Calongea, a new genus of hypogeous Pezizaceae (Pezizales) Rosanne A. Healy1, Gregory Bonito2, James M. Trappe3 1 Department of Plant Biology, University of Minnesota, 1445 Gortner Avenue, St. Paul, Minnesota 55108 U.S.A..mailto:healy089@umn.edu 2 Department of Biology, Duke University, Durham, North Carolina 27708 U.S.A. mailto:gmb2@duke.edu 3 Department of Forest Ecosystems and Society, Oregon State University, Corvallis, Oregon 97331-5752 U.S.A. mailto:trappej@onid.orst.edu RESUMEN HEALY, R. A., G. BONITO & J. M. TRAPPE (2009). Calongea, un nuevo género de hipogea Pezizaceae. Anales Jard. Bot. Madrid 66(1): (en inglés). El análisis filogenético del ITS y LSU rDNA de especies Europeas y Norte Americanas de Pachyphloeus revelan un género nuevo de trufa. Los datos moleculares de este estudio además de las secuencias obtenidas de una búsqueda BLAST en GenBank indican que Pachyphloeus prieguensis es un miembro de la familia Pezizaceae pero no está relacionada a ningún otra especie hipogea o epigea en dicha familia. Encontramos diferencias morfológicas en las hifas del peridio y gleba así como en las esporas que morfológicamente distinguen este de Pachyphloeus. Proponemos que el nuevo género monotípico Calongea, con C. prieguensis comb. nov., como la especie tipo, en honor a uno de los descubridores originales, Prof. Francisco de Diego Calonge. ABSTRACT HEALY, R. A., G. BONITO & J. M. TRAPPE (2009). Calongea, a new genus of hypogeous Pezizaceae. Anales del Jardin Botanico Madrid 66(1): (in English) Phylogenetic analysis of the ITS and LSU rDNA of Pachyphloeus species from Europe and North America revealed a new truffle genus. These molecular analyses plus sequences downloaded from a BLAST search in GenBank indicated that Pachyphloeus prieguensis is in the Pezizaceae but well outside of the genus Pachyphloeus. Morphological differences in the peridial and glebal hyphae and spores distinguish this genus from Pachyphloeus. We here propose the monotypic new genus Calongea, with the type species C. prieguensis comb. nov., in honor of Healy & al. Calongea, a new genus of Pezizaceae 2 Prof. Francisco de Diego Calonge, who has long studied the hypogeous fungi of Spain and participated in describing the type species of Calongea. Key words: Pachyphloeus, ITS, LSU, truffle, Pezizales, new genus, Ascomycota INTRODUCTION Pachyphloeus prieguensis Mor.-Arr., J. Gómez & Calonge was described in 1996 from deciduous woodlands with calcareous soil in Spain (Moreno-Arroyo & al., 1996). Some of its distinctive features include pallid, pink-tinged veins and labyrinthiform, mostly hollow canals within a tuberous, minutely verrucose, foul-smelling, dark reddish-brown to blackish-brown ascoma. One challenge in identifying or describing Pezizalean ascomycetous truffles is that their relationships are confused by convergent morphologies. In their adaptation to a hypogeous habit, they have gained features such as odors adapted for spore dispersal through mycophagy and lost features such as apothecia and forcible spore discharge adapted for air dispersal of spores. Consequently, these sporocarps often resemble others in their ecological niche more than they resemble their most closely related epigeous relatives (Trappe & Claridge, 2005; Læssøe & Hansen, 2007). During a phylogenetic study of Pachyphloeus species with use of ITS and LSU rDNA, P. prieguensis sequences consistently fell outside of the genus when analyzed with maximum parsimony and maximum likelihood optimization criteria. In addition, the ITS and LSU sequences of P. prieguensis were not very similar to any accessions held in Genbank’s DNA sequence database (82% and 92% similarity, respectively). These revelations prompted us to reexamine the morphology of P. prieguensis. In so doing, we found differences in the peridial structure, gleba and spore ornamentation that distinguish this genus from Pachyphloeus. MATERIALS AND METHODS Dried pieces of ascomata were rehydrated in 3% KOH and either viewed with bright field microscopy or dehydrated in an ethanol series to 100%, critical point dried in a DCP-1 Denton Critical Point Drying Apparatus (Denton Vacuum Inc., Cherry Hill, NJ), mounted on aluminum stubs with double-sided tape, silver painted around the specimen edges, spatter-coated for 120 sec with Au/Pd, and visualized with 15 kV in a JEOL 5800LV SEM. All images were digitally Healy & al. Calongea, a new genus of Pezizaceae 3 captured. Other morphology was studied with bright field microscopy. Spore measurements were taken from 50 randomly selected spores, under 100X oil immersion. Molecular