Mycologia, 95(4), 2003, pp. 765–772. 2003 by The Mycological Society of America, Lawrence, KS 66044-8897
Mattirolomyces tiffanyae, a new trufﬂe from Iowa, with ultrastructural evidence for its classiﬁcation in the Pezizaceae
R. A. Healy1
Bessey Microscopy Facility, Iowa State University, Ames, Iowa 50011-1020
Abstract: A new species of hypogeous Pezizales, Mattirolomyces tiffanyae, is described and illustrated. Its asci are typically three-spored, an unusually small number in the non-Tuber Pezizales. Ascus septal pore ultrastructure consists of a uni- or bi-convex band, which suggests an afﬁnity with the Pezizaceae. Secondary spore-wall development is similar to that of Peziza, and several species of hypogeous Pezizaceae. Key words: Hydnobolites, hypogeous ascomycete, septal pore, Terfezia, Terfeziaceae
Trufﬂes, hypogeous Ascomycota, are difﬁcult to identify due to the evolutionary convergence of morphological characters among taxa that share a similar habitat and mode of spore dispersal. Trufﬂes develop below the soil surface and remain below the soil surface, or sometimes emerge at maturity. Most are known or suspected to be ectomycorrhizal (Maia et al 1996, Trappe and Maser 1977), and spores either are released passively into the soil from disintegrated asci and ascocarps or dispersed through mammal mycophagy ( Johnson 1996, Maser and Maser 1987, Maser et al 1978, Trappe and Maser 1977). Salient features, such as the mode of ascus dehiscence, morphology of the ascus tip, and amyloid reactions of asci in Melzer’s solution that phylogenetically are useful in the classiﬁcation of epigeous Ascomycota, usually are altered in hypogeous Ascomycota so that relationships cannot be discerned on the basis of these characters. Morphological and molecular evidence is accumulating for the independent derivation of trufﬂes, with the exception of Elaphomyces, from at least three families within the epigeous Pezizales (Kimbrough 1994, O’Donnell et al 1997, Percudani et al 1999). Septal pore ultrastructure is one morphological character that appears to correlate well with molecular
Accepted for publication April 1, 2003. 1 E-mail: firstname.lastname@example.org
sequence analysis of the18S rDNA in the Pezizales. The Pezizales have a unique lamellate structure associated with septal pores of vegetative hyphae. Septal pore ultrastructure at the base of the ascus appears to be consistent within families of the Pezizales (Kimbrough 1994) and has been interpreted to link several trufﬂes to epigeous families (Li and Kimbrough 1994, Kimbrough et al 1991, 1996). Another useful taxonomic character is spore-wall development. Early secondary spore-wall development appears to be consistent among taxa within the Pezizaceae (Dyby and Kimbrough 1987, Merkus 1975, 1976). The ﬁnal stages of spore-wall ornamentation differ among species (Dyby and Kimbrough 1987). A new species of Mattirolomyces Fischer was found during studies of trufﬂes and false trufﬂes in Iowa. It is here described and illustrated. To help establish the phylogenetic afﬁnities of this taxon, the ultrastructure of septal pores and spore-wall development were examined.
