Novel Tuber spp. (Tuberaceae, Pezizales) in the Puberulum Group from Mexico

Gonzalo GUEVARA-GUERRERO Summary: Recent taxonomic and molecular studies on Tuber in North America have revealed several new Gregory BONITO species. Here we propose Tuber bonitoi, T. brunneum, T. pseudoseparans and T. tequilanum as new species. Efrén CÁZARES-GONZÁLEZ These species are described from Mexico and differ in a combination of morphologically features including Rosanne HEALY ascospore size, pellis cell size, peridium thickness and ascoma color. These new taxa have phylogenetic affi- nity to Tuber separans, described from the United States. Phylogenetic placement of these novel Tuber species Rytas VILGALYS in the /puberulum clade are presented. James TRAPPE Keywords: Ascomycota, systematics, taxonomy, white truffles, phylogeny, biodiversity.

Résumen: Recientes estudios taxonómicos y moleculares en el género Tuber de Norteamérica han revelado Ascomycete.org, 7 (6) : 367-374. varias nuevas especies. Proponemos aquí Tuber bonitoi, T. brunneum, T. pseudoseparans y T. tequilanum como Novembre 2015 nuevas especies. Estas especies son descritas de México y difieren en una combinación de características Mise en ligne le 30/11/2015 morfológicas en el tamaño de la ascospora, tamaño de las células del epicutis, grosor del peridio y color de ascomas. Estos nuevos taxones tienen afinidad filogenética T.a separans descrita de Estados Unidos. La ubi- cación filogenética de estas especies nuevas deTuber en el clado /puberulum es presentado. Palabras clave: Ascomycota, sistemática, taxonomía, trufas blancas, filogenia, diversidad.

Introduction T. zhongdianense X. Y. He, Hai M. Li & Y. Wang, T. sinosphaerosporum L. Fan, J. Z. Cao & Yu Li, T. lijiangense L. Fan & J. Z. Cao, T. vesicoperi- dium L. Fan, T. microsphaerosporum L. Fan & Y. Li, T. alboumbilicum The genus Tuber P. Micheli ex F. H. Wigg. is one of the most impor- Y. Wang & Shu H. Li, T. pseudosphaerosporum L. Fan, T. panzhihua- tant truffle lineages given its ecological and economic value. Some nense X. J. Deng & Y. Wang, and T. microverrucosum L. Fan & C. L. Hou, species of Tuber (e.g. T. melanosporum Vittad., T. aestivum (Wulfen) but also several undescribed species with light color ascoma and Spreng, T. borchii Vittad.) are cultivated worldwide and contribute reticulate spores (BONITO et al., 2009, 2010; CHEN & LIU, 2007; FAN et al., to a multimillion dollar industry (HALL et al., 2007; MARTIN & BONITO, 2012; HALASZ et al., 2005; JEANDROZ et al., 2008; KINOSHITA et al., 2011; 2012; MELLO et al., 2006; PACIONI, 1999; PALENZONA et al., 1972; PAOLOCCI PAYEN et al., 2014; WANG et al., 2007). BONITO et al. (2009) further asses- et al., 2006; PAYEN et al., 2014). Tuber species are part of forest food sed diversity of these fungi through phylogenetic study on Tuber webs with links to vertebrate and invertebrate taxa, which play a from North America and showed that many species appear endemic role in truffle spore propagule dispersal ASER(M et al., 2008; MCGRAW to North America and include many taxa pending formal descrip- et al., 2002; TRAPPE et al., 2009). In addition, Tuber spp. form ectomy- tion. Tuber separans appeared as a complex of species distributed corrhizal associations with both conifers and woody flowering across the western United States, where it was described, into cen- plants and help their plant hosts acquire mineral nutrition and water tral and eastern Mexico (BONITO et al., 2009; CÁZARES et al., 1992; GUE- however this mutualist