Tuber regimontanum, new species of truffle from Mexico
Tuber regimontanum, una nueva especie de trufa de México
Gonzalo Guevara1, Gregory Bonito2, Efrén Cázares3, Julio Rodríguez4, Rytas Vilgalys2, James M. Trappe3
1 Instituto Tecnológico de Cd. Victoria, Av. Portes Gil 1301 Pte. C.P. 87010, A.P. 175. Cd. Victoria, Tam., México.
2 Department of Biology, Duke University Durham, NC 27708, USA.
]]> 3 Oregon State University, Dept. Forest Science, Richardson Hall 321, Corvallis, Oregon, 97331, USA.4 Facultad de Ciencias Biológicas, UANL Av. Pedro de Alba s/n cruz con Av. Manuel L. Barragán, Ciudad Universitaria. 66450 San Nicolás de los Garza, N.L., México
* Autor para correspondencia: guevaragg@hotmail.com
Recibido 22 de enero 2008
Aceptado 18 de junio 2008
Abstract
Tuber regimontanum is described as a new species from the N of Mexico (Nuevo Leon). It is close to T. melanosporum and T. indicum but these species have smaller spores then the described species. Molecular analysis of the the ITS ribosomal has shown these threes species are significatly differents. With the new species are until now six Tuber species known from Mexico.
Key words: Hypogeous fungi, Quercus, Tuber.
]]>Resumen
Se describe Tuber regimontanum como especie nueva del norte de México (Nuevo León). Esta especie es afín a T. melanosporum y T. indicum, pero estas dos especies presentan esporas mas pequeñas que la especie que se describe. Molecularmente y basándose en un análisis del ITS ribosomal estas tres especies difieren significativamente. Con la nueva especie son ya seis las especies de Tuber conocidas de México.
Palabras clave: Hongos hipogeos, Quercus, Tuber.
Introduction
In Mexico only five species of Tuber are known (Table 1) and two undescribed. In August of 2007 several black truffles in the genus Tuber were collected in a Quercus forest in the region of Sierra of Picachos in Nuevo León. Due to the uncertainty of its taxonomic affiliation to either T melanosporum or T. indicum, a ribosomal DNA study was performed. We conclude that this is a new species and we have described its morphological characteristics and phylogenetic placement.
Materials and methods
The methods of Gilkey (1939,1954), Castellano et al. (1989) and Pegler et al. (1993) were used for their morphological description. Hand cut sections were mounted in KOH 5% and Melzer reagent for microscopic examination. Compound microscope, stereoscope and Scanning Electron Microscope (SEM) were used for the microscopic analysis. For molecular analyses DNA was extracted with CTAB 2x buffer using the chloroform extraction technique. The ribosomal ITS and LSU regions were PCR amplified using a standard set of primers (ITS5 & LR5) and thermocycle conditions (Bertini et al. 1999; Gardes et al. 1991; Vilgalys & Hester, 1990). Four µl of each PCR product was loaded into a 1.0% agarose gel buffered with TAE buffer and stained with 2µl SYBR safe (Invitrogen, Carlsbad, CA) per 80 ml TAE buffer. Gel electrophoresis products were viewed on a GelDoc XR imager (Bio–Rad Laboratories, Inc., Hercules, CA). PCR products were cleaned using Qiagen Quick–Clean columns and used for PCR sequencing reaction. Sanger sequencing was performed using Big Dye chemistry version 3.1 (Applied Biosystems, Foster City, CA) with either the primer ITS5 (forward) or LR5 (reverse). DNA sequences were determined on an ABI3700 (Applied Biosystems, Foster City, CA). Unambiguous regions were excluded from the final ITS alignment and the dataset was analyzed using unweighted parsimony methods in PAUP 4.0b 10 (Swofford, 2001), using 1000 random addition sequences and 5000 bootstrap replicates. The North American species T. lyonii Butters, was chosen as an outgroup based on preliminary data on phylogenetic relationships across the genus Tuber. The ITS and LSU sequences of T. regimontanum and T. lyonii produced in this study were deposited in the GenBank under the access number EU375838 and EU3 94704, respectively.
