- Email: [email protected]
Chaetosphaeria tortuosa, the newly discovered teleomorph of Menispora tortuosa, with a key to known Menispora species
m y c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 104 – 109
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycres
Chaetosphaeria tortuosa, the newly discovered teleomorph of Menispora tortuosa, with a key to known Menispora species Martina RE´BLOVA´a,*, Keith A. SEIFERTb, George P. WHITEc a
Department of Plant Taxonomy and Biosystematics, Institute of Botany, Academy of Sciences, 252 43 Pru˚honice, Czech Republic Biodiversity (Mycology and Botany), Environmental Science Team, Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, Canada c Madawaska Highlands Biodiversity Project, 95 Cooper Hill Road, Griffith, ON K0J 2R0, Canada b
article info
abstract
Article history:
Chaetosphaeria tortuosa is described as the newly discovered teleomorph of Menispora tor-
Received 2 June 2005
tuosa, based on specimens from Canada and the Czech Republic, and single spore isolations
Accepted 1 September 2005
from both morphs. The fungus produces superficial, more or less globose, papillate, dark
Corresponding Editor:
brown to black smooth perithecia (200–)220–250 ! (220–)230–260 mm. The asci are unituni-
David L. Hawksworth
cate, 8-spored, cylindrical-fusiform, (110–)120–133(–145) ! 12–14 with a distinct apical, nonamyloid annulus 1–1.5 mm high, 3.5–4 mm wide. The ascospores are fusiform, 19–
Keywords:
24 ! 5–6 mm, hyaline, 3-septate, smooth, and 2-seriate in the ascus. The morphology of
Ascomycota
the teleomorph and anamorph are similar to that of C. ovoidea (anamorph: M. glauca), dif-
Chaetosphaeriales
fering in dimensions of asci and ascospores, and in the disposition and morphology of the
Conidial fungi
phialides of the anamorphs. The generic concept and phylogeny of Menispora is briefly dis-
Hyphomycetes
cussed, and a key to the 11 species currently accepted in the genus is provided.
Pleomorphic fungi
ª 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Menispora tortuosa is a common lignicolous fungus in Northern Temperate, deciduous forests. Its conspicuous colonies develop between the bark and sapwood of dead trees, hidden from sight until the bark is removed. The colonies form irregularly shaped patches up to several cm across, starting out light green and changing to grayish turquoise, eventually almost black. They have a peculiar texture because the slimy conidia, which impart the green colour, are covered by a layer of coiled, brown setae that eventually dominate the colony appearance as they continue to develop and the conidia are dispersed. In Ontario, Canada, the fungus is one of the first
hyphomycetes to sporulate in the spring, especially on fallen branches or logs of Acer saccharum that have been lying on the ground for three or more years. Colonies can be found through most of the summer, then a new flush begins after the first autumn frost. In 2003, we noticed an inconspicuous, black pyrenomycete sometimes associated with colonies of M. tortuosa, and speculated that this might be its undescribed teleomorph. Subsequently, we also discovered the same teleomorph in the Czech Republic. The perithecia were dark brown to black, opaque, globose to subglobose and superficial and included cylindrical-fusiform asci with a shallow apical ring, with 3-septate, hyaline and fusiform ascospores arranged in two series. This
* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.09.003
Chaetosphaeria tortuosa, the teleomorph of Menispora
description matches the genus Chaetosphaeria. Isolations of single- and mass-ascospores on potato-carrot medium (PCA; Gams et al. 1998) yielded sporulating cultures of M. tortuosa that were identical to those isolated from conidia. So far, there is a perfect correlation between Menispora and Chaetosphaeria (Chaetosphaeriaceae, Chaetosphaeriales). The type species of Menispora, M. glauca is the anamorph of C. ovoidea, and M. caesia is the anamorph of C. pulviscula (Booth 1957). Both species produce a Phialophora-like synanamorph in culture (Constantinescu et al. 1995; Re´blova´ 1998), but we have not observed this in cultures derived from conidia of M. tortuosa or ascospores of its teleomorph. Because no teleomorph has been described for M. tortuosa and the link between the two morphs was confirmed in vitro and in vivo, a new holomorph species and a new anamorph-teleomorph connection in the genus Chaetosphaeria is proposed. The new species is described and illustrated and a key to species accepted in Menispora is provided.
Materials and methods Dried herbarium specimens were rehydrated in water and studied in water, Melzer’s reagent or 90 % lactic acid. Singleascospore isolates were obtained from fresh material with the aid of a single-spore isolator (Meopta, Czech Republic). Cultures were grown on potato-carrot agar (PCA). Colony characters were derived from cultures grown on PCA and placed at 25 C in the dark for 14 d. Cultures are maintained in the Canadian Collection of Fungal Cultures, Ottawa (DAOM) and the Centraalbureau voor Schimmelcultures, Utrecht (CBS). All measurements were made in Melzer’s reagent. Means G standard errors (S.E.) are based on 25 measurements and are given for ascospore, ascus and conidial dimensions. The length/breadth ratios (L/B) for asci, ascospores and conidia are given. Images were captured in Melzer’s reagent using differential interference microscopy (DIC) and phase contrast (PC) and processed using Adobe Photoshop 6.0 CE.
