Sequence alignment and phylogenetic analysis

Sequences were aligned with MAFFT v. 6.850b, using the ‘–genafpair’ option but default settings otherwise (Katoh et al. 2005). All introns and exons were aligned separately. Regions containing many leading or trailing gaps were removed from the ITS and LSU alignments prior to tree building. Phylogenetic analysis under the maximum-likelihood (ML) criterion (Felsenstein 1981) was conducted with RAxML v. 7.2.8, using its novel rapid bootstrap option combined with the autoMRE bootstopping criterion (Pattengale et al. 2009) with subsequent search for the best tree under the GTRMIX approach (Stamatakis et al. 2008). The resulting best-known ML tree was rooted using the midpoint-rooting method (Farris 1972, Hess & de Moraes Russo 2007). Bootstrapping under the maximum-parsimony (MP) criterion (Fitch 1971) was done with PAUP v. 4.0b10 (Swofford 2002), treating gaps as missing data, collapsing branches of zero minimum length, and using, per bootstrap replicate, five rounds of random sequence addition followed by TBR branch swapping, saving only one tree per round. In MP bootstrapping, 1 000 replicates were conducted. Search for the best MP tree(s) was done in the same manner but using 1 000 rounds of random sequence addition, saving no more than ten trees per round, and the strict consensus of tree all most-parsimonious trees determined.

The relative performance of the four loci (ITS, LSU, ACT and TUB) in phylogenetic inference for the group was assessed as follows. ML bootstrap analyses of the four alignments were conducted separately (using the same settings as above), the support values from each gene mapped to the best ML tree from combined analysis using RAxML, and each average bootstrap support determined, both absolute and relative to the number of variable characters per alignment. Under MP, partitioned Bremer support (Baker & DeSalle 1997, Baker et al. 1998) was determined using the ‘bremer.tcl’ script (Göker et al. 2009b) in conjunction with PAUP (heuristic-search settings were as above but with 100 rounds for each Bremer search), visualised using a heatmap as implemented in the opm package for R (Vaas et al. 2012) and summed up over all nodes for each gene, both absolute and relative to the number of parsimony-informative characters per alignment (partitioned Bremer support trees are available upon request). The suitability of the four loci for molecular taxonomy of the group was investigated using OPTSIL (Göker et al. 2009a, Stielow et al. 2011) with the revised classification of the group (as detailed below) as reference partition, thus optimizing sequence dissimilarity thresholds for F values between 0.0 and 1.0 (with a step width of 0.05), and measuring the resulting best agreement between the clustering and the reference partition. The F value determines the shape of the clusters; we here considered the full range between single-linkage (0.0) and complete-linkage (1.0) clustering; see Göker et al. (2009a) for details. The underlying distance matrices were calculated with PAUP, using uncorrected (‘p’) distances.

Taxonomy

Alloconiothyrium Verkley, Göker & Stielow, gen. nov. — MycoBank MB800756

Type species. Alloconiothyrium aptrootii Verkley, Göker & Stielow.

Etymology. Named after its morphological resemblance to Coniothyrium in contrast to the phylogenetic distance between both genera.

Conidiomata pycnidial or eustromatic. Conidiogenous cells holoblastic, annellidic. Conidia olivaceous-brown and irregular in outline, surface roughened. Sexual morph unknown.

Alloconiothyrium aptrootii Verkley, Göker & Stielow, sp. nov. — MycoBank MB800757; Fig. 3

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Fig. 3

Alloconiothyrium aptrootii (CBS 980.95^T, ex-type culture). a. Colony on OA; b. colony on MEA; c, d. conidia on OA; e, f. conidiogenous cells showing distinct annellations; g. chlamydospores. — Scale bars = 10 μm.

Etymology. Named after André Aptroot, who collected the soil sample from which the species was isolated.

Conidiomata pycnidial, 300–450 μm diam and with a single cavity, or eustromatic and consisting of complexes reaching 1 mm diam, with several cavities, the outer surface black, glabrous or covered by grey mycelium. Conidiomatal wall composed of an outer layer of brown, thick-walled textura angularis and an inner layer of hyaline, thick-walled textura angularis-globulosa, the outer surface sometimes covered by a diffuse web of brown hyphae. Conidiogenous cells discrete, often positioned on clumps of cells that protrude into the cavity, broadly ampulliform, holoblastic, annellidic, often with an elongated neck showing several distinct percurrent proliferations, 4–9 × 3–4 μm. Conidia globose to irregularly ellipsoid, initially hyaline, after secession olivaceous-brown, mature conidial wall orange-brown, the outer surface verruculose giving the conidium an irregular outline, with 1 large oil-droplet 1–1.5 μm diam, 0-septate, 3–4(–5) × 2.5–3(–3.5) μm, average L/W ratio 1.2 ± 0.2. Chlamydospores formed in the mycelium, terminal or intercalary, usually solitary, globose, mostly 6–8.5 μm diam, with a smooth brown wall and 1–2 large oil-droplets. Sexual morph unknown.

Colonies on OA reaching 37–40 mm diam in 10 d, with an even, glabrous, colourless margin. Immersed mycelium mostly colourless, but with some faint buff to honey in the centre after 10 d. Aerial mycelium very diffuse, white or absent. Reverse concolourous. Conidiomata developing after 10–15 d. Colonies on MEA reaching 36–40 mm diam in 10 d, with an even, buff margin. Immersed mycelium greenish olivaceous to olivaceous, fading to buff at margin, mostly covered by a moderately dense layer of woolly to floccose grey to white aerial mycelium. Reverse in the centre isabelline, fading over cinnamon to buff at the margin.

Specimens examined. PAPUA NEW GUINEA, Central Province, Varirata Nat. Park near Port Moresby, isolated by A. van Iperen from a soil sample, Oct. 1995, A. Aptroot, holotype CBS H-21035, living ex-type culture CBS 980.95; isolated from the same soil sample CBS 981.95.

