378 Mycol. Res. 104 (3) : 378–381 (March 2000). Printed in the United Kingdom. Picoa lefebvrei and Tirmania nivea, two rare hypogeous fungi from Spain G. MORENO, J. DI! EZ and J. L. MANJO! N Departamento de BiologıU a Vegetal, Facultad de Ciencias, Universidad de AlcalaU , 28871 AlcalaU de Henares, Madrid, Spain. Accepted 10 May 1999. Picoa lefebvrei and Tirmania nivea are reported for the second and first time from Spain and Europe, respectively. They are illustrated and compared with material and descriptions of the related P. juniperi, T. pinoyi and P. lefebvrei is compared with material from Israel. The collection of T. nivea was originally recorded as T. pinoyi, which should, therefore, be removed from the European Catalogue of hypogeous fungi. The Iberian Peninsula provides a link between the African and European continents, which may allow fungi to migrate from desert and semiarid regions of north Africa to Europe. Plants belonging to the Cistaceae, are common inhabitants of many sites in the Mediterranean basin, particularly species of Helianthemum, Xolantha, Cistus, Halimium and Fumana. Most of these plants establish mycorrhizas, and have a specific mycota associated with them, in particular, hypogeous ascomycetes (Tuber, Terfezia, Tirmania and Picoa spp.). Due to their commercial value, these fungi not only serve as food for the local populations, but also serve as an alternative income source. These mycorrhizal fungi play an important role in the maintenance of Mediterranean shrub-lands by preventing erosion and desertification. For these reasons it is important to study these fungi and understand their biology and taxonomy more fully. In the present study, we follow the previous works of Moreno, Gala! n & Ortega, 1986 ; Moreno, Gala! n & Montecchi, 1991 ; Moreno & Gala! n, 1993 ; Moreno, Kreisel & Gala! n, 1997 ; Gala! n & Moreno, 1998, and describe two rare and poorly described hypogeous fungi, one of them not previously reported from Spain. MATERIALS AND METHODS Microscopic examinations and measurements of sporocarp elements were carried out in tap water, 2 % aqueous KOH, Melzer’s reagent and Congo Red. The photomicrographs were made with a Nikon microscope (Optiphot model) with phase-contrast and automatic photography. The SEM photographs were taken on a Zeiss DSM-950 microscope after preparation as follows : fragments of the gleba were mounted in concentrated ammonium hydroxide (28–30 %) and allowed to soak for 10 min. They were then dehydrated in aqueous ethanol (70 %) for 30 min and later immersed in pure acetone for 30 min. This was followed by critical point drying and then each specimen was sputter-coated with gold-palladium. Voucher specimens were deposited in AH (herbarium of Alcala! University, Spain). Picoa lefebvrei (Pat.) Maire, Annales Mycologici 4 : 332. 1906. (Figs 1–16) Phaeangium lefebvrei Pat., Journal de Botanique 8 : 155–156 (1894). Terfezia schweinfurthii Henn., Hedwigia 40 : 100 (1901). Ascoma hypogeous, gregarious 0±7–1±5 cm diam., subglobose, brown, reddish-brown to dark brown. Peridium with a mycelial tomentum, reddish-brown, with more or less irregular pyramidal rounded warts. Gleba off-white, very crumbly, with fertile pockets separated by pale off-white veins. Odour strong, pleasant. Peridial tomentum composed of cylindrical, septate, yellow to pale brown, hyphae 10–12 µm diam., the walls up to 2–3 µm thick, with vesicles and granules on its surface. Peridium 180–300 µm thick. Ectal excipulum ca 200 µm thick, composed of globose to isodiametric cells, 20–40 µm diam., brownish-yellow, cells near the