Taxonomy Chaetosphaeria tortuosa Re´blova´, Seifert & G.P. White, sp. nov. (Figs 1–14.) Anamorph: Menispora tortuosa Corda, Icon. Fung. 3: 8 (1839). Eriomenella tortuosa (Corda) Peyronel, Nuova G. Bot. Ital., n.s. 25: 447 (1918). Menispora obtusa Sacc. & Berl., Atti R. Istit. Ven. Sci., Lett., Arti, Ser. VI 3: 741 (1885). Synonymy according to Hughes & Kendrick (1963). Perithecia gregaria, non stromatica, fusca vel atra, globosa vel subglobosa, glabra, (200–)220–250 mm alta, (220–)230–260 mm diam; ostiolum periphysatum. Paries perithecii fragilis, 30–35 mm crassus, bistratosus. Paraphyses persistentes, copiosae, septatae, 3–3.5 mm latae prope basim, sursum ad 1.5–2 mm, ascos superantes. Asci unitunicati, cylindrico-fusiformis, (110–)120–133(–145) ! 12– 14 mm, longit.:latit. 10:1. Ascospores fusiformes, 19–24 ! 5–6 mm, longit.:latit. 4:1, hyalinae, 3-septatae, leves. Anamorphe Menispora tortuosa.
105
Typus: Canada: Ontario: Denbigh (45 09.656# N 77 19.377# W), on decayed wood beneath the bark of Acer saccharum, 1 Nov. 2004, G.P. White (DAOM 234417 – holotypus; cultura viva; DAOM 234417, CBS 117554). Colonies in vivo spreading, inconspicuous to conspicuous, about 1 mm deep, irregular in outline but can be several cm in the longest dimension, light green to greyish turquoise, comprising a dense carpet of independent conidiophores superficially resembling a sporodochium, with a tacky mass of slimy conidia surmounted by coiled setae that terminate the conidiophores. No setae independent from conidiophores are produced. Perithecia solitary or gregarious, usually near the anamorphic part of the colony but not among the conidiophores, dark brown to black, opaque, smooth to slightly roughened, superficial, globose to subglobose, (200–)220– 250 mm high, (220–)230–260 mm diam, not collapsing when dry, papillate; ostiole periphysate. Perithecial wall fragile, 30– 35 mm thick, two-layered; outer wall textura angularis, composed of brown, thin-walled, polyhedral cells with opaque walls; and inner layer of thinner-walled, subhyaline to hyaline elongated and compressed cells. Paraphyses persistent, septate, copious, not constricted at the septa, branched, anastomosing, 3–3.5 mm wide near the base, tapering to 1.5–2 mm, longer than the asci. Asci unitunicate, 8-spored, cylindrical-fusiform, rounded to obtuse at the apex, (110–)120–133(– 145) ! 12–14 mm (mean G S.E. Z 126.5 G 1.5 ! 12.8 G 0.1), L/B 10:1, shortly stipitate, apical annulus distinct, shallow, non amyloid, 1–1.5 mm high, 3.5–4 mm wide. Ascospores fusiform, 19–24 ! 5–6 mm (mean G S.E. Z 21.6 G 0.2 ! 5.7 G 0.1), L/B 4:1, hyaline, 3-septate, over-mature ascospores may develop up to 5 transverse septa, smooth, 2-seriate in the ascus. Conidiophores macronematous, septate, brown, paler towards the apex, smooth, up to 700 mm long, 5–6 mm wide at the base, sparsely branched in the lower part, which is straight, upper part unbranched, strongly flexuous or irregularly coiled, terminating in a sterile subhyaline rounded cell ca. 3–3.5 mm diam; lateral branches up to 300 mm long. Phialides develop in the lower part of the main axis of the conidiophore or its lateral branches, cylindrical with a narrowing straight apex, 15–24 ! 3.5–5 mm (mean G S.E. Z 20 G 0.8 ! 4.2 G 0.2), subhyaline becoming hyaline towards the conidiogenous aperture, arising from short, septate, pale brown metulae (10.5–)13–22.5 ! 4–5 mm below the septa, aggregated in dense groups of two to nine that often appear digitate; the branches with phialides are directed upwards; conidiogenous aperture 1–1.5 mm wide; collarette shallow, indistinct. Conidia falcate with parallel walls, rounded at each end, 18–22 ! 3.5–4 mm (mean G S.E. Z 20 G 0.8 ! 3.7 G 0.1), L/B 5.5:1, 3-septate, hyaline, each polar cell with a single straight or slightly curved setulum, 8–12 mm long, subterminally inserted on the concave side. Colonies in vitro growing slowly on PCA at 25 C, about 2 mm radial growth in 7 d, white, appressed, planar, with little aerial mycelium, reverse white; sporulating between 7–14 d, setae developed by 7 d, concentrated near inoculum. Cardinal temperatures for growth: minimum 10 C, optimum 25–30 C, maximum O 30 C; setae produced most abundantly at 15 C. The morphology of conidiophores, phialides and conidia is identical to that observed on the natural substratum,
106
M. Re´blova´ et al.
Figs 1–9 – Chaetosphaeria tortuosa. Figs 1–2. Asci. Fig. 3. Paraphyses. Fig. 4. Ascospores. Fig. 5. Longitudinal section of the perithecial wall. Figs 6–9. Menispora tortuosa anamorph, from nature. Figs 6, 8–9. Conidiophores. Fig. 7. Conidia. DIC: Figs 1–2, 4–6, 7 (partly), 9; PC: Figs 3, 7 (partly), 8. Figs 1–2, 5 from M.R. 2925; Figs 3–4 from DAOM 234417 (holotype); Figs 6–9 from M.R. 2894. Bars Z 10 mm.