Notes — The fungus is only known from a soil sample collected in Papua New Guinea, and all other coniothyrium-like fungi studied here are relatively distantly related. The annellidic conidiogenous cells and the verruculose conidia remind of Coniothyrium palmarum, the type species of the genus, but that species is characterised by 2-celled conidia and is also genetically distinct, and belongs in the Leptosphaeriaceae (de Gruyter et al. 2009).

Dendrothyrium Verkley, Göker & Stielow, gen. nov. — MycoBank MB800758

Type species. Dendrothyrium variisporum Verkley, Göker & Stielow.

Etymology. Named after the branched, tree (= dendron)-like conidiophores occurring in the conidiomata of the type species.

Conidiomata pycnidial or eustromatic. Conidiogenous cells discrete or integrated in conidiophores that are branched at the base, phialidic, terminal cells of the conidiophore occasionally also percurrently proliferating. Conidia 1-celled, olivaceous-brown, thin- and smooth-walled. Sexual morph unknown.

Dendrothyrium longisporum Verkley, Göker & Stielow, sp. nov. — MycoBank MB800759; Fig. 4

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Fig. 4

Dendrothyrium longisporum (CBS 582.83^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c. conidia on OA; d. conidiogenous cells on OA; e. conidia on OA. — Scale bars = 10 μm.

Etymology. Named after the comparatively long conidia of this species.

Conidiomata pycnidial, globose, 140–170 μm diam, with a single, central ostiolum 10–20 μm. Conidiomatal wall composed of textura angularis with pale yellowish brown cells and darker cells around the ostiolum, sometimes overlaid with a diffuse web of thin-walled brown hyphae. Conidiogenous cells discrete or integrated in simple, 1–2-septate, 10–17 μm long conidiophores, phialidic, doliiform to ampulliform, with a distinct periclinal thickening, 3.5–6(–8) × 2–3 μm. Conidia consistently cylindrical-ellipsoid, initially hyaline, soon after secession with a olivaceous-brown, thin, smooth wall, with minute granules and no oil-droplets, 0-septate, (3.5–)4–5(–6) × 1.5–2 μm, average L/W ratio 2.8 ± 0.4. Sexual morph unknown.

Colonies on OA reaching 28–32 mm diam in 10 d, with a smooth, glabrous margin. Immersed mycelium colourless, faintly yellowish to ochreous in the centre where scattered pycnidia emerge after 5–7 d. Aerial mycelium only in the centre, fluffy, white. Reverse concolourous. Colonies on MEA reaching 23–25 mm diam in 10 d, with an even to slightly undulating buff margin; immersed mycelium buff to ochreous in the centre, where also numerous densely aggregated pycnidia are formed after 5–7 d, colony surface mostly hidden under a mat of pure white, woolly-tufty aerial mycelium. Reverse in the centre chestnut, fading over fulvous to ochreous or buff near the margin.

Specimens examined. CANADA, Manitoba, Grand Beach, isolated from Arceuthobium pusillum, 25 July 1981, J. Reid, holotype CBS H-10965, living ex-type culture CBS 582.83. – GERMANY, Monheim, from leaf spot in Triticum aestivum, June 1984, M. Hossfeld 111, living culture CBS 824.84 (preserved as Coniothyrium cerealis).

Notes – See following species.

Dendrothyrium variisporum Verkley, Göker & Stielow, sp. nov. — MycoBank MB800760; Fig. 5

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Fig. 5

Dendrothyrium variisporum (CBS 121517^T, ex-type culture). a. Colony on OA; b. colony on MEA; c–e. conidia and conidiogenous cells on OA. — Scale bars = 10 μm.

Etymology. Named after the variation in the shape of the conidia.

Conidiomata eustromatic, often merged to complexes reaching 400–500 μm diam with several discrete or fused cavities, dark brown to black; sporocarps on the agar surface appearing grey due to numerous colourless hyphal outgrowths. Conidiomatal wall relatively thick, composed of a single layer of textura angularis with hyaline to pale yellow, relatively thick-walled cells 4–7 μm diam. Outer surface sometimes overgrown by a diffuse web of brown, glabrous hyphae oriented parallel to the wall surface. Conidiogenous cells integrated in 1–4-septate acropleurogenous conidiophores that are simple or branched at the base, 10–18(–25) × 2.5–4 μm, phialidic, terminal cells cylindrical and slightly attenuating to the apex where sometimes one or more percurrent proliferations can be seen. Conidia variable in shape, subglobose, ellipsoid or obovoid, sometimes curved or with a broad, blunt end, initially hyaline, soon after secession with an olivaceous-brown, thin, smooth wall, contents with 1–3 minute oil-droplets, 0-septate, 3–4(–4.5) × 1.5–2.5(–3) μm, average L/W ratio 1.6 ± 0.3. Sexual morph unknown.

Colonies on OA reaching 35–38 mm diam in 10 d, with an even, glabrous and colourless margin. Immersed mycelium colourless, aerial mycelium absent. Reverse concolourous. Pycnidia formed after 7–10 d in concentrical zones. Colonies on MEA reaching 26–28 mm diam in 10 d, with an even to slightly ruffled, colourless margin. Immersed mycelium buff to ochreous but mostly hidden under a dense mat of woolly-floccose, pure white to buff, later in the centre greyish aerial mycelium. Reverse in the centre umber, fading over sienna to luteous to buff near the margin. Pycnidia formed after 10–14 d.

Specimens examined. SWITZERLAND, Zürich, isolated as endophyte of Erica carnea, July 1981, O. Petrini, living culture CBS 197.82, CBS H-10964 (dried culture). – SYRIA, isolate from declined grape vine, K.A. Halim 35, holotype CBS H-21036, living ex-type culture CBS 121517.

