A new species of Coniochaeta with a key to the species known in Argentina

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1 Mycol. Res. 103 (6): (1999) Printed in the United Kingdom 689 A new species of Coniochaeta with a key to the species known in Argentina A. I. ROMERO 1, C. C. CARMARA N 1 * AND L. E. LORENZO Departamento de Ciencias Biolo gicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabello n2, 4 piso, Ciudad Universitaria, Nun ez, 1428 Buenos Aires, Argentina Departamento de Bota nica, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Unidad Postal Universidad, 8400 San Carlos de Bariloche, Rı o Negro, Argentina Coniochaeta rhopalochaeta sp. nov. was isolated from decorticated wood of Bulnesia retama in Argentina. It is distinguished principally by the ascomata with mace-like capitate setae. C. scatigena is first recorded for Argentina. A key to the seven known Argentine species of Coniochaeta is provided. Asci of these species were studied with fluorescence microscopy; size of the fluorescent apical ring could be a useful taxonomic character. Coniochaeta contains about 35 species (Hawksworth et al., 1995), occurring mostly on dung, wood and soil, with one species recently reported from wood submerged in fresh water (Crane & Shearer, 1995). Its taxonomic position is still unclear, most workers (Hawksworth, 1978; Hawksworth & Yip, 1981; Mahoney & LaFavre, 1981; Checa et al., 1988) agree with its placement in the Coniochaetaceae, but there is no agreement as to which order (Sordariales or Xylariales) it is better included in. As Rogers (1994) pointed out it is an isolated family whose relationship with other ascomycetes is obscure. Mahoney & LaFavre (1981) gave a complete list and commented on the different substrata occupied by these species. Eighteen species (Mahoney & LaFavre, 1981; Kamiya, Uchiyama & Udagawa, 1995) have been isolated in axenic culture, developing both teleomorph and anamorph stages on agar media. Several different genera have been described for the anamorph of Coniochaeta species: Lecythophora-like (Gams & McGinnis, 1983; Kamiya et al., 1995), Nodulisporium-like (Hawksworth, 1978), Geniculosporium-like (Udagawa & Horie, 1982), Hormonema (Udagawa & Sugiyama, 1982), Phialophoralike (Rogers & Grand, 1971) and Paecilomyces-like (Van der Linde, 1991). Information on the taxonomy and distribution of this group in South America is limited to studies on coprophilous ascomycetes from Venezuela (Dennis, 1970), Uruguay (Garcı a Zorro n, 1973), Chile (Udagawa, 1980; Muroi & Udagawa, 1984) and Peru (Muroi, Udagawa & Otani, 1987). In Argentina the genus is not well known. No species were described by Spegazzini; the first records were by Godeas & * PRHIDEB publication number 106. Marchand (1979) from soil and Lorenzo (1990, 1992) has described and illustrated three other species. Coniochaeta pulveracea is known from Buenos Aires province (Romero, 1998). An undescribed species of Coniochaeta with distinctive perithecial setae was found on wood in San Juan Province, and is here described as new. A review of species known from Argentina is provided with key to taxa. MATERIALS AND METHODS The materials considered here are deposited in the BAFC and IMI herbaria (abbreviations according to Holmgren, Holmgren & Barnett, 1990) and Centro Regional Universitario Bariloche (BCRU). The specimens were studied under bright field microscopy, the samples being mounted in KOH and phloxine, and with an epifluorescence microscope (EFM) using 5% calcofluor (Romero & Minter, 1988) for the observation of specific structures. The cultures of the new species were made on oatmeal agar (OM) taking ascospores from the perithecia and exposing them to a 12 h photoperiod. The rest of the studied strains were from the BAFC culture collections. The drawings were made using a camera lucida and the photographs were taken with Tri X Pan 400 ASA film. The key to species was only made on the basis of the Argentine collections. RESULTS AND DISCUSSION Coniochaeta rhopalochaeta A. I. Romero & Carmara n sp. nov. (Figs 1 15) Etym.: rhopalon (Greek) club, with reference to the setae being mace-shaped.