Chaetosphaeria tortuosa, the teleomorph of Menispora
107
Figs 10–14 – Chaetosphaeria tortuosa; Menispora tortuosa anamorph, from PCA culture. Fig. 10. Conidiophore (14 d). Figs 11–12. Conidiophores (1 m). Fig. 13. Phialide. Fig. 14. Conidia; arrows indicate a setulum at each end. DIC: Figs 11–12, 14 (partly); PC: 10, 13–14 (partly). Figs 10–14 from DAOM 234417 (ex-type culture). Bars: Z 10 mm.
but the colonies are less dense and lack the green colouration typical of well-developed in vivo colonies. Conidiophores to 800 mm long, 5–6 mm diam, branches to 250 mm long. Phialides cylindrical, 20–35 ! 4–4.5 mm (mean G S.E. Z 22.7 G 1.8 ! 4.2 G 0.1), hyaline, singly or in sparse groups of two to five, arising from short subhyaline metulae (11.5–)17–24 ! 4–5 mm, conidiogenous aperture 1–1.5 mm wide, with a shallow, indistinct collarette. Conidia cylindrical, 19–23 ! (3–)3.5–4 mm (mean G S.E. Z 20.1 G 0.3 ! 3.7 G 0.1), L/B 5.5:1, setulae 6–11 mm long. Habitat. Saprobic on decayed wood and bark of deciduous trees; so far known from temperate zone of Northern Hemisphere. Hosts. Acer, Betula, Fagus, Fraxinus, Populus, Quercus (Hughes & Kendrick 1963; Holubova´-Jechova´ 1973). Other material examined. Canada: Ontario: Stittsville, Heritage Corners (45 11.9# N 75 58.8# W), on inner side of the decayed bark of Acer sacchariferum, 2 Nov. 2003, K. A. Seifert & S. L. River (CBS 117553, DAOM 234417). Czech Republic: Southern Moravia: obora hot, on inner side of a decayed bark of Fraxinus Soutok near Lanz angustifolia, 23 Oct. 2004, M. Re´blova´ M.R. 2894 (CBS 117552, PRM 903807).
Discussion Chaetosphaeria tortuosa is the third species of the genus to be linked with an anamorph in Menispora. It is reminiscent
of C. ovoidea, the teleomorph of M. glauca, but differs by having smaller asci and ascospores. The anamorph, M. tortuosa, is also similar to M. glauca. Both have 3-septate conidia with polar, subterminal setulae, but M. tortuosa has dense aggregates of phialides having a digitate appearance and a straight phialidic tip. The phialides of M. glauca arise singly on one to several supporting cells along the conidiophore and the phialides terminate with an apex that is strongly curved downwards, away from the main conidiophore stalk (‘recurved phialides’; cfr. Hughes & Kendrick 1963). In cultures of C. tortuosa, we observed delayed formation of conidial setulae (Fig. 14, see arrows), which develop only after conidium secession. Our specimens on the natural substratum had only mature conidiophores and all conidia were setulate. Phialides produced in vitro are longer than those produced in vivo. Our preliminary phylogenetic analyses of Menispora based on nuc rLSU and (ITS) sequences (Re´blova´ & Winka 2000; Re´blova´ & Seifert unpubl.) include all sampled species (viz. M. caesia, M. ciliata, M. glauca, M. manitobaensis and M. tortuosa) in a well-supported monophyletic clade (100 % bootstrap, 92 % jackknife). These species are all morphologically typical of the presently accepted generic concept of Menispora, i.e. they all produce hyaline septate or aseptate phialoconidia, with or without setulae, and setiform conidiophores with laterally arranged phialides, sometimes with ‘recurved’ phialides. The Menispora clade occurs within a robust clade also
M. Re´blova´ et al.
108
containing representatives of the genera Codinaea, Codinaeopsis, Dictyochaetopsis, Menisporopsis, and Thozetella. Four species of Menispora, namely M. britannica (Kirk 1985), M. gamundiae (Arambarri & Cabello 1995), M. fuegiana (Arambarri & Godeas 1994), and M. uncinata (Hughes & Kendrick 1968), represent a morphological pattern slightly different from the type species, i.e. sterile setae independent of the conidiophores are accompanied by a shorter layer of conidiophores with a terminal mono- or polyphialide. In overall habitat, these four species resemble some species of Codinaea rather than Menispora; species of both genera are known to be anamorphs of Chaetosphaeria, and both display the similar degree of variability. Unfortunately, these Menispora species
were unavailable for molecular studies and a revised generic delimitation of Menispora must await their inclusion. If both morphological patterns are to be included in Menispora, then the uniting feature seems to be the unique character of a phialide tip, which lacks a conspicuous collarette and is often strongly or slightly curved downwards. Menispora is one of 16 ‘form’ genera linked with the life-cycles of Chaetosphaeria species (Booth 1957; Holubova´-Jechova´ 1973; Constantinescu et al. 1995; Re´blova´ 1998). The monograph of the genus published by Hughes & Kendrick (1963, 1968) included five species. Presently, we accept eleven species in Menispora and present the following key with the qualifier that our phylogenetic revision is still in progress.