Notes — The branched, acropleurogenous conidiophores (Fig. 5d) that can be provided with annellidic terminal apertures are the most distinctive feature of this species. Dendrothyrium longisporum is a close relative, but morphologically quite distinct from D. variisporum by the pycnidial sporocarps with a well-developed ostiolum, more consistently cylindrical-ellipsoid conidia (average L/W ratio 2.8 vs 1.6 in D. variisporum) and the absence of branched conidiophores or annellidic conidiogenesis. Despite these differences, the multi-locus phylogenetic analysis supports the placement of the two fungi in a single genus. In contrast to D. longisporum, the two strains assigned to D. variisporum do not group in a monophyletic cluster in the multi-locus phylogeny. CBS 197.82 is nonetheless considered to be conspecific with the ex-type strain, mainly based on good agreement in phenotypic characters and because there is no support for the non-monophyly of D. variisporum (Fig. 2). Based on the material available it can be postulated that the genus Dendrothyrium is a widely dispersed genus of endophytes and (weak) plant pathogens with a wide host spectrum.

Paraconiothyrium Verkley, Stud. Mycol. 50: 327. 2004

Type species. Paraconiothyrium estuarinum Verkley & Manuela Silva, Stud. Mycol. 50: 327. 2004.

A description of the type species was provided by Verkley et al. (2004). Main features of this species are summarised in Table 3.

Conidiomata eustromatic, simple or complex, or pycnidial. Conidiogenous cells discrete or integrated, phialidic or holoblastic, annellidic. Conidia aseptate, sometimes 1-septate, thin- to relatively thick-walled, smooth-walled or verruculose, hyaline when liberated, later brown.

Paraconiothyrium archidendri Verkley, Göker & Stielow, sp. nov. — MycoBank MB800761; Fig. 6

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Fig. 6

Paraconiothyrium archidendri (CBS 168.77^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c–e. conidia on OA. — Scale bars = 10 μm.

Etymology. Named after the host genus, Archidendron, from which the species was isolated.

Conidiomata pycnidial, globose, with a single ostiolum 10–30 μm diam, initially glabrous and pale brown, or pilose and appearing grey, later black due to conidia produced inside, 250–350(–400) μm diam, the surface of the wall provided with hyaline to pale brown hyphal outgrowths. Conidiomatal wall composed of single layer of relatively thick-walled, pale yellowish textura angularis with cells mostly 5–10 μm diam. Conidiogenous cells discrete, globose to doliiform, holoblastic, occasionally annellidic with 1–3 percurrent proliferations, 3.5–5(–6.5) × 2.5–4 μm. Conidia variable in shape, subglobose or ellipsoid, more rarely obovoid, ends rounded, sometimes one end more or less blunt, initially hyaline, soon after secession olivaceous-brown, contents with several small oil-droplets (< 0.5 μm diam) near each end, conidial wall at maturity relatively thick, smooth, sometimes minutely verruculose, 0-septate, 3.5–6 × 2.5–3.5(–4) μm, average L/W ratio 1.5 ± 0.2. Sexual morph unknown.

Colonies on OA reaching 50–55 mm diam in 10 d, with an even, glabrous and colourless margin; immersed mycelium ochreous to cinnamon, aerial mycelium absent. Reverse concolourous. Conidiomata developing after 20–25 d. Colonies on MEA reaching 50–53 mm diam in 10 d, with an even, colourless to buff margin; immersed mycelium not visible from above, entirely hidden under a dense moderately high mat of woolly-floccose, white to greyish, in the centre weakly citrine to hazel aerial mycelium; conidiomata not observed. Reverse predominantly ochreous to fulvous, in the centre olivaceous-black with rust patches or circular zones. Conidiomata developing after 20–25 d.

Specimen examined. BURMA, E. of Yezin, Kyaukthanbut Village, on leaf spot in Pithecellobium bigeminum (= Archidendron bigeminum), Oct. 1976, M.M. Thaung, isolated by H.A. van der Aa 5654B, holotype CBS H-21037, living ex-type culture CBS 168.77.

Notes — The only strain available of this species sporulated tardily on OA and MEA with small numbers of sporocarps. It was isolated from leaf spots on the leguminose tree Archidendron bigeminum in Burma, and more material needs to be collected in order to assess its ecology and geographic distribution. In the multi-locus phylogeny, Parac. archidendri clusters with Parac. fuckelii, but this grouping is not supported by bootstrapping. The two taxa do share annellidic conidiogenesis (not seen in the other Paraconiothyrium species) and a relatively low conidium L/W ratio (1.3–1.5) compared to other members of Paraconiothyrium (≥ 1.7).

Paraconiothyrium fuckelii (Fuckel) Verkley & Gruyter, Stud. Mycol. 75: 25. 2012. — Fig. 7

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Fig. 7

Paraconiothyrium fuckelii (CBS 797.95). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c, d. conidia on OA; e. conidiogenous cells and conidia on OA. — Scale bars = 10 μm.

Basionym. Coniothyrium fuckelii Sacc., Nuovo Giorn. Bot. Ital. 7: 318. 1875 (asexual morph).

=Sphaeria coniothyrium Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 115. 1870 (sexual morph).

≡ Leptosphaeria coniothyrium (Fuckel) Sacc., Nuovo Giorn. Bot. Ital. 7: 317. 1875.

More synonyms are provided in Domsch et al. (2007), who described the sexual morph. Below only a description of the asexual morph in vitro is given.

Conidiomata pycnidial 300–400 μm diam and with a single cavity, more often eustromatic and consisting of complexes up to 1.2 mm diam, with several cavities, the outer surface black, glabrous, but often covered by a diffuse web of white to greyish hyphae. Conidiomatal wall composed of an outer layer of textura angularis with somewhat thickened, brown walls, and an inner layer of textura angularis-globulosa with somewhat thickened, hyaline walls. Conidiogenous cells discrete or integrated in short, simple, 1–2-septate conidiophores, broadly ampulliform to globose, holoblastic, often annellidic with 1 or 2 percurrent proliferations noticeable by the distinct scars on a somewhat elongated neck, hyaline, 4–10(–13) × 3–5 μm. Conidia variable in shape, subglobose to ellipsoid or obovoid, rarely more cylindrical, initially hyaline with mostly 1–3(–5) small oil-droplets (< 1 μm diam), soon after secession olivaceous-brown, conidial wall smooth, orange-brown, 0-septate, 3–4 × 2–3(–3.5) μm, average L/W ratio 1.4 ± 0.2.