2 A new species of Coniochaeta lm 20 lm Figs 1 8. Coniochaeta rhopalochaeta. Fig. 1. Macroscopic morphology of ascomata. Fig. 2. Peridial setae. Fig. 3. Ascus, paraphyses and ascospores. Fig. 4. Peridial setae. Fig. 5. Asci in phloxine. Fig. 6. Apical portion of young ascus in phloxine. Fig. 7. Ascospores with germ slit. Fig. 8. Asci with fluorescence microscopy. Note the apical ring. Ascomata subglobosa vel pyriformia, ostiolata, nigra, exterius granulosa, µm diam., gregaria aut aggregata, superficialia. Peridium tenue, fuscum brunneum, ex textura angularis, µm crassitudine; stratum exterius cum rhopalochaeta brunnea in apice inflata et atra. Paraphyses filiformes, abundantes, septatae, 2 3 µm latis. Asci unitunicati, cylindrici, cum annulo apicali, cum iodo non caerulescenti, octospori, µm. Ascosporae uniseriatae in asco, unicellulares, ellipsoideae, brunneae, muris laevigatis, sulco germinalis recto, completo, laterali, µm. Species homothallica. Status conidieus Lecythophora. Cellulae conidiogenae phialiditer crescentes, breves, simplices, hyalinae, formae diversae, collarulis apicalibus inconspicuis, vel collarulae nullae. Conidia copiosa, hyalina, unicellularia, forma diversa, plerumque elongato-ellipsoidea, µm. Cellulae inflatae similes chlamydosporarum, hyalinae, frequentes. Holotypus BAFC , Argentina, province of San Juan, Prov Route 141, at 100 km SE from the Parque Provincial Ichigualasto, Valle de la Luna, Carmara n, C., July 1994 supra lignum decorticatum Bulnesia retama (Hook.) Griseb. Culture derived from the holotype BAFC cult. 272 and IMI Ascomata subglobose to pyriform, ostiolate, black, externally granulose, µm diam., clustered, superficial. Peridium thin, dark brown, µm thick composed of textura angularis, of isodiametric cells with thin, brown walls. Setae short, mace-like arising from the outer cells, very dark (almost black), globose or subglobose apices. Paraphyses abundant, filiform, septate, unbranched, 2 3 µm wide, slightly longer than the asci. Asci fasciculate, unitunicate, 8-spored,

3 A. I. Romero, C. C. Carmara n and L. E. Lorenzo 691 Figs Coniochaeta rhopalochaeta. Fig. 9. Colony on OA. Note black zones show the prescence of perithecia. Fig. 10. Mycelium with EFM. Figs Conidiogenous cells. Note the minute collarettes. Fig. 14. Conidia. Fig. 15. Conidiogenous cells with EFM. (Scale bars: fig. 9 1 cm, figs µm.) cylindric, with a truncate apex, and a small apical ring, J-, µm. Ascospores uniseriate, unicellular, dark brown, ellipsoid in front view, µm, lenticular in lateral view, smooth, with a lateral longitudinal germ slit extending from pole to pole, refractive inclusion drops often visible in young spores. On decorticated wood of Bulnesia retama. Homothallic. Colony on OM spreading slowly, reaching 5 cm diam. at 20 after 12 d. Mycelial mat close-knit and mostly submerged with low aerial growth. Colony surface more or less glabrous, slimy, white initially, later pinkish (PL9 between 7C 7D and 8C 8D Ceres, Maerz & Paul, 1930), finally orange with dark points corresponding to the perithecia. Conidiogenesis enteroblastic (phialidic). Conidiogenous cells intercalary or discrete with narrow apices, µm. Terminally flared collarettes present or lacking, usually inconspicuous. Conidia produced consecutively in small loose clusters, frequently budding to give rise to smaller ones, unicellular, varied in size and shape, mostly oblong-ellipsoid to cylindric, µm. Chlamydospore-like cells terminal or intercalary, in short chains. Review of the literature shows that although ascus and ascospore characteristics are very important, setae are still a prominent feature of most Coniochaeta species. Most of the described setae (Mahoney & LaFavre, 1981) are dark brown to black, straight