptadelphia and suggests a relationship with the Trichosphaeriales. The monophyly of the Trichosphaeriaceae and the relationships between the Trichosphaeriaceae, Annula-tascaceae and the Sordariales can be questioned, but a conclusive assessment requires phylogenetic analysis of more related taxa. The genus Wallrothiella is redescribed based on new collections of W. congregata and is characterized by the production of tiny, globose ascospores in cylindrical asci. The new collection is proposed as a neotype for the genus.

Key words: Aquaticola, life history, LSU rDNA, phylogeny, systematics

	INTRODUCTION

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Brachysporium Sacc. is a hyphomycetous, dematiaceous genus of approximately 12 saprobic, wood-inhabiting species (Ellis 1966, 1971; Hughes 1951, 1955; Holubová-Jechová 1972). The species produce dry, apical heads of multiseptate conidia that secede by rhexolytic fragmentation of denticles. Brachysporium species rarely have been grown in axenic culture, and no teleomorph connections have been reported. The phylogenetic relationships of this genus are unknown. Rhexolytic secession is a rare character in hyphomycetes, in which conidia secede by the fracture of an intercalary cell or isthmus, in this case a narrow, cylindrical denticle. Species of the plant-pathogenic Pyricularia Sacc. and Nakataea Hara (both with teleomorphs in the Magnaporthaceae [Cannon 1994] and considered synonymous by Sivanesan in Kirk et al. 2001) and the lignicolous saprobic Pleurothecium Höhn. (teleomorph in Carpoligna F.A. Fern. & Huhndorf) have rhexolytic conidial secession and are morphologically similar to species of Brachysporium. They differ in conidial shape and degree of pigmentation of the conidiophores and conidia, but the similarities in conidium ontogeny suggest hypotheses for phylogenetic relationships among these fungi.

We frequently noted superficial, dark, glabrous perithecia in fresh or herbarium collections of six Brachysporium species (B. abietinum Hol.-Jech., B. brevius Hol.-Jech., B. obovatum [Berk.] Sacc., B. nigrum [Link] S. Hughes, B. pendulisporum S. Hughes, B. polyseptatum [Preuss] S. Hughes) from temperate Europe and North America. These had more or less leathery to fragile perithecial walls, persistent paraphyses and unitunicate long-stipitate asci, each containing a distinct, refractive, nonamyloid apical annulus and eight hyaline, ellipsoidal to fusiform, 0–1-septate ascospores. The proximity of conidiophores and perithecia suggested they might be parts of one life cycle. We isolated and germinated single ascospores of the teleomorphs associated with B. nigrum, B. obovatum and B. polyseptatum. These yielded slow-growing colonies that usually remained sterile, but identical colonies were derived from ascospores and conidia of B. nigrum. Cultures originating from conidia of B. nigrum eventually formed conidia in the presence of contaminating fungi.

Based on characters of the perithecia, asci, ascospores and conidiogenesis, the Trichosphaeriaceae (Trichosphaeriales, Barr 1990) is an appropriate classification of the Brachysporium holomorphs. In this study, we provide a taxonomic description for the teleomorphs of these six Brachysporium species, and explore the systematic position and phylogenetic relationships of two of the species using phylogenetic analyses of large-subunit rDNA.

Taxonomic affinities of the teleomorphs. – The teleomorphs of Brachysporium species all have a similar morphology and anatomy, similar to that of several genera discussed below that have been included in or confused with Chaetosphaeria Tul. & C. Tul. (or its synonyms). The holoblastic, denticulate conidiogenesis seen in Brachysporium species, however, is inconsistent with that of chaetosphaeriaceous anamorphs, which all have phialidic conidiogenesis (Réblová and Winka 2000, Réblová 2000). Despite these characteristic teleomorphs and anamorphs, we could not conclusively attribute these fungi to any known genus of nonstromatic, perithecial ascomycetes. Among comparatively well-known ascomycetes, they can be compared with phenotypically similar teleomorphs such as Aquaticola W.H. Ho et al, Chaetosphaeria and Wallrothiella Sacc. They also merit comparison with some less familiar, older genera that no longer are widely used, namely Zignoëlla (Sacc.) Sacc. and Zignoina Cooke.

Saccardo (1883) introduced the genus Zignoëlla with three subgenera distinguished by ascospore septation; subg. Eu-Zignoëlla. (ascospores 3-septate), subg. Zignaria (ascospores initially 1-septate) and subg. Zignoina (ascospores initially aseptate). Booth (1957) considered Zignoëlla a taxonomic synonym of Chaetosphaeria and transferred Z. pulviscula (Currey) Sacc., the lectotype species (Clements and Shear 1931), to Chaetosphaeria. Cooke (1885) described Zignoina with the single species Zignoina subcorticalis Cooke, with a description that matched Zignoëlla subg. Zignoina (Saccardo 1883). There is no reference in the protologue of Zignoina either to Saccardo’s subgenus or to any species that Saccardo (1883) included in Zignoëlla and its subgenera. Although it is possible that Cooke intended to change the rank of Saccardo’s subgenus, he actually proposed a new genus typified by Z. subcorticalis. We observed no complete perithecia on the type material of Z. sub-corticalis (AUSTRALIA: on inner side of decayed bark, K 78713), only a few remnants of the lower halves of empty and crushed perithecia immersed in the substrate. In the protologue, Z. subcorticalis (Cooke 1885) was described as, "Sparsa. Peritheciis semi-immersis, pertusis, atris, opacis. Asci cylindricis, octosporis. Sporidiis ellipticis, hyalinis, continuis (12 x 4 µm)." Because critical characters are missing in this diagnosis, we have chosen not to adopt Zignoina for teleomorphs linked to Brachysporium. Saccardo (1891) did not accept Cooke’s genus Zignoina and instead expanded Zignoëlla subg. Zignoina with several species, including Zignoëlla groenendalensis Sacc. et al. Our examination of the type material of Z. groenendalensis revealed that it is identical to the teleomorph of Brachysporium nigrum described below. The same fungus also was described later as Wallrothiella melanostigmoides Feltgen (Feltgen 1903).

Wallrothiella was erected by Saccardo (1882) and included Sphaeria congregata Wallr., a species described from Salix wood with apically collapsing, glossy black perithecia and tiny, globose ascospores. This was selected as the lectotype species, as Wallrothiella congregata (Wallr.) Sacc., by Clements and Shear (1931). The genus was considered a synonym of Trichosphaeria Fuckel in the broadly delimited Sphaeriaceae (von Arx and Müller 1954), then attributed to the Niessliaceae (Barr 1990, Eriksson and Hawksworth 1998). On the basis of a North American collection, Samuels and Barr (1997) considered W. congregata a member of the Trichosphaeriaceae, but the type material was not found. We neotypify, redescribe and illustrate W. congregata in this paper based on two recent European collections. Because of similarities in morphology, we initially considered Wallrothiella a possible genus for the teleomorphs of Brachysporium species. However, Wallrothiella differs from the Brachysporium teleomorphs in the structure of the perithecial wall and the morphology of the paraphyses, ascospores and asci. So far, the anamorph of W. congregata is unknown.

Species of Aquaticola colonize submerged, decayed wood in freshwater habitats in Asia (Ho et al 1999). The two described species have hyaline, aseptate, ellipsoidal ascospores and minute, dark perithecia similar to teleomorphs of Brachysporium. According to phylogenetic analysis of nuclear large subunit rDNA (LSU rDNA), Aquaticola is related to the Annulatascaceae (Ranghoo et al 1999). No anamorphs are known in this genus.

Carpoligna is a monotypic genus of perithecial ascomycetes typified by C. pleurothecii F.A. Fern. & Huhndorf, which has hyaline, 3-septate ascospores in unitunicate asci with a well-developed, nonamyloid, apical annulus (Fernández et al 1999). The anamorph is Pleurothecium recurvatum (Morgan) Höhn., which produces slimy heads of 3-septate, holoblastic conidia on a curved rachis of denticles. Although attributed to the Sordariales by Kirk et al (2001), the analysis of LSU rDNA by Réblová and Winka (2001) placed C. pleurothecii at the end of a long branch in a well-supported clade with species of Ascotaiwania Sivan. & H.S. Chang, with unclear ordinal affinities.

Nakataea sigmoidea (Cavara) Hara (teleomorph Magnaporthe salvinii [Catt.] R.A. Krause & R.K. Webster) and Pyricularia grisea (Cooke) Sacc. (teleomorph M. grisea [T.T. Hebert] M.E. Barr) are important plant pathogens with conidiophores, conidia and rhexolytic secession similar to species of Brachysporium. Despite the similarity in anamorphs, the teleomorphs are rather different and unlikely to be confused with Brachysporium-like holomorphs. Magnaporthe R.A. Krause & R.K. Webster species have erumpent perithecia with elongated necks and produce lightly pigmented, allantoid, 3-septate ascospores in unitunicate asci with a well-developed, non-amyloid, apical annulus (Barr 1977). The family Magnaporthaceae is of uncertain affiliation within the Sordariomycetes (Kirk et al 2001). Two species of Magnaporthe have phialidic anamorphs belonging to Harpophora W. Gams (Scott and Deacon 1983, Land-schoot and Jackson 1989, Gams 2000), suggesting some heterogeneity in the generic concept.

	MATERIALS AND METHODS

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Herbarium material and fungal strains. – Dried herbarium specimens were rehydrated in 3% (aq.) KOH and subsequently studied in water, Melzer’s reagent, cotton blue in lactic acid and 90% lactic acid. All measurements were made in lactic acid. Means ± standard errors are given for spore and ascus dimensions and are based on 20–25 measurements. The length/width ratios (L/W) for asci are given for pars sporifera.

Single-ascospore and conidial isolates were obtained from fresh material with the aid of a single-spore isolator (Meopta). Colonies were grown on cornmeal agar (CMA, Difco), oatmeal agar (OA, Gams et al 1998), potato-dextrose agar (PDA, Difco), potato-carrot agar (PCA, Gams et al 1998) and Blakeslee’s malt-extract agar (MEA, Pitt 1979). Colony characters were taken from cultures grown on MEA or PCA for 21 d at room temperature (about 25 C) in incident light. Color codes in descriptions (with the form 3A-B8, i.e., plate number, column, row) and capitalized color names refer to Kornerup and Wanscher (1978).