Colonies on OA reaching 70–75 mm diam in 10 d, with an even, glabrous and colourless margin. Immersed mycelium in the centre faintly hazel or ochreous, aerial mycelium absent or diffuse, pure white. Reverse concolourous. Colonies on MEA reaching 60–65 mm diam in 10 d, with an even to slightly ruffled colourless margin mostly covered under the aerial mycelium. Immersed mycelium completely hidden under a dense but not high mat of woolly to woolly-floccose, glaucous grey to pale grey-olivaceous aerial mycelium. Reverse bay, fading over sienna to luteous at the margin.

Specimens examined. DENMARK, loc. unknown, isolated from root of gymnosperm, May 1969, D.S. Malla S 7(45), living culture CBS 584.69; Geelskov, on canes of Rubus sp., A.M. Dahl-Jensen, Dec. 1995, isol. G. Verkley 338, living culture CBS 797.95. – GERMANY, München, Feldberg, isolated from Picea abies with cankers, Oct. 1985, O. Kandler, living culture 653.85. – THE NETHERLANDS, from sputum of man, Nov. 1971, isolated by M. Luykx, living culture CBS 764.71B.

Notes — Wollenweber & Hochapfel (1937) included the pathogens on Rosaceae in their concept of Coniothyrium fuckelii, and this is the core of the phylogenetic species here recognised under the name Paraconiothyrium fuckelii. In the literature the name for the sexual morph Leptosphaeria coniothyrium has mostly been used, but as has been established in previous molecular studies, the species is not congeneric with the type species of Leptosphaeria, L. doliolum, which resides in the Leptosphaeriaceae (Verkley et al. 2004, de Gruyter et al. 2009). According to Domsch et al. (2007) this species has a world-wide distribution.

Paraconiothyrium sp. 1 (‘Microsphaeropsis pseudaspera’?) — Fig. 8

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Fig. 8

Paraconiothyrium sp. (‘Microsphaeropsis pseudaspera’?) (CBS 113682). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c–e. conidia on OA. — Scale bars = 10 μm.

Conidiomata pycnidial, globose to elliptical in surface view, with one or two ostioli, 8–12 μm diam, dark olivaceous-brown to black, pilose, 200–350(–400) μm diam, the surface provided with brown hyphal outgrowths emerging from a dense web of hyaline to dark brown hyphae growing parallel over the wall surface. Conidiomatal wall composed of an outer layer of relatively thin-walled brown textura angularis with cells mostly 4–6 μm diam, and an inner layer of similar but smaller, hyaline cells. Conidiogenous cells discrete, doliiform to broadly ampulliform, phialidic, with an indistinct periclinal thickening, 4–5(–6) × 3–4 μm. Conidia variable in shape, globose to subglobose or ellipsoid, more rarely obovoid, initially hyaline, contents with mostly 1–3(–5) small oil-droplets (< 1 μm diam), soon after secession olivaceous-brown, conidial wall smooth, orange-brown, 0-septate, 3–4.5(–5) × 2–3 μm, average L/W ratio 1.3 ± 0.2. Sexual morph unknown.

Colonies (CBS 113682) on OA reaching 41–46 mm diam in 10 d, with an even, glabrous colourless margin. Immersed mycelium colourless, with numerous pycnidia formed in distinct concentrical zones after 4–5 d. Reverse concolourous, appearing grey-olivaceous where pycnidia develop. Colonies on MEA reaching 32–36 mm diam in 10 d, with an even, glabrous, buff margin. Immersed mycelium in the centre olivaceous to olivaceous-black, buff in a submarginal zone, covered in the centre by olivaceous to olivaceous buff, woolly-floccose aerial mycelium, in a submarginal zone abruptly changing to pure white. Reverse in the centre umber to sienna, with dull luteous areas, fading to pale luteous at the margin.

Specimens examined. SPAIN, Santiago de Compostela, isolated from air sample, 15 Mar. 2002, M.J. Aira, deposited by A.M. Stchigel, living culture CBS 113682 (preserved as Microsphaeropsis pseudaspera). – THE NETHERLANDS, from nail of human, Apr. 1987, living culture CBS 251.87.

Notes — CBS 113682 was identified as Microsphaeropsis pseudaspera, a fungus described by Sutton (1974) from dead branches of Eucalyptus in Portugal. The conidiogenous cells and conidia of this isolate agree well with those described for this coelomycete based on material in planta. The clinical strain from the Netherlands is very similar in colony characters and other phenotypic traits, as are the sequences of CBS 251.87 generated in this study. Whether the name M. pseudaspera definitively applies to this material can only be confirmed by sequencing of the type material or recollecting from Eucalyptus.

ParaphaeosphaeriaO.E. Erikss.

Type species. Paraphaeosphaeria michotii (Westend.) O.E. Erikss., Ark. Bot., ser. 2, 6: 406. 1967.

Câmara et al. (2001) provide descriptions of sexual and asexual morphs of Paraph. michotii and Paraph. pilleata, while other species treated there under Paraphaeosphaeria were transferred later to Neophaeosphaeria and Phaeosphaeriopsis (Câmara et al. 2003). None of the amerosporic coniothyrium-like fungi associated with these sexual morphs has been assigned a formal name.

Asexual morphs classified in Paraphaeosphaeria can be described as follows:

Conidiomata eustromatic or pycnidial. Conidiogenous cells discrete or integrated, phialidic, or annellidic with one or two percurrent proliferations. Conidia aseptate or 1-septate, smooth to verrucose.