]]>Results
Tuber regimontanum Guevara, Bonito & Rodríguez sp. nov Figs. 1–5
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Ascomata 10–40 x 8–18 mm, globosa, subglobosa vel lobata, atrobrunnea vel nigra, verrucis pyramidalibus usque ad 2–3 mm latis et 1–1.5 mm altis. Gleba solida, maturitate atrobrunnea vel negra venis albis marmoratis. Peridium 100–300 µm crassum, epicute pseudoparenchymatisque subcute intertextis. Sporae fusiformes vel ellipsoideae, 33–55 (–62) x 23–31 µm, maturitate porphyreae, spinis 2–5 x 1–2 µm. Asci globosi, subglobosi vel ovoidei, hyalini, acaules, sporis 1–4. Species interjecta Tuber melanosporum et T. indicum. (Holotypus Guevara 909, ITCV hic designatus).
Ascomata 10–40 x 8–17 x 8–18 mm, globose, subglobose to lobulated, dark brown to black, dry surface, rough with pyramidal verrucae 2–3 mm of wide x 1–1.5 mm of high, without basal mycelium or rhizomorphs. Gleba solid, white to light brown or dark, black when dried, with white veins toward the peridium, marbled. Odor fragrant and pleasant, flavor fungi–like. KOH 5% and FeSO4 10% negative or lightly black after 10min. Peridium 100–300 µn thick. Outer layer apseudoparenchyma of 100–250 µm wide, hyphae 5–30 µm diam. angular or isodiametric, reddish brown to dark coffee, thick walled, without intracelular contents. Inner layer of 30–50 µm thick, difficult to distinguished from the outer layer, interwoven hyaline hyphae. Veins formed by hyaline interwoven hyphae. Ascospores 33–55 (–62) x 23–31 µm without ornamentation, reddish brown; 1 spore in an asci are 40–55 (–62) x 30–31 µn, with two 37–42 x 25–26 µn, with three 33–37 x 23–26 µn, with four 28–35 x 18–22 µn, broadly fusiform to ellipsoid. Spines 2–5 x 1–2 µm, thin, robust toward the base, hyaline in youth, straw yellow to reddish brown when mature, thick walled. Asci globose to subglobose or ovoid, without pedicel, thick walled, 2–5 µm thick, hyaline.
Habitat. Hypogeous, gregarious, under a Quercus polymorpha forest, on the side of intermittent stream, soil pH 8.
Collections examined. Nuevo León, Sierra of Picachos, Mpio. de Higueras, August 23,2007, Guevara 909 (Holotype ITCV, isotypes OSU and DUKE).
Discussion. Tuber regimontanum is similar macro–and microscopically to the European species T. melanosporum Vitt. and the Chinese species T. indicum Cooke & Massee. T. melanosporum has spores 28–32 x 16–21 µm, blackish brown, while T. indicum, 26 x 17µm, brown–blackish (Montecchi & Sarasini, 2000). The molecular results using the ITS nuclear ribosomal DNA, demonstrated that T. regimontanum is significantly different from these two species at the molecular level (Fig. 5).
Etymology: Latin, regi– (region) and montanum (mountain), from the "mountainous regions" with respect to the Cd. of Monterrey where its citizens are denominated "regimontanos".
Acknowledgements
]]> The senior author would like to thank the DGEST (Dirección General de Educación Superior Tecnológica) for their financial support. The participation of Gregory Bonito, Efren Cazares, James Trappe and Rytas Vilgalys was supported by U.S. National Science Foundation Grants DEB–0641297 and DEB–0710213. We also thank Rosanne Healy for assistance with scanning electron microscopy at the University of Duke and sponsored by the NSF (Grant DBI–0098534).
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