Observations on the genus Cresporhaphis (Trichosphaeriaceae), with a key to the known species, and C. ulmi sp. nov.

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1 Mycol. Res. 105 (1): (January 2001). Printed in the United Kingdom. 122 Observations on the genus Cresporhaphis (Trichosphaeriaceae), with a key to the known species, and C. ulmi sp. nov. Vicent CALATAYUD 1 and Begon a AGUIRRE-HUDSON 2 CEAM, C Charles R. Darwin 14, Parc Tecnolo gic, ES Paterna, Vale ncia, Spain. 58 Willowhayne Drive, Walton on Thames, Surrey KT12 2NR, UK. vicent ceam.es Received 10 February 2000; accepted 17 April A taxonomic and ecological reassessment of the genus Cresporhaphis is presented, with a key to all 7 species currently known, including C. ulmi sp. nov. from Spain, on twigs of Ulmus minor. Species are facultatively lichenized with chlorococcoid algae, Trentepohlia and other algae, or saprobic. INTRODUCTION The generic name Cresporhaphis was introduced by Aguirre- Hudson (1991) to accommodate several species with ampulliform and usually sessile perithecia-like ascomata, collapsing laterally when mature, functionally unitunicate asci, a hamathecium consisting of paraphyses and occasionally periphyses, and falcate to fusiform ascospores. In that study, five species were accepted in the genus, all known from Europe. Barr (1993) then added a new taxon from North America: C. rhoina (Ellis & Everh.) Barr 1993 (basionym: Sphaeria rhoina Ellis & Everh. 1885, as rhuina ). The species are distinguished primarily by the size of the ascomata and ascospores, ascospore septation, and their arrangement within the ascus, iodine reaction of the hymenial gelatin, the morphology of the ascus apex in lactophenol cotton-blue, and by the presence of conidiomata, microconidia and or macroconidia. The genus was originally described as lichenized since detailed microscopic examination of thalli revealed the presence of algal cells in close association with the fungal filaments surrounding the ascomata, even in old herbarium collections. Although the precise identity of the photobiont was not established, this was stated as being chlorococcoid globose algae different from Trentepohlia. Nevertheless, Barr (1993) did not mention a thallus when the new combination of C. rhoina was made, although Ellis & Everhart (1888) described Coelosphaeria fusariospora (now included within the concept of C. rhoina) as one of the species standing ambiguously between lichens and fungi. Also, two more recent collections of Cresporhaphis wienkampii (Lahm) M. B. Aguirre 1991 from Great Britain (Aguirre-Hudson & Coppins 1999; see also below) were found to be loosely associated with Trentepohlia. A new species described here as C. ulmi, does not have an apparent thallus either, so that the genus Cresporhaphis is evidently facultatively lichenized, with some of the species being entirely bark saprobes. MATERIALS AND METHODS Microphotographs of the new taxon were taken with Olympus SZH10 and BX-50 stereo- and compound-microscopes, respectively, the latter equipped with differential interference contrast optics (DIC). Drawings were made with the help of a drawing tube. Hand-made and microtome sections of ascomata and conidiomata were mounted primarily in water but, to increase tissue differentiation, some were stained with lactophenol-cotton blue. In the ascospore size, the range (given in non-italics and outside parenthesis) was calculated after rejecting 10% of the lowest and highest values measured; the averages are included in italics, and the extremes are given within parentheses. Conidial development terminology follows Hennebert & Sutton (1994). Type or authentic material of C. rhoina was requested for examination from NY, but no specimens were received; the information on this species included in the key is based on literature descriptions (Ellis & Everhart 1885, 1888, Barr 1993). TAXONOMY Aguirre-Hudson (1991) tentatively referred the new genus Cresporhaphis to the order Trichosphaeriales, although she did not observe hyphomycetous anamorphs associated with the genus. These had been reported in other genera traditionally