A key to the species accepted in Menispora 1
Setae independent of conidiophores absent or rarely present; conidiophores terminating in a sterile, whip-like extension with lateral branches in their lower part; phialides borne on short metulae along the main axis of the conidiophore or its branches, singly or in groups, terminally or laterally. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Setae independent of conidiophores regularly present; conidiophores arising from the substratum and developing a shorter layer around setae, terminating in a mono- or poly-phialide; phialides terminal rarely lateral, with one terminal or additional lateral aperture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2(1) Conidia with subterminally inserted setulum on each polar cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Conidia without setulae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3(2) Conidia 3-septate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Conidia aseptate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4(3) Phialides arising singly, terminal on 1–3-septate metulae; phialide apex strongly curved downwards away from the
main stipe; conidia 16–26 ! 3–4.5 mm; Phialophora-like synanamorph; ascospores fusiform, 3-septate, 21–29 ! 4– 5.5(–6) mm; asci (100–)115–150 ! 8.5–11.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . glauca (teleomorph: C. ovoidea) Phialides arising in groups of 2–9, the cluster having a digitate appearance, terminal or lateral on short, branched or unbranched metulae; phialide apex straight or curved very gently; conidia 18–25 ! 3.5–4 mm; ascospores fusiform, 3-septate, 19–24 ! 5–6 mm; asci (110–)120–133(–145) ! 12–14 mm . . . . . . . . . . . . . . tortuosa (teleomorph: C. tortuosa) 5(3) Apex of phialide curved strongly downwards towards the main stipe; phialides arising singly, terminal or rarely in
lateral series on one side of the conidiophore; conidia 11.5–21 ! 2.5–3.5 mm . . . . . . . . . . . . . . . . . . . . . . . . .ciliata Apex of the phialide straight or slightly curved downwards towards the main stalk . . . . . . . . . . . . . . . . . . . . . . . . 6 6(5) Conidia 14–16 ! 3–3.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gamsii
Conidia 17–23 ! 3.5–4 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . convoluta 7(2) Conidia 3-septate, 17–21.5 ! 4–4.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . manitobaensis
Conidia aseptate, 15.5–21.5 ! 3–3.5 mm; Phialophora-like synanamorph; ascospores fusiform, 1-septate later 3-septate, 19–23(–25) ! 3–3.5(–4) mm; asci 90–115 ! 7–8.5 mm . . . . . . . . . . . . . . . . caesia (teleomorph: C. pulviscula) 8(1) Conidia aseptate, with subterminally inserted setulum at each end cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Conidia 1-septate, lacking setulae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 9(8) Setae sinuous, sometimes coiled apically, rounded at the apex; conidia 18.5–23.5 ! 4–5 mm, setulae 2–3 mm
long . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . uncinata Setae straight, not coiled, acute at the apex; conidia 15–18 ! 2–2.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . britannica 10(8) Setae straight to sinuous; conidia 22–27 ! 2–2.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fuegiana
Setae sinuous and coiled above; conidia 23–26 ! 2.5–3 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gamundiae
Acknowledgments This project was partly supported by the Research Project of the Institute of Botany, Academy of Sciences of the Czech
Republic (AV0Z6005908). We are grateful to Robert Shoemaker and Sarah Hambleton for presubmission reviews of this manuscript.
Chaetosphaeria tortuosa, the teleomorph of Menispora
references
Arambarri AM, Cabello MN, 1995. Menispora gamundiae sp. nov., a new hyphomycete from Argentina (Buenos Aires province). Mycological Research 99: 377–378. Arambarri AM, Godeas AM, 1994. Menispora fuegiana sp. nov. a new hyphomycete from Tierra del Fuego (Argentina). Mycotaxon 52: 95–98. Booth C, 1957. Studies of pyrenomycetes: I. Four species of Chaetosphaeria, two with Catenularia conidia. II. Melanopsamma pomiformis and its Stachybotrys conidia. Mycological Papers 68: 1–27. Constantinescu O, Holm K, Holm L, 1995. Teleomorph-anamorph connections in ascomycetes: the anamorphs of three species of Chaetosphaeria. Mycological Research 99: 585–592.