association is more complex than it seems VARA et al., 2013). The morphology of these southern collections only to be e.g. T. magnatum, and not much information on nutritional loosely fit the description T. separans. Closer morphological and mo- transfer exists (BIDARTONDO et al., 2004; KOVACS & JAKUCS, 2006; LALLI et lecular analysis has revealed cryptic diversity in the southern part al., 2015; TRAPPE et al., 2009; WALKER et al., 2005; WURZBURGER et al., of the T. separans lineage in Mexico. In this study we propose four 2001), related to this, T. aestivum could have detrimental effects on southern taxa as novel species for North America based on morpho- oaks and hazel seedlings (TORDA et al., 2014). Within the genus Tuber, logical and phylogenetic analyses: Tuber bonitoi, T. brunneum, the /puberulum clade is one of the most species diverse and geo- T. pseudoseparans and T. tequilanum. graphically widespread of the eleven recognized clades (BONITO et al., 2013; PAYEN et al., 2014). Truffles belonging to the /puberulum clade tend to be small to medium sized, white to brown in color with Material and Methods alveolate-reticulate spore ornamentation. Tuber borchii (aka ‘bian- chetto’) is one species in this clade that has economic importance Sample collection.— In this study more than 100 fruiting bodies (LOTTI et al., 2010). Despite their ecological and economical impor- of Tuber (ascomata) were studied from northeastern México. Specific tance, studies on this clade of truffles have been limited due to the localities are listed in the “specimens examined” sections in the in- taxonomic difficulty to delimitate species based on morphology, dividual descriptions. Specimens were preserved following recom- the small sized ascomata (of most species in this group), and their mendations of CASTELLANO et al. (1989), and when possible duplicate generally low economic value (HALASZ et al., 2005; MELLO et al., 2000; splits have been deposited in the herbaria José Castillo Tovar (ITCV, BONITO et al., 2010). index herbariorum), Oregon State University (OSC), Duke University The taxonomy of Tuber puberulum Berk. & Broome and related (DUKE). Previously accessioned herbarium specimens of Tuber, in- species e.g. T. separans Gilkey has been controversial in the past (GIL- cluding type collections from OSC and ITCV, were also examined du- KEY, 1939, 1954; HARKNESS, 1899). Recent molecular phylogenetic stu- ring this study. dies from North America, Europe and Asia have shown that the Morphological observations.— Morphological data were ob- /puberulum clade is comprised of the species T. separans, T. borchii, tained by the methods of CASTELLANO et al. (1989), GILKEY (1939), and T. californicum Harkn., T. sphaerosporum Gilkey, T. puberulum, T. liui PEGLER et al. (1993). Examined characters included ascoma size, sur- A S. Xu, T. dryophilum Tul. & C. Tul., T. oligospermum Tul. & C. Tul., T. ra- face texture and color, peridial structure; spore length and width paeodorum Tul. & C. Tul., T. maculatum Vittad., T. foetidum Vittad., (excluding ornamentation), length/width ratio (Q), shape, wall thick- T. cruposum R. Hesse, T. whetstonense J.L. Frank, D. Southw. & Trappe, ness, number of reticular meshes along and across spore axes, T. latisporum Juan Chen & J. G. Liu, T. huizeanum L. Fan & C. L. Hou, height of the meshes, color and ascus size, shape, wall thickness, T. cistophilum P. Alvarado, G. Moreno, Manjón, Gelpi & J. Muñoz, and number of spores per ascus. Hand-cut sections were mounted 