2 V. Calatayud and B. Aguirre-Hudson 123 included in the order (Kendrick & Murase 1994). However, Barr (in Barr & Cannon 1994) noted the heterogeneous nature of the Trichosphaeriales, but confirmed (Barr, pers. comm.) that Cresporhaphis was a true member of the Trichosphaeriaceae (see also Barr 1993). This opinion was later reflected in Hawksworth et al. (1995). When the Trichosphaeriales and Trichosphaeriaceae were circumscribed by Barr (1983, 1990, respectively), she noted that their asci sometimes bore apical annuli, occasionally bluing in iodine. She did not mention how the ascospores were liberated from the asci, so presumably the taxa in the group exhibited a range of dehiscence mechanisms. Aguirre- Hudson (1991) noted that the asci of some species of Cresporhaphis showed a longitudinal creasing when empty. In the new species, dehiscence is apparently by an apical fissure (see Fig. 6). Many of the species of Cresporhaphis are very rare, and we are pleased to report another UK collection of C. wienkampii (Scotland: East Lothian, East Linton, by River Tyne, on Salix, 26 Feb. 2000, B. J. Coppins s.n., E). C. rhoina appears to be closely related to Cresporhaphis muelleri (Duby) M. B. Aguirre 1991, but on the basis of the published accounts, we have decided to keep both taxa as distinct species for the moment. Since two more species are now included in Cresporhaphis, a revised key is provided below, as well as a detailed description of the new taxon. Cresporhaphis ulmi Calatayud & M. B. Aguirre, sp. nov. (Figs 1 13) Etym.: ulmi, growing on Ulmus. C. macrospora similis sed ascomatibus, ascis et ascosporibus minoribus differt, alius µm alius (33 )38 50( 55) µm; thallus absens, et conidia ellipsoidea vel oblonga. Typus: Spain: region of Arago n, province of Teruel, between Puebla de Arenoso and Olba, close to Los Lucas, beside the main road, c. 2 km E of Olba, c N, 0 37 W, alt. c. 700 m., on suberose outgrowths of Ulmus minor twigs, 14 March 1999, V. Calatayud (MA-Fungi holotypus; herb. Calatayud 10- isotypus). Thallus not observed. Ascomata perithecioid, black, smooth, numerous, scattered singly, but occasionally confluent and laterally collapsed, semi-immersed in the substratum, globose to ampulliform, µm diam, ostiole situated in a small papilla (Figs 1 2). Exciple c µm thick, consisting of isodiametric cells, c µm diam, or somewhat elongated, forming a textura angularis; cell walls brown, becoming darker towards the ostiole, and of paler appearance towards the hamathecium (Figs 3 4). Hamathecium consisting of simple, hyaline, guttulate, septate, thin-walled paraphyses and periphyses; paraphyses 2 4 µm wide (Figs 7, 13); periphyses 2( 3) µm wide; hymenial gelatin I-. Asci clavate, stalked, thin-walled, not thickened at the apex, sometimes curved, (55 ) µm, 8-spored (Figs 5, 12); dehiscence apparently fissurate (Fig. 6). Ascospores colourless, thin-walled, falcate, with rounded apices, 1- or 3-septate, occasionally 2-septate, non-halonatae, smooth, guttulate, (33 ) ( 55) µm [length width ratio: ; 112 ascospores measured] (Figs 8 10). Conidiomata pycnidial, c µm, reddish brown, but dark brown around the ostiole. Exciple consisting of a few layers of isodiametric brownish cells, forming a textura angularis. Conidiogenous cells cylindrical, somewhat tapered towards their apex, c µm. Conidia c µm, ellipsoid to oblong, hyaline, aseptate, produced singly and apically (Fig. 11). Additional material examined: Spain: region of Arago n, province of Teruel, c. 1 km SW of Mora de Rubielos, c N, 0 41 W, alt. c m., on the trunk of several Ulmus minor trees, 30 Aug. 1999, V. Calatayud & M. J. Sanz (herb. Calatayud 11). Distribution: The species is so far known from two localities in the Iberian Peninsula, both in the region of Arago n. Interestingly, the specimens from the non-type locality were collected from the bark of several live trees affected by Dutch elm disease (Ophiostoma novo-ulmi), the bark of which was partly detached from the trunks. Key to the species of Cresporhaphis 1 Ascospores aseptate, occasionally 1-septate, mostly less than 30 µm in length Ascospores always 1- to multiseptate, mostly exceeding 30 µm in length (1) Ascomata confluent; hymenial gelatin bluish-amber in iodine; conidiomata not observed Ascomata solitary; hymenial gelatin bluish or not in iodine; conidiomata pycnidial (2) Thallus greyish white, individual ascomata µm diam, asci cylindrical, µm, ascus apex slightly pierced by a pore; ascospores biseriately arranged in the ascus, aseptate, µm; on trunks of Acer muelleri Thallus absent; individual ascomata µm diam, asci oblong, ascus apex unpierced; ascospores bi- or tri-seriately arranged in the ascus, aseptate to delicately 1-septate, 20 30( 45) (1 ) µm; on bark or periderm of Populus and Rhus.. rhoina 4(2) Ascomata semi-immersed in the bark, µm diam; hymenial gelatin unchanged in iodine; ascus apex pierced by a narrow and short pore; ascospores always aseptate, µm; conidia aseptate, bacilliform µm; on twigs of Acer acerina. Ascomata superficial, µm diam; hymenial gelatin bluish-green in iodine; asci without an apical thickening; ascospores usually aseptate, but occasionally 1 3 septate, (20 )25 30( 35) µm; conidia aseptate, cylindrical to filiform, curved, µm; on trunks of Salix, Robinia and Quercus wienkampii. 