109
Gams W, Hoekstra ES, Aptroot A, 1998. CBS Course of Mycology. 4th edn. Centraalbureau voor Schimmelcultures, Baarn. Holubova´-Jechova´ V, 1973. Lignicolous hyphomycetes from Czechoslovakia 4. Menispora. Folia Geobotanica et Phytotaxonomica 8: 317–336. Hughes SJ, Kendrick WB, 1963. Microfungi IX. Menispora Persoon. Canadian Journal of Botany 41: 693–718. Hughes SJ, Kendrick WB, 1968. New Zealand fungi 12. Menispora, Codinaea, Menisporopsis. New Zealand Journal of Botany 6: 323–375. Kirk PM, 1985. New or interesting microfungi XIV. Dematiaceous hyphomycetes from Mt. Kenya. Mycotaxon 23: 305–352. Re´blova´ M, 1998. Revision of three Melanomma species described by L. Fuckel. Czech Mycology 50: 163–183. Re´blova´ M, Winka K, 2000. Phylogeny of Chaetosphaeria and its anamorphs based on morphological and molecular data. Mycologia 92: 939–954.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycres
Chaetosphaeria tortuosa, the newly discovered teleomorph of Menispora tortuosa, with a key to known Menispora species Martina RE´BLOVA´a,*, Keith A. SEIFERTb, George P. WHITEc a
Department of Plant Taxonomy and Biosystematics, Institute of Botany, Academy of Sciences, 252 43 Pru˚honice, Czech Republic Biodiversity (Mycology and Botany), Environmental Science Team, Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, Canada c Madawaska Highlands Biodiversity Project, 95 Cooper Hill Road, Griffith, ON K0J 2R0, Canada b
article info
abstract
Article history:
Chaetosphaeria tortuosa is described as the newly discovered teleomorph of Menispora tor-
Received 2 June 2005
tuosa, based on specimens from Canada and the Czech Republic, and single spore isolations
Accepted 1 September 2005
from both morphs. The fungus produces superficial, more or less globose, papillate, dark
Corresponding Editor:
brown to black smooth perithecia (200–)220–250 ! (220–)230–260 mm. The asci are unituni-
David L. Hawksworth
cate, 8-spored, cylindrical-fusiform, (110–)120–133(–145) ! 12–14 with a distinct apical, nonamyloid annulus 1–1.5 mm high, 3.5–4 mm wide. The ascospores are fusiform, 19–
Keywords:
24 ! 5–6 mm, hyaline, 3-septate, smooth, and 2-seriate in the ascus. The morphology of
Ascomycota
the teleomorph and anamorph are similar to that of C. ovoidea (anamorph: M. glauca), dif-
Chaetosphaeriales
fering in dimensions of asci and ascospores, and in the disposition and morphology of the
Conidial fungi
phialides of the anamorphs. The generic concept and phylogeny of Menispora is briefly dis-
Hyphomycetes
cussed, and a key to the 11 species currently accepted in the genus is provided.
Pleomorphic fungi
ª 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Menispora tortuosa is a common lignicolous fungus in Northern Temperate, deciduous forests. Its conspicuous colonies develop between the bark and sapwood of dead trees, hidden from sight until the bark is removed. The colonies form irregularly shaped patches up to several cm across, starting out light green and changing to grayish turquoise, eventually almost black. They have a peculiar texture because the slimy conidia, which impart the green colour, are covered by a layer of coiled, brown setae that eventually dominate the colony appearance as they continue to develop and the conidia are dispersed. In Ontario, Canada, the fungus is one of the first
hyphomycetes to sporulate in the spring, especially on fallen branches or logs of Acer saccharum that have been lying on the ground for three or more years. Colonies can be found through most of the summer, then a new flush begins after the first autumn frost. In 2003, we noticed an inconspicuous, black pyrenomycete sometimes associated with colonies of M. tortuosa, and speculated that this might be its undescribed teleomorph. Subsequently, we also discovered the same teleomorph in the Czech Republic. The perithecia were dark brown to black, opaque, globose to subglobose and superficial and included cylindrical-fusiform asci with a shallow apical ring, with 3-septate, hyaline and fusiform ascospores arranged in two series. This
* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.09.003
Chaetosphaeria tortuosa, the teleomorph of Menispora
description matches the genus Chaetosphaeria. Isolations of single- and mass-ascospores on potato-carrot medium (PCA; Gams et al. 1998) yielded sporulating cultures of M. tortuosa that were identical to those isolated from conidia. So far, there is a perfect correlation between Menispora and Chaetosphaeria (Chaetosphaeriaceae, Chaetosphaeriales). The type species of Menispora, M. glauca is the anamorph of C. ovoidea, and M. caesia is the anamorph of C. pulviscula (Booth 1957). Both species produce a Phialophora-like synanamorph in culture (Constantinescu et al. 1995; Re´blova´ 1998), but we have not observed this in cultures derived from conidia of M. tortuosa or ascospores of its teleomorph. Because no teleomorph has been described for M. tortuosa and the link between the two morphs was confirmed in vitro and in vivo, a new holomorph species and a new anamorph-teleomorph connection in the genus Chaetosphaeria is proposed. The new species is described and illustrated and a key to species accepted in Menispora is provided.
Materials and methods Dried herbarium specimens were rehydrated in water and studied in water, Melzer’s reagent or 90 % lactic acid. Singleascospore isolates were obtained from fresh material with the aid of a single-spore isolator (Meopta, Czech Republic). Cultures were grown on potato-carrot agar (PCA). Colony characters were derived from cultures grown on PCA and placed at 25 C in the dark for 14 d. Cultures are maintained in the Canadian Collection of Fungal Cultures, Ottawa (DAOM) and the Centraalbureau voor Schimmelcultures, Utrecht (CBS). All measurements were made in Melzer’s reagent. Means G standard errors (S.E.) are based on 25 measurements and are given for ascospore, ascus and conidial dimensions. The length/breadth ratios (L/B) for asci, ascospores and conidia are given. Images were captured in Melzer’s reagent using differential interference microscopy (DIC) and phase contrast (PC) and processed using Adobe Photoshop 6.0 CE.