367 in 5% KOH and Melzer’s reagent for light microscopy. Spore measu- Peridium 200–550 μm thick; pellis a pseudoparenchyma 50– rements of Tuber spp. in KOH compared to those in water showed 300 μm thick, the cells 5–53 μm broad, isodiametric to slightly an- no KOH effect (J. Trappe, unpublished data). Microscopic structures gular, the widest surrounded by smaller cells resulting in a “rosette were measured and photographed under a light microscope and like” appearance (Fig. 2 C), pubescent in some areas or grouped to stereomicroscope. form warts, hyaline to yellowish orange in KOH, the walls 2 μm thick; DNA sequencing and phylogenetic analyses.— Molecular pro- dermatocystidia 35–88 × 5–6 μm, single, clustered or turf, tapered tocols follow those of GUEVARA et al. (2008). DNA was extracted from to the tip, some sinuate, fragile, thin and irregular-walled, septate; truffle fruiting bodies with the chloroform extraction technique subpellis 75–400 μm thick, hyaline interwoven hyphae, 5– 10 μm using CTAB 2X DNA extraction buffer. The ITS region was amplified broad at the septa, thin to thick (1–3 μm) walled. Gleba of hyaline, with the primer pair ITS1f-LR5 (GARDES & BRUNS, 1993; VILGALYS & HES- parallel to interwoven, thin-walled hyphae, 3.5–10 μm broad at the TER, 1990; WHITE et al., 1990). PCR products were cleaned of single septa, the cells commonly cylindrical to slightly inflated. stranded oligonucleotides enzymatically with antarctic phospha- Ascospores broadly ellipsoid, subglobose or sometimes globose; tase and endonuclease digestion (New England Biolabs). Sanger se- thick walled, excluding their alveolate-reticulate ornamentation, in quencing was performed by Big Dye chemistry v3.1 with the 1-spored asci 35–50 × 33–45 μm (Q=1.0–1.51), 2-spored 30 –48 × forward primer ITS5 (WHITE et al., 1990) and reverse primers LR5. DNA 25–38 μm (Q=1.06–1.35), 3-spored 23–43 × 20–33 μm (Q=1.0–1.43), sequences were determined on an ABI 3700 capillary sequencer. and 4-spored 22–35 × 20–33 μm (Q=1.0–1.4), the walls up to 2 μm DNA sequences were viewed and manually edited in Sequencher thick and yellowish in KOH; reticulum with (6–)7–9 meshes along 4.0. Sequences were queried against GenBank with the BLASTN al- the spore length and 6–7 across, the alveolar walls 5 μm tall. Asci gorithm to verify that sequences belonged to Tuber. Sequences 82–100 × 50–82 μm, globose, subglobose to broadly ellipsoid, ses- were aligned with MUSCLE (EDGAR, 2004). Alignments were manually sile to pedicellate and bifurcate in some, the walls up to 2 μm thick, checked and ambiguous regions were excluded in Mesquite 2.5 with 1, 2 or 3 layers, hyaline in KOH. (MADDISON & MADDISON, 2009). Phylogenetic analyses were conducted with maximum likelihood Distribution, habitat and season: Mexico, Veracruz, under Pinus (ML) in PAUP* (SWOFFORD, 2002). The best fit nucleotide substitution hartwegi, in a mountainous forest, with Abies religiosa. September. model was based on the Akaike information criterion and was im- Specimens examined: HOLOTYPE. MEXICO. Veracruz, Cofre de Pe- plemented in PAUP* 4d106 (SWOFFORD, 2002). ML bootstrap support rote, road Los Conejos-La Peña, Municipality of Xalapa, elev. 3000 m, based on 1000 replicates was assessed with RAxML (STAMATAKIS et al., 18 Sept. 2007, leg. E. Cázares and R. Vilgalys, Trappe 32421 (OSU). 