Paraphaeosphaeria angularis Verkley & Aa, sp. nov. — MycoBank MB800765; Fig. 9

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Fig. 9

Paraphaeosphaeria angularis (CBS 167.70^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c–e. conidia on OA. — Scale bars = 10 μm.

Etymology. Named after the angular shape of the conidia.

Conidiomata pycnidial, globose, ostiolum absent or with a single undifferentiated ostiolum 15–20 μm diam, pale olivaceous-brown, black due to mature conidia inside, 150–350(–450) μm diam. Conidiomatal wall composed of an outer layer of relatively thick-walled, yellowish brown textura angularis with cells 5–12 μm diam, and an inner layer of similar structure with hyaline and smaller cells. Conidiogenous cells discrete, globose to doliiform, phialidic, with an indistinct periclinal thickening, 4–7.5 × 4–6 μm. Conidia ellipsoid or elongated-ellipsoid, with a more or less clear angular outline, initially hyaline with 2–5 small oil-droplets (1–1.5 μm diam), then smoky greyish brown with relative dark, amorphous contents mostly showing no oil-droplets, often a brighter longitudinal band can be seen, conidial wall glabrous and moderately thick, 0-septate, 4.5–7(–8) × 3–4 μm, average L/W ratio 1.9 ± 0.2. Occasionally 2-celled conidia 8 × 5 μm are observed. Sexual morph unknown.

Colonies on OA reaching 53–56 mm diam in 10 d, with an even, glabrous and colourless margin. Immersed mycelium colourless, fully covered by a very diffuse mat of pure white finely felted aerial mycelium, and pycnidia developing after 3–5 d in a pattern of radiating and branching rows. Reverse concolourous, greyish where pycnidia are formed. Colonies on MEA reaching 43–46 mm diam in 10 d, with an even to slightly undulating, glabrous margin. Immersed mycelium buff, appearing darker and olivaceous or greyish in the centre where pycnidia develop, colony largely covered by a high, tufty to woolly-floccose mat of dirty white, in the centre more greyish, aerial mycelium. Reverse cinnamon to ochreous, darker where pycnidia are formed.

Specimen examined. BRAZIL, Bahia, Salvador, isolated from Saccharum officinarum, Oct. 1969, C. Ram, isol. H.A. van der Aa no. 1870, holotype CBS H-11085, living ex-type culture CBS 167.70.

Notes — A relatively high average conidial L/W ratio and especially the peculiar bright longitudinal band that can be observed over the mature conidium wall (difficult to record in photomicrographs) characterize this unique species, which is only known from a strain isolated from Saccharum officinarum in Brazil. The sexual morph is currently unknown, but since the species groups in a well-supported cluster with the pleomorphic species, Paraph. michotii and Paraph. pilleata, it would not be unlikely that it exists. The asexual morphs of these two close relatives of Paraph. angularis are otherwise highly similar (summarised in Table 3). All three species are associated with monocots.

Paraphaeosphaeria arecacearum Verkley, Göker & Stielow, sp. nov. — MycoBank MB800762; Fig. 10

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Fig. 10

Paraconiothyrium arecacearum (CBS 158.75^T, ex-type culture). a. Colony on OA; b. colony on MEA; c–e. conidia on OA; f. amorphous hyphal exsudate on OA. — Scale bars = 10 μm.

Etymology. Named after the occurrence in association with genera of the family Arecaceae (= Palmae).

Conidiomata pycnidial, globose, glabrous, mostly with a rather undifferentiated single ostiolum, pale olivaceous or greenish, soon black due to mature conidia inside, 130–350 μm diam. Conidiomatal wall composed of an outer layer of relatively thick-walled, pale yellow to olivaceous textura angularis with cells mostly 4–7.5 μm diam, and an inner layer of hyaline thin-walled textura angularis-globulosa. Conidiogenous cells discrete, globose, doliiform to broadly ampulliform, phialidic with a distinct periclinal thickening, 4–6.5 × 3–4.5 μm. Conidia ellipsoid to obovoid-pyriform, initially hyaline, soon after secession olivaceous-brown, predominantly with two persistent polar oil-droplets (1.5–2 μm diam), conidial wall glabrous, 0-septate, (3–)3.5–6(–8.5) × 2–3 μm, average L/W ratio 2.0 ± 0.4. Sexual morph unknown.

Colonies on OA reaching 70–75 mm diam in 10 d, with an even to slightly ruffled, glabrous and colourless margin. Immersed mycelium colourless, aerial mycelium absent or very scanty, felted, pure white, vegetative hyphae exudating orange-brown material in amorphous masses (up to 25 μm wide) over variable length along the hyphae. Pycnidia developing after 3 d in a pattern of branching and radiating rows (but evenly distributed and numerous), in the centre also concentrated in concentrical zones after 10 d. Reverse concolourous, but appearing grey due to pycnidial development. Colonies on MEA reaching 65–69 mm diam in 10 d, with a somewhat ruffled, colourless glabrous margin. Immersed mycelium buff, appearing grey due to developing pycnidia that are completely covered by a dense, woolly-floccose to tufty, pure white to faintly greyish or luteous mat of aerial mycelium. Reverse buff to pale luteous, with greyish brown concentrical zones where the pycnidia develop.

Specimens examined. IVORY COAST, isolated from Cocos nucifera, Dec. 1975, J. Mouchacca, living culture CBS 614.75. – SURINAM, isolated from soil under Elaeis guineensis, Mar. 1974, J.H. van Emden, holotype CBS H-11048, living ex-type culture CBS 158.75.

Notes — This species is notable for its rapid growth rate and sporulation. The two cultures that are known thus far are both associated with tropical palms. Coniothyrium palmarum, the type species of Coniothyrium (Leptosphaeriaceae) is frequently found on palms as well, but that species can easily be distinguished from Paraph. arecacearum by the annellidic conidiogenesis and verrucose and 0–1-septate conidia 6–8.5 × 4–5 μm (Sutton 1980).