MATERIALS AND METHODS
Fresh material. Water mounts of sections from fresh ascocarps were used to study peridial and glebal anatomy of ascocarps and of asci. Fifty mature ascospores were measured, and asci were observed for color change in Melzer’s solution. Descriptions of this material follow suggestions and terminology of Weber et al (1997). Microscopy. Two specimens were prepared for TEM following Curry and Kimbrough (1983). Resin-embedded blocks were thin-sectioned to 55 nm with a Diatome diamond knife (Diatome-U.S., Fort Washington, Pennsylvania) on a Reichert Ultracut S ultramicrotome (Leica, Wien, Austria) and 3–4 sections picked up in a drop of water with a single slot copper grid and deposited on a Formvar coated rack by the method of Rowley and Moran (1975). Sections were stained with uranyl acetate and lead citrate and viewed with 80 kV on a JEOL 1200EX scanning transmission electron microscope ( JEOL USA Inc., Peabody, Massachusetts) (TEM). Images were captured on Kodak Electron Image Film SO163 (Eastman Kodak, Rochester, New York) or captured digitally with an SIS Megaview III camera (Lakewood, Colorado). For each set of thin sections, a section 1 m thick was collected and placed on a glass slide, stained with toluidine blue O, mounted in Permount on a drop of xylene and coverslipped. Thick sections were studied with bright-ﬁeld optics for median sections through septae at the bases of asci, and those so identiﬁed were circled on a digital image
MYCOLOGIA room-like odor, the surface ﬁnely pubescent, the mycelial hairs sparse, septate, short, up to 113 m 13.8 m, otherwise the surface even, white, unchanging. Not staining where handled or damaged. No rudimentary basal stipe or mycelial tuft (FIG. 1). Peridium of textura intricata (FIG. 2). Peridial hyphae 5– 17.5 m in diam at the septum, often inﬂated, continuous with and similar to hyphae of sterile veins in gleba. Walls of outermost peridial hyphae up to 2 m thick. Gleba solid, white, with fertile pockets that dry to cream-colored and sterile veins remaining unchanged. Asci inoperculate, non-amyloid in Melzer’s solution, irregularly arranged in fertile pockets of gleba, 53–63 m, short pedicelellipsoidal, 110–139 m late, 1–3 (4)-spored, with usually 3 uncrowded, globose spores irregularly uniseriate to inordinate (FIG. 3). Paraphyses absent. Spores within same ascus frequently in different stages of development (not shown). Spores hyaline, with one guttule, globose, 22.5–26.8 m diam, with a mean of 24.6 m diam excluding ornamentation, 29–,33.6 m diam, with a mean of 31.3 m diam including ornamentation; hyaline in water, unchanging to pale yellow in Melzer’s solution. Mature spores ornamented with ﬂat-topped spines and ridges forming a partial reticulum, irregular in thickness, 2.5–5 m high, with a mean of 3.7 m (FIG. 4). Habitat.—Hypogeous to emergent in Hayden-Lester-Storden association soil formed from glacial till, in mixed deciduous upland woods composed of Quercus macrocarpa Michx., Acer saccharum Marsh., Carya ovata (Mill.) K. Koch, Prunus serotina Ehrh., Tilia americana L., and Ulmus L. sp., or Q. alba L. and Ostrya virginiana (Mill.) K. Koch. Paratypes: U.S.A. Iowa, Story County, McFarland Park (42 06 00 N, 93 34 30 W), near Ames, col. R. Healy, 23 Aug 1998 RH 237 (ISC); Story County, Hickory Grove Park (41 59 30 N, 93 21 30 W), near Nevada, col. R. Healy, 1 Sep 1998 RH 251 (ISC); 7 Sep 1998 RH 257 (ISC); 17 Sep 1998 RH 274 (ISC); 6 Aug 1999 RH 520 (ISC); Reactor Woods (42 02 30
of the section. The circled septae then were used as a guide when searching thin sections with TEM for septae at the base of asci. For light microscopy, one specimen was ﬁxed in FAA and processed for parafﬁn embedding (Ruzin 1999). Sections 8 m thick were stained with iron hematoxylin and mounted in Permount for viewing of general morphology. Light micrographs were taken on a Leitz Orthoplan compound microscope with a Leica WILD MPS 52 camera system (Leica, Wien, Austria) or digitally captured with a Zeiss AxioCam HRc camera system (Thornwood, New York). For scanning electron microscopy (SEM), 50 m sections were cut from parafﬁn-embedded blocks, deparafﬁnized in xylene, transferred to 100% ETOH, critical-point dried in a DCP-1 Denton Critical Point Drying Apparatus (Denton Vacuum Inc., Cherry Hill, New Jersey), mounted on aluminum stubs, with double-sided tape, painted around the edges with silver paint, sputter-coated with Au/Pd and viewed with 15 kV in a JEOL 5800LV SEM ( JEOL USA Inc., Peabody, Massachusetts). Images were digitally captured.