The cultures are maintained at the Institute of Botany, in Prhonice and the Canadian Collection of Fungal Cultures (DAOM), Agriculture & Agri-Food Canada, Ottawa. Specimens with M.R. numbers are preserved at the Institute of Botany, Prhonice. The isolates used in this study and their sources are listed in TABLE I.

DNA extraction, amplification and sequencing. – DNA was isolated using a FastDNA^TM Kit and the FastPrep^TM FP120 (BIO 101 Inc.) using mycelium removed from PDA cultures. PCR and cycle sequencing reactions were performed on a Techne Genius thermocycler (Techne Cambridge Ltd.). PCR reactions were performed using Ready-To-Go^TM Beads (Amersham Canada Ltd.) in 25.0 µL volumes, each containing 20–100 ng of genomic DNA, 1.5 units Taq DNA Polymerase, 10.0 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl₂, 200 µM of each dNTP, 0.2 µL of each primer (50 µM) and stabilizers including BSA. The reaction profile included an initial denaturation for 3 min at 94 C, followed by 30 cycles of 1 min denaturation at 94 C, 1.5 min annealing at 58 C, 2 min extension at 72 C, with a final extension of 10 min at 72 C. Amplicons were purified using Gene Clean II Kit (Q-BIOgene) following the manufacturer’s directions. The cycle-sequencing reaction mixture comprised one-eighth strength (1.0 µL) BigDye^TM V2.0^TM Terminator Cycle Sequencing Ready Reaction Kit sequencing mix (ABI Prism/Applied Biosystems), an equal volume of the reaction booster halfBD (Bio/Can Scientific, Mississauga, Ontario); 0.5 µL sequencing primer (5.0 µM); 20–50 ng purified DNA; and sterile water to a final volume of 5.0 µL. Reactions were brought to 20.0 µL, purified by ethanol/sodium acetate precipitation and resuspended as recommended for processing on an ABI PRISM^TM 310 DNA sequencer, ABI 373 Stretch DNA Sequencer or ABI PRISM^TM 3100 Genetic Analyzer (Applied Biosystems, Foster City, California). A portion of the large ribosomal subunit (28S) DNA was amplified and sequenced using primers LROR and LR8 and cycle-sequenced using primers LROR, LR3R, LR17, LR16, LR6 and LR8.

Sequence data analyses. – Phylogenetic relationships were examined using 58 partial LSU rDNA sequences from 11 different orders of ascomycetes. Saccharomyces cerevisiae was used as outgroup. Homologous LSU rDNA sequences from 47 taxa were retrieved from GenBank; accession numbers are given on FIG. 1. The dataset included three new sequences of the Brachysporium holomorphs (two ascospore isolates, one conidial isolate) and eight new sequences of representatives of the Hypocreales, Ophiostomatales and Microascales (TABLE I).

View larger version (53K): [in this window] [in a new window]   FIG. 1. One of four equally parsimonious trees from a heuristic analysis of LSU rDNA sequences from 11 ascomycetous orders. Branches with thin lines were not present in a strict consensus of the four MPTs. Bootstrap and jack-knife values (>50%) from 1000 replicates are included at the nodes. The asterisk (*) indicates taxa represented by the anamorph in the phylogeny. Branch lengths are drawn to scale (1759 steps, CI 0.368, RI 0.594, RC = 0.217, HI = 0.632).

Several LSU sequences of members of the Annulatascaceae available from GenBank were not included in the analysis because they required insertions in the stem parts of the secondary structure to align with the remainder of the dataset.

The phylogenetic analyses were performed with PAUP* 4.0 (Swofford 2000) using parsimony (heuristic search with 100 random sequence additions). Support for the branches was tested with 1000 replicates of bootstrap and jackknife analysis. Constraint analyses were run using the Kishino-Hasegawa test as implemented in PAUP*, with the Trichosphaeriaceae, Annulatascaceae or various interpretations of the Sordariales (see Results) forced to be distinct and monophyletic.

	RESULTS

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Phylogenetic analysis. – A maximum parsimony analysis (PA), with gaps treated as missing data, was performed using the 358 phylogenetically informative characters in the alignment of 1259 bp. Four most-parsimonious trees (MPT) were obtained, one of which is shown in FIG. 1. The Hypocreales (68% bootstrap support/67% jackknife support), Microascales (99/99), Diatrypales/Xylariales (94/94), Diaporthales (100/100) and Ophiostomatales (93/91) were well-supported clades at the order level. The Sordariales was a poorly supported clade with two major lineages, the strongly supported Chaetosphaeriaceae (100/100) and a lineage containing three other sordariaceous families, the Chaetomiaceae, Lasiosphaeriaceae and Sordariaceae. Wallrothiella subiculosa Höhn. was basal to the Sordariales (cf. Hughes and Dickinson 1968), and appeared extralimital to both the Chaetosphaeriaceae lineage and the clade including the other three sordariaceous families; its systematic position remains unclear (Réblová and Winka 2000). Two well-supported clades with ambiguous ordinal affiliations were the Ascotaiwania/Carpoligna group (100/100), previously noted by Ranghoo et al (1999), and the Ceratocystis group (92/92).

The three sequences of Cryptadelphia formed a well-supported clade (99/99) within a poorly supported Trichosphaeriales. The representatives of the Annulatascaceae (Annulatascus hongkongensis W.H. Ho et al, "Aquaticola hongkongensis" (nom. nud. Ranghoo et al 1999, Ascolacicola austriaca Réblová et al) were mixed with members of the Trichosphaeriaceae (Rhamphoria delicatula Niessl and the three Cryptadelphia sequences). Aquaticola hongkongensis and R. delicatula were placed at the base of the Trichosphaeriales clade. Clohiesia corticola K.D. Hyde, currently classified in the Annulatascaceae, was placed in the Sordariales, basal to the Chaetosphaeriaceae.

Constraint analyses (CA) were performed to test the monophyly of the Trichosphaeriaceae and Annulatascaceae. When the Trichosphaeriaceae was treated as monophyletic, the two trees (not shown) were two steps longer and the Kishino-Hasegawa (KH) test did not reject them as significantly worse than the MPTs (P = 0.638 and 0.415). The two CA trees (not shown) with the Annulatascaceae forced to be monophyletic were 12 steps longer but still considered an acceptable hypotheses for the phylogeny (P = 0.2013). Three CAs were run to assess the inclusion in the Sordariales of (i) the Trichosphaeriaceae and Annulatascaceae, (ii) the Trichosphaeriaceae and Annulatascaceae minus Rhamphoria, and (iii) the Trichosphaeriaceae and Annulatascaceae minus Rhamphoria and Cryptadelphia. The CA forcing a monophyletic Sordariales including all the members of the Trichosphaeriaceae and Annulatascaceae resulted in two trees 25 steps longer than the MPTs, both of which were rejected by the KH test (P = 0.0018 and 0.0038). When Rhamphoria was excluded from the Sordariales, the CA resulted in a single tree 18 steps longer than the MPT that was accepted by the KH test (P = 0.1033). However, a CA with both Cryptadelphia and Rhamphoria excluded from the Sordariales, but the putative members of the Annulatascaceae included, yielded four trees 22 steps longer than the MPT, three of which were rejected and one of which was accepted (P = 0.481, 0.0427, 0.0462, 0.0535).

In some of our analyses with a larger selection of sequences from the Chaetosphaeriaceae, and Porosphaerella cordanophora E. Müll. & Samuels (AF178563), the Ophiostomatales was paraphyletic within the Trichosphaeriales clade but never with significant bootstrap or jackknife support (not shown).

Some correlation was noted between groups of ascomycetes and particular modes of conidiogenesis. Four clades represent taxa with holoblastic, denticulate conidiogenesis: (i) the Trichosphaeriales; (ii) the monophyletic Carpoligna/Ascotaiwania clade; (iii) the Ophiostomatales and (iv) the Diatrypales/Xylariales. Perithecial ascomycetes with typically enteroblastic (phialidic or basipetal) conidiogenesis belonged to these four clades: (i) the Sordariales, (ii) the Microascales, (iii) the Ceratocystis group and (iv) the Diaporthales. Two remaining clades include species that produce either phialidic or holoblastic conidia, namely the Hypocreales and the Magnaporthaceae.

Anamorph-teleomorph connections. – We were successful in isolating conidia and ascospores from Cryptadelphia groenendalensis and its anamorph Brachysporium nigrum. The culture derived from conidia of M.R. 1495-99 eventually began to sporulate after several months on oatmeal agar at room temperature (about 25 C) in the presence of an unidentified Fusarium contaminant accidentally introduced by fungus-feeding mites. Parts of the colonies then were transformed into a dense, velvety carpet of conidiophores, somewhat powdery because of the profusely produced dark brown conidia. Most conidiophores were a similar dark brown to those produced in nature, but in light sectors of the colony the conidiophores were less pigmented. In contrast to the unbranched conidiophores found in vivo, and a strictly terminal conidiogenous zone, the conidiophores produced in vitro often had one or two short branches and conidia were produced singly or in clusters at nodes along the length of the conidiophores. Another subculture grown on PCA at room temperature sporulated after 2 mo in the presence of a fungal contaminant (Chloridium virescens [Pers.] W. Gams & Hol.-Jech.) in the absence of mites. Conidiophores were formed sparsely at margins of the colony, and conidia were produced singly on conidiophores on short apical denticles (FIG. 17). On both agar media there was a great variation in conidium size, shape and septation, but typical three-septate conidia with the two central cells darkest were common. The culture derived from ascospores of M.R. 1497-99 never sporulated, although the cultural characters otherwise were identical with those of the culture derived from conidia.

View larger version (150K): [in this window] [in a new window]   FIGS. 15–18. Cryptadelphia groenendalensis. 15. Ascospores. 16. Mature asci with ascospores. 17. Conidia of the Brachysporium nigrum anamorph, from culture. 18. Conidia of B. nigrum, from nature. FIGS. 15, 16, 18: DIC. Scale bars: 15–18 = 10 µm. FIGS. 15, 16, 18 from MR 2455-02; Fig.17 from culture M.R. 1495-99.

Because the cultures isolated from conidia of B. nigrum and ascospores of C. groenendalensis were similar, the LSU sequences differed by only 1 bp and the conidial isolate eventually sporulated, we concluded that the connection between these two morphs was well-documented. The similarity of the cultures derived from C. polyseptata and C. obovata to the cultures of C. groenendalensis, the similarity of the LSU sequence of the C. polyseptata culture to C. groenendalensis and hence to a fungus with a Brachysporium anamorph and their placement in a monophyletic group in the analysis, led us to believe that an anamorph-teleomorph connection between C. polyseptata and B. polyseptatum was the most likely explanation. Thus, we suggest that teleomorph-anamorph relationships exist between the other four pairs of morphs presented in this study, although the connections remain to be proven by culturing of ascospores and conidia.