analyses were done with DNA extracted from previously unexposed portions of mature gleba taken from the inside of air-dried ascocarps. DNA was extracted with 24:1 chloroform:isoamyl alcohol and precipitated in isopropanol. Both the internal transcribed spacer region (ITS1, 5.8S, and ITS2) and three divergent domains (D1, D2, D3) of the ribosomal large subunit (LSU) locus were amplified by use of the universal fungal primer set ITS5 - LR5 (Bertini & al., 1999; Vilgalys & Hester, 1990). PCR conditions and the handling of PCR products were as described in Healy & al. (in press). Sanger sequencing was performed on an ABI3700 (Applied Biosystems, Foster City, CA) using Big Dye chemistry version 3.1 (Applied Biosystems, Foster City, CA) with the forward primer ITS5 and reverse primer LR5. DNA sequences were assembled and manually edited with Sequencher 4.0 (Gene Codes, Ann Arbor, MI) and queried against the NCBI public database GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the BLASTN algorithm to compare with other sequences and to verify that sequences were of the target group. Sequences from top blast hits (LSU) were combined with our data and were aligned manually using Mesquite 4.0 (Maddison & Maddison, 2002). Ambiguously aligned regions were excluded from the analyses. Phylogenetic analyses were conducted by the maximum parsimony and maximum liklihood methods. Parsimony analyses were carried out by a heuristic search in PAUP 4.0b10 with 1000 random addition sequences and 5000 bootstrap replicates (Swofford, 2001). Two independent maximum likelihood analyses based on a general-time-reversible 6-parameter model of evolution were run using the software program GARLI and included 100 bootstrap replications (Zwickl, 2006). The truffle species Genea harknessii from within the Pyronemataceae was chosen as an outgroup based on previous studies focused on the systematics of the Pezizomycetes (Hansen & Pfister, 2006). Sequences for Calongea prieguensis produced in this study were deposited in GenBank under accession numbers FJ228462 and FJ228463. Herbarium abbreviations are according to the Index Herbariorum (http://sweetgum.nybg.org/ih/herbarium_list.php). RESULTS OF PHYLOGENETIC ANALYSIS Phylogenetic relationships of 27 ingroup Pezizaceae taxa based on maximum likelihood analyses of LSU rDNA are depicted in Fig. 1. This most likely tree resulted from analyses that Healy & al. Calongea, a new genus of Pezizaceae 4 Figure 1 Most likely tree from maximum likelihood analyses of LSU rDNA with 27 ingroup Pezizaceae. Bootstrap values for significantly supported nodes (>70 %) are labeled with parsimony bootstrap on top and likelihood values below. Taxon labels include the Latin binomial followed by the Genbank accession number and geographic origin (when known). 5 Healy & al. Calongea, a new genus of Pezizaceae included 794 characters, of which 187 were parsimony-informative (parsimony analysis) and 556 were constant. Bootstrap values for significantly supported nodes (>70 %) are labeled with parsimony bootstrap on top and likelihood values below. Blast search results turned up no close matches with P. prieguensis LSU or ITS sequences. TAXONOMIC TREATMENT Calongea Healy, Bonito & Trappe, gen. nov. Figs. 2-5 A Pachyphloeo differt exipulo duplici; stratum exteriore pilis brevibus cellulis apicalibus versiformibus, hi in canalibus cavis similita praesentes; stratum interiore hyphis filamentis et cellulis inflatis; canalibusque cavitatibus glebae epithecio peridioideo inclusis; sporae spinis variabiliter dispositis, frequenter curvis, sine perisporis; et ordinatione DNA ITS et LSU . Species typicus: Calongea prieguensis. Differing from Pachyphloeus by its combination of a 2-layered peridium: outer layer of thick-walled cells giving rise to hairs with versiform tips; inner layer a mix of filamentous hyphae and inflated cells; a peridium-like epithecium with versiform hairs lining the canals and cavities; spores with spines of varying length, width and spacing, the spine bases connected by low ridges to form an irregular reticulum; and ITS and LSU DNA not congruent with other genera in the Pezizaceae. Etymology: We dedicate this genus to Prof. Francisco de Diego Calonge, whose mycological career encompassed study of many groups, including hypogeous fungi, and who provided to J. Trappe hospitality, good fellowship, good stories, lessons on Spanish fungi, history, customs, and culture, and outstanding food and wine, both in Madrid and on memorable field trips. Calongea prieguensis (Mor.