Taxonomy Chaetosphaeria tortuosa Re´blova´, Seifert & G.P. White, sp. nov. (Figs 1–14.) Anamorph: Menispora tortuosa Corda, Icon. Fung. 3: 8 (1839). Eriomenella tortuosa (Corda) Peyronel, Nuova G. Bot. Ital., n.s. 25: 447 (1918). Menispora obtusa Sacc. & Berl., Atti R. Istit. Ven. Sci., Lett., Arti, Ser. VI 3: 741 (1885). Synonymy according to Hughes & Kendrick (1963). Perithecia gregaria, non stromatica, fusca vel atra, globosa vel subglobosa, glabra, (200–)220–250 mm alta, (220–)230–260 mm diam; ostiolum periphysatum. Paries perithecii fragilis, 30–35 mm crassus, bistratosus. Paraphyses persistentes, copiosae, septatae, 3–3.5 mm latae prope basim, sursum ad 1.5–2 mm, ascos superantes. Asci unitunicati, cylindrico-fusiformis, (110–)120–133(–145) ! 12– 14 mm, longit.:latit. 10:1. Ascospores fusiformes, 19–24 ! 5–6 mm, longit.:latit. 4:1, hyalinae, 3-septatae, leves. Anamorphe Menispora tortuosa.
105
Typus: Canada: Ontario: Denbigh (45 09.656# N 77 19.377# W), on decayed wood beneath the bark of Acer saccharum, 1 Nov. 2004, G.P. White (DAOM 234417 – holotypus; cultura viva; DAOM 234417, CBS 117554). Colonies in vivo spreading, inconspicuous to conspicuous, about 1 mm deep, irregular in outline but can be several cm in the longest dimension, light green to greyish turquoise, comprising a dense carpet of independent conidiophores superficially resembling a sporodochium, with a tacky mass of slimy conidia surmounted by coiled setae that terminate the conidiophores. No setae independent from conidiophores are produced. Perithecia solitary or gregarious, usually near the anamorphic part of the colony but not among the conidiophores, dark brown to black, opaque, smooth to slightly roughened, superficial, globose to subglobose, (200–)220– 250 mm high, (220–)230–260 mm diam, not collapsing when dry, papillate; ostiole periphysate. Perithecial wall fragile, 30– 35 mm thick, two-layered; outer wall textura angularis, composed of brown, thin-walled, polyhedral cells with opaque walls; and inner layer of thinner-walled, subhyaline to hyaline elongated and compressed cells. Paraphyses persistent, septate, copious, not constricted at the septa, branched, anastomosing, 3–3.5 mm wide near the base, tapering to 1.5–2 mm, longer than the asci. Asci unitunicate, 8-spored, cylindrical-fusiform, rounded to obtuse at the apex, (110–)120–133(– 145) ! 12–14 mm (mean G S.E. Z 126.5 G 1.5 ! 12.8 G 0.1), L/B 10:1, shortly stipitate, apical annulus distinct, shallow, non amyloid, 1–1.5 mm high, 3.5–4 mm wide. Ascospores fusiform, 19–24 ! 5–6 mm (mean G S.E. Z 21.6 G 0.2 ! 5.7 G 0.1), L/B 4:1, hyaline, 3-septate, over-mature ascospores may develop up to 5 transverse septa, smooth, 2-seriate in the ascus. Conidiophores macronematous, septate, brown, paler towards the apex, smooth, up to 700 mm long, 5–6 mm wide at the base, sparsely branched in the lower part, which is straight, upper part unbranched, strongly flexuous or irregularly coiled, terminating in a sterile subhyaline rounded cell ca. 3–3.5 mm diam; lateral branches up to 300 mm long. Phialides develop in the lower part of the main axis of the conidiophore or its lateral branches, cylindrical with a narrowing straight apex, 15–24 ! 3.5–5 mm (mean G S.E. Z 20 G 0.8 ! 4.2 G 0.2), subhyaline becoming hyaline towards the conidiogenous aperture, arising from short, septate, pale brown metulae (10.5–)13–22.5 ! 4–5 mm below the septa, aggregated in dense groups of two to nine that often appear digitate; the branches with phialides are directed upwards; conidiogenous aperture 1–1.5 mm wide; collarette shallow, indistinct. Conidia falcate with parallel walls, rounded at each end, 18–22 ! 3.5–4 mm (mean G S.E. Z 20 G 0.8 ! 3.7 G 0.1), L/B 5.5:1, 3-septate, hyaline, each polar cell with a single straight or slightly curved setulum, 8–12 mm long, subterminally inserted on the concave side. Colonies in vitro growing slowly on PCA at 25 C, about 2 mm radial growth in 7 d, white, appressed, planar, with little aerial mycelium, reverse white; sporulating between 7–14 d, setae developed by 7 d, concentrated near inoculum. Cardinal temperatures for growth: minimum 10 C, optimum 25–30 C, maximum O 30 C; setae produced most abundantly at 15 C. The morphology of conidiophores, phialides and conidia is identical to that observed on the natural substratum,
106
M. Re´blova´ et al.
Figs 1–9 – Chaetosphaeria tortuosa. Figs 1–2. Asci. Fig. 3. Paraphyses. Fig. 4. Ascospores. Fig. 5. Longitudinal section of the perithecial wall. Figs 6–9. Menispora tortuosa anamorph, from nature. Figs 6, 8–9. Conidiophores. Fig. 7. Conidia. DIC: Figs 1–2, 4–6, 7 (partly), 9; PC: Figs 3, 7 (partly), 8. Figs 1–2, 5 from M.R. 2925; Figs 3–4 from DAOM 234417 (holotype); Figs 6–9 from M.R. 2894. Bars Z 10 mm.