	TAXONOMY

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The undescribed ascomycetes with Brachysporium anamorphs, represented by the ascospore and conidial isolates of B. nigrum and ascospore isolate of B. polyseptatum, form a monophyletic group in the phylogenetic analysis. No particular relationships with Aquaticola, Chaetosphaeria or Wallrothiella were evident. Therefore, the new genus Cryptadelphia is proposed for holomorphs with Brachysporium anamorphs.

Cryptadelphia Réblová & Seifert, gen. nov.

Perithecia nonstromatica, subimmersa usque superficialia, conica vel subglobosa, nigra, glabra. Paries perithecii coriaceus usque fragilis, bistratosus; stratum externum carbonaceum e cellulis angularibus, internum hyalinum, e cellulis elongatis, compressis compositum. Ostiolum periphysatum. Paraphyses persistentes, cylindricae, septatae, ramosae. Asci unitunicati, 8-spori, cylindrici usque clavati, longistipitati, apice non amyloideo, annulo refringente praediti. Ascosporae hyalinae, fusiformes, ellipsoideae vel oblongelimoniformes, primum 0-septatae, postea septo mediano divisiae.

Anamorphe: Brachysporium, conidiophora macronematosa, nonramosa, fusca vel atra, cellulae conidiogenae holoblasticae, polyblasticae, denticulatae, sympodialiter proliferentes, conidia solitaria, euseptata, fusca vel atra, rhexolytice secedentia.

Species typica C. groenendalensis (Sacc., E. Bomm. & M. Rouss.) Réblová & Seifert.

Perithecia nonstromatic, superficial, globose to subglobose, conical around the ostiole, dark brown to black, glabrous. Perithecial wall more or less leathery to fragile, composed of two regions; outer region of carbonaceous, dark brown, angular to rectangular cells ca. 3.0–4.0(–5.0) µm diam; inner region of hyaline, thinwalled, elongated, compressed cells. Ostiolar canal periphysate. Paraphyses persistent, hyaline, cylindrical, tapering distally, septate, branching. Asci unitunicate, cylindrical-clavate, long-stipitate, 8-spored, refractive apical annulus distinct, nonamyloid. Ascospores hyaline, fusiform to ellipsoidal to oblonglemon-shaped, at first nonseptate, followed by formation of a median septum while the ascospores are still in the ascus. Anamorphs classified in Brachysporium. Conidiophores macronematous, unbranched, dematiaceous. Conidiogenous cells integrated, terminal, sympodially proliferating, polyblastic, denticulate. Conidia solitary, euseptate, dematiaceous, secession rhexolytic.

Type species. – C. groenendalensis (Sacc., E. Bomm. & M. Rouss.) Réblová & Seifert.

Anamorph. – Brachysporium Sacc., Michelia 2:28. 1880, emend. E.W. Mason & S. Hughes, Naturalist 1951:45. 1951.

Etymology. – Cr yptós hidden (Gk), adelphé sister (Gk), i.e., the hidden sister of Brachysporium.

Commentary. – The teleomorphs of Cryptadelphia are very similar in appearance and, although there are differences in ascospore and ascus dimensions and possibly ascospore septation, construction of a key based on teleomorph characters is difficult. The differences among the species are easily visible in the conidia and conidiophores of the Brachysporium anamorphs. Our Cr yptadelphia/Brachysporium key (modified from Ellis 1971) relies on anamorph characters.

The asci of Cryptadelphia species are unitunicate. We occasionally observed ripe asci with the outer layer of the lower, sterile part (stipe) disintegrating. These asci floated in the centrum, and disintegration was confined to the stipes. The stipe disintegration was observed in C. groenendalensis (FIG. 19B) and C. polyseptata (FIG. 35). We are unsure whether this is an artifact of slide preparation or part of the biology of the fungus.

View larger version (52K): [in this window] [in a new window]   FIG. 19. Cryptadelphia groenendalensis. A. Mature asci with paraphyses. B. Ascus stipe disintegrating along the outside of the vertical axis. C. Ascospores. D. Conidia and conidiophores of the Brachysporium nigrum anamorph, from nature. E. Longitudinal section of the perithecial wall. F. Habit sketch of perithecia and conidiophores, from nature. Scale bar = 10 µm. FIGS. 19A–F from Zignoëlla groenendalensis (Holotype, PAD).

View larger version (150K): [in this window] [in a new window]   FIGS. 28–37. Cryptadelphia polyseptata. 28, 29. Ascospores. 30. Paraphyses and young ascus. 31–34. Mature asci with ascospores. 35. Ascus stipe with signs of outer wall disintegration. 36. Conidia of the Brachysporium polyseptatum anamorph, from nature. 37. Conidiophore, from nature (arrow indicates conidiogenous locus). FIGS. 28, 30, 32, 33, 36, 37: DIC; 29, 31, 34, 35: Phase contrast. Scale bars: 28, 29, 35–37 = 10 µm; 30–34 = 20 µm. FIGS. 28–37 from PRM 900539 (Holotype).

	KEY TO SPECIES OF CRYPTADELPHIA AND BRACHYSPORIUM

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1. Conidia 2-septate 2 1. Conidia more than 2-septate 3     2. Middle cells of conidia pale brown, apical cell darkest, (8.0–)9.0–11.5 µm wide C. obovata     2. Middle and apical cells of conidia dark brown, 11.0–14.0 µm wide B. brittanicum 3. Conidia 3-septate 4 3. Conidia more than 3-septate 9     4. Conidia versicolorous, one or two polar cells (sub)hyaline, middle cells brown 5     4. Conidia concolorous, all cells of the same brown color C. brevior 5. Conidia ellipsoidal 6 5. Conidia obovoidal to clavate 7     6. Conidia regularly 3-septate, 17.0–23.0 µm long C. groenendalensis     6. Conidia occasionally developing 4th septum, 24.0–29.0(–33.5) µm long C. abietis     7. Conidia up to 24 µm long (18.0–25.0 µm) B. dingleyae 7. Conidia longer than 24 µm 8     8. Middle and apical cells of conidia mid to dark brown, 9.5(–11.0) µm wide C. polyseptata     8. Middle and apical cells of conidia yellowish-brown, 10.0–17.5(–21.0) µm wide B. helgolandicum 9. Conidia 4-septate 10 9. Conidia 5-septate B. novaezelandiae     10. Conidia broadly fusiform to ellipsoidal B. masonii     10. Conidia limoniform, pendulous 11 11. Conidia 29.0–41.0 x (14.5–)15.0–16.0(–17.0) µm C. pendulispora 11. Conidia 41.0–51.0 x 20.0–25.0 µm B. pulchrum

Cryptadelphia abietis Réblová & Seifert, sp. nov. FIGS. 2–8

View larger version (132K): [in this window] [in a new window]   FIGS. 2–8. Cryptadelphia abietis. 2. Ascospores. 3. Mature asci containing ascospores. 4. Ascal apex with annulus. 5. Mature ascus with ascospores. 6. Conidia of the Brachysporium abietinum anamorph, from nature. 7, 8. Conidiophores and conidia, from nature. FIGS. 2, 4–8: DIC; 3: Phase contrast. Scale bars: 2–4, 6–9 = 10 µm; 5 = 20 µm. FIGS. 2–8 from PRM 900536 (Holotype).

Anamorphe: Brachysporium abietinum. Conidia ellipsoidea, 3–4-septata, 24.0–29.0(–33.5) ( = 31.8 ± 0.8) x 10.0–12.5(–13.5) ( = 12.0 ± 0.2) µm, cellulis polaribus subhyalinis usque hyalinis, cellulis centralis brunneis.

Anamorph. – Brachysporium abietinum Hol.-Jech., Folia Geobot. Phytotax. 7:221. 1972.

Perithecia semi-immersed to nearly superficial with only the base immersed, solitary, globose to subglobose, conical around the ostiole, papillate, 190–240 µm diam, 250–270 µm high, dark brown to nearly black, glabrous. Perithecial wall 24.0–28.0 µm thick, leathery to fragile, consisting of two regions. Paraphyses branching, hyaline, septate, 3.0–5.0 µm wide near the base, tapering to 1.5–2.0 µm, longer than the asci. Asci cylindrical-clavate, 86.0–110.0 ( = 98.0 ± 1.8) µm long in pars sporifera, 8.0–10.0 ( = 8.8 ± 0.2) µm wide, with long, slender stipe, 20.0–34.0 ( = 28.0 ± 1.4) µm long, L/W 14.5:1, broadly rounded to truncate at the apex, with distinct refractive apical annulus, ca. 3.0 µm diam, 2.0 µm high, containing eight obliquely uniseriate spores. Ascospores ellipsoidal to fusiform to oblonglemon-shaped, straight or inequilateral, (13.0–)14.0–17.0(–18.0) ( = 15.8 ± 0.2) µm long, (4.5–)5.0–6.0(–7.0) ( = 5.7 ± 0.1) µm wide, at first 1-celled, followed by formation of a median septum, smooth-walled.

Colonies on the natural substrate irregularly effuse, dark brown, hair y, conidiophores interspersed among the perithecia. Conidiophores mononematous, macronematous, solitary, erect, straight or slightly flexuous, cylindrical, unbranched, dark brown near the bottom, paler toward the apex, 1–5-septate, darker brown at the septa, 200–370 µm long, 5.0–7.0 µm wide above the base, 5.0–6.0 µm wide in the middle, tapering to 3.0–4.0 µm. Conidiogenous cells integrated, terminal, proliferating sympodially, polyblastic, denticulate, hyaline. Conidia ellipsoidal, 24.0–29.0 (–33.5) ( = 31.8 ± 0.8) µm long, 10.0–12.5(–13.5) ( = 12.0 ± 0.2) µm wide, 3–septate, with two long, central cells, sometimes with a fourth septum subdividing the short apical cell, not constricted or slightly constricted at the septa, versicolorous, middle cells brown, polar cells hyaline to subhyaline, smooth-walled; conidia borne on short hyaline pedicels singly or in groups at the apex of the conidiophore.

Etymology. – Abietis (L), referring to Abies alba.

Illustrations and descriptions. – Brachysporium abietinum was fully described and illustrated by Holubová-Jechová (1972:221, Pl. 5-b, Pl. 6-5) and Hughes (1977a:1, FIGS. 1–3).