During a project conducted 1996–2000 to ﬁnd and catalogue trufﬂes and false trufﬂes in Iowa, an undescribed species was collected at several sites in Story County. It is here described and documented as a new species. Mattirolomyces tiffanyae Healy, sp. nov. FIGS. 1–25
Stereothecium subglobosum, usque ad 17 15 mm, odore fungi, pagina puberula, ceterum aequata, alba, immutabili. Peridium ex constans textura intricata. Gleba a peridio non separabilis, solida, alba, marsupio fertilibus in sicco cremicoloribus, venis sterilibus immutabilibus. Asci ellipsoidei, haud amyloidei, persistentes, unispori ad quadrispori. Paraphyses carentes. Sporae hyalinae, uniguttulatae, globosae, 29–33.6 m diam ornamentis inclusis, verrucatis ad porcatis et ex partibus reticulatis, ornamentis 2.5–5 m altis. Holotypus hic designatus: U.S.A., Iowa: Story County, 18 Aug 1998, Healy, R. 231 (HOLOTY PE: ISC; ISOTY PE: BPI, OSC)
Fruit body structure. Ascocarp a stereothecium, often with small holes indicating mycophagous activity, subglobose, the largest 17 15 mm, with a mush-
5 mm. Figs. 2–3. Bright-ﬁeld microscopy (BFM). 2. Peridium; bar FIGS. 1–10. M. tiffanyae 1. Sectioned ascocarp; bar 50 m. 3. Ascus; bar 25 m. 4. Scanning electron microscopy of mature ascospore in a sectioned ascus; bar 5 m. Figs. 5–8, 10. Transmission electron microscopy (TEM). 5. Lamellate structures in septal pore of glebal hypha with globose Woronin bodies (W); bar 500 nm. 6. Lamellate structures in septal pore of glebal hypha; bar 250 nm. 7. Ascus (A) with bi-convex band in septal pore (arrow), and vesicles in mound of differentiated ascosplasm overlaying septal pore; bar 3 m. 8. Higher magniﬁcation of Fig. 7, bi-convex band and vesicles in ascus (A); bar 250 nm. Fig. 9. BFM of toluidine blue O stained asci (A) produced from ascogenous hyphal cell; bar 20 m. 10. Same ascogenous cell as in Fig. 9, tipped slightly counterclockwise, with bi-convex band in septum at the base of ascus (A). Note Woronin bodies (W) in cell subtending ascus, and seven nuclei (N); bar 3 m.
HEALY: MATTIROLOMYCES TIFFANYAE
MYCOLOGIA the latter stages of secondary wall formation and usually involve variability in the secondary wall (Gibson and Kimbrough 1988, Read and Beckett 1996, Wu and Kimbrough 1991). Therefore, the latter stages of secondary spore-wall development were examined in more detail than early stages and previous wall development was not examined. After primary wall formation, spore-wall development closely follows the process reported in species of Peziza (Dyby and Kimbrough 1987, Merkus 1975), Terfezia claveryi Chatin ( Janex-Favre and Parguey-Leduc 1985) T. leptoderma Tul. and Tul. ( Janex-Favre et al 1988), and Hynobolites cerebriformis Tul. and Tul. (Kimbrough et al 1991). No more than one nucleus per spore was observed. As in spores of other Euascomycetes, the inner delimiting membrane becomes the sporoplasmalemma and the outer delimiting membrane becomes the investing membrane (Carroll 1969, Mainwaring 1967, 1972). The outer delimiting membrane evaginates into the epiplasm (FIGS. 13, 14). The perisporal sac, the space between the investing membrane and the primary wall, sometimes contains electron-dense condensed material (FIG. 14). The perisporal sac enlarges as the investing membrane continues to elongate and evaginate into the epiplasm (FIGS. 15–19). The perisporal sac contains ﬂocculose material (FIGS. 15–19) that increases in density as it accumulates (FIG. 16). Vacuoles increase in number and size in the epiplasm surrounding the spores and contain electron-dense bodies (FIGS. 15–19). As vacuolization proceeds, the tonoplasts align with the investing membrane so that eventually the spore appears to be surrounded by a double membrane (FIGS. 17, 19, 20). Secondary wall formation begins with an epispore precursor, which develops as short ﬁbrils perpendicular to the primary wall (FIGS. 18–20). Hemispherical