or bent, unbranched, rigid hairs with a sharp apex. They may be scattered over the perithecial wall or concentrated on its upper portion. Some species are described as lacking setae. Perithecia of C. pulveracea are reported as non-setose, but careful examination showed short, brown, blunt bristles. In the collection of C. rhopalochaeta the perithecia appear non-setose under the lens, but the ascomata have small, distinctive, mace-like structures with a short stipe and swollen apex. These are differentially pigmented, with a brown base and black inflated apex (Figs 2, 4). The contributions on anamorphs of Coniochaeta (Hawksworth, 1978; Hawksworth & Yip, 1981; Mahoney & LaFavre, 1981; Van der Linde, 1991) suggest that the genus is heterogeneous. It is unusual for species of the same genus to show both enteroblastic and holoblastic ontogeny. Even among the taxa with enteroblastic conidiogenesis, it is peculiar that a Paecilomyces conidial state (as described for Coniochaeta cypraespora by Van der Linde, 1991) and a Hormonema conidial state (Udagawa & Sugiyama, 1982) should be referrable to species in the same teleomorph genus.

4 A new species of Coniochaeta 692 Figs Coniochaeta asci observed with EFM. Figs C. hansenii. Figs C. scatigena. Figs C. ligninaria. Figs C. pulveracea. Fig. 26. C. extramundana. Fig. 27. C. rhopalochaeta, ascospores with germ tube observed with EFM bright field at low intensity at the same time. (Scale bars: figs µm, figs µm.) Udagawa & Sugiyama s description and drawing (1982) of their Hormonema state seems to represent a Lecythophora species. The remaining known anamorphs are Phialophora-like. According to Gams & McGinnis (1983), however, within this genus, as delimited by Schol-Schwarz (1970), the P. hoffmannii group presents different characteristics from the accepted generic concept. This group includes slimy pink to olivaceousbrown colonies and conidiogenous cells with reduced collarettes. Gams & McGinnis (1983) reintroduce Lecythophora Nannf. for these species. Udagawa & Takada (1989) proposed a new species, C. arxii, which lacked an anamorph stage in culture but expressed colonial morphology and colouration similar to other Lecythophora species. Kamiya et al. (1995) described two new species of Conichaeta with Lecythophora anamorphs. We have studied cultures of some of these species taking into account the Argentine species.

5 A. I. Romero, C. C. Carmara n and L. E. Lorenzo 693 conidia were observed. Perithecia were present as well. We consider that this is the only species with a true Phialophoralike conidial state. The colony of C. tetraspora was yeast-like, slimy with a very strong yellow pigment (Plate 10, L 6 8, Ta-Ming-Spanish Yellow, Maerz & Paul, 1930). Conidogenous cells and conidia were consistent with those described by Godeas & Marchand (1979) and Cain (1961) and perithecia were formed. The anamorphs of this species and C. ligniaria described by Rogers (1965) are best placed in Lecythophora. In the case of C. rhopalochaeta the anamorph is also a Lecythophora, with a yeast-like, slimy, orange (PL9 between 7C 7D and 8C 8D Ceres, Maerz & Paul, 1930) colony. Perithecia, conidiogenous cells, conidia, and chlamydospores were formed. The anamorphs of C. pulveracea and C. hansenii are still unknown. C. rhopalochaeta is unique on account of a combination of bristle morphology, asci and ascospore features, and cultural characteristics. Slide preparations of the unopened asci in calcofluor show that the apical ring is strongly fluorescent (Figs 16 27). The reaction is similar in all studied species except for the size of the ring. It appears larger (stronger) in C. hansenii, i.e. in the species with polysporous asci, than in the octosporous species. Among these, we