Mycologia, 95(1), 2003, pp. 134–140. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897 Soil ascomycetes from Spain. XIII. Two new species of Apiosordaria Dania Garcı́a During a continuous survey of soil ascomycetes from Spain, two interesting species of Apiosordaria were isolated. Their morphological characteristics differentiate them from all previously described species of the genus (Krug et al 1983, Guarro and Cano 1988, Stchigel et al 2000). In the present report these species are fully described, illustrated, and proposed as new. Instituto de Investigaciones Fundamentales en Agricultura Tropical ‘‘Alejandro de Humbolt’’, calle 1 esq.2, Santiago de las Vegas, Boyeros, C. de La Habana, Cuba A. M. Stchigel J. Guarro1 Facultat de Medicina i Ciéncies de la Salut, Universitat Rovira i Virgili, C/ Sant Llorenç 21, 43201, Reus, Tarragona, Spain MATERIALS AND METHODS The soil samples were collected from several places in Els Gorgs de la Febró, Tarragona, Spain. The vegetation is mostly composed of Quercus ilex L. and Pinus halepensis Mill. The area has a Mediterranean climate with an annual average temperature of 15–36 C and an annual precipitation up to 600 mm. Soil was mainly collected from the A0 horizon with sterile polyethylene bags. These were sealed with a rubber band, and labeled. On returning to the laboratory samples were stored at 4–7 C until used. Approx 1 g of the sample was treated with 2% (p/v) phenol (Phenol crystal, Panreac, Barcelona, Spain) for 10 min. The supernatant was discarded and the solid phase resuspended in 10 mL of sterilized water. The suspensions were cultured on potato carrot agar (PCA, potato 20 g, carrot 20 g, agar 20 g) at 15 C in the dark. Strains were grown on oatmeal agar (OA; Difco, Detroit, Michigan, USA), PCA and malt extract agar (MEA, Difco, Detroit, Michigan, USA). Cultures were made and structures were measured as described previously by Stchigel et al (1998). Color notations in parentheses are from Kornerup and Wansher (1984). Photomicrographs were obtained with a Leitz Dialux 20 EB microscope. Scanning electron microscopy techniques were described previously by Figueras and Guarro (1988). Abstract: Apiosordaria hispanica sp. nov. and Apiosordaria globosa sp. nov. isolated from soil samples collected in Tarragona, Catalonia, Spain, are described and illustrated. Both species are morphologically close to A. otanii. The ascospores of A. hispanica have tuberculate walls, while those of A. otanii have small warts. Apiosordaria globosa differs from those species by the globose upper cell of the ascospores, which has a small apical protrusion with sub-apical germ pore when young. In A. hispanica and A. globosa the lower cells of the ascospores are slightly warted, while in A. otanii the lower cell of the ascospores is smoothwalled. Key Words: Ascomycota, Lasiosphaeriaceae, Mediterranean region, soil borne fungi INTRODUCTION The genus Apiosordaria Arx & Gams (Lasiosphaeriaceae, Ascomycota) is characterized by dark ascomata, non-amyloid asci and two-celled ascospores (Arx and Gams 1967). These are composed of a dark and ornamented upper cell and a hyaline lower cell (Krug et al 1983). The presence of spines, warts or pits in the upper cell of the ascospores is the principal character that differentiates this genus from other related genera of the Lasiosphaeriaceae. Triangularia Boedijn and Zopfiella Winter both have a smooth upper cell, although they also have unequal two-celled ascospores (Guarro and Cano 1988). The genus Apiosordaria comprises 21 species, including mainly soil-borne and coprophilous species (Krug et al 1983, Stchigel et al 2000). TAXONOMY Apiosordaria