sporocarp surface are darker and thicker, while cells near the interior are whitish-yellow and thinner walled. Ental excipulum ca 100 µm thick, composed of cylindrical, hyaline, interwoven hyphae, 3–10 µm diam. Asci 90–140¬40–62 µm, club-shaped, with a pedicel very variable in length (up to 50¬8 µm), 2–8-spores per ascus. Ascospores globose to oval, hyaline, 22–28¬20–24 µm, with a large lipid guttule, at maturity uniformly warty, sometimes with scattered large warts and also dense small warts. Spore ornamentation weakly cyanophilic. Specimens examined : Spain : Torres de la Alameda (Madrid), stony calcareous grass-land, associated with Helianthemum spp., leg. J. L. Manjo! n & J. Dı! ez, 22 May 1998, AH 19560. SEM 347. Fuentiduen4 a G. Morenzo, J. Dı! ez and J. L. Manjo! n 379 Figs 1–16. Picoa lefebvrei (AH 19560). Fig. 1. Habit of ascoma ‘ in situ.’ Fig. 2. Peridium detail, pyramidal flat warts. Fig. 3. Section of peridium. Fig. 4. Section of peridium, detail showing the outside cells. Figs 5–6. Tomentum hyphae of ascoma (MO). Figs 7–8. Details of tomentum hyphae (SEM). Figs 9–10. Pedicellate ascus. Fig. 11. Eight-spored ascus. Figs 12–13. Ellipsoidal spores bearing small warts. Figs 14–15. Warty spores (SEM). Fig. 16. Detail of spore warts (SEM). Fig. 17. Picoa juniperi (AH 19561). Smooth spore (SEM). Bars ¯ 0±5 cm (Figs 1, 2) ; 100 µm (Fig. 3) ; 20 µm (Figs 4, 9, 10) ; 10 µm (Figs 5–7, 11–13) ; 5 µm (Figs 8, 14, 15, 17) ; 1 µm (Fig. 16). Two rare hypogeous fungi 380 Figs 18–22. Tirmania nivea (MA-Fungi 37352). Fig. 18. Asci bearing short pedicels. Fig. 19. Ascus detail. Fig. 20. Ellipsoidal, smooth spores (MO). Figs 21–22. Ellipsoidal, smooth spore (SEM). Bars ¯ 20 µm (Figs 18–20) ; 2 µm (Figs 21–22). del Tajo (Madrid), gypsy shrub-land, associated with Helianthemum squamatum (L.) Dum. Cours., leg. L. Go! mez, 20 Mar. 1997, AH 22369 and 22368. Israel : Botsuvha, Israeli desert with Helianthemum kahiricum Delil., 2 Feb. 1985, in AH, duplicate in Montecchi Herbarium. Also studied : Picoa juniperi Vittad : Spain : Priego. Co! rdoba, under Quercus ilex ssp. ballota, leg. J. Go! mez, Apr. 1991, AH 19561. (Fig. 17). After the microscopic and macroscopic study of P. lefebvrei and P. juniperi, we conclude that P. lefebvrei is distinguished by ascoma with reddish tones, peridium with a reddish-toned hyphal tomentum, and by hyaline spores, that are generally globose with warty ornamentation. Macroscopically, the ascoma of P. juniperi is characterized by its black peridium without reddish tones, with or without a sparse mycelium, and regular pyramidal warts. It has generally oval, smooth spores. P. lefebvrei was reported by Alsheikh & Trappe (1983 b) from material collected in Kuwait. They also examined samples from Tunisia, Algeria, Libya and Iraq. They were the first to report that the spores were ornamented at maturity. Alsheikh & Trappe (1983 b), based on spore ornamentation and the hyphal tomentum, placed P. lefebvrei in Phaeangium, erected by Patouillard (1894). We disagree with the separation of this genus from Picoa. The literature on hypogeous fungi includes several examples of genera with both smooth and ornamentated spores, such as Tirmania. According to our observations, P. juniperi and P. carthusiana Tul. & Tul. also have a hyphal tomentum. The spores of P. lefebvrei were described as smooth by Patouillard (1894) ‘ Les spores sont incolores, lisses, ovoides ’, and Maire (1906) ‘ l’epispore est toujours absolument lisse ’. It is likely that both authors studied immature ascoma. Patouillard (1894), however, noticed strong similarities between