Paraphaeosphaeria minitans (W.A. Campb.) Verkley, Göker & Stielow, comb. nov. — MycoBank MB800766

Basionym. Coniothyrium minitans W.A. Campb., Mycologia 39: 191. 1947.

≡ Paraconiothyrium minitans (W.A. Campb.) Verkley, Stud. Mycol. 50: 332. 2004.

A detailed description of the fungus, of which the sexual morph is unknown, is provided by Domsch et al. (2007). In the past a number of CBS strains have been identified as Coniothyrium minitans (Table 1, Fig. 1), but the sequence data indicate they belong to a number of different taxa. Most of these strains were obtained from soil samples, and critical characteristics of this species like infective capability of sclerotia of Sclerotinia, were not documented.

Paraphaeosphaeria neglecta Verkley, Riccioni & Stielow, sp. nov. — MycoBank MB800767; Fig. 11

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Fig. 11

Paraphaeosphaeria neglecta (CBS 124078^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse of CBS 124077 on the right; c, d. conidia on OA; e. conidiogenous cells on OA. — Scale bars = 10 μm.

Etymology. Named for the fact that this fungus was not recognised as distinct within the Paraphaeosphaeria (Paraconiothyrium) sporulosum complex.

Conidiomata pycnidial, globose, glabrous, with a single ostiolum 20–30(–50) μm diam, black due to mature conidia inside, the wall yellowish brown but cells surrounding the ostiolum darker, conidiomata 240–350 μm diam. Conidiomatal wall composed of an outer layer of yellow-brown, relatively thick-walled textura angularis, and an inner layer of similar structure but with hyaline, thin-walled cells. Conidiogenous cells discrete or positioned on clumps of cells that protrude into the cavity, globose, doliiform to broadly ampulliform, phialidic with a distinct periclinal thickening, 4–6 × 3–5 μm. Conidia highly variable in shape, subglobose, ellipsoid to obovoid-pyriform, or more cylindrical, initially hyaline, soon after secession olivaceous-brown, mostly with two polar oil-droplets (1.5–2 μm diam), and rarely with a few additional smaller ones, conidial wall glabrous or minutely roughened, 0-septate, (3–)3.5–6(–8.5) × 2–3 μm, average L/W ratio 1.7 ± 0.4 (CBS 124078^T; 1.5 ± 0.3 for CBS 303.77). Sexual morph unknown.

Colonies on OA reaching 45–50 mm diam in 10 d, with an even, glabrous and colourless margin. Immersed mycelium initially colourless, then luteous sometimes with sienna centre, with rather diffuse but high, tufty, pure white aerial mycelium. Pycnidia developing in discontinuous concentrical zones or scattered after 7–10 d. Reverse concolourous. Colonies on MEA reaching 34–39 mm diam in 10 d, with an even to undulating, glabrous margin. Immersed mycelium entirely hidden under a dense mat of woolly-floccose, white to pale luteous, sometimes also glaucous to glaucous grey aerial mycelium. Reverse mostly sienna, fading to pale luteous at the margin. Pycnidia absent or developing after 12–15 d.

Specimens examined. CHILE, Valdivia, South Chilean Forest, isolated from rotten wood, June 1978, A.E. Gonzáles, living cultures CBS 335.78 and CBS 337.78 (CBS H-10913, H-10923). – FRANCE, Brest, from cankered Juniperus sp., July 1975, M. Morelet, living culture CBS 359.75; isolated from Taxus baccata, 5 Nov. 1975, I. Vegh, living culture CBS 303.77; same substrate, 14 May 1976, I. Vegh 9793, living culture CBS 305.77; isolated from Cupressocyparis leylandii, 1 Apr. 1977, I. Vegh 10145, living culture CBS 307.77. – GERMANY, Ülzen, from Erica carnea, Aug. 1970, L. Kiewnick, living culture CBS 434.71A; Freiburg, H. Courtois, living culture 431.77 (CBS H-10916, dried culture); Bavendorf, Ravensburg, on dead branches of Pyrus malus, Aug. 1981, R. Weiler (CBS H-10915, CBS H-10926), living culture CBS 452.81 isolated by H.A. van der Aa 7867. – ITALY, Latina, from wood of Actinidia chinensis var. hort. 16A, L. Riccioni, holotype CBS H-21039, living ex-type culture CBS 124078 (ER 1503); isolated from the same material CBS 124077 (ER 1501). – THE NETHERLANDS, Oostvoorne, on Pyrola rotundifolia, 3 Apr. 1971, H.A. van der Aa 2526 (CBS H-10734), living culture CBS 434.71B; Baarn, on leaf of Azalea sp., Mar. 1972, H.A. van der Aa 3012, living culture CBS 300.72; Wageningen, on Azalea sp., Feb. 1972, H. van Kesteren, living culture CBS 302.72 isolated by H.A. van der Aa 2998; loc. unknown, J.C. Went 1021a, isolated from acid mull soil, with very well decomposed leaves, Feb. 1961, living culture CBS 180.61 (VKM F-2659); Eese Estate near Steenwijk, on seed of Quercus robur, 13 Oct. 1983, H.A. van der Aa 8895, living culture CBS 683.83; Utrecht, ear of human, 15 Dec. 2005, living culture CBS 119637 isolated by J. Vlooswijk.

Notes — Nine of the isolates formerly identified as Parac. sporulosum in CBS belong to a new species, for which the name Paraph. neglecta is proposed. Paraphaeosphaeria neglecta and Paraph. sporulosa are not sister taxa and material available in this study suggests that, although both species are ubiquitous fungi occurring in soil and on various plants in different habitats, only Paraph. neglecta may be truly cosmopolitan, at least besides Europe also occurring in the Americas. Records of Paraph. sporulosa from outside Europe should be confirmed by sequence studies. Paraphaeosphaeria neglecta may have a preference for plants, as only one soil isolate belonged to that species. One isolate originated from the human ear. The colonies of Paraph. sporulosa and Paraph. neglecta look very similar especially on OA, and there is also overlap in conidial sizes. In Paraph. sporulosa conidia are 3.5–5(–6) × 3–4 μm, average L/W ratio 1.5 ± 0.2, and in Paraph. neglecta (3–)3.5–6(–8.5) × 2–3 μm, average L/W ratio 1.7 ± 0.4. Conidia for some strains of Paraph. neglecta show a minutely roughened outer wall surface, a feature not observed in Paraph. sporulosa. A further difference pertains to percurrent proliferation in the conidiogenous cells, which was observed in Paraph. sporulosa and not in Paraph. neglecta, but it should be noted that this character has proven not to be very reliable in coelomycetes.