Known distribution. – Canada (Ontario), Czech Republic, Slovak Republic (Hughes 1977a).

Habitat. – Saprobic on decayed coniferous wood, Abies alba, Picea abies.

Holotype. – CZECH REPUBLIC. Southern Bohemia: umava Mountains National Park, Záto, Boubínsk prales National Nature Reserve, decayed wood of a trunk of Picea abies, 17 Aug 1999, M. Réblové M.R. 1476-99 (PRM 900536).

Additional specimens examined. – CANADA. Ontario: Blakeney, decaying wood of unidentified conifer, 17 Jul 1952 (DAOM 28643a). CZECH REPUBLIC. Southern Bohemia: umava Mountains National Park, elezná Ruda, glacial cirque of the Certovo jezero lake, decayed wood of a trunk of Abies alba, 12 Aug 1999, M. Réblová M.R. 1446-99; ibid., Modrava, Mount Studená hora, decayed decorticated wood of a root of Picea abies, 26 Aug 2000, M. Réblová M.R. 1761-00*. SLOVAK REPUBLIC. Central Slovakia. Bánská Bystrica, Kremnické pohorie Mountains, Badínsk prales forest, in valley of the Badínsk potok, decayed wood of a trunk of A. alba, 9 Aug 1969, V. Holubová-Jechová (PRM 710006*, holotype of Brachysporium abietinum); Bánská Bystrica, Cierny Balog, Slovenské Rudohorie Mountains, Dobrocsk prales forest, decayed wood of a trunk of P. abies, 8 Aug 1969, V. Holubová-Jechová M.R. 2548-00*.

Commentary. – Cryptadelphia abietis is similar to C. brevior and C. obovata in ascospore size but differs clearly by the versicolorous, 3-septate conidia of its anamorph. Brachysporium abietinum differs from the somewhat similar B. nigrum by larger and paler conidia and its coniferous substrate. The minority of conidia of B. abietinum that develop a fourth septum (see below) are similar to those of Brachysporium masonii S. Hughes, which differs by having consistently 4-septate, somewhat broader conidia and an angiosperm wood substrate.

Hughes (1977a) compared Canadian material of B. abietinum with the type material from the Czech Republic and found that some conidia in the Canadian collection developed a fourth septum and that the central cells then were unequally septate. Such conidia frequently were observed in recently collected Bohemian material (FIG. 7, PRM 900536; M.R. 1446–99). We did not find the teleomorph on the Canadian specimen, which has abundant perithecia of Chaetosphaeria abietis (Höhn.) W. Gams & Hol.-Jech.

The connection between these two morphs has not been confirmed by culturing ascospores and conidia.

Cryptadelphia brevior Réblová & Seifert, sp. nov. FIGS. 9–12

View larger version (168K): [in this window] [in a new window]   FIGS. 9–14. 9–12. Cryptadelphia brevior. 9, 10. Ascospores. 11. Mature asci containing ascospores, with remnants of paraphyses. 12. Conidia of the Brachysporium brevius anamorph, from nature. 13, 14. Cryptadelphia obovata. 13. Ascospores. 14. Conidia of the Brachysporium obovatum anamorph, from nature. FIGS. 9, 11, 12, 13, 14: DIC; 10: Phase contrast. Scale bars: 9–14 = 10 µm. FIGS. 9–12 from PRM 647191 (Holotype); 13, 14 from PRM 900538 (Holotype).

Anamorphe: Brachysporium brevius. Conidia ellipsoidea usque cylindracea, 3-septata, 17.5–22.5 ( = 20.0 ± 0.5) x (7.0–)8.0–10.0(–12.5) ( = 9.6 ± 0.5) µm, cellulis brunneis.

Anamorph. – Brachysporium brevius Hol.-Jech., Folia Geobot. Phytotax. 7:222. 1972.

= Brachysporium noblesiae G.P. White & Illman, Fungi Canadenses No. 327. 1990.

Perithecia nearly superficial with the base slightly immersed, solitary, globose to subglobose, conical around the ostiole, papillate, 180–220 µm diam, 250–320 µm high, dark brown to nearly black, glabrous, finely roughened. Perithecial wall 19.0–24.0 µm thick, leathery to fragile, consisting of two regions. Paraphyses branching, hyaline, septate, 3.0–4.0 µm wide near the base, tapering to 1.5–2.0 µm, longer than the asci. Asci cylindrical-clavate, 88.0–100.0(–110.0) ( = 94.5 ± 1.9) µm long in pars sporifera, (7.0–)8.0–9.0 ( = 8.4 ± 0.2) µm wide, with long, slender stipe, 18.0–27.0 ( = 21.0 ± 1.1) µm long, L/W 13.5:1, broadly rounded to truncate at the apex, with distinct refractive apical annulus, ca. 3.0 µm diam, 2.0–2.5 µm high, containing eight obliquely uniseriate spores. Ascospores ellipsoidal to fusiform to oblonglemon-shaped, straight or inequilateral, 15.0–17.0 ( = 15.8 ± 0.3) µm long, 5.0–6.0 ( = 5.3 ± 0.1) µm wide, 1-celled, formation of a median septum not observed, smooth-walled.

Colonies on the nature substrate effuse, dark brown, hairy, conidiophores interspersed among the perithecia. Conidiophores mononematous, macronematous, solitary, erect, straight or slightly flexuous, cylindrical, unbranched, dark brown near the bottom, paler toward the apex, 5-septate, darker brown at the septa, 60.0–130.0(–150.0) µm long, ca. 5.0 µm wide above the base, 4.0–5.0 µm wide in the middle, tapering to 3.0–5.0 µm. Conidiogenous cells integrated, terminal, sympodially proliferating, polyblastic, denticulate, hyaline. Conidia ellipsoidal to short cylindrical, slightly tapering at the base, broadly rounded at the top, 17.5–22.5 ( = 20.0 ± 0.5) µm long, (7.0–)8.0–10.0(–12.5) ( = 9.6 ± 0.5) µm wide, rounded at each end, 3-septate, septa equidistant, not constricted or gently constricted at the septa, at maturity concolorous, mid brown, darker brown at the septa; conidia borne on short subhyaline pedicels singly or in groups at the apex of the conidiophore.

Etymology. – Brevior (L), shorter, for the shorter conidiophore of this species, chosen to match the epithet of the anamorph.

Illustrations and descriptions. – Brachysporium brevius was fully described and illustrated by Holubová-Jechová (1972:222, Pl. 5-d, Pl. 6-6) and White and Illman (1990:1, 2, FIGS. 1–4, as B. noblesiae).

Known distribution. – Canada (Ontario), Czech Republic, Slovak Republic (White and Illman 1990, as B. noblesiae).

Habitat. – Saprobic on decayed wood of Fagus sylvatica.

Holotype. – CZECH REPUBLIC. Moravia: Vsetín, Rajnochovice, Hostnské vrchy Mountains, ernava forest, decayed wood of a trunk of Fagus sylvatica, 17 Aug 1966, V. Holubová-Jechová (PRM 647191, also holotype of Brachysporium brevius).

Additional material examined. – CANADA. Ontario: Blossom Park, Ottawa, undetermined decayed wood, 28 Jul 1981, G.P. White (DAOM 181293* holotype of B. noblesiae). CZECH REPUBLIC. Southern Bohemia: umava Mountains National Park, Záto, Boubínsk prales National Nature Reserve, decayed wood of Fagus sylvatica, 30 Jun 1971, V. Holubová-Jechová M.R. 2552-02Moravia: Louná nad Desnou, Hrub Jeseník Mountains, Mount Mraveneník, decayed wood of a trunk of F. sylvatica, 3 Aug 1971, V. Holubová-Jechová M.R. 2553-02*. SLOVAK REPUBLIC. Central Slovakia: Detva, Slovenské Rudohorie, Mount Kalamárka, decayed wood of a branch of F. sylvatica, 7 Aug 1969, V. Holubová-Jechová M.R. 2554-02*.

Commentary. – Brachysporium brevius is readily distinguished from other Brachysporium species in having conidia with three equidistant septa and concolorous, moderate brown conidial cells with dark brown septa ca. 2.0 µm thick. This is the only species of Cryptadelphia where a median septum was not observed in the ascospores. So far, the teleomorph is known only from the type collection. Examination of more material might demonstrate this character, which is typical of other known Cryptadelphia species.

The connection between these two morphs has not been confirmed by culturing ascospores and conidia.

Cryptadelphia groenendalensis (Sacc., E. Bomm. & M. Rouss.) Réblová & Seifert, comb. nov. FIGS 15–19

Basionym. – Zignoëlla groenendalensis Sacc., E. Bomm. & M. Rouss., Atti R. Ist. Veneto Sci., Venezia Ser. 6, Vol. 2:435. 1884.

= Wallrothiella melanostigmoides Feltgen, Vorstud. Pilz-Flora Luxemb. 1., Nachträge 3:285. 1903, non Trichosphaeria melanostigmoides (Feltgen) Munk sensu Munk, Dansk Bot. Arkiv 17(1):184. 1957.

= Trichosphaeria notabilis Mouton sensu Munk, Dansk Bot. Arkiv 17(1):183. 1957.

Anamorph. – Brachysporium nigrum (Link) S. Hughes, Can. J. Bot. 36:742. 1958.

For full synonymy of the anamorph refer to Hughes (1958).

Perithecia nearly superficial with base slightly immersed, solitary or in small groups, globose to subglobose, conical around the ostiole, papillate, (150–) 170–210 µm diam, (160–)210–220 µm high, dark brown to nearly black, glabrous. Perithecial wall 22.0–26.0(–30.0) µm thick, leathery to fragile, consisting of two regions. Paraphyses persistent, branching, hyaline, septate, 3.0–5.0 µm wide near the base, tapering to 1.5–2.0 µm, longer than the asci. Asci cylindrical-fusiform, (72.0–)85.0–118.0(–128.0) ( = 91.7 ± 1.1) µm long in pars sporifera, (7.5–)8.0–10.0 (–11.0) ( = 8.7 ± 0.1) µm wide, with long, slender stipe, (12.0–)20.0–35.0(–44.0) ( = 27.3 ± 0.8) µm long, with age disintegrating externally along the vertical axis and becoming longer up to 52.0–66.0 µm, L/W 13.5:1, broadly rounded to truncate at the apex, with distinct refractive apical annulus, ca. 3.0 µm diam, 2.0 µm high, containing eight obliquely uni-or biseriate spores. Ascospores ellipsoidal to fusiform to oblonglemon-shaped, straight or inequilateral, (14.0–)15.0–18.0(–20.0) ( = 16.0 ± 0.1) µm long, (4.0–)5.0–6.0(–7.0) ( = 5.7 ± 0.04) µm wide, at first 1-celled, with a median septum forming at maturity, not constricted at the septum, smooth-walled.