-Arr., J. Gómez & Calonge) Healy, Bonito & Trappe Basionym: Pachyphloeus prieguensis Mor.-Arr., J. Gómez & Calonge, Bol. Soc. Micol. Madrid 21: 87-88. 1996. TYPE: [Spain] Córdoba, Priego, 4 April 1992 (MA). Illustrations: Moreno-Arroyo & al., Figs. 1-6 (1996); Montecchi & Sarasini, 210-211 (2002); Moreno-Arroyo & al. 158-160 (2005) GenBank Accession Numbers FJ228463, FJ228462 Healy & al. Calongea, a new genus of Pezizaceae Figure 2. Bright field microscopy, Calongea prieguensis excipular hairs. Scale bar = 50 μm. Figures 3-6. Scanning electron micrographs. Figure 3. Hollow glebal canal lined with hairs in C. prieguensis. Scale bar = 250 μm. Figure 6 Healy & al. Calongea, a new genus of Pezizaceae 7 4. Higher magnification of Fig. 3 showing details of hairs along the glebal canals. Scale bar = 25 μm. Figure 5 Spore of C. prieguensis. Scale bar = 10 μm. Figure 6. Spore of an undescribed, North American Pachyphloeus species. Scale bar = 5 μm. Ascomata 1- 3 cm broad, subglobose to irregular. Peridium reddish brown in youth, by maturity blackish brown, nearly smooth or minutely verrucose. Gleba cream colored in youth, by maturity yellow to yellowish pink or light vinaceous, the context thick and containing elongated, labyrinthiform canals and chambers pallid in youth, by maturity pinkish to brown. Odor strong, most unpleasant, sweet-foetid; rehydrated herbarium specimens smell like rotting cardboard. Peridium of two layers: outer layer 83-165 μm thick including the minute warts formed in places by mounded cells; cells in face view smoky-brown with golden-brown walls up to 7.4 µm thick, isodiametric to subrectangular, length x width ca 40-41 x 13 μm to 50 x 41 μm, interspersed with occasional cells up to 100 x 78 μm. Surface cells between the warts giving rise to unbranched, hyaline to smoky-brown hairs up to 60 μm long, with one to several septa and walls 0.5-1.3 μm thick; apical cells of the hairs globose to capitate, cylindrical or ventricose, 1050 μm long and 10-50 μm broad at the widest point (Fig. 2). The inner peridial layer is of hyaline, filamentous, interwoven cells up to 8.3 μm broad and with walls 0.5 μm thick interspersed with inflated cells up to 20 μm broad with walls up to 3.3 μm thick; this layer grades into the interwoven hyphae of the gleba. The peridium and outer glebal vein tissue together are 50-149 μm thick. Gleba with sterile veins, hollow canals, and fertile pockets of tissue. The sterile veins are of textura angularis similar to the peridium. Glebal canals lined with tissue similar to the outer peridium, with large, thick-walled cells and thinner-walled, septate hairs 60 to 100 μm long (Figs. 3, 4), varying from filiform and ± 8 μm broad at the septum with apical cells inflated up to 28 μm and with the diversity of apical cells as on the peridial hairs. Asci irregularly arranged in fertile pockets of tissue, oblong to pyriform, fresh measurements according to Moreno-Arroyo & al. (1996) 65-125 (140) x 30-50 μm; inamyloid in Melzer’s solution, even after pre-treatment with 3% KOH, producing eight irregularly biseriate to inordinately arranged spores. Filamentous, interwoven to parallel hyphae among asci 5-8.3 μm in diameter, but inflated near the borders with peridium-like tissue of sterile veins, and that line canals. The glebal filaments merge with the sterile vein tissue and become indistinguishable from Healy & al. Calongea, a new genus of Pezizaceae 8 it. Interspersed among the narrow, filamentous hyphae and the asci are large-celled filamentous hyphae with cells swollen up to 58 x 50 μm. These cells have hyaline to brown walls up to 2-5 μm thick, and walls are thickest at the septae whereas cells they connect may be hyaline and thin-walled. Spores globose, (16-) 17-19 (-22) μm (avg. 17.7 μm) including ornamentation, and (13-) 15-18 (-19.5) (avg. 15.6 μm) excluding ornamentation. Initially hyaline, at maturity the walls olivaceous brown and inamyloid, the ornamentation of flexuous, often bowed-over spines varying on a given spore in spacing and from 0.5-2.5 μm tall and 0.5-2 μm broad (Fig. 5); a low, irregular reticulum connects the bases of neighboring spines. Collections examined SPAIN. Asturias: Córdoba 15-IV-1990, J. Gómez, Trappe 12832, GenBank FJ228463 (OSC); ITALY. Laconi, 2-V-1999, Fantini 1980, GenBank FJ228462 (OSC). Collections of Pachyphloeus species examined: ITALY. Venezia: Cuneo, Roascio, Pachyphloeus citrinus under mixed conifer and broadleaf forest, 12-IX-2000, M. Machioni JRWL2197, EU543196 (OSC); Pachyphloeus melanoxanthus under Ostrya and Quercus, I-1999, Macchioni 1860, EU543194 (OSC); MEXICO. Nuevo Leon: Santiago, El Ranchito, Pachyphloeus marroninus under Quercus polymorpha, 14-IX-1983, García 3757, EU427551 (UNL); UNITED STATES. Iowa: Story County, Ames, YMCA Woods, Pachyphloeus cf carneus under Quercus macrocarpa, 9-VIII-2000, Healy 756, EU543206 (ISC), Story County, Hickory Grove Park, Pachyphloeus cf melanoxanthus under Quercus alba, Tilia americana, Carya ovata, 28-VII-2000, Healy 735, EU543193 (ISC); Webster County, Woodman Hollow State Preserve, Pachyella clypeata on log in riparian deciduous woods, 18-IX-2005, Healy, EU543195 (ISC); Winneshiek County, Upper Iowa Public Fishing Access, Pachyphloeus marroninus under Quercus alba and Q. macrocarpa, 2-X-1998, Healy 286, EU427550 (ISC, OSC); Woodbury County, Stone State Park, Pachyphloeus cf carneus under Quercus macrocarpa, 8-VIII-1999, Healy 525, EU543203 (ISC), Woodbury County, Stone State Park, Pachyphloeus virescens, 27-VII-2000, Healy 729, EU543198 (ISC); Washington: Thurston County, Fort Lewis, Pachyphloeus thysellii under Pseudotsuga menziesii, 18-VIII-1993, Trappe 13082, EU543197 (OSC). Healy & al. Calongea, a new genus of Pezizaceae 9 Calongea prieguensis and Pachyphloeus species compared The genus Pachyphloeus was described in 1844, typified by P. melanoxanthus (Tul.) Tul. & C. Tul., with the following characteristics: 1.) a thick, fleshy peridium with small warts; 2.) a thick margined orifice stuffed with hyphae, where the interstitial veins come to the surface at the apex of the ascoma and separate into fissures; 3.) a narrow point of attachment; 4.) sterile veins that marble the gleba, and initially differ in color from the fertile tissue but later the color becomes more uniform overall; and 5.) a well-ordered series of asci with eight globose spores ornamented with spines, irregularly arranged in the ascus, colored at maturity (Tul. & Tul., 1844). Since this original description of the genus, other features that unite species in the genus include a peridium composed of a single layer of thick-walled textura angularis (Pegler & al., 1993; Montecchi & Sarasini, 2000; Hansen & al., 2001; Healy, 2002). The structure of the peridium is said to be constant throughout a given genus (Eckblad, 1968). The erecting of a new genus to accommodate Calongea prieguensis unifies the type of peridium present on species of Pachyphloeus. In contrast, C. prieguensis has a duplex peridium composed of a mixture of textura globulosa and textura angularis in the outer layer, subtended by a mixture of interwoven filamentous and inflated hypae. Unlike Pachyphloeus, the peridium of C. prieguensis has short hairs with apical cells of variable shapes and diameters, distinct from their subtending cells. These are also present on the cells lining the hollow (rather then stuffed) canals. The peculiar hairs on the excipulum of Calongea have not been reported in other species. Although some species such as P. conglomeratus, P. lateritius and P. austro-oregonensis have slender, hyphoid hairs of uniform diameter on the peridium (Berk. & Broome, 1846; Fogel & States, 2002; Frank & al., 2006 respectively), and many species have a basal tuft of mycelium. Asci of most Pachyphloeus species are arranged in a well-ordered to irregular palisade with paraphyses. The canals between opposing hymenia are stuffed with extensions of these paraphyses, and the sterile veins are composed of textura angularis similar to cells of the peridium. In contrast, Calongea asci are irregularly arranged in the gleba, and canals are open, not stuffed. In addition, the large-celled, brown-walled filamentous hyphae in the fertile tissue of Calongea have not been reported from any species of Pachyphloeus. The asci of P. conglomeratus are disorganized in the gleba, but unlike Calongea, this species has a solid gleba. Healy & al. Calongea, a new genus of Pezizaceae 10 Pachyphloeus austro-oregonensis has empty canals between opposing hymenia, and in places where the excipulum invaginates the gleba (Frank & al., 2006). However, Calongea canals are very different, in that they are lined by excipular-like rather than hymenial tissue. The ornamentation of Pachyphloeus spores, generally of regular capitate to acute spines, is of rigid, straight (not curved) short to long spines evenly distributed on the spores. Spines on any given spore are uniform in length. Spine development appears to be the same among species of Pachyphloeus, until nearly mature, when variations occur. The spine apices may partially to fully cover the spores in some species, forming the perisporium (Healy, 2002). The distribution, height, and width of