Paraphaeosphaeria sardoa Verkley, W. Gams & Aa, sp. nov. — MycoBank MB800769; Fig. 12

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Fig. 12

Paraphaeosphaeria sardoa (CBS 501.71^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c–e. conidiogenous cells on OA; f, g. conidia on OA. — Scale bars = 10 μm.

Etymology. Named after Sardinia, where this fungus was collected.

Conidiomata pycnidial, single, or eustromatic and more complex, globose, glabrous, superficial or immersed in the agar, 300–450(–600) μm diam, initially pale but soon appearing dark brown to black due to mature conidia inside. Conidiomatal wall composed of hyaline to very pale yellowish textura angularis with moderately thickened walls, lined by a layer of globose hyaline thin-walled cells. Conidiogenous cells globose to broadly ampulliform, hyaline, discrete, or integrated in short, simple, 1–2-septate conidiophores, but more often positioned on clumps of cells that protrude into the cavity, phialidic, with a distinct periclinal thickening, 5–10 × 4.5–6 μm. Conidia variable in shape, subglobose, ellipsoid, obovoid, or pyriform, often irregular in outline, initially hyaline, after secession olivaceous-brown, verruculose, with mostly 6–12 oil-droplets up to 1 μm diam, 0-septate, (4.5–)5–6(–7) × (3–)3.5–4.5(–5) μm, average L/W ratio 1.4 ± 0.2. Sexual morph unknown.

Colonies on OA reaching 40–44 mm diam in 10 d, with an even, glabrous and colourless margin; immersed mycelium colourless, forming a glaucous pigmentation in sectors or irregular patches, or locally faintly olivaceous after 10–14 d; the surface covered by a diffuse layer of woolly to tufty, first pure white, later locally glaucous aerial mycelium. Reverse concolourous. Conidiomata developing scattered or in dense clusters mostly colourless sectors after 5–7 d. Colonies on MEA reaching 38–40 mm diam in 10 d, with an even, colourless margin; immersed mycelium not visible from above, entirely hidden under a dense but relatively low, woolly-floccose mat of aerial mycelium that shows glaucous and rosy buff to salmon areas; conidiomata developing on the agar surface underneath the aerial mycelium, scattered or in dense clusters. Reverse predominantly ochreous, locally with rust patches, olivaceous black where pycnidia are numerous.

Specimen examined. ITALY, Sardinia, on dead leaves of Smilax aspera, 18 May 1971, W. Gams, isolated by H.A. van der Aa 2568, holotype CBS H-21040, living ex-type strain CBS 501.71.

Notes — Only a single isolate was available of this species. More isolates need to become available in order to assess its ecology and geographic distribution.

Paraphaeosphaeria sporulosa (W. Gams & Domsch) Verkley, Göker & Stielow, comb. nov. — MycoBank MB800768; Fig. 13

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Fig. 13

Paraphaeosphaeria sporulosa (CBS 218.68, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c, d. conidia on OA. — Scale bars = 10 μm.

Basionym. Coniothyrium fuckelii var. sporulosum W. Gams & Domsch, Nova Hedwigia 18: 9. 1969.

≡ Coniothyrium sporulosum (W. Gams & Domsch) Aa, Verh. Kon. Ned. Akad. Wetensch., tweede sect., 68: 3. 1977.

≡ Paraconiothyrium sporulosum (W. Gams & Domsch) Verkley, Stud. Mycol. 50: 332. 2004.

Conidiomata pycnidial, single, or eustromatic and more complex, globose, superficial or immersed in the agar, glabrous, 120–250(–350) μm diam, initially pale but soon appearing dark brown to black due to mature conidia inside. Conidiomatal wall composed of hyaline to pale yellowish brown textura angularis, lined by a thin layer of globose hyaline cells. Conidia released through one, rarely two, well-developed ostioli 15–25 μm diam that are somewhat darker then the surrounding wall and lined with hyaline short clavate periphyses. Conidiogenous cells globose to ampulliform, hyaline, discrete or positioned on aggregated clumps of cells that protrude into the cavity, phialidic or with 1–2 percurrent proliferations, mostly 4.5–7 × 3.5–4.5(–5) μm. Conidia subglobose, ellipsoid or obovoid-pyriform, initially hyaline, after secession olivaceous-brown, glabrous, with one large (1.5–2 μm diam) and often also 1–2 additional smaller oil-droplets, 0-septate, 3.5–5(–6) × 3–4 μm, average L/W ratio 1.5 ± 0.2. Sexual morph unknown.

Colonies on OA reaching 42–50 mm diam in 10 d, with an even, glabrous and colourless margin. Immersed mycelium colourless but in most strains soon showing a pure yellow to pale luteous, more rarely brownish pigmentation, the surface mostly glabrous but in the centre and sometimes also elsewhere with tufts of pure white aerial mycelium. Reverse concolourous, appearing olivaceous-brown where numerous pycnidia are formed. Conidiomata developing after 7–10 d. Colonies on MEA reaching 30–35 mm diam in 10 d, with an even to slightly ruffled, colourless to buff margin. Immersed mycelium barely visible from above, appearing olivaceous in the centre, covered by a dense mat of woolly-floccose, dirty white to rosy buff or ochreous aerial mycelium. Reverse mostly pale luteous to ochreous, in the centre with darker areas of umber and chestnut.