Colonies on the natural substrate effuse dark brown, hairy, conidiophores interspersed among the perithecia. Conidiophores mononematous, macronematous, solitary, erect, straight or slightly flexuous, cylindrical, unbranched, dark brown, paler toward the apex, 1–7-septate, darker brown at the septa, 140–420 µm long, 6.5–8.5 µm wide at the base, 5.0–7.0 µm wide in the middle, tapering to 3.0–4.0 µm. Conidiogenous cells integrated, terminal, sympodially proliferating, polyblastic, denticulate, hyaline. Conidia ellipsoidal, 17.0–23.0 ( = 18.9 ± 0.7) µm long, 8.0–11.0 ( = 9.8 ± 0.2) µm wide, 3-septate, not constricted or gently constricted at the septa, versicolorous, two middle cells brown, polar cells hyaline to subhyaline to very pale brown, smooth-walled; conidia borne on short subhyaline pedicels singly or in groups at the apex of conidiophore.

Characteristics in culture. – Colonies after 21 d on Blakeslee’s MEA at room temperature 1–2 mm diam, convex, sterile, with moderately dense somewhat radiating aerial mycelium, grayish brown (5D3) in mass, the reverse dark brown to black (5–6F8), the margin discrete to slightly gnawed.

Illustrations and descriptions. – Berlese (1894:94, Pl. 85, FIG. 4), Feltgen (1903:285), Munk (1948:6, Pl. 2, FIG. 1; 1957:183, as Trichosphaeria cf. notabilis). Brachysporium nigrum was fully described and illustrated by Ellis (1966:46: FIG. 35), Holubová-Jechová (1972: 219, Pl. 5-a, Pl. 6-2,4,7) and Hughes and Kokko (1975:1, 2, FIGS. 1–8).

Habitat. – Saprobic on decaying wood and bark of deciduous trees, rarely on conifers, recorded from Abies alba, Acer platanoides, A. pseudoplatanus, Betula verrucosa, Carpinus betulus, Castanea sativa, Fagus sylvatica, F. grandifolia, Fraxinus excelsior, Fraxinus sp., Lonicera sp., Populus sp. Quercus petraea, Quercus sp., Tilia cordata, and Ulmus glabra (Hughes 1955, Ellis 1966, Holubová-Jechová 1972, Hughes and Kokko 1975).

Known distribution. – Belgium, Canada (British Columbia, Ontario, Quebec, Saskatchewan, Czech Republic, Denmark, Germany (W. Gams, personal communication), Great Britain, Hungary, Ukraine and USA (New York, Vermont) (Hughes 1955; Ellis 1966; Holubová-Jechová 1972; Hughes and Kokko 1975; Révay 1985, 1986).

Holotypes. – BELGIUM. Groenendal near Brussels, decayed wood of Fagus sylvatica, M. Rousseau (PAD, as Zignoëlla groenendalensis). LUXEMBOURG. Kockelscheuer, decayed wood of a branch of Quercus sp., Aug 1903, J. Feltgen (LUX 043444, as Wallrothiella melanostigmoides).

Additional specimens examined. – CZECH REPUBLIC. Southern Bohemia: umava Mountains, Volary, Boubínsk prales National Nature Reserve, decayed wood of F. sylvatica, 17 Aug 1999, M. Réblová M.R. 1606-99, 1800-99, 2455-00; ibid., Volary, Medvdice National Nature Reserve, decayed wood of Ulmus glabra, 18 Sep 1997, M. Réblová M.R. 1011-97, 1016-97, 1414-99, 1448-99, 1522-99, 1907-01, 1925-00ibid., Stoec, Mount Spálenite, decayed wood of Fraxinus excelsior, 16 Aug 1999, M. Réblová M.R. 1497-99, 1744-00, 2497-00; ibid., Práily, Mount danidla, decayed wood of F. sylvatica, 26 Aug 1998, M. Réblová M.R. 1409-98, 1443-98, 2092-00, 2411-00. Moravia: flood plain forest Ranpurk near Lanhot, decayed wood of Carpinus betulus; 15 Oct 1997, M. Réblová M.R. 1125-97; 16 Oct 1997, M.R. 1131-97Mikulov, Milovice, Mount picák, decayed wood of C. betulus, 2 Oct 1999M. Réblová M.R. 1495-99*. DENMARK. Mols: Jutland, decayed wood of Quercus sp., 28 Apr 1943, A. Munk 33; 29 Sep 1943, A. Munk 21 (C, as Trichosphaeria cf. notabilis). UKRAINE. Ruthenia: Carpathian Mountains, Rachiv, Kvasi, on the right bank of the Tisa river, decayed wood of F. sylvatica, 29 Jun 1997, M. Réblová M.R. 951-97; Massif Borava, Guklivij, decayed wood of A. alba, 21 Jul 1998, M. Réblová M.R. 1314-99.

Living cultures. – Derived from specimens M.R. 1495-99 (DAOM 231136, single conidium), M.R. 1497-99 (DAOM 231137, single ascospore).

Commentary. – Cryptadelphia groenendalensis can be distinguished easily from other Cryptadelphia species by its longer ascospores and asci in pars sporifera and by the characters of its B. nigrum anamorph. The conidia of B. nigrum resemble those of B. abietinum but differ by being shorter and 3-septate, with more or less equal polar cells; the species occurs on wood and bark of angiosperms rather than gymnosperms.

Munk (1948, 1957) studied two species of Trichosphaeria, T. cf. notabilis Mouton and T. cf. melanostigmoides (Feltgen) Munk. The material that Munk (1957) used to propose a combination for T. melanostigmoides (DENMARK. Mols: Jutland, decayed wood, 29 Apr 1943, A. Munk 16, C!), belongs to a fungus related to Didymopsamma moravica Petr. (Petrak 1925, Réblová 2000). The specimen that Munk (1948, 1957) used for the description and illustration of Trichosphaeria cf. notabilis sensu Munk (DENMARK. Sjælland, Tokkekøb Hegn, decayed wood of Fagus sylvatica, 19 Aug 1963, A. Munk, C!), is conspecific with Z. groenendalensis. Examination of the holotype material of Trichosphaeria notabilis Mouton (Mouton 1900; BELGIUM. Liège, Bois de chêne, decayed wood of Quercus sp., V. Mouton 208, BR 98446.88!) revealed a fungus whose affinity lies with Phaeotrichosphaeria Sivan. rather than with any of the fungi described here.

Cryptadelphia obovata Réblová & Seifert, sp. nov. FIGS. 13–14

Perithecia superficialia, basi subimmersa, conica usque subglobosa, papilla minuta, (180–)270–300 µm diam, (180–)250–300 µm alta, nuda. Asci cylindrici usque clavati, parte sporifera (76.0–)80.0–94.0 ( = 82.3 ± 1.4) x (8.0–)8.5–10.0 ( = 9 ± 0.08) µm, stipite angusto (14.5–)19.0–30.0 (–34.0) ( = 24.8 ± 1.1) µm longo, apice non amyloideo, annulo refringente praediti. Ascosporae ellipsoideae usque fusiformes usque limoniformes, (12.5–)13.0–

16.0 ( = 14.5 ± 0.1) x 5.0–6.0 ( = 5.5 ± 0.05) µm, 0–1-septatae, septo mediano, non constrictae, leves, hyalinae.

Anamorphe: Brachysporium obovatum. Conidia obovoidea usque obpyriformes, 2-septata, (16.0–)18.5–24.0 ( = 19.7

± 0.9) x (8.0–)9.0–11.5 ( = 9.5 ± 0.4) µm, cellula basali hyalina usque subhyalina, cellulis superioribus brunneis.

Anamorph. – Brachysporium obovatum (Berk.) Sacc., Syll. Fung. 4:427. 1886.

For full synonymy of the anamorph refer to Hughes (1958).

Perithecia superficial with base slightly immersed, solitary, globose to subglobose, conical around the ostiole, papillate, (180–)270–300 µm diam, (180–) 250–300 µm high, dark brown to nearly black, glabrous, finely roughened. Perithecial wall 24.0–30.0 µm thick, leathery to fragile, consisting of two regions. Paraphyses branching, hyaline, septate, 3.0–4.0 µm wide near the base, tapering to 1.5–2.0 µm, longer than the asci. Asci cylindrical-fusiform, (76.0–) 80.0–94.0 ( = 82.3 ± 1.4) µm long in pars sporifera, (8.0–)8.5–10.0 ( = 9.0 ± 0.08) µm wide, with long, slender stipe, (14.5–)19.0–30.0(–34.0) ( = 24.8 ± 1.1) µm long, L/W 12.5:1, broadly rounded to truncate at the apex, with distinct refractive apical annulus, ca. 2.5–3.0 µm diam, 1.5–2.0 µm high, containing eight obliquely uni- or biseriate spores. Ascospores ellipsoidal to fusiform to oblonglemon-shaped, straight or inequilateral, (12.5–)13.0–16.0 ( = 14.5 ± 0.1) µm long, 5.0–6.0 ( = 5.5 ± 0.05) µm wide, at first 1-celled, with a median septum at maturity, not constricted at the septum, smooth-walled.

Colonies on the natural substrate effuse, dark brown, hairy, conidiophores interspersed among the perithecia. Conidiophores mononematous, macronematous, solitary, erect, straight or slightly flexuous, cylindrical, unbranched, dark brown near the bottom, paler toward the apex, 5-septate, darker brown at the septa, 150–250 µm long, ca. 5.0 µm wide above the base, 4.0–5.0 µm wide in the middle, tapering to 3.0–4.0 µm. Conidiogenous cells integrated, terminal, sympodially proliferating, polyblastic, denticulate, hyaline. Conidia obovoidal to obpyriform, (16.0–)18.5–24.0 ( = 19.7 ± 0.9) µm long, (8.0–)9.0–11.5 ( = 9.5 ± 0.4) µm wide at the broadest point, 2-septate, not constricted or gently constricted at the septa, basal cell smaller, hyaline to subhyaline, middle cell pale brown, terminal cell largest, darker brown, smooth-walled; conidia borne on short pedicels concolorous with the basal cell, singly or in groups at the apex of the conidiophore.