5(1) Ascospores multiseptate; on trunks of Pinus pinicola Ascospores 1- and 3- septate; on various other substrata (5) Ascospores µm; ascus apex pierced by a broad conical pore; conidia filiform, c µm; on trunks of Quercus macrospora Ascospores (33 )38 50( 55) µm; asci not thickened at the apex; conidia ellipsoid to oblong, c µm; on the trunk and corky outgrowths of twigs of Ulmus ulmi

3 Observations on Cresporhaphis 124 Table 1. The main distinguishing characters amongst Cresporhaphis species with septate ascospores. C. macrospora C. pinicola C. ulmi Thallus Pulverulent, whitish Smooth, ash-grey Absent Ascomatal width (µm) Asci (µm) c Ascus apex Thickened, forming a small cap Thickened, forming a small cap, Not-thickened pierced by a broad conical pore unpierced Ascospores (µm) 1-, 3-septate , 9-septate , 3-septate Conidiomata (µm) Pycnidial, c ; conidia filiform and curved, c Not observed Pycnidial, c ; conidia ellipsoid to oblong, c Ecology Probably lichenized, growing on Probably lichenized, growing on Saprophytic on Ulmus minor the bark furrows of Quercus robur Pinus sp. Distribution Central Europe and Sweden Portugal Spain Literature Aguirre-Hudson (1991) Aguirre-Hudson (1991) Present paper Figs 1 9. Cresporhaphis ulmi (holotypus). Fig. 1. Habit. Fig. 2. Transverse section of an ascoma. Fig. 3. Detail of the wall of an ascoma in transverse section. Fig. 4. Detail of the wall of an ascoma in superficial view. Fig. 5. Asci in Lugol s solution. Fig. 6. Empty ascus after discharging the ascospores (DIC). Fig. 7. Paraphyses (DIC). Figs 8 9. Ascospores (DIC). Scale bars: Fig. 1 1 mm; Fig µm; Figs µm. Ecology: The perithecium-like ascomata of this fungus grow on the bark and suberose outgrowths of twigs of the smallleaved elm (Ulmus minor). Cork formation is normal in this tree species, and it is not caused by the fungus. Since we were unable to observe algae in close association with fungal filaments of Cresporhaphis ulmi, and this did not cause any visible injury to the tree, we regard the species as saprophytic. Interestingly, C. ulmi was absent from non-suberose, but smooth, zones of the twigs and new shoots. During the examination of many elm trees while searching for C. ulmi, a similar ecology was observed for several other unidentified fungi. In fact, their abundance caused difficulties when trying to ascertain the conidiomata belonging to C. ulmi. These were scarce, while amongst the most confusing was a coelomycete with narrowly fusiform, slightly falcate conidia, of c µm, but produced in 1 to 3 loci per conidiogenous cell. Also the empty cavity of some old ascomata was filled by an opportunistic Fusarium-like fungus with colourless, falcate, 3 7 septate conidia, µm, which could be mistaken for macroconidia of C. ulmi. In addition to these, other mitosporic fungi were observed, one of the most frequent being a dematiaceous hyphomycete. When growing

4 V. Calatayud and B. Aguirre-Hudson Cresporhaphis macrospora produces aseptate, filiform macroconidia, while C. ulmi has ellipsoid to oblong microconidia. Both taxa also differ in their ecological preferences: C. macrospora grows on Quercus robur, and C. ulmi was collected on Ulmus minor. A third species of Cresporhaphis, C. pinicola (G. Samp.) M. B. Aguirre 1991, also known from the Iberian Peninsula, has septate ascospores of a similar size to those of C. ulmi. However, in the former the ascospores are 5 9-septate, and it is known only from pines. The ascospores of the rest of the species so far included in the genus are mainly simple, although C. wienkampii can occasionally have 1- or 3-septate ascospores (Aguirre-Hudson 1991), and C. rhoina faintly 1- septate ascospores (Barr 1993). Van den Boom & Giralt (1996) also mentioned