Specimens examined. GERMANY, Kitzeberg, isolated from wheat field soil, 1963, W. Gams C353, living culture ex-isotype CBS 218.68 (CBS H-6956, isotype of Coniothyrium fuckelii var. sporulosum). Other isolates examined are listed in Table 1.

Notes — In the literature Paraph. sporulosa has been reported as a cosmopolitan soil fungus (Domsch et al. 2007), but this study sheds doubt on whether those records were based on correct identifications. Isolates in CBS from outside Europe all proved to belong to Paraph. neglecta or to other species outside the Montagnulaceae. For morphological differences with the closely related Paraph. neglecta, see the note under that species.

Paraphaeosphaeria verruculosa Verkley, Göker & Stielow, sp. nov. — MycoBank MB800770; Fig. 14

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Fig. 14

Paraphaeosphaeria verruculosa (CBS 263.85^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c, d. conidia on OA. — Scale bars = 10 μm.

Etymology. Named after the moderately roughened outer wall of the conidia.

Conidiomata pycnidial, globose, ostiolum 10–15 μm diam or absent, black due to mature conidia inside, 140–200 μm diam, predominantly formed in the aerial mycelium (on OA). Conidiomatal wall composed of an outer layer of a relatively thick-walled, orange-brown textura angularis (cells 4–8 μm diam) and an inner layer of hyaline, thin-walled textura angularis. Conidiogenous cells discrete, globose to broadly ampulliform, phialidic, 4–6 × 3–4 μm. Conidia globose, subglobose to ellipsoid, initially hyaline with mostly 3–7 small oil-droplets (< 1 μm diam), after secession olivaceous-brown and mostly with fewer but larger oil-droplets (1.5–2 μm diam), mature conidial wall orange-brown, verruculose, 0-septate, (3–)4–5(–6) × (2.5–)3–3.5(–5) μm, average L/W ratio 1.3 ± 0.2. Sexual morph unknown.

Colonies on OA reaching 50–54 mm diam in 10 d, with an even, glabrous and colourless margin. Immersed mycelium first colourless, becoming chestnut, often with a greenish haze, in the centre. Aerial mycelium very diffuse, felty, locally also with long, grey tufts. Reverse concolourous. Pycnidia developing after 12–18 d. Colonies on MEA reaching 40–44 mm diam in 10 d, with an even, colourless margin. Immersed mycelium entirely hidden under a dense, high mat of floccose aerial mycelium which is for the most pure white, but in an irregularly outlined central area grey to grey-olivaceous. Reverse chestnut in the centre (irregular outline), surrounded by umber fading to ochreous, margin pale luteous.

Specimens examined. CHILE, Valdivia, isolated from wood logs of Pinus radiata stored outdoors for 1–8 months, Nov. 1984, H.L. Peredo, living culture CBS 682.84. – COLOMBIA, Cundinamarca, Monserrate, isolated from páramo soil, after burning, Mar. 1980, W. Gams, living culture CBS 354.80. – GERMANY, Bayerischer Wald, from needle of Picea abies, Mar. 1985, H. Butin, holotype CBS H-21041, living ex-type culture CBS 263.85.

Notes — This species is characteristic for having globose to subglobose conidia at maturity with an elegant verruculose ornamentation of the outer conidium wall. It may be widely distributed in soils, and shows a preference for conifers. More material needs to become available in order to better understand its ecology.

Paraphaeosphaeria viridescens Verkley, Göker & Stielow, sp. nov. — MycoBank MB800764; Fig. 15

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Fig. 15

Paraconiothyrium viridescens (CBS 854.73^T, ex-type culture). a. Colony on OA; b. colony on MEA, also showing reverse on the right; c. conidia on OA; d. conidiogenous cells on OA; e. conidia on OA. — Scale bars = 10 μm.

Etymology. Named after the green pigment this fungus produces in culture.

Conidiomata pycnidial, with a single cavity but lacking a differentiated ostiolum, black due to mature conidia inside, 250–450 μm diam. Conidiomatal wall composed of an outer layer of yellow to very pale orange-brown and thin-walled textura angularis with relatively large cells 5–12 μm diam, and an inner layer of hyaline, thin-walled textura angularis-globosa. Conidiogenous cells discrete, globose, doliiform to broadly ampulliform, phialidic with a distinct periclinal thickening, occasionally percurrently proliferating to form a neck-like protrusion, 5–7.5 × 4–5 μm. Conidia consistently ellipsoid, initially hyaline, soon after secession with a greenish yellow, thin, smooth wall, contents with 1–2 oil-droplets (< 1 μm diam) at each end, 0-septate, (3–)4–4.5(–5) × 1.8–2.2 μm, average L/W ratio 2.0 ± 0.2. Sexual morph unknown.

Colonies on OA reaching 52–55 mm diam in 10 d, with an even, glabrous colourless margin. Immersed mycelium colourless to very faintly yellow, later with a green pigment and with numerous pycnidia developing in distinct concentrical zones after 3–5 d. Reverse concolourous. Colonies on MEA reaching 40–43 mm diam in 10 d, with an even to slightly ruffled, buff margin. Immersed mycelium in the centre dark herbage green (often with bluish tinges), darkening to dull green, covered by an appressed, diffuse to more dense mat of finely felted to floccose, greyish aerial mycelium. Reverse in the centre greenish olivaceous to olivaceous, quite abruptly changing to buff at the margin.

Specimen examined. MONTENEGRO, Lake of Skadar, isolated from freshwater, Oct. 1973, M. Muntañola-Cvetkovic, No. SK 1-32, holotype CBS H-21038, living ex-type culture CBS 854.73.

Notes — The species is noted for producing a green pigment diffusing in the agar and conidia with a consistently ellipsoid shape and relatively high L/W ratio (2.0 ± 0.2), and a relatively faintly green yellowish wall at maturity. CBS 854.73^T originates from fresh water and it remains unclear if the fungus also occurs in soils or plants, as do most of its close relatives.