Etymology. – Obovatus (L), obovate, for the shape of the conidia, chosen to match the epithet of the anamorph.

Illustrations and descriptions. – Brachysporium obovatum was fully described and illustrated by Ellis (1966:44, FIG. 33), Hughes (1955:264), Holubová-Jechová (1972:218, Pl. 5-c, Pl. 6-1,3), Hughes and Kokko (1977:1, 2, FIGS. 1–4) and Révay (1986:74).

Habitat. – Saprobic on decayed wood and bark of deciduous trees, rarely on conifers, recorded from Acer pseudoplatanus, Alnus glutinosa, Betula alleghaniensis, B. verrucosa, Carpinus betulus, Fagus sylvatica, Fraxinus excelsior, Pinus sp., Populus sp., Prunus avium, P. spinosa, Quercus cerris, Q. petraea, Q. robur, Quercus sp., Sambucus nigra, Tilia cordata, and Ulmus sp. (Ellis 1966, Holubová-Jechová 1972, Hughes and Kokko 1977).

Known distribution. – Austria, Belgium, Canada (Ontario and Quebec), Czech Republic, Germany, Great Britain, Hungary, Italy, Slovak Republic and USA (New York) (Ellis 1966, Holubová-Jechová 1972, Hughes and Kokko 1977, Révay 1986).

Holotype. – CZECH REPUBLIC. Moravia: Beclav, Lanhot, Cahnov forest, decayed wood of a trunk of Q. robur, 4 Oct 1977, V. Holubová-Jechová M.R. 1817- 01 (PRM 900538).

Additional specimens examined. – CZECH REPUBLIC. Northern Bohemia: Doksy, Mount Mal Bezdz, decayed wood of a trunk of F. sylvatica, 26 Apr 1973, V. Holubová-Jechová M.R. 2555-02; Southern Bohemia. umava Mountain National Park, Volary, Medvdice National Nature Reserve, decayed wood of Ulmus glabra, 15 Aug 1999, M. Réblová M.R. 1798-99. Moravia: ÿár nad Sázavou, Mount ákova hora (ca. 750 m), decayed wood of a trunk of F. sylvatica, 25 Jul 1970, V. Holubová-Jechová M.R. 2556-02Perov, ebracka Nature Reserve, decayed wood of a branch of Fraxinus excelsior, 14 Nov 1972, V. Holubová-Jechová M.R. 1808-01 Chropyne, Horní les forest, on decayed wood of twigs of Quercus robur, 12 Nov 1972, Z. Pouzar M.R. 2558-02*; Náme nad Oslavou, Sedlec, Duby forest, decayed wood, 2 Jul 1990, V. Holubová-Jechová M.R. 2559-02*; Veverská Bítka, Prádelna, decayed wood of Tilia cordata, 20 Aug 1974, V. Holubová-Jechová M.R. 2560-02*; Hustopece, Ivan, Horní les forest, decayed wood of a branch of Q. robur, 31 Aug 1971, V. Holubová-Jechová M.R. 1809-01; Lednice, decayed wood of a trunk of Q. robur, 21 Aug 1974, V. Holubová-Jechová M.R. 2563-02*. SLOVAK REPUBLIC. Central Slovakia: Hrhov, Slovensk kras Mountains, Mount Doln vrch, decayed wood of roots of Carpinus betulus, 12 May 1977, V. Holubová-Jechová M.R. 2564-02*; Southern Slovakia: Nitra, Mochovce, Mount Zudrok (ca. 270 m) decayed wood of a trunk of Quercus cerris, 5 Aug 1975, V. Holubová-Jechová M.R. 1810-01; Prievidza, Nitrica, decayed wood of Fagus sylvatica, 9 Jul 1976, V. Holubová-Jechová M.R. 2566-02*; Velk Krtí, Nová Ves, Mount Hol vrok, decayed wood of a stump of Quercus petraea, 14 Aug 1975, V. Holubová-Jechová M.R. 2567-02*.

Commentary. – Cryptadelphia obovata resembles C. abietis and C. brevior in ascospore shape and size but differs by having shorter asci. The characters of conidia of B. obovatum, B. abietinum and B. brevius, respectively, can easily distinguish the otherwise similar holomorphs. In the type material of C. obovata, four mature and four immature ascospores often were observed in a single ascus.

Brachysporium obovatum resembles the freshwater species B. helgolandicum Schaumann (Schaumann 1973), which differs by having conidia that are 2–3-septate, pale yellowish-brown with a subhyaline basal cell, borne on a flexuous ca. 7.0–18.0 µm long pedicel. Brachysporium obovatum is also similar to B. britannicum S. Hughes, which differs by having dark brown conidia. Teleomorphs have yet to be discovered for B. helgolandicum and B. britannicum.

The connection between these C. obovatum and its putative anamorph has not yet been confirmed by culturing ascospores and conidia.

Cryptadelphia pendulispora Réblová & Seifert, sp. nov. FIGS. 20–27

View larger version (147K): [in this window] [in a new window]   FIGS. 20–27. Cryptadelphia pendulispora. 20, 21. Ascospores. 22, 23. Mature asci with ascospores and paraphyses. 24. Ascal apex with annulus. 25. Conidiophore with conidium of the Brachysporium pendulisporum anamorph, from nature. 26, 27 Conidia, from nature. FIGS. 20, 22–27: DIC, 21: Phase contrast. Scale bars: 20–27 = 10 µm. FIGS. 20–27 from DAOM 44914a (Holotype).

Anamorphe: Brachysporium pendulisporum. Conidia fusiformis usque limoniformes, 4-septata, 29.0–41.0 ( = 35.3 ± 1.1) x (14.5–)15.0–16.0(–17.0) ( = 15.3 ± 0.1) µm, cellulis polaribus hyalinis usque subhyalinis, cellulis centralis brunneis.

Anamorph. – Brachysporium pendulisporum S. Hughes, Can. J. Bot. 33:266. 1955.

Perithecia semi-immersed to nearly superficial with the base slightly immersed, solitary, globose to subglobose, conical around the ostiole, papillate, 190–270 µm diam, 250–260 µm high, dark brown to nearly black, glabrous, finely roughened. Perithecial wall 22.0–28.0 µm thick, leathery to fragile, consisting of two regions. Paraphyses branching, hyaline, septate, 3–4 µm wide near the base, tapering to 1.5–2.0 µm, longer than the asci. Asci cylindrical-clavate, (74.0–) 77.0–92.0(–97.0) ( = 83.2 ± 1.3) µm long in pars sporifera, (8.0–)9.0–10.0 ( = 8.9 ± 0.2) µm wide, with long, slender stipe, 15.0–30.0(–44.0) ( = 25.5 ± 0.2) µm long, L/W 12.5:1, broadly rounded to truncate at the apex, with distinct refractive apical annulus, ca. 3.5(–4.0) µm diam, 1.0–1.5 µm high, containing eight obliquely uni- or biseriate spores. Ascospores ellipsoidal to fusiform to oblonglemon-shaped, straight or inequilateral, (12.0–)13.0–14.0 (–16.0) ( = 13.4 ± 0.2) µm long, 5.0–6.0(–7.0) ( = 5.6 ± 0.1) µm wide, at first 1-celled, with a median septum at maturity, not constricted at the septum, smooth-walled.

Colonies on the natural substrate effuse, dark brown, hairy, conidiophores interspersed among the perithecia. Conidiophores mononematous, macronematous, solitary or in groups, erect, straight or slightly flexuous, cylindrical, unbranched, dark brown near the bottom, paler toward the apex, 1–8-septate, darker brown at the septa, 100–290 µm long, 5.5–7.0 µm wide above the base, 4.0–5.5 µm wide in the middle, tapering to 3.5–5.0 µm. Conidiogenous cells integrated, terminal, sympodially proliferating, polyblastic, denticulate, hyaline. Conidia fusoid to limoniform, 29.0–41.0 ( = 35.3 ± 1.1) µm long, (14.5–)15.0–16.0(–17.0) ( = 15.3 ± 0.1) µm wide, 4-septate, not constricted or gently constricted at the septa, versi-colorous, central cells brown to dark brown, polar cells small, subhyaline, smooth-walled; conidia borne on short subhyaline to pale brown pedicels, singly or in groups at the apex of the conidiophore.

Etymology. – Pendulus- (L), pendulous, drooping, -spora (L), spore, for the appearance of conidia, chosen to match the epithet of the anamorph.

Illustrations and descriptions. – Brachysporium pendulisporum was described fully and illustrated by Hughes (1955:266; 1977b:1, 2, FIGS. 1–5), Ellis (1971: 223, FIGS. 153 g), and Matsushima (1987:6, FIG. 395).

Known distribution. – Canada (Quebec), USA (New York) (Hughes 1977b, Matsushima 1987).

Habitat. – Saprobic on decaying wood and bark of angiosperms.

Holotype. – CANADA. Quebec: Old Chelsea, decayed wood, 21 Oct 1954, S.J. Hughes (DAOM 44914, also holotype of Brachysporium pendulisporum).

Additional specimen examined. – USA. New York State: Alleghany State Park near Star Lake, decayed wood, 7 Sep 1961, S.J. Hughes (DAOM 147404).

Commentary. – Cryptadelphia pendulispora cannot be distinguished from C. obovata by ascus or ascospore dimensions, but their anamorphs, B. pendulisporum and B. obovatum have different conidium septation and dimensions. The connection between these two morphs has not yet been confirmed by culturing ascospores and conidia.

Cryptadelphia polyseptata Réblová & Seifert, sp. nov. FIGS. 28–37

Perithecia superficialia, basi subimmersa, conica usque subglobosa, papilla minuta, (150–)180–240 µm diam, 230–250 µm alta, nuda. Asci cylindrici usque clavati, parte sporifera (83.0–)88.0–108.0 ( = 95 ± 1.7) x (7.0–)8.0–9.0 (

= 7.8 ± 0.1) µm, stipite angusto 19.0–30.0 ( = 24.0 ± 1.4) µm longo, apice non amyloideo, annulo refringente praediti. Ascosporae ellipsoideae usque fusiformes usque limoniformes, 12.0–15.0 (–17.0) ( = 14.0 ± 0.1) x 5.0–6.0 ( = 5.4 ± 0.05) µm, 0–1-septatae, septo mediano, non constrictae, leves, hyalinae.