see a progressive decrease in size from C. ligniaria, C. rhopalochaeta, C. pulveracea to C. scatigena where the apical ring is reduced and is only conspicuous in young asci. With preparations in calcofluor, particularly those incubated in damp chambers, it was also possible to see clearly the germ tube coming out through the slit on the ascospore wall (Fig. 27). Notes on the Argentine species Fig. 28. Five species are known from Argentina: C. tetraspora (Godeas & Marchand, 1979) C. ligniaria (Lorenzo, 1990), C. extramundana, C. hansenii (Lorenzo, 1992) and C. pulveracea (Romero, 1998). C. scatigena is recorded for the first time from Argentina. The overall colony appearance of C. extramundana was not slimy, characterized by dark pigments as described by Mahoney & LaFavre (1981) and conidiogenous cells and 1. C. extramundana Mahoney & LaFavre, Mycologia 73: 931 (1981). Lorenzo s (1992) description and illustration of this species from Argentina was the second record of this species; the first being from arid chaparral soil in California, U.S.A. (Mahoney & LaFavre, 1981). Specimen examined: Argentina, Neuque n, Dpto. Los lagos, Estancia Fortı n Chacabuco, dung of red deer (Cervus elaphus), March 1989, Lorenzo, L. (BCRU 00171). BAFC cult from BCRU C. hansenii (Oudem.) Cain, Univ. Toronto Studies, Biol. ser. 38: 63 (1934). Key to Coniochaeta species reported in Argentina 1. Asci multispored C. hansenii 1. Asci 4- or 8-spored Asci 4-spored C. tetraspora 2. Asci 8-spored at maturity Ascospores in polar view (end views) cruciform; anamorph Phialophora-like C. extramundana 3. Ascospores lenticular in polar and lateral view; anamorph Lecythophora-like or unknown Ascomata obviously setose Ascomata with setae hardly visible under the lens (20 ) Ascospores µm in face view. Anamorph Lecythophora C. ligniaria 5. Ascospores µm in face view. Anamorph unknown C. scatigena 6. Ascomata covered with blunt setae. Ascospores broadly ellipsoidal, µm in face view. Anamorph unknown C. pulveracea 6. Ascomata with mace-like setae. Ascospores ellipsoidal, µm. Anamorph Lecythophora...C. rhopalochaeta

6 A new species of Coniochaeta 694 This species was first described and illustrated for Argentina by Lorenzo (1992). It was reported from Chile by Muroi & Udagawa (1984). According to Mahoney & LaFavre (1981) it is an exclusively coprophilous species and it seems to have a large geographic distribution. It shares the features of polysporous asci and coprophilous habit with other species such as C. polysperma Furuya & Udagawa, C. multispora Cain and C. philocoproides Griffiths. Specimen examined: Argentina: Rı o Negro, Dpt. Bariloche, S. C. de Bariloche, Llao Llao, dung of Lepus capensis, Sep. 1989, Lorenzo. L. (BAFC ). 3. C. ligniaria (Grev.) Massee, Grevillea 16: 37 (1887). This species was collected by Lorenzo (1990) in Argentina. In South America it is also known from Uruguay (Garcı a Zorro n, 1973) and Peru (Muroi et al., 1987). It is reported from wood, Pisum sativum seeds, and Triticum rhizosphere, and is widespread (Hawksworth & Yip, 1981). Specimen examined: Argentina: Neuque n, Dpt. Bariloche, Nat. Route 237, at 30 km from the city of S. C. Bariloche, Rinco n Chico, on dung of Lepus europaeus, Nov. 1988, Lorenzo, L. (BAFC ). 4. C. pulveracea (Ehrh.) Munk, Dansk Bot. Arkiv 12: 11 (1948). This taxon was first recorded for Argentina by Romero (1998). Specimen examined: Argentina: Buenos Aires, Partido de Ramallo, Ramallo, stump of Eucalyptus viminalis, Aug. 1982, Romero, A. I. (BAFC ). 