2008) and Bayesian inference and posterior probabilities (HUELSEN- ISOTYPE Guevara 913a (ITCV 913a). BECK, 2001) were carried out on the CIPRES Science Gateway (MILLER et al., 2010). Levels of interspecific ITS variation were determined by aligning sequences from the species of interest to their closest sister Comments: Tuber bonitoi is a beautiful and perhaps the largest white truffle species in Mexico.Tuber brunneum differs in its brown taxa in MUSCLE (EDGAR, 2004). The alignments were then manually color, pellis with slightly smaller cells, slightly longer and narrower edited, but no regions were excluded. Uncorrected P values resul- ascospores, and more numerous spores per ascus. Tuber separans ting from these ITS alignments were then calculated in PAUP* 4d106 differs in its overall thinner peridium, smaller pellis cells, and smaller (SWOFFORD, 2002). Sequences produced in this study are deposited ascospore range (Table 1). in GenBank under accession numbers KT897472-KT897487 (Table 2). Tuber brunneum Guevara, Bonito & Trappe, sp. nov. – Fig. 2 E–G – Results Mycobank MB814688 – Genbank KT897474. Holotypus hic designatus: R. Vilgalys, J.Trappe 33835 (OSU). Phylogenetic analysis of 54 referenced taxa included 494 charac- ters, 208 of which were parsimony-informative. As previous studies Etymology: In reference to the brown color of the ascoma. have shown, the /puberulum and /maculatum clades, were distinct from each other in ML and BI analyses (Fig 1). The designation of Ascomata 14–17 mm broad, globose to subglobose or irregular, T. bonitoi, T. brunneum, T. pseudoseparans and T. tequilanum as new dark brown, white to cream in some areas. Peridium <0.5 mm, species is supported by ITS rDNA analysis and morphological cha- smooth to velvety, irregularly roughened, verruculose in small areas racters. up to 8 warts per mm, white furrows and depressions with white- cream surface hairs. Gleba solid, light brown to brown, marbled with white veins that emerge as depressions. Odor pleasant. Taste Taxonomy not recorded. Peridium 180–400 μm thick; pellis a pseudoparenchyma 50– Tuber bonitoi Guevara & Trappe, sp. nov. – Fig. 2 A–D – Mycobank 300 μm thick, the cells 5–30 μm broad, versiform, isodiametric or MB 814687 – Genbank KT897472. angular, hyaline, yellowish in KOH, the walls 2.0 μm thick; dermato- Holotypus hic designatus: J. Trappe 32421 (OSU). cystidia 37–67 × 2–5 μm, claviform, constricted, sinuate, branched, some tapered to the tip, clustered, fragile, hyalines, thin-walled, sep- Etymology: bonitoi in honor to the American mycologist Gregory tate; subpellis 75–210 μm thick, of hyaline, periclinal to interwoven Bonito for his contributions to knowledge of Tuber spp. in North hyphae 2–5 μm broad at the septa, cell walls 1–2 μm thick. Gleba America (and worldwide) and being a mentor for others who study of hyaline, interwoven, thin-walled hyphae, 2.5–6 μm broad at the the group. septa, the cells commonly cylindrical interwoven to inflated in some areas, 2–5μm broad. Ascospores broadly ellipsoid to subglobose or Ascomata 30–47 mm broad, globose, subglobose, irregular or sometimes globose; excluding their alveolate-reticulate ornamen- gibbose, very brittle, yellow cream, light brown to red-orange brown tation, in 1-spored asci 38–57 × 30–43 μm (Q=1.0–1.5), 2-spored in some areas, with a pinkish stain, finely verruculose in some areas, (21–) 35–43 × 21–35 μm (Q=1.0–1.4), 3-spored 23–43 × 23–33 μm 9–10 warts/mm. Peridium thin (< 0.6 mm), smooth to tomentose (Q=1.0–1.30), 4-spored 23–33 × 20–30 μm (Q=1.0–1.25), and 5-spo- (in depressions), roughened to irregular, dry, with furrows, not de- red 25–33 × 20–25 μm (Q=1.0–1.32), the walls up to 2 μm