some specimens of Cresporhaphis (as C. aff. macrospora) collected from Olea europea in Portugal. The material was re-examined by us and it shows affinities not only with C. macrospora, but also with C. ulmi. Nevertheless, in the Portuguese material, the ascospores tend to be somewhat larger ( µm) than in C. ulmi, and are 1 (2 )septate and straight. Furthermore, we could not find paraphyses within the ascomata, which, on the other hand, seemed to be associated with algae. Unfortunately, the material was too scarce for a reliable comparison with C. ulmi, but on the basis of the differences so far observed we consider it appropriate to exclude it from C. ulmi. ACKNOWLEDGEMENTS Figs Cresporhaphis ulmi (holotype). Fig. 10. Ascospores. Fig. 11. Conidia. Fig. 12. Two mature asci. Fig. 13. Paraphyses. Scale bar 20 µm. on bark of the trunk, C. ulmi was associated with nitrophilous lichen communities dominated by Phaeophyscia orbicularis, Physcia adscendens and Xanthoria parietina. Remarks: Cresporhaphis ulmi is closely related to C. macrospora (Eitner) M. B. Aguirre 1991 in having 1 3-septate ascospores, but the new species has smaller ascomata, asci and ascospores (see Table 1). In C. ulmi the ratio of 1-septate versus 3-septate ascospores varied between different ascomata. When a large number of ascospores from different ascomata were studied, 3-septate ones were more abundant. Nevertheless, we have also examined some ascomata in which 1- septate ascospores were predominant; in this case, they were usually accompanied by many simple, immature ascospores. Also the type of conidia is different in these two species: Prof. D. L. Hawksworth (MycoNova, London) is thanked for his valuable comments, and Dr B. J. Coppins (Royal Botanic Garden, Edinburgh) for reviewing the manuscript and for details of his collection of C. wienkampii. The first author is indebted to Dr M. J. Sanz (CEAM, Vale ncia) for her help collecting material of C. ulmi. The second author would like to thank Dr J. C. David and Mr K. J. Hudson (CABI Bioscience, Egham), and Dr B. M. Spooner (Royal Botanic Gardens, Kew), for technical assistance; also Dr W. Hominick (Director of Biodiversity and Biosystematics, CABI Bioscience, Egham) for permitting the consultation of herbarium material on the premises; and Dr G. Renobales (Facultad de Farmacia, Universidad del Pais Vasco) for the loan of a compound microscope. REFERENCES Aguirre-Hudson, B. (1991) A taxonomic study of the species referred to the ascomycete genus Leptorhaphis. Bulletin of the British Museum (Natural History), Botany 21: Aguirre-Hudson, B. & Coppins, B. J. (1999) Cresporhaphis wienkampii. British Lichen Society Bulletin 85: 50. Barr, M. E. (1983) The ascomycete connection. Mycologia 75: Barr, M. E. (1990) Prodromus to nonlichenized, pyrenomycetous members of class Hymenoascomycetes. Mycotaxon 39: Barr, M. E. (1993) Redisposition of some taxa described by J. B. Ellis. Mycotaxon 46: Barr, M. E. & Cannon, P. F. (1994) Discussion 3: Calosphaeriales, Clavicipitales, Coryneliales, Diaporthales, Diatrypales, Halosphaeriales, Hypocreales, Meliolales, Ophiostomatales, Phyllacorales, Sordariales, Trichosphaeriales, and Xylariales. In Ascomycete Systematics: problems and perspectives in the nineties (D. L. Hawksworth, ed.): [NATO ASI Series A: Life Sciences Vol. 269.] Plenum Press, New York. Ellis, J. B. & Everhart, B. M. (1885) New species of fungi. Journal of Mycology 1:

5 Observations on Cresporhaphis 126 Ellis, J. B. & Everhart, B. M. (1888) New species of fungi from various localities. Journal of Mycology 4: Hawksworth, D. L., Kirk, P. M., Sutton, B. C. & Pegler, D. N. (1995) Ainsworth & Bisby s Dictionary of the Fungi. 8th edn. CAB International, Wallingford. Hennebert, G. L. & Sutton, B. C. (1994) Unitary parameters in conidiogenesis. In Ascomycete Systematics: problems and perspectives in the nineties (D. L. Hawksworth, ed.): [NATO ASI Series A: Life Sciences Vol. 269.] Plenum Press, New York. Kendrick, W. B. & Murase, G. (1994) Can we recognize monophyletic groups among Hyphomycetes? In Ascomycete Systematics: problems and perspectives in the nineties (D. L. Hawksworth, ed.): [NATO ASI Series A: Life Sciences Vol. 269.] Plenum Press, New York. van den Boom, P. P. G. & Giralt, M. (1996) Contribution to the flora of Portugal, lichens and lichenicolous fungi I. Nova Hedwigia 63: Corresponding Editor: D. L. Hawksworth