Chaetosphaeria tortuosa, the teleomorph of Menispora
107
Figs 10–14 – Chaetosphaeria tortuosa; Menispora tortuosa anamorph, from PCA culture. Fig. 10. Conidiophore (14 d). Figs 11–12. Conidiophores (1 m). Fig. 13. Phialide. Fig. 14. Conidia; arrows indicate a setulum at each end. DIC: Figs 11–12, 14 (partly); PC: 10, 13–14 (partly). Figs 10–14 from DAOM 234417 (ex-type culture). Bars: Z 10 mm.
but the colonies are less dense and lack the green colouration typical of well-developed in vivo colonies. Conidiophores to 800 mm long, 5–6 mm diam, branches to 250 mm long. Phialides cylindrical, 20–35 ! 4–4.5 mm (mean G S.E. Z 22.7 G 1.8 ! 4.2 G 0.1), hyaline, singly or in sparse groups of two to five, arising from short subhyaline metulae (11.5–)17–24 ! 4–5 mm, conidiogenous aperture 1–1.5 mm wide, with a shallow, indistinct collarette. Conidia cylindrical, 19–23 ! (3–)3.5–4 mm (mean G S.E. Z 20.1 G 0.3 ! 3.7 G 0.1), L/B 5.5:1, setulae 6–11 mm long. Habitat. Saprobic on decayed wood and bark of deciduous trees; so far known from temperate zone of Northern Hemisphere. Hosts. Acer, Betula, Fagus, Fraxinus, Populus, Quercus (Hughes & Kendrick 1963; Holubova´-Jechova´ 1973). Other material examined. Canada: Ontario: Stittsville, Heritage Corners (45 11.9# N 75 58.8# W), on inner side of the decayed bark of Acer sacchariferum, 2 Nov. 2003, K. A. Seifert & S. L. River (CBS 117553, DAOM 234417). Czech Republic: Southern Moravia: obora hot, on inner side of a decayed bark of Fraxinus Soutok near Lanz angustifolia, 23 Oct. 2004, M. Re´blova´ M.R. 2894 (CBS 117552, PRM 903807).
Discussion Chaetosphaeria tortuosa is the third species of the genus to be linked with an anamorph in Menispora. It is reminiscent
of C. ovoidea, the teleomorph of M. glauca, but differs by having smaller asci and ascospores. The anamorph, M. tortuosa, is also similar to M. glauca. Both have 3-septate conidia with polar, subterminal setulae, but M. tortuosa has dense aggregates of phialides having a digitate appearance and a straight phialidic tip. The phialides of M. glauca arise singly on one to several supporting cells along the conidiophore and the phialides terminate with an apex that is strongly curved downwards, away from the main conidiophore stalk (‘recurved phialides’; cfr. Hughes & Kendrick 1963). In cultures of C. tortuosa, we observed delayed formation of conidial setulae (Fig. 14, see arrows), which develop only after conidium secession. Our specimens on the natural substratum had only mature conidiophores and all conidia were setulate. Phialides produced in vitro are longer than those produced in vivo. Our preliminary phylogenetic analyses of Menispora based on nuc rLSU and (ITS) sequences (Re´blova´ & Winka 2000; Re´blova´ & Seifert unpubl.) include all sampled species (viz. M. caesia, M. ciliata, M. glauca, M. manitobaensis and M. tortuosa) in a well-supported monophyletic clade (100 % bootstrap, 92 % jackknife). These species are all morphologically typical of the presently accepted generic concept of Menispora, i.e. they all produce hyaline septate or aseptate phialoconidia, with or without setulae, and setiform conidiophores with laterally arranged phialides, sometimes with ‘recurved’ phialides. The Menispora clade occurs within a robust clade also
M. Re´blova´ et al.
108
containing representatives of the genera Codinaea, Codinaeopsis, Dictyochaetopsis, Menisporopsis, and Thozetella. Four species of Menispora, namely M. britannica (Kirk 1985), M. gamundiae (Arambarri & Cabello 1995), M. fuegiana (Arambarri & Godeas 1994), and M. uncinata (Hughes & Kendrick 1968), represent a morphological pattern slightly different from the type species, i.e. sterile setae independent of the conidiophores are accompanied by a shorter layer of conidiophores with a terminal mono- or polyphialide. In overall habitat, these four species resemble some species of Codinaea rather than Menispora; species of both genera are known to be anamorphs of Chaetosphaeria, and both display the similar degree of variability. Unfortunately, these Menispora species
were unavailable for molecular studies and a revised generic delimitation of Menispora must await their inclusion. If both morphological patterns are to be included in Menispora, then the uniting feature seems to be the unique character of a phialide tip, which lacks a conspicuous collarette and is often strongly or slightly curved downwards. Menispora is one of 16 ‘form’ genera linked with the life-cycles of Chaetosphaeria species (Booth 1957; Holubova´-Jechova´ 1973; Constantinescu et al. 1995; Re´blova´ 1998). The monograph of the genus published by Hughes & Kendrick (1963, 1968) included five species. Presently, we accept eleven species in Menispora and present the following key with the qualifier that our phylogenetic revision is still in progress.