hispanica D. Garcı́a, Stchigel et Guarro, sp. nov. FIGS. 1–9 Mycelium ex hyphis dilute brunneis vel dilute olivaceobrunneis, septatis, ramosis, anastomosantibus, laevibus, 1–6 mm diam composito. Coloniae in agaro cum decocto tuberorum et carotarum (PCA) expansae, planae, ex mycelio vegetativo submerso et aereo, brunneae; reversum brunneum. Ascomata superficialia vel immersa, dispersa vel in catervis parvis aggregata, ostiolata, brunnea vel atrobrunnea, pyriformia vel subglobosa, 350–420 3 265–330 mm, pilosa; pilis laxis, longis, flexuosis, dilute flavo-brunneis, septatis, 2–3 mm diam composito. Collum conicum, nigrum, 78–88 (–113) mm longum, 127–137 mm latum ad basim, papilliforme. Peridium membranaceum, pilosum, 6–9 stra- Accepted for publication June 17, 2002. 1 Corresponding author, Email: umb@fmcs.urv.es 134 GARCÍA FIGS. 1–2. ET AL: TWO NEW SPECIES OF APIOSORDARIA 135 Apiosordaria hispanica FMR 7398. 1. Ascus with ascospores. 2. Ascospores. Scale bars: 15 20 mm, 25 10. mm. tiorum compositum, 17–20 mm crassitunicatum, ex textura intricata vel textura angularis. Asci in fasciculo ad centrum dispositi, 8-spori, cylindrici, stipitati, 146–232 3 15–22 mm, cum muris tenuibus. Paraphyses numerosae, hyalinae, filiformes, septatae, 0.5–2 mm diam. Periphyses hyalinae, filiformes, septatae, 0.5–1 mm diam. Ascosporae uniseriatae, primum unicellulares, clavatae, hyalinae, deinde transverse septatae ad bicellulares; cellula superior obovoidea cum apice acuminato, brunnea, crassitunicata, 23–28 (–30) 3 18– 22 (–23) mm, tuberculata, cum foramine germinali singulari, apicali vel sub-apicali, 0.5–2 mm diam praedita; cellula inferior hyalina vel sub-hyalina, conica, 1–5 (–6) mm longa. Status conidialis nullus. Mycelium composed of pale brown to pale olivebrown, septate, branched, smooth-walled hyphae, 1– 6 mm diam. Colonies on PCA growing rapidly, attaining 84–86 mm diam in 14 d at room temperature (22–25 C), flat, thin, consisting of submerged mycelium and sparse aerial hyphae, zonate and slightly radiate, brown (M. 5E6); ascomata produced after one mo; reverse brown (M. 5F5). Ascomata immersed 136 MYCOLOGIA FIGS. 3–9. Apiosordaria hispanica FMR 7398. 3. Ascoma. 4–6. Asci. 7. Detail of ascospore wall. 8, 9. Ascospores. Scale bars. 3 5 200 mm, 4 5 40 mm, 5–75 20 mm, 8, 9 5 10 mm. GARCÍA ET AL: TWO NEW SPECIES OF to superficial, ostiolate, subglobose to pyriform, dark brown to black, 350–420 3 265–330 mm, tomentose, covered with long, slender, pale yellowish-brown, smooth, septate, unbranched or inconspicuously branched hypha-like hairs, 2–3 mm diam, with a conspicuous neck; neck 78–88 (–113) mm long, 127–137 mm wide at the base, with brown, papillate cells disposed around the ostiole. Peridium membranaceous, pilose, 6–9-layered, 17–20 mm thick, brownish-orange to brown; outer layer with textura intricata, composed by brown cells, 2–5 mm diam; inner layer composed by thin-walled polygonal cells, 9–15 mm diam. Asci 8spored, fasciculate, cylindrical, thick-walled when young, becoming thin-walled at maturity, 146–232 3 15–22 mm, rounded at the apex, with a thin apical ring, stipitate, evanescent. Paraphyses hyaline, filiform, septate, 0.5–2 mm wide. Periphyses hyaline, filiform, septate, unbranched, 0.5–1 mm wide. Ascospores uniseriate, clavate, hyaline, and 1-celled when young, becoming two-celled by the formation of a transverse septum; upper cell obovoid, truncate at the base and with a slightly acuminate apex, brown, thick-walled, 23–28 (–30) 3 18–22 (–23) mm, uniformly ornamented with numerous rounded warts of 0.5–2 mm diam, with an apical to sub-apical germ pore, 0.5–2 mm diam; lower cell sub-hyaline, conical and slightly