Anamorphe: Brachysporium polyseptatum. Conidia obovoidea, 3–4-septata, 24.0–29.0 ( = 27.3 ± 0.5) x 9.5(–11.0) ( = 9.6 ± 0.1) µm, cellula basali subhyalina, cellulis superioribus brunneis.

Anamorph. – Brachysporium polyseptatum (Preuss) S. Hughes, Can. J. Bot. 33:264. 1955.

= Brachysporium bloxami (Cooke) Sacc., Syll. Fung. 4: 426. 1886.

For full synonymy of the anamorph refer to Hughes (1955, 1958).

Perithecia nearly superficial with base slightly immersed, solitary or in small groups, globose to subglobose, conical around the ostiole, papillate, (150–) 180–240 µm diam, 230–250 µm high, dark brown to nearly black, glabrous, finely roughened. Perithecial wall 24.0–26.0(–30.0) µm thick, leathery to fragile, consisting of two regions. Paraphyses branching, hyaline, septate, 3.0–4.0 µm wide near the base, tapering to 1.5–2.0 µm, longer than the asci. Asci cylindrical-clavate, (83.0–)88.0–108.0 ( = 95 ± 1.7) µm long in pars sporifera, (7.0–)8.0–9.0 ( = 7.8 ± 0.1) µm wide, with long, slender stipe, 19.0–30.0 ( = 24.0 ± 1.4) µm long, L/W 15.0:1, broadly rounded to truncate at the apex, with distinct refractive apical annulus distinct, ca. 3.0–4.0 µm diam, 2 µm high, containing eight obliquely uni- or biseriate spores. Ascospores ellipsoidal to fusiform to oblonglemon-shaped, straight or inequilateral, 12.0–15.0(–17.0) ( = 14.0 ± 0.1) µm long, 5.0–6.0 ( = 5.4 ± 0.05) µm wide, at first 1-celled, with a median septum at maturity, not constricted at the septum, smooth-walled.

Colonies on the natural substrate effuse, dark brown, hairy, conidiophores interspersed among the perithecia. Conidiophores mononematous, macronematous, solitary, erect, straight or slightly flexuous, cylindrical, unbranched, dark brown near the bottom, paler toward the apex, 1–7-septate, darker brown at the septa, 250–350 µm long, 5.0–7.0 µm wide above the base, 4.0–5.0 µm wide in the middle, tapering to 3.0–5.0 µm. Conidiogenous cells integrated, terminal, sympodially proliferating, polyblastic, denticulate, hyaline. Conidia obovoidal, 24.0–29.0 ( = 27.3 ± 0.5) µm long, 9.5(–11.0) ( = 9.6 ± 0.1) µm wide, with broadly rounded apical cell, 3–4-septate, not constricted or slightly constricted at the septa, versicolorous, basal cell small, subhyaline, other cells larger and mid to dark brown, smooth-walled; conidia borne on short hyaline pedicels, singly or in groups at the apex of the conidiophore.

Characteristics in culture. – Colonies after 21 d on Blakeslee’s MEA at room temperature about 5 mm diam, planar, sterile, with sparse dense mycelium, the obverse and reverse dark brown (5–6F8) to black, the margin discrete, submerged. Colonies after 21 d on PCA at room temperature about 2 mm diam, planar to convex, sterile, covered with low, lanose white to gray aerial mycelium, densest on the inoculum block, obverse and reverse dark brown (6F4), the margin diffuse, entire.

Etymology. – Pol’ys (Gk), many, -septata (L), septate, for the multiple septa of the conidia, chosen to match the epithet of the anamorph.

Illustrations and descriptions. – Brachysporium polyseptatum was fully described and illustrated by Hughes (1951:46, FIG. C, as B. bloxami; 1955:264, FIG. 2) and Ellis (1971:223, FIG. 153 c, as B. bloxami).

Known distribution. – Czech Republic. The anamorph is known from Germany, Great Britain and Hungary (Ellis 1966, 1971; Révay 1993).

Habitat. – Saprobic on decaying wood and bark, and mycosaprobic on old basidiomata. Recorded from Acer pseudoplatanus, Alnus sp., Betula sp., Castanea sativa, Fagus sylvatica, Fraxinus excelsior, Pinus sylvestris, Prunus avium, Quercus robur, Trametes molis (Hughes 1951, Ellis 1966).

Holotype. – CZECH REPUBLIC. Central Bohemia: Kersko, decayed wood of a branch of Quercus robur, 19 Aug 1972, Z. Pouzar (PRM 900539).

Additional specimens examined. – UNITED KINGDOM. Surrey: Ashstead Commons, on Quercus sp., 20 Apr 1946, S.J. Hughes (DAOM 34455, IMI 4584). CZECH REPUBLIC. Western Bohemia: Luické hory Mountains, Mount Koprivník, decayed wood of a trunk of F. sylvatica, 30 Jun 1977, V. Holubová-Jechová M.R. 2548-02Kyjov (distr. Decín), Krásná Lípa, in a valley Kyjovské údolí, decayed wood of a branch of A. pseudoplatanus, 7 Jun 1977, V. Holubová-Jechová M.R. 2549-02*; Central Bohemia: Nebeská skála, decayed wood of a trunk of F. sylvatica, 4 Jul 1972, V. Holubová-Jechová M.R. 2550-02evnice near Prague, in valley of Moklick potok, on decayed basidiomata of Trametes molis, 24 Oct 1988, V. Holubová-Jechová M.R. 2551-02Southern Bohemia. umava Mountains National Park, Cenkova Pila, Povydrí National Natural Reserve, decorticated wood of Alnus viridis, 27 Aug 2000, M. Réblová M.R. 1668-00.

Living culture. – Derived from ascospores of specimen M.R.1668-00 (DAOM 231136).

Commentary. – Cr yptadelphia polyseptata has the shortest ascospores of the Cryptadelphia species described here. Brachysporium polyseptatum is readily distinguished from B. dingleyae G.P. White & Illman, which also produces obovoidal, 3-septate conidia; in B. dingleyae, the conidia are shorter and darker (18.0–25.2 x 9.3–14.5 µm; Hughes 1965).

Wallrothiella Sacc., Syll. Fung. 1:455. 1882.

Perithecia nonstromatic, superficial, solitary or in small groups, minute, globose to subglobose, conical around the ostiole, papillate, dark brown. Perithecial wall leathery, composed of three regions. Paraphyses persistent, broadly cellular, longer than the asci. Asci unitunicate, cylindrical-clavate, stipitate, 8-spored, uniseriate, apical annulus shallow, indistinct, nonamyloid, visible as two minute bodies. Ascospores globose, hyaline, non-septate. Anamorphs unknown.

Type species. – Wallrothiella congregata (Wallr.) Sacc.

Wallrothiella congregata (Wallr.) Sacc., Syll. Fung. 1: 455. 1882. FIGS. 38–45

View larger version (137K): [in this window] [in a new window]   FIGS. 38–45. Wallrothiella congregata. 38–40. Mature asci containing ascospores. 41, 42. Asci with paraphyses. 43. Longitudinal section of the perithecial wall, detail. 44, 45. Longitudinal section of the perithecial wall. FIGS. 38, 41, 43–45: DIC; 39–42: Phase contrast. Scale bars: 38–40 = 10 µm; 41, 42 = 20 µm; 43–45 = 100 µm. FIGS. 43–45 from PRM 900546 (Neotype).

Perithecia nearly superficial, base slightly immersed, solitary or in small groups, globose to subglobose, conical around the ostiole, papillate, 170–275 µm diam, 200–375 µm high, dark brown, glabrous.

Ostiolar canal periphysate. – Perithecial wall leathery, 42–45 µm thick at the bottom, becoming thicker at sides and toward the top 55–75 µm, consisting of three regions; outer region of dark, olivaceous-gray to brown, thick-walled, angular cells; middle region of almost hyaline to very pale brown, thick-walled angular cells (textura prismatica to textura epidermoidea); inner region of pale brown, thinner-walled, more elongated angular cells. Paraphyses hyaline, broadly cellular, constricted at the septa, ca. 4.0–5.0 µm wide near the base, tapering to 2.5–3.0 µm, longer than asci. Asci cylindrical-clavate, 22.0–30.0 ( = 26.0 ± 0.8) µm long in pars sporifera, (3.0–)4.0–4.5(–5.0) ( = 4.0 ± 0.1) µm wide, stipitate, stipe (7.0–)10.0–19.0 ( = 14.0 ± 1) µm long, L/W 10.0: 1, dissolving at maturity at the bottom, truncate at the apex, with apical annulus shallow, indistinct, non-amyloid, visible as two minute bodies, containing eight uniseriate spores. Ascospores globose, 2.0–2.5 (–3.0) ( = 2.5 ± 0.1) µm long, 2.0–2.5 ( = 2.3 ± 0.07) µm wide, hyaline, 1-celled, with a tendency to collapse inwards upon drying, smooth-walled, no germ pores or slits visible.

Anamorph. – Unknown.

Habitat. – Saprobic on decayed wood.

Neotype. – FRANCE: Pyrénées Atlantiques Mountains, 09 Prat Communal, Misseau de Loumet ca. 1000 m, on decayed wood of Quercus sp. and old hymenium of a basidiomycete (Corticiaceae), 7 Aug 2002, J. Fournier J.F. 02129 (PRM 900546, designated here).

Other specimen examined. – UKRAINE. Ruthenia: Carpathian Mountains, Rachiv, Kvasi, on the right bank of the Tisa River, decayed wood of Picea abies, 29 Jun 1997, M. Réblová M.R. 935–97.

Known distribution. – France, Germany, Ukraine (Wallroth 1833).

Commentary. – Wallrothiella congregata differs from generic concept of Cryptadelphia in the structure of the perithecial wall and morphology of paraphyses, ascospores and asci. The stipe dissolves and the asci float in the centrum at maturity. Ascospore germination was not observed on PCA or CMA.