P. lefebvrei and P. juniperi, confirming that both species belong in Picoa. Pacioni & El-Kholy (1994) described P. lefebvrei from Egypt. Although these authors remarked on the warty spores, it seems they studied immature ascocarps and the SEM photographs reported in their work show smooth spores. Regarding the chorology of P. lefebvrei, this species is typically north African and west Asian (Alsheikh & Trappe 1983 b). In Europe, it has been reported from France (Civrysur-Serein, Yonne), by Riousset et al. (1989, 1996) associated with Helianthemum nummularium (L.) Miller and from Spain (Campaspero, Valladolid), by Calonge et al. (1995) among grass and calcareous soil. The collections reported, herein, from near Madrid, Spain are the second record of this species from the Iberian Peninsula. The reddish tones in the peridium were not reported by Alsheikh & Trappe (1983 b), and they described shorter asci that are stipitate. There is no doubt, however, about its identification. We consider colour of the peridium as a variable G. Morenzo, J. Dı! ez and J. L. Manjo! n feature, and we have observed the peridium to be brown, reddish-brown, and dark brown. We also consider the size of the asci to be highly variable in this species, hence it is not a reliable diagnostic character. Although we consider P. lefebvrei as belonging to Picoa, others do not. Dr M. A. Castellano (personal communication) has said ‘ Dr Trappe (Oregon University) and I are adamant that Phaeangium is the proper place for P. lefebvrei and it should not be aligned with P. juniperi ’. According to Dr Castellano, P. juniperi is special within Picoa and is not similar to P. carthusiana. P. juniperi seems to be an unusual fungus and is not related to other species traditionally placed in Picoa. Molecular work confirms this (pers. comm.). There is a lack of information about the mycorrhizal fungi of semiarid areas in the southern Europe. For this reason, and although there could be some doubts about the proper taxonomic place of P. lefebvrei, we believe that the data reported in this paper to be significant. We hope molecular studies will clarify its taxonomic position. Tirmania nivea (Desf. : Fr.) Trappe, Transactions of the British Mycological Society 57 : 88 (1971). (Figs 18–22) Tuber niveum Desf., Flora Atlantica : 436 (1798) : Fr., Systema Mycologicum 2 : 292 (1823). Terfezia ovalispora Pat., in Dybowski, Naturaliste 12 : 268 (1890) ; nom. nud. Tirmania ovalispora (Pat.) Pat., EU numeUration des Champignons observeUs en Tunisie : 9 (1892), comb. Tirmania cambonii Chatin, La Truffe : 81 (1892). Tirmania africana Chatin, Bulletin de la SocieUteU Botanique de France 38 : 62 (1892). Terfezia africana (Chatin) Maire & Werner, MeUmoires de la SocieUteU des Sciences Naturelles du Maroc 45 : 17 (1937). According to Moreno-Arroyo et al. (1997), T. nivea is characterized by subglobose to ovoid ascoma that are up to 10 cm diam, with a peridium that is without tomentum, fleshy, off-white when young, becoming yellow to cinnamoncoloured, and finally brownish-red at maturity. The gleba is solid, white to pale yellow, with several sterile, off-white veins. We observed the following microscopic features : Asci amyloid (4–6)–8–spored, club-shaped 95–110¬45–50 µm, with a pedicel (up to 25¬8 µm). Spores 16–18¬12±5–14 µm, oval to ellipsoidal, hyaline, smooth (SEM) with a large lipid guttule. Specimen examined : Spain : Tabernas Desert, Almeria, in sandy soils, leg. P. Rodrı! guez, 29 May 1995, MA-Fungi 37.352. SEM 348. Tirmania nivea is easily identified by its large, white to pale yellow, pear-shaped