thick, red- tachable. Gleba marbled, solid, with white to grey veins. Odor fra- dish brown to brown in KOH, reddish brown in Melzer’s reagent; grant, of nuts or bread when dried. Taste not recorded. reticulum with 3–9 meshes along the spore length and 3–7 across, 368 the alveolar walls 1–2.5 μm tall, with subreticulation inside the al- Distribution, habitat and season: Mexico, Jalisco, in a pine-oak veolus in many. Asci 68–132 × 42–97 μm, subglobose to broadly el- forest under Quercus magnolifolia. In fall at 1470 m. lipsoid, pedicel lacking to prominent, the walls 1-3 layers, hyaline in Specimens examined: HOLOTYPE. MEXICO. Jalisco, La Primavera, KOH, yellow in Melzer’s reagent. Cells around asci cylindrical to iso- Municipality of Guadalajara, elev. 1470 m, 29 Sept. 009, leg. R. Vilga- diametric in some areas 2.5-6 μm wide at septum, thin walled, hy- lys, Trappe 33835 (OSU). PARATYPES. 28 Sept. 2009, Trappe 33837 lines in KOH. (OSU); 29 Sept. 2009, Trappe 33836, 33837, 33820, 33822, 33827,

Figure 1 – Phylogeny of the Tuber puberulum and T. maculatum clades based Maximum Likelihood analyses and Bayesian inference of ITS rDNA a GTR+I+G model of nucleotide substitution. One of three most likely trees (-l = 5440.027) Reference taxa are provided followed by their GenBank accession number. Sequences from holotype collections are labelled. The /maculatum clade is shown in gray; the /puberulum clade in black. Orange astrices indicate nodes with posterior probabilities >98 and bootstrap values >70. 369 33828, 33830, 33831, 33834 (OSU); 2 Oct. 2009, Trappe 33839b Tuber tequilanum Guevara, Bonito & Trappe, sp. nov. – Fig. 2 L–O (OSU). – Mycobank MB814690 – Genbank KT897482. Holotypus hic designatus: México. M. Castellano, Trappe 33796 (OSU). Comments: Tuber brunneum can be recognized by its brown color Etymology: The epithet tequilanum pertaining to origin or place, with white depressions. Aside from color difference,T. separans dif- in reference to the locality of the type collection along the road to fers in having a narrower spore range, and fewer spores per ascus. Tequila volcano in the Municipality of Tequila, Jalisco, Mexico. Tuber bonitoi differs in having a thicker peridium, with larger pellis cells in the upper range. Tuber pseudoseparans differs in its white as- Ascomata 10–20 mm broad, subglobose, ellipsoid or irregular, coma, thinner peridium, slightly larger pellis cells, and overall larger fragile, white to yellowish cream, light brown when handling in ascospores (Table 1). some areas. Peridium thin (<0.3 mm), smooth to villous, with de- pressions that connect with veins. Gleba marbled, solid, dark brown with white veins. Odor pleasant. Taste not recorded. Tuber pseudoseparans Guevara, Bonito & Trappe, sp. nov. – Fig. 2 Peridium 120–230 μm thick; pellis a pseudoparenchyma 50– H–K – Mycobank MB814689 – Genbank KT897480. 70 μm thick, the cells 4–20 μm broad, versiform to angular or iso- diametric, some areas prostrate interwoven cells, hyaline to Holotypus hic designatus: E. Cázares, Trappe 33778 (OSU). yellowish in KOH, the walls >1 μm thick; dermatocystidia 25–75 × 4–7 μm, branched, clustered, claviform, some tapered to the tip, fra- Etymology: The epithet pseudoseparans given in reference to the gile, with irregular cell wall, thin-walled, septate, hyaline in KOH; sub- false similarity with T. separans. pellis 80–140 μm thick, of hyaline, periclinal to interwoven hyphae 2–5 μm broad at the septa, thick walled. Gleba of hyaline, loosely Ascomata 10–12 mm broad, subglobose