spore spines of Calongea is irregular, and the spines themselves are often curved rather than straight. No evidence was seen of a perisporium. Pachyphloeus spores most similar to C. prieguensis are of an undescribed species from North America that also lacks a perispore, and has straight rather then the more usual capitate spines of a Pachyphloeus species (Fig. 6). However, unlike Calongea, and in keeping with other Pachyphloeus, the spines are rigid, and uniform in length, diameter, and distribution. The odor of fresh C. prieguensis was described as unpleasant (Moreno-Arroyo & al., 1996), and this was the case even in rehydrated specimens. The odor of fresh Pachyphloeus specimens ranges from mild to strong, usually of a potato-like odor, but whether it is agreeable or not may be disputable. Pachyphloeus citrinus from England was described as having a strong odor “like that of rotting sea-weed” (Berkeley & Broome, 1846, p.79), and P. melanoxanthus was described as “nauseous” (Berkeley & Broome, 1967, p. 359). However, other references describe the odor from various species as “mild” to “strong”, but not unpleasant (eg. Montecchi & Sarasini, 2000; Colgan & Trappe, 2004; Frank & al., 2006,). We find the odor of species we have collected to be interesting but not unpleasant. A recent effort to delimit the Pezizaceae, on the basis of parsimony analyses of LSU rDNA, included the following fourteen hypogeous genera in the family: Amylascus, Cazia, Eriomyces, Hydnobolites, Hydnotryopsis, Kalaharituber, Mattirolomyces, Mycoclelandia, Pachyphloeus, hypogeous Peziza, Ruhlandiella, Sphaerozone, Terfezia and Tirmania (Læssøe & Hansen, 2007). Our results based on both molecular and morphological characters yielded the discovery of yet another hypogeous lineage in the Pezizaceae and adds to our knowledge of truffle biodiversity. The proposed new genus Calongea brings the number of hypogeous genera in this family to fifteen. Healy & al. Calongea, a new genus of Pezizaceae 11 A comparison with other genera in the family The clade of hypogeous fungi that Calongea falls into includes Ruhlandiella, Terfezia, Tirmania, and a Peziza. In contrast to Calongea, Ruhlandiella lacks a peridium, the paraphyses and epithecium are gelatinous, and the spores are alveolate reticulate (Montecchi & Sarasini, 2000). The duplex structure of the peidium is similar to Peziza, but Peziza spores are ellipsoidal in shape. Among other genera with globose spores, Terfezia and Tirmania have a similar tissue type in their peridia, but they have a solid gleba, rather than hollow canals. Spore ornamentation resembles that of Terfezia leptoderma and T. olbiensis in variation of spine width, and distribution, and the wartiness along the spore wall surface between spines (see electron micrographs in Montecchi & Sarasini, 2000), which we interpret as incipient spines on Calongea spores. Molecular character analysis Previous studies based on LSU rDNA have had trouble resolving the higher order relationships between the major clades within the Pezizaceae (Hansen and Pfister, 2006; Læssøe and Hansen, 2007). Our results demonstrate a similar lack of resolution with this locus. Although we can say with confidence that Calongea and Pachyphloeus are distinct from each other, we have not been able to determine the closest epigeous or hypogeous relative of Calongea. The use of more genetic markers (e.g. RNA polymerase I & II), the discovery of new species, and greater taxon sampling could help resolve the relationships between Calongea and other genera within the Pezizaceae. ACKNOWLEDGEMENTS The authors are indebted to Efren Cazares for translation of the abstract. Healy thanks Dr. HT Horner and staff at the Nanotechnology, Imaging and Microscopy Facility at Iowa State University for use of their equipment for the SEM portion of this project. The authors thank Dr. Rytas Vilgalys for consultation and for generously sharing his lab and equipment to do molecular analysis. Trappe’s participation in the study was supported by the U.S. Forest Service, Pacific Northwest Research Station. These herbaria deserve thanks for efficiently processing our loan requests: Iowa State University Botany Department (ISC), Oregon State University Department of Botany and Plant Pathology (OSC), and Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas (UNL). Healy & al. Calongea, a new genus of Pezizaceae 12 REFERENCES Berkeley, M.J. & Broome, E.C. 1846. Notices of British Hypogeous Fungi. The Annals and Magazine of Natural History 18: 73-82. 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