5. C. scatigena (Berk. & Broome) Cain, Univ. Toronto Stud., Biol. ser 38: 62 (1934). This is the first report for Argentina. It has also been reported by Muroi & Udagawa (1984) from Chile. The Argentine material agrees with the description in Checa et al. (1988). Ascospore measurements are within the given range: face view µm wide, lateral view 12 µm wide, µm long. As has been pointed out by these authors, this species is close to C. ligniaria and perhaps the main difference seems to be the larger size of all the structures of C. scatigena than C. ligniaria. For a full description and illustrations see Checa et al. (1988). Specimen examined: Argentina: Neuque n, Parque Nacional Lanı n, Paso Trome n, dung of Lepus europaeus, March 1995, Lorenzo, L. (BCRU 817). 6. C. tetraspora Cain, Can J. Bot. 39: 1231 (1961). This species was isolated (Godeas & Marchand, 1979) from soil associated with coihue (Nothofagus dombeyi), from Argentina. Reported exclusively from soil by Mahoney & LaFavre (1981) but Hawksworth & Yip (1981) reported it on dung of cow and on conifer wood. It seems to be a cosmopolitan species. C. tetraspora and C. nodulisporioides D. Hawksw., are the only species with four-spored asci. The most distinctive feature separating them is the anamorph, phialidic Lecythophora-like in C. tetraspora and polyblastic Nodulisporium-like in C. nodulisporioides (Hawksworth, 1978). Specimen examined: Argentina: Rı o Negro, Dpt. Bariloche, Lago Gutie rrez, isolated from soil, Aug. 1976, Godeas, A. & Marchand, S. (BAFC cult. 2803). The authors would like to express their gratitute to Dr Paul Cannon for making valuable comments on the manuscript. REFERENCES Cain, R. F. (1961). Studies of soil fungi III. New species of Coniochaeta, Chaetomidium and Thielavia. Canadian Journal of Botany 39, Checa, J., Barrasa, J. M., Moreno, G., Fort, F. & Guarro, J. (1988). The genus Coniochaeta (Sacc.) Cooke (Coniochaetaceae, Ascomycotina) in Spain. Cryptogamie, Mycologie 9, Crane, J. L. & Shearer, C. A. (1995). A new Coniochaeta from fresh water. Mycotaxon 54, Dennis, R. W. G. (1970). Fungus flora of Venezuela and adjacent countries. Kew bulletin additional series III. Verlag Von J. Cramer. 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7 A. I. Romero, C. C. Carmara n and L. E. Lorenzo 695 Udagawa, S. (1980). Some new or noteworthy coprophilous Pyrenomycetes from South America. Transactions of the Mycological Society of Japan 21, Udagawa, S. & Horie, Y. (1982). Two new species of terrestrial Ascomycetes from Eastern Nepal. Reports on the Cryptogamic study in Nepal, March Miscellaneous Publication of the National Science Museum, Tokyo, Udagawa, S. & Sugiyama, Y. (1982). New records and new species of ascomycetous microfungi from Nepal, a preliminary report on the expedition of Reports on the Cryptogamic study in Nepal. Miscellaneous Publication of the National Science Museum, Tokyo, March, Udagawa, S. & Takada, M. (1989). A new species of Coniochaeta. Studies in Mycology 31, Van der Linde, E. J. (1991). Coniochaeta cypraespora sp. nov. with a Paecilomyces conidial state. Mycological Research 95, (Accepted 7 August 1998) BMS Millennium Symposium on Tropical Mycology April 2000: The British Mycological Society will hold a special symposium, covering all aspects of tropical mycology with field trips and a museum interface thereafter. Forty eight speakers from home and abroad will be invited to contribute in a formal programme which will be supported by identification workshops and poster sessions. Prof. Jack Rogers of Washington State University, Pullman, will deliver the Benefactor s lecture. The symposium will be held at the Liverpool John Moores University, with accommodation at the University of Liverpool. The Council of the British Mycological Society has generously offered five bursaries to the sum of 500 each for deserving delegates wishing to attend. Further details about the meeting can be obtained from Prof. Roy Watling; r.watling rbge.org.uk.