or irregular, white, interwoven, thin-walled hyphae, 2.5–5 μm broad at the septa, the cream, light brown in some areas when handling. Peridium thin, cells commonly cylindrical. Ascospores broadly fusiform; excluding <0.2 mm, undetachable, smooth to velvety, irregularly roughened, their alveolate-reticulate ornamentation, in 1-spored asci 42–55 × furrows and depressions continuing as vein into the gleba, with 30–38 μm (Q=1.28–1.57), 2-spored 32–48 × 25–35 μm (Q=1.14–1.5), 3-spored 22–42 × 18–30 μm (Q=1.05–2.0), 4-spored 20–40 × 16– white-cream hyphal hairs, dry. Gleba solid, light brown, marbled 31 μm (Q=1.05–1.5), and 5-spored 17–32 × 14–26 μm (Q=1.05–1.53), with white veins that emerge as depressions on peridium. Odor thick walled, reddish brown in KOH; reticulum with 5–7 (–8) meshes pleasant. Taste not recorded. along the spore length and 3–5 (–8) across, the alveolar walls up to Peridium 125–190 μm thick; pellis a pseudoparenchyma 75– 4 μm tall, some spores with incomplete or irregular reticulation. Asci 125 μm thick, the cells 5–38 μm broad, versiform, isodiametric, squa- 69–90 × 50–84 μm, broadly ellipsoid to subglobose, pedicel lacking red, rectangular or angular, in some areas prosenchyma, in others to prominent, the wall formed by 2-3 layers, hyaline in KOH, the cells anticline, hyaline to yellowish in KOH, thick walled (>1.0 μm); der- around the asci commonly cylindrical, prostrated or interwoven, 3– matocystidia 15–80 × 4–10 μm, claviform, constricted, sinuate, bran- 5 μm broad, thin walled, hyaline in KOH. ched, some tapered to the tip, clustered, fragile, hyalines in KOH, Distribution, habitat and season: Mexico, Jalisco, in a pine-oak septate, thin-walled, many with irregular cell walls; subpellis 30– forest under Quercus magnolifolia. In fall at 1575 m. 100 μm thick, of hyaline, periclinal to interwoven hyphae 3–10 μm

broad at the septa, cell wall 1–2 μm thick. Gleba of hyalines, inter- Specimens examined: HOLOTYPE. MEXICO. Jalisco, Municipality woven, thin-walled hyphae, 3–5 μm broad at the septa. Ascospores of Tequila, Volcano Tequila road Km. 6, elev. 1575-1855 m, 28 Sept. broadly ellipsoid to fusiform; excluding their alveolate-reticulate or- 2009, M. Castellano, Trappe 33796 (OSU). PARATYPE. Trappe 33794, namentation, in 1-spored asci 46–65 × 33–46 μm (Q=1.21–1.6), 2- 33795, 33798, 33810, 33812 (OSU); Km. 20, Trappe 33803, 33805, spored (21–) 34–52 × 24–40 μm (Q=1.21–1.55), 3-spored 23–50 × 33806 (OSU); Volcano Tequila road Km. 11, 2170 m, 25 Sept. 2009, 19–35 μm (Q=1.0–1.78), 4-spored 20–41 × 18–30 μm (Q=1.0–1.6), Trappe 33755, 33758 (OSU); Sierra de Quila, El Estuche Grande, 27 and 5-spored 20–37 × 15–30 μm (Q=1.06–1.5) the walls > 2 μm Sept. 2009, Trappe 33790 (OSU); Municipality of Guadalajara, Parque La Primavera, 2 Oct. 2009, Trappe 33843, 33844, 33846, 33852 (OSU). thick, yellow, orange yellow in KOH; reticulum with 3–8 meshes along the spore length and 2– 6 across, the alveolar walls 2–2.5 μm Comments: Tuber tequilanum is distinctive in its stark white color tall. Asci 73–100 × 55–83 μm, globose, subglobose to broadly ellip- of its peridium, which browns upon handling or disturbance. In ad- soid, pyriform, pedicel lacking to prominent, the walls 2–3 layers (as dition, T. separans differs from T. tequilanum in its overall shorter in T. lyonii), hyalines in KOH, yellow in Melzer’s reagent, hyphae ascospores, larger pellis cells in the upper range, and asci in a single around the asci prostrated or interwoven, cylindrical, 5-7.5 μm wide layer. Tuber bonitoi has a thicker peridium with larger pellis cells, and at the septa, thin walled, hyaline in KOH. a wider range of ascospore sizes. Tuber pseudoseparans differs in its overall larger ascospores (Table 1). Distribution, habitat and season: México, Jalisco, hypogeous in a pine-oak forest under Quercus magnolifolia. In fall at 2185 m. Discussion