N, 93 39 20 W), Ames, col. C. Notis 28 Aug 1999 RH 556 (ISC); col. R. A. Healy 3 Oct 1999 RH 601 (ISC). Etymology. It is my pleasure to name this taxon for Dr. Lois Hattery Tiffany. For more than half a century at Iowa State University, she fostered a broad range of mycological studies with many students but always has maintained a particular interest in the hypogeous fungi. Septal pore ultrastructure. Septal pores in glebal hyphae were viewed with TEM. Ascogenous hyphae were indistinguishable from other glebal hyphae and therefore only identiﬁed as such where they were connected to an ascus. Some glebal hyphae have ‘‘Peziza type septal pore’’ structures (Kimbrough 1994) of alternating electron-dense and electron-translucent bands (FIGS. 5, 6). Median sections through septal pores at the base of asci display bi-convex bands (FIG. 8, a higher magniﬁcation of FIG. 7; and FIG. 11, a higher magniﬁcation of FIGS. 9, 10) and uni-convex bands (FIG. 12) similar to those found in the Pezizaceae (Curry and Kimbrough 1983) and Terfeziaceae ( Janex-Favre et al 1988). Woronin bodies are spherical (FIG. 5), consistent with those of the Pezizaceae (Kimbrough 1994) and sometimes observed in ascogenous hyphae (FIG. 10). Vesicles are observed within a hemispherical mound of differentiated ascoplasm separated from the main portion of the ascus by a membrane and situated over the septal pore at the base of the ascus (FIGS. 7, 8, 10, 11). Similarly, the ascoplasm situated over septal pores with uni-convex bands is differentiated and separated from the remainder of the ascoplasm (FIG. 12). Secondary spore-wall development. Spore-wall development up to secondary wall formation tends to be similar in the Pezizales (Dyby and Kimbrough 1987, Merkus 1975, 1976). Major differences occur during
→ 250 FIGS. 11–12. TEM of septal pores. 11. Higher magniﬁcation of FIG. 10, bi-convex band at base of ascus (A); bar nm. 12. Uni-convex band at base of older ascus (A); bar 250 nm. FIGS. 13–25. TEM of secondary spore wall development in M. tiffanyae. 13. young spore with electron-translucent primary wall (PW) and outpouching (outlined region) of the investing membrane (IM) into the epiplasm (EP); bar 3 m. 14. Higher magniﬁcation of outlined region in Fig. 13, electron-dense material between IM and PW; bar 250 nm. 15. Elongation and evagination of IM into EP to create perisporal sac (PS); bar 3 m. 16. Spore with voluminous PS, ﬁlled with ﬂocculose substance and surrounded by epiplasmic vacuoles (V); bar 3 m. 17. Higher magniﬁcation of outlined region in Fig. 16, showing alignment of tonoplasts (T) with IM; bar 700 nm. 18. Development of epispore precursor with electron-dense bodies (DB) in V and PS; bar 3 m. 19. Higher magniﬁcation of region outlined in Fig. 18, showing DB in PS and development of epispore precursor (arrow) of short ﬁbrils perpendicular to PW; bar 500 nm. 20. Deposition of DBs on epispore precursor (arrow); bar 250 nm. 21. Striate DB separated from epispore (ES) by zone of electron translucence (ETZ); bar 250 nm. 22. Nearly mature spine with striate ES, column of ﬁbrils separated from electron-dense cap by ETZ; bar 500 nm. 23. BFM of nearly mature spore showing spine caps (arrows) stained dark in toluidine blue O, and copious EP; bar 10 m. 24. Spore spine of compact ﬁbrils with margins of loosely arranged ﬁbrils; bar 500 nm. 25. Mature spore spine of compact ﬁbrils with margins of loosely arranged ﬁbrils overlaid by collapsed IM and T; bar 500 nm.