warted, 1–5 (–6) mm long. Anamorph unknown. Colonies on OA and MEA growing rapidly, attaining 55–70 mm diam in 14 d at room temperature (22–25 C), zonate and slightly radiate, cottony, brown to olive-brown (M. 5E6 to M. 4E8); reverse dark olive brown. Ascomata produced after one mo on OA; on MEA, ascomata not produced. At 15 and 35 C growing rapidly on PCA and OA; on MEA colonies restricted, ascomata not produced. Specimen examined SPAIN, TARRAGONA: Vilaplana, Els Gorgs de la Febró, soil, 25-IX-1996, col. AM Stchigel and M Calduch (HOLOTY PE: FMR 7398). Apiosordaria hispanica is morphologically similar to Apiosordaria vermicularis (Morinaga et al) Krug, Udagawa & Jeng (Morinaga et al 1978), Apiosordaria yaeyamensis Morinaga, Utatsu & Minoura (Morinaga et al 1979), Apiosordaria otanii Udagawa (Udagawa 1990) and Apiosordaria microcarpa Udagawa & Muroi (Udagawa and Muroi 1981). All have 8-spored asci and the upper cell of the ascospores more or less spinulose. Apiosordaria vermicularis can be easily differentiated from A. hispanica by its non-ostiolate ascomata and longer lower cell of the ascospores (12– 20 mm), which is frequently 1-septate (Morinaga et al 1978). Apiosordaria microcarpa and A. yaeyamensis have ostiolate ascomata, but the upper cell of the ascospores is smaller (15–19 3 10–12.5 mm and 10– 12.5 3 6–8 mm, respectively) (Udagawa and Muroi 1981, Morinaga et al 1979). In Apiosordaria otanii, APIOSORDARIA 137 the upper cell of the ascospores has very small warts and the lower cell is smooth-walled (Udagawa 1990), while A. hispanica has ascospores with the upper cell ornamented with large tubercles and the lower cell slightly warted. Apiosordaria globosa D. Garcı́a, Stchigel et Guarro, sp. nov. FIGS. 10–20 Mycelium ex hyphis dilute brunneis vel dilute olivaceo-brunneis, septatis, ramosis, anastomosantibus, laevibus, 1–6 mm diam composito. Coloniae in PCA expansae, planae, ex mycelio vegetativo submerso et aereo, brunneae; reversum brunneum. Ascomata superficialia vel immersa, dispersa vel in catervis parvis aggregata, ostiolata, brunnea vel atrobrunnea, pyriformia vel subglobosa, 600–770 3 320–540 mm, dense pilosae, pilis laxis, longis, flexuosis, dilute flavo-brunneis, septatis, 1–3 mm diametro composito. Collum conicum, nigrum, 220–280 mm longum, 210– 250 mm latum ad basim, papilliformes. Peridium membranaceum, pilosum, 6–9 stratiorum compositum, 15–19 mm crassitunicatum, ex textura intricata vel textura angularis. Asci 8-spori in fasciculo ad centrum dispositi, cylindrici, stipitati, 195–250 3 24–28 mm, cum muris tenuibus. Paraphyses numerosae, hyalinae, filiformes, septatae, 0.5–2 mm diam. Periphyses hyalinae, filiformes, septatae, 0.5–1 mm diam. Ascosporae uniseriatae, primum unicellulares, clavatae, hyalinae, deinde transverse septatae ad bicellulares; cellula superior globosa, atrobrunnea vel nigra, crassitunicata, 25–27 (–29) 3 (22–)23–27 mm, verruculosa, cum foramine germinali singulari et sub-apicali, 1–2 mm diam praedita; cellula inferior hyalina vel subhyalina, conica, (1–) 4–6 mm longa. Status conidialis nullus. Mycelium composed of pale brown to pale olivebrown, septate, branched, smooth-walled hyphae, 1– 6 mm diam. Colonies on PCA growing rapidly, attaining 80–86 mm diam in 14 d at room temperature, flat, thin, consisting of submerged mycelium and sparse aerial hyphae, zonate and slightly radiate, brown (M. 5E6); ascomata produced after one mo; reverse brown (M. 5F5). Ascomata immersed to superficial, ostiolate, subglobose to pyriform, dark brown to black, 600–770 3 320–540 mm, tomentose, covered with long, slender, pale brownish-yellow, smooth, septate, hypha-like hairs, 1–3 mm diam, unbranched or inconspicuously branched, with a conspicuous neck; neck 220–280 mm long, 210–250 mm wide at the base, with brown, papillate cells around the ostiole. Peridium membranaceous, pilose, 6–9-layered, 15–19 mm thick, brownish orange to brown; outer layer with textura intricata, composed of brown irregular cells, 2–5 mm diam; inner layer composed of thin-walled polygonal cells, 9–17 mm diam. Asci 8- 138 FIGS. 10–11. 