Wallroth (1833) described Sphaeria congregata from decayed wood of Salix sp. The fungus was characterized as having gregarious, subglobose, black, glossy, finely roughened, papillate perithecia that collapsed apically. In the protologue, Wallroth (1833) cryptically noted "Sphaeria atomus Desm.?". Sphaeria atomus is a foliicolous species described originally from leaves of Fagus sylvatica, later known as Sphaerella atomus (Desm.) Cooke (Grevillea 3:169. 1874, non Sphaeria atomus Desm. sensu Rabenh., Hedwigia 5. 1866 nec Sphaerella atomus [Desm.] Fuckel sensu Fuckel, Jahrb. Nassau. Ver. Naturk. 23–24:99. 1870 = Sphaerella fagi Auersw., Mycol. Europaea 5, 6:6. 1869). Later, Saccardo (1882) transferred S. congregata to his new genus Wallrothiella, as W. congregata, chosen by Clements and Shear (1931) as the lecto-type species. He appended the original description of S. congregata, adding full details of the asci (cylindrical, minute, 20 x 2 µm) and ascospores (globose, 1-seriate in the ascus, 2 µm diam). Saccardo was probably the last to examine the type material of W. congregata. Von Arx and Müller (1954) could not locate it. We found two collections labeled Sphaeria congregata in STRC and PC. The collection preserved in PC is a duplicate of the STRC collection made by Montagne (1858). Unfortunately, the material in both collections is depauperate and neither contained a fungus matching the description of W. congregata (Wallroth 1833, Saccardo 1882). The two recent European collections match the circumscription of W. congregata given by Saccardo (1882) with one exception. The perithecia did not collapse apically as noted in the protologue, instead not collapsing or only a few collapsing laterally upon drying. The reported length of the asci of W. congregata (Saccardo 1882) is shorter than in the present collection, but because Saccardo presented only one measurement and not a range, this difference probably is not significant. In the new collections, the mature asci were seen floating in the centrum, following the early disintegration of the stipes, leaving the ascus in pars sporifera 22.0–30.0 µm long.

Wallrothiella presently accommodates a diverse assortment of about 41 taxa, of which some were already transferred to other genera, e.g. W. arceuthobii (Peck) Sacc. (= Caliciopsis arceuthobii [Peck] M.E. Barr, Barr et al 1986), W. eunotiaespora (Cooke & Harkn.) Berl. & Voglino (= Botryosphaeria quercuum [Schwein.] Sacc., Shoemaker 1964, Sivanesan 1984), W. minima (Fuckel) Sacc. (= Chaet. myriocarpa C. Booth, Booth 1957, 19580 and W. melanostigmoides (= Cryptadelphia groenendalensis, this study). Wallrothiella subiculosa and its Pseudogliomastix protea (Sacc.) W. Gams anamorph (Hughes and Dickinson 1968, Gams 1971) remain of uncertain phylogenetic and taxonomic disposition (Réblová and Winka 2000). Further revision and reclassification of the remaining species of Wallrothiella is required.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY KEY TO SPECIES OF... DISCUSSION LITERATURE CITED

 
The holomorph genus Cryptadelphia is characterized by a unique combination of teleomorphs and anamorphs. The teleomorphs are similar to those of several genera in the Chaetosphaeriaceae and Trichosphaeriales. The Brachysporium anamorphs, with sympodial, denticulate conidiogenesis and rhexolytic secession of the multiseptate conidia, are unlike any of the phialidic anamorphs of the Chaetosphaeriaceae. The morphological characters of these species, especially those of the teleomorphs, suggest an affinity with the Trichosphaeriales, which generally is supported by phylogenetic analysis of partial LSU rDNA sequences.

True Brachysporium species apparently have not been cultured before. The conidia are slow to germinate, and the resulting colonies grow slowly on agar media. Because these fungi tend to grow on rotten wood, often there are spores of other fungi present that are quicker to germinate and spread on isolation plates. Only the anamorph-teleomorph connection between C. groenendalensis and its anamorph B. nigrum has been convincingly documented here, although the ascospore isolate did not develop conidia. The consistent phylogenetic placement of these two cultures, which differed at only one of 1200 bp sequenced, was further support for their connection. Only a colony derived from ascospores was isolated for C. polyseptata, but the close phylogenetic relationship of this culture with those of C. groenendalensis convinced us that we were observing conspecific anamorphs and teleomorphs for the other species we have placed in Cryptadelphia, even though we were unable to prove this by isolating ascospores and conidia. The second possibility seems very unlikely, that a single pyrenomycete, growing on a diversity of wood types, but still phylogenetically most closely related to Brachysporium, actually mightbe a parasite of several Brachysporium species. This possibility can be rejected by the sequence differences between the two ascospore isolates obtained, and the correlating phenotypic differences in characters, such as ascospore length and septation and ascospore dimensions.

A similar example of frequent co-ocurrence of teleomorphs and anamorphs is known for Helminthosphaeria Fuckel/Diplococcium Grove. In that case, neither anamorph nor teleomorph could be grown in pure culture but the genetic link between the two never has been doubted (Samuels et al 1999).

Cryptadelphia species could be confused morphologically with some species of Chaetosphaeria if no anamorph is present on the substrate, although the two genera obviously are distinct phylogenetically. Chaetosphaeria species have a thinner perithecial wall, ca. 15–20 µm thick, a thin and shallow apical annulus, a generally shorter ascus stipe, which is persistent and usually obtuse at the base, and anamorphs with phialidic conidiogenesis. Morphological characteristics and the similarities in LSU rDNA sequences suggest a relationship between Cryptadelphia and Aquaticola at the family level. Aquaticola morphologically differs from Cryptadelphia in having species with ascospores that are always nonseptate, with the irregular occurrence of a mucilagenous sheath, a light to dark pigmented peridium and a long perithecial neck. The anamorphs of Aquaticola spp. are unknown. Cryptadelphia also is reminiscent of Trichosphaeria Fuckel (Fuckel 1870), based on T. pilosa (Pers. : Fr.) Fuckel, the lectotype species (Clements and Shear 1931).

Cryptadelphia differs from Trichosphaeria in the absence of any perithecial vestiture, a long ascus stipe and delayed formation of the median septum in the ascospores. Trichosphaeria includes fungi with superficial, small, setose perithecia sitting in a subiculum, cylindrical to oblong, 8-spored asci and hyaline, 1-celled, rarely appendiculate ascospores (von Arx and Müller 1954). It is the type genus of the Trichosphaeriaceae (Barr 1990, Eriksson and Hawksworth 1998). The anamorphs of Trichosphaeria spp. are as yet unknown, and we can only hypothesize that their conidiogenesis will be holoblastic and denticulate.

Based on morphological and molecular characters, we tentatively place Cryptadelphia in the Trichosphaeriaceae, Trichosphaeriales. The current classification of Trichosphaeriales recognizes only one family, the terrestrial Trichosphaeriaceae. The freshwater family Annulatascaceae is usually classified in the Sordariales (Wong et al 1998, Campbell et al 2003, Eriksson et al 2003). As noted by Réblová and Winka (2001), morphological data and similarities in LSU rDNA sequences suggest that the Annulatascaceae are more appropriately placed in the Trichosphaeriales. One exception is Clohiesia corticola, classified in the Annulatascaceae by Eriksson et al (2003) but more closely related to the Chaetosphaeriaceae (Réblová and Winka 2001). The data presented here offer support for the view that the Annulatascaceae are not part of the Sordariales, but the constraint analysis did not conclusively reject the inclusion of the Annulatascaceae and Cryptadelphia in the Sordariales if Rhamphoria was excluded. We accept the concept of Trichosphaeriaceae proposed by Barr (1990), but our data suggest that the family may be paraphyletic with the Annulatascaceae, suggesting that their delimitation might require adjustment or that the two families should be combined. Barr (1990) included four genera in the Trichosphaeriaceae including Rhamphoria (with a Phaeoisaria anamorph, see Müller and Samuels 1982), but this genus is now considered of uncertain position in the Sordariomycetes by Eriksson et al (2003). In our analysis, Rhamphoria delicatula was a sister group to Aquaticola in the Tri-chosphaeriales clade including Cryptadelphia. Constraint analyses clarify that Rhamphoria should not be included in the Sordariales, but confirmation of its phylogenetic relationship with the Trichosphaeriaceae awaits cultivation and sequence analysis of Trichosphaeria pilosa (Pers. : Fr.) Fuckel, the type species of the genus, family and order. The tendency of the morphologically very different Ophiostomatales, usually considered one of the sister groups of the Diaporthales (Spatafora and Blackwell 1994), to be paraphyletic within the Trichosphaeriales clade in analyses of larger datasets has not been noted in other studies and is another indication that more intensive sampling of these fungi will be required before a stable phylogeny can be achieved.

Following the redescription of Wallrothiella here, we cannot regard W. subiculosa and its Pseudogliomastix protea anamorph (Hughes and Dickinson 1968, Gams 1971, Gams and Boekhout 1985) as congeneric with W. congregata. However, in ascus, ascospore and centrum morphology, W. subiculosa is somewhat similar to species of Cryptadelphia, differing by its phialidic anamorph, strongly cupulate perithecia and distinctive perithecial anatomy. Wallrothiella subiculosa has been classified variously in the Trichosphaeriaceae (Barr 1990) or the Niessliaceae (Samuels and Barr 1997). On the basis of LSU rDNA sequence analysis, W. subiculosa is related to the Sordariales, Chaetosphaeriaceae (Réblová and Winka 2000).

In this paper, we have used a molecular phylogenetic approach to infer relationships of Cryptadelphia, Aquaticola, Chaetosphaeria, Wallrothiella sensu stricto and Wallrothiella subiculosa. The phylogenetic affinities of these taxa are not obvious based on the morphology of their teleomorphs because of the highly modified or convergent morphologies of their ascomata, asci and ascospores. Fungi with small, dark, nonstromatic perithecia, simple perithecial walls, unitunicate asci and hyaline 0–1-septate ascospores appear to be derived in at least three Ascomycete orders. However, their anamorphs present two distinct modes of conidiogenesis, phialidic and holoblastic-denticulate, respectively, and provide a useful character for their classification into families, in addition to characters of perithecia, ascospores and asci alone.

	ACKNOWLEDGMENTS

 
This project was partly supported by the Grant Agency of Academy of Sciences (GAAV B6005106) and COBICE project to M.R. We thank these herbaria for their loan of the type and other material: C, BR, DAOM, G, H, K, LUX, PAD, PC, STRC, UPS. We are grateful to Gerry Louis-Seize for the sequences of C. groenendalensis, Sarah Hambleton for advice on sequencing and phylogenetic analysis, Jacques Fournier for collecting the specimens of W. congregata and Robert A. Shoemaker and S. Hambleton for reviews of the manuscript.

	FOOTNOTES

 
Accepted for publication August 26, 2003.

For the Department of Agriculture and Agri-Food, ©Minister of Public Works and Government Services Canada, 2004 Government of Canada. Used with permission.

¹ Corresponding author. E-mail: reblova{at}ibot.cas.c3

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