HEALY: MATTIROLOMYCES TIFFANYAE
Comparison of secondary spore-wall development in the epigeous and hypogeous Pezizaceae and Terfeziaceae Species Developmental event in secondary spore wall formation a b nr c nr d e /
Evagination of investing membrane into epiplasm Electron-dense condensed material in perisporal sac Flocculose material in perisporal sac Epiplasmic vacuoles form around spores, tonoplasts align with investing membrane Epispore precursor of short ﬁbrils arranged perpendicular to primary wall Hemispherical electron-dense material deposited on primary wall to form secondary wall ornament precursors Ornament precursors separated from primary wall by thin zone of electron translucence Ornament capped by electron-dense material Electron-dense cap separated from ornament by zone of electron translucence Epispore becomes striate Mature ornament composed of compact interwoven ﬁbrils surrounded by loosely interwoven ﬁbrils, over which investing membrane collapses
a: Mattirolomyces tiffanyae; b: Terfezia claveryi ( Janex-Favre and Parguey-Leduc 1985); c: Terfezia leptoderma ( Janex-Favre et al 1988); d: Hydnobolites cerebriformis (Kimbrough et al 1991); e: Peziza species (Dyby and Kimbrough 1987); f: Reported as elements of the endoplasmic reticulum rather than tonoplasts. nr not reported.
electron-dense condensed material is deposited on top of the epispore precursor (FIGS. 18–20). It is separated from the epispore by a thin zone of electron translucence and appears to be striate (FIG. 21). This material is deposited in irregularly spaced mounds of variable breadth until spines or ridges (FIGS. 22–24) are formed. The tips of the spines and ridges sometimes are capped with electron-dense material (FIG. 22) that also stains densely with toluidine blue O (FIG. 23). The cap material is similar in appearance to that deposited on the epispore (FIG. 21). The electron-dense cap is separated from the spine column by a thin zone of electron translucence (FIG. 22). The electron-dense cap is compact and condensed (FIG. 22), while the underlying secondary wall obviously is ﬁbrillar (FIG. 22). The epispore becomes compact (FIG. 21) and striated with electron-dense and electron-translucent zones (FIG. 22) as ornamentation proceeds. The electron-dense cap material is not apparent on the most mature spores (FIG. 24). Late in development of the secondary wall, the spine differentiates into an inner, compact, moderately electrondense column of ﬁbrils surrounded by more loosely arranged ﬁbrils, less electron dense than the inner column (FIG. 25), and the tonoplast/investing membrane complex degenerates and collapses over the spines and ridges (FIG. 25). The spines and ridges of mature spores are viewed readily with SEM (FIG. 4). As in other taxa of the Pezizaceae, the mature spore contains a large, single, central, lipid droplet
(FIG. 23) (Dyby and Kimbrough 1987). Secondary wall development coincides with the vacuolization and disappearance of epiplasm. The epiplasm is interpreted here as it has been by other investigators (Mainwaring 1972, Merkus 1976) to contribute material destined for the secondary wall. In a deviation from spore development in the epigeous Pezizaceae, there is sometimes a considerable amount of epiplasm remaining at spore maturity (FIG. 23).
Fischer erected the genus Mattirolomyces (Terfeziaceae) to accommodate a taxon similar in appearance to Terfezia that differed in ascocarp shape and ascus characters. Ascocarps in Mattirolomyces are relatively spherical rather than turbinate in shape because they lack the base usually present in Terfezia, and the asci are 2–3 times longer than wide, with uncrowded, irregularly uniseriate to biseriate arranged spores rather than the ellipsoidal to globose asci, with crowded, inordinately arranged spores of Terfezia (Fischer 1938). Trappe (1971) subordinated Mattirolomyces to a subgenus of Terfezia because the delimiting characters also were found in some species of Terfezia. Trappe’s circumscription of Terfezia subgenus Mattirolomyces included these species, each described with a pallid gleba, elongate asci with uncrowded spores, and spores with a partial to complete reticulum: T. austroafricana Marassas and Trappe, T. decaryi Heim,