A key to the species accepted in Menispora 1
Setae independent of conidiophores absent or rarely present; conidiophores terminating in a sterile, whip-like extension with lateral branches in their lower part; phialides borne on short metulae along the main axis of the conidiophore or its branches, singly or in groups, terminally or laterally. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Setae independent of conidiophores regularly present; conidiophores arising from the substratum and developing a shorter layer around setae, terminating in a mono- or poly-phialide; phialides terminal rarely lateral, with one terminal or additional lateral aperture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2(1) Conidia with subterminally inserted setulum on each polar cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Conidia without setulae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3(2) Conidia 3-septate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Conidia aseptate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4(3) Phialides arising singly, terminal on 1–3-septate metulae; phialide apex strongly curved downwards away from the
main stipe; conidia 16–26 ! 3–4.5 mm; Phialophora-like synanamorph; ascospores fusiform, 3-septate, 21–29 ! 4– 5.5(–6) mm; asci (100–)115–150 ! 8.5–11.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . glauca (teleomorph: C. ovoidea) Phialides arising in groups of 2–9, the cluster having a digitate appearance, terminal or lateral on short, branched or unbranched metulae; phialide apex straight or curved very gently; conidia 18–25 ! 3.5–4 mm; ascospores fusiform, 3-septate, 19–24 ! 5–6 mm; asci (110–)120–133(–145) ! 12–14 mm . . . . . . . . . . . . . . tortuosa (teleomorph: C. tortuosa) 5(3) Apex of phialide curved strongly downwards towards the main stipe; phialides arising singly, terminal or rarely in
lateral series on one side of the conidiophore; conidia 11.5–21 ! 2.5–3.5 mm . . . . . . . . . . . . . . . . . . . . . . . . .ciliata Apex of the phialide straight or slightly curved downwards towards the main stalk . . . . . . . . . . . . . . . . . . . . . . . . 6 6(5) Conidia 14–16 ! 3–3.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gamsii
Conidia 17–23 ! 3.5–4 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . convoluta 7(2) Conidia 3-septate, 17–21.5 ! 4–4.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . manitobaensis
Conidia aseptate, 15.5–21.5 ! 3–3.5 mm; Phialophora-like synanamorph; ascospores fusiform, 1-septate later 3-septate, 19–23(–25) ! 3–3.5(–4) mm; asci 90–115 ! 7–8.5 mm . . . . . . . . . . . . . . . . caesia (teleomorph: C. pulviscula) 8(1) Conidia aseptate, with subterminally inserted setulum at each end cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Conidia 1-septate, lacking setulae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 9(8) Setae sinuous, sometimes coiled apically, rounded at the apex; conidia 18.5–23.5 ! 4–5 mm, setulae 2–3 mm
long . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . uncinata Setae straight, not coiled, acute at the apex; conidia 15–18 ! 2–2.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . britannica 10(8) Setae straight to sinuous; conidia 22–27 ! 2–2.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fuegiana
Setae sinuous and coiled above; conidia 23–26 ! 2.5–3 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gamundiae
Acknowledgments This project was partly supported by the Research Project of the Institute of Botany, Academy of Sciences of the Czech
Republic (AV0Z6005908). We are grateful to Robert Shoemaker and Sarah Hambleton for presubmission reviews of this manuscript.
Chaetosphaeria tortuosa, the teleomorph of Menispora
references
Arambarri AM, Cabello MN, 1995. Menispora gamundiae sp. nov., a new hyphomycete from Argentina (Buenos Aires province). Mycological Research 99: 377–378. Arambarri AM, Godeas AM, 1994. Menispora fuegiana sp. nov. a new hyphomycete from Tierra del Fuego (Argentina). Mycotaxon 52: 95–98. Booth C, 1957. Studies of pyrenomycetes: I. Four species of Chaetosphaeria, two with Catenularia conidia. II. Melanopsamma pomiformis and its Stachybotrys conidia. Mycological Papers 68: 1–27. Constantinescu O, Holm K, Holm L, 1995. Teleomorph-anamorph connections in ascomycetes: the anamorphs of three species of Chaetosphaeria. Mycological Research 99: 585–592.
109
Gams W, Hoekstra ES, Aptroot A, 1998. CBS Course of Mycology. 4th edn. Centraalbureau voor Schimmelcultures, Baarn. Holubova´-Jechova´ V, 1973. Lignicolous hyphomycetes from Czechoslovakia 4. Menispora. Folia Geobotanica et Phytotaxonomica 8: 317–336. Hughes SJ, Kendrick WB, 1963. Microfungi IX. Menispora Persoon. Canadian Journal of Botany 41: 693–718. Hughes SJ, Kendrick WB, 1968. New Zealand fungi 12. Menispora, Codinaea, Menisporopsis. New Zealand Journal of Botany 6: 323–375. Kirk PM, 1985. New or interesting microfungi XIV. Dematiaceous hyphomycetes from Mt. Kenya. Mycotaxon 23: 305–352. Re´blova´ M, 1998. Revision of three Melanomma species described by L. Fuckel. Czech Mycology 50: 163–183. Re´blova´ M, Winka K, 2000. Phylogeny of Chaetosphaeria and its anamorphs based on morphological and molecular data. Mycologia 92: 939–954.