ascoma, and by its shortly pedicellate, strongly amyloid asci, and ellipsoid, smooth spores (Alsheikh & Trappe, 1983 a). It is closely related to T. pinoyi (Maire) Malençon. T. pinoyi, however, differs from T. nivea by its 381 generally globose and larger spores (15–20 µm), reticulated with a small mesh. This ornamentation is hard to observe with light microscopy but can be observed with SEM. SEM photographs of T. pinoyi spores were published by Pacioni & El-Kholy (1994). The material from the Tabernas Desert, Almeria, is the first record of T. nivea for Europe, but it was published as T. pinoyi by Moreno-Arroyo et al. (1997). Both species have similar distribution (Alsheikh & Trappe, 1983 a). Although MorenoArroyo et al. (1997) indicated ‘ esporas esfe! ricas o algo elipsoidales de 16–24 µm de dia! m.’, when examining the herbarium material we observed ellipsoid spores (never spherical) that were decidedly smooth and somewhat larger. We propose, therefore, the removal of the Spanish record of T. pinoyi from the European catalogue of hypogeous fungi. A C K N O W L E D G E M E N TS We express our gratitude to Dr M. A. Castellano, Corvallis, Oregon USA, and Dr N. L. Bougher, CSIRO, Australia, for critical scientific review, and review of the English. To Mr A. Montecchi, Italy, for his valuable comments and suggestions. The valuable assistance of J. A. Pe! rez and A. Priego at the ‘ Servicio de Microscopı! a Electro! nica ’ of the University of Alcala! . Finally, we want to thank DGICYT (project PB95-0165), INIA (project SC98-030) and ‘ Vicerrectorado de Investigacio! n de la Univ. de Alcala! ’ (EO28}98) for financial support. REFERENCES Alsheikh, M. & Trappe, J. M. (1983 a). Desert truffles : the genus Tirmania. Transactions of the British Mycological Society 81, 83–90. Alsheikh, M. & Trappe, J. M. (1983 b). Taxonomy of Phaeangium lefebvrei, a desert truffle eaten by birds. Canadian Journal of Botany 61, 1919–1925. Calonge, F. D., Garcı! a, F, Santos, J. C. & Juste, P. (1995). Contribucio! n al estudio de los hongos de Valladolid y provincias limı! trofes. III. Algunas especies hipogeas interesantes. Boletin Sociedad MicoloU gica de Madrid 20, 291–299. Gala! n, R. & Moreno, G. (1998). Ruhlandiella berolinensis, an exotic species in Europe. Mycotaxon 68, 265–271. Maire, R. (1906). Notes mycologiques. Annales Mycologici 4, 329–399. Moreno, G. & Gala! n, R. (1993). Hydnangium pila Pat., an older name for Martellia mediterranea Moreno, Gala! n & Montecchi. Mycotaxon 47, 157–159. Moreno, G., Gala! n, R. & Montecchi, A. (1991). Hypogeous fungi from peninsular Spain II. Mycotaxon 42, 201–238. Moreno, G., Gala! n, R. & Ortega, A. (1986). Hypogeous fungi from continental Spain. I. Cryptogamie-Mycologie. Paris 7, 201–229. Moreno, G., Kreisel, H. & Gala! n, R. (1997). Setchelliogaster rheophyllus comb. nova (Bolbitiaceae, Agaricales), an Iberic and North African fungus species. Feddes Repertorium 108, 567–573. Moreno-Arroyo, B., Go! mez, J. & Calonge, F. D. (1997). Aportaciones a la micoflora hipogea Ibe! rica. BoletıU n Sociedad MicoloU gica de Madrid 22, 91–95. Pacioni, G. & El-Kholy, H. (1994). Tartufi del deserto Egiziano. Micologia e Vegetazione Mediterranea 9, 69–84. Patouillard, N. (1894). Les te' rfez de la Tunisie. Journal de Botanique 8, 153–156. Riousset, L., Riousset, G., Jalade, M. & Chevalier, G. (1989). Prima raccolta in Francia di Phaeangium lefebvrei Patouillard 1894. Il Fungo : 9–11. Riousset, L., Riousset, G., Jalade, M. & Chevalier, G. (1996). Prima raccolta in Francia di Picoa lefebvrei (Patouillard) Maire 1906. Il Fungo suppl. no 3 : 15–17.