10 mm. MYCOLOGIA Apiosordaria globosa FMR 7400. 10. Ascus with ascospores. 11. Ascospores. Scale bars: 105 20 mm, 11 5 spored, fasciculate, cylindrical, thick-walled when young becoming thin-walled when mature, 195–250 3 24–28 mm, rounded at the apex, with a thin apical ring, stipitate, evanescent. Paraphyses hyaline, filiform, septate, unbranched, 0.5–2 mm wide. Periphyses hyaline, filiform, septate, 0.5–1 mm wide. Ascospores uniseriate, broadly fusiform, hyaline and 1-celled when young, becoming two-celled by the formation of a transverse septum; upper cell globose, dark brown to black and opaque, with a small subapical protuberance, which disappears when ascospores mature, thick-walled, 25–27 (–29) 3 (22–) 23–27 mm, uniformly ornamented with small warts; lower cell sub-hyaline, conical, (1–) 4–6 mm long, slightly warted; germ pore sub-apical, 1–2 mm diam. Anamorph unknown. Colonies on OA growing rapidly, attaining 80–86 mm diam in 14 d at room temperature, zonate and slightly radiate, cottony, with immersed and sparse aerial mycelium, olive-brown (M. 4E5); reverse dark brown; ascomata produced after one mo. Colonies on MEA restricted, elevated, cottony and radiately wrinkled, olive-brown (M. 4E5); reverse olive-brown (M. 4E8), ascomata not produced. At 15 and 35 C, the growth rate is similar to that at room temperature on all media used; ascomata not produced. GARCÍA ET AL: TWO NEW SPECIES OF APIOSORDARIA 139 FIGS. 12–20. Apiosordaria globosa FMR 7400 12. Ascoma. 13. Asci. 14, 15. Detail of ascus. 16. Detail and optical section of ascospored wall. 17, 18. Ascospores. 19, 20. Ascospores (SEM). Scale bars: 12 5 200 mm, 13 and 155 20 mm, 14 and 16–20 5 10 mm. 140 MYCOLOGIA Specimen examined. SPAIN, TARRAGONA: Vilaplana, Els Gorgs de la Febró, from soil, 25-IX-1996, col. AM Stchigel, M Calduch (HOLOTY PE: FMR 7400). Apiosordaria globosa is very similar to A. otanii (Udagawa 1990). Both have cylindrical, 8-spored asci and small warts on the upper cell of the ascospores. However, they can be differentiated by the shape and size of the upper cell of the ascospores, which are broadly ellipsoidal with a truncate base, 20–24 3 15– 18 mm in A. otanii, and globose, measuring 25–27 (–29) 3 23–27 mm in the new taxon. In addition, A. otanii produces a Chr ysosporium-like anamorph, which is lacking in the new species, and the lower cell of the ascospores is smooth, while in A. globosa it is slightly warted. ACKNOWLEDGMENTS This work was supported by Fundació Ciència i Salut (Reus). The senior author is grateful for the fellowship grant from Universitat Rovira i Virgili (Tarragona), Spain. LITERATURE CITED Arx JA von, Gams W. 1967. Über Pleurage verruculosa und die zugehörige Cladorrhinum-Konidienform. Nova Hedwigia 13:199–208. Figueras MJ, Guarro J. 1988. A scanning electron microscopic study of ascoma development in Chaetomium malaysiense. Mycologia 80:298–306. Guarro J, Cano J. 1988. The genus Triangularia. Trans Br Mycol Soc 91:587–591. Kornerup A, Wanscher JH. 1984. Methuen handbook of colour. 3rd ed. London: Eyre Methuen. 252 p. Krug JC, Udagawa S-I, Jeng RS. 1983. The genus Apiosordaria. Mycotaxon 17:533–549. Morinaga T, Minoura K, Udagawa S-I. 1978. 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