HEALY: MATTIROLOMYCES TIFFANYAE T. terfezioides (Matt.) Trappe and T. spinosa Harkn. (Marasas and Trappe 1973). Of these, T. spinosa is the only species from North America. Results from recent molecular analyses that included T. terfezioides among the taxa sampled support the reinstatement of the genus Mattirolomyces (Diez et al 2002, Norman and Egger 1999, Percudani et al 1999), an interpretation that Trappe agrees with (pers comm). Diez et al (2002) noted that Terfezia differs from Mattirolomyces in ecology and in geographic distribution. Terfezia is found in arid to semi-arid environments and is mycorrhizal with herbaceous species of Cistus and Helianthemum. Mattirolomyces terfezioides is found in temperate forests, and there is evidence that it forms ectendomycorrhizae with Robinia pseudoacacia, a woody plant (Bratek et al 1996). Before the folding of Mattirolomyces into Terfezia, M. terfezioides was the only species described. A reassessment of Terfezia is needed to determine whether any other species should be transferred to Mattirolomyces. T. spinosa differs from M. terfezioides mainly in larger spore size and longer spore spines (Gilkey 1947, Montecchi and Sarasini 2000). Mattirolomyces tiffanyae differs from M. terfezioides and from species currently classiﬁed as Terfezia subgenus Mattirolomyces in the small number of spores per ascus, the large spore size, and the spore ornamentation of blunt spines and ridges forming a partial reticulum, the mesh of which varies in thickness. These same characters, along with a solid gleba distinguish M. tiffanyae from Hydnobolites, which has hollow or hyphastuffed glebal canals that are lined with a hyphal layer similar to that of the peridium. Delastria rosea Tul. and Tul.is similar to M. tiffanyae in ascus shape and number of spores per ascus but differs in the alveolate-reticulate ornamentation of its spores, the wide variation of spore sizes and ornamentation within a single ascocarp and the color of its gleba, which is pinkish to brown and stains pinkish when touched. It is hypothesized that many of the Terfeziaceae are hypogeous Pezizaceae (Kimbrough et al 1991). There is morphological evidence to support classiﬁcation of Hydnobolites (Kimbrough et al 1991) in the Pezizaceae and molecular evidence to support classiﬁcation of Mattirolomyces (as T. terfezioides), Pachyphloeus and Terfezia (Percudani et al 1999) in the Pezizaceae. Results from this study give ultrastructural evidence to support the classiﬁcation of M. tiffanyae in the Pezizaceae. It is unusual within the epigeous Pezizales for asci to produce fewer than eight spores but less unusual among hypogeous Pezizales. Among the Terfeziaceae, Delastria rosea Tul. and Tul. is the only species with fewer than ﬁve spores per ascus. It is possible that the less mature spores in asci of M. tiffanyae,
with multiple stages of spore development, were in the process of aborting. However, abortion of spores would not explain why initially there were fewer than ﬁve spores per ascus. An investigation of spore ontogeny up to the time of spore delimitation would be informative regarding the fate of postmitotic nuclei in this taxon, which could shed light on why so few spores develop within a given ascus. Secondary spore-wall development in M. tiffanyae is similar to that found in the Pezizaceae and Terfeziaceae (Table I), although wall ornamentation of species of Peziza were less complex than Mattirolomyces. Deposition of both the epispore and the ornaments is by direct precipitation, as found also in the Pezizaceae (Wu 1991) and in Hydnobolites, a member of the Pezizaceae previously classiﬁed in the Terfeziaceae (Kimbrough et al 1991), rather than the gradual condensation reported in the Humariaceae (Wu and Kimbrough 1991). Among members of the Pezizaceae and Terfeziaceae, secondary spore-wall development of M. tiffanyae is most similar to that of H. cerebriformis but differs in the lack of a complete reticulum and in the variable thickness of the ridges that form a partial reticulum. It also is similar to that in T. leptoderma and T. claveryi but differs most notably in the latter stages, where an electron-dense cap forms on the spine tips and ridges of M. tiffanyae but covers the spine tips and valleys on spores of Terfezia. The ﬁnal stage of ornamentation in M. tiffanyae, where the ﬁbrillar columns are compact and dense in the center and more loosely arranged along the periphery, is similar to that found in H. cerebriformis but not reported in species of Terfezia.
The author gratefully acknowledges ﬁnancial support for this study from an Iowa State University Professional Advancement Grant and a grant from the Iowa Science Foundation. Christine Notis is thanked for her collection, Dr. Lynn Clark and Dr. James Trappe are thanked for their help with the Latin diagnosis and for helpful comments and suggestions for improving this manuscript, Deborah Lewis is thanked for assistance with accession of ascocarps, and Dr. Trappe is thanked for information on T. decaryi. The Bessey Microscopy Facility at Iowa State University is thanked for the use of its equipment.
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