Specimens examined: HOLOTYPE. MEXICO. Jalisco, Municipality Species in the /puberulum clade are generally recognized by their of Tequila, Sierra de Quila north of “El Vallado”, elev. 2185 m, 26 Sept. white to pale color and small size, and native species belonging to 2009, leg. E. Cázares, Trappe 33778 (OSU). PARATYPE. Trappe 33774 this group are found throughout North America, Europe, and Asia, (OSU). and parts of Northern Africa and South America (BONITO et al., 2010, 2013; JEANDROZ et al., 2008; PAYEN et al., 2014). In some cases, it ap- pears that species belonging to the /puberulum clade have been Comments: Tuber pseudoseparans has overall larger ascospores introduced into other continents unintentially e.g. T. rapaeodorum than the other species described here. In addition, T. separans differs in Australia and New Zealand (BONITO et al., 2010). from T. pseudoseparans in having fewer spores per ascus, T. bonitoi By compiling collection and environmental sequence data, BONITO differs in its thicker pellis, and T. brunneum differs in its brown as- et al. (2010) estimated that around 25 undescribed taxa belong to coma. (Table 1). the /puberulum clade. Species delimitation based soley on morpho- 370 Figure 2 – A–D. Tuber bonitoi. A. Ascoma (bar=1 cm). B. Ascospore (bar=10 μm). C. Cross section of peridium of a pseudoparechymatous pellis with “roseta” (arrow) (bar=10 μm). D. Cystidia with irregular cell wall (arrow), Trappe 32421 (OSU, holotype). E–H. T. brunneum. E. Ascoma (bar=1 cm). F. Ascospore/1 asci (bar=10 μm ). G. Cross section of peridium of the pseudoparenchymatous pellis, Trappe 33835 (OSU, holo- type). H–K. T. pseudoseparans. H. Ascoma (bar 1 cm). I. Ascospore (bar=10 μm). J. Cross section of peridium of pseudoparechymatous cells. K. Cystidia, Trappe 33778 (OSU, holotype). L–O. T. tequilanum. L. Ascoma (bar=1 cm). M. Ascospore (bar=10 μm). N. Cross section of peridium of the pseudoparechymatous pellis. O. Cystidia, Trappe 33796 (OSU, holotype).

logy has been controversial for fungi and for truffles. For truffles wi- discovered (BONITO et al., 2010, 2013; KINOSHITA et al., 2011; WANG et thin the /puberulum group, molecular divergences between taxa al., 2013). appear to be easier to detect than are the morphological diver- Plant hosts of truffles in the /puberulum group include angios- gences. Recently, many new truffle species in this clade have been perms and conifers (BIDARTONDO et al., 2004; BONITO et al., 2010). Three described from vouchered collections in Asia. We expect that the of the new species described here, Tuber brunneum, T. pseudosepa- high richness and shallow branching patterns within the /puberu- rans and T. tequilanum, were found under Quercus magnolifolia bet- lum group are due to both its wide geographic distribution and its ween 1470 and 2185 m elevation, but T. bonitoi was found under recent radiation, estimated at between 65 and 53 million years (BO- Pinus teocote, P. montezumae and Abies religiosa, at 3000 m asl. Host NITO et al., 2013; PAYEN et al., 2014). preferences have been observed for truffle species within the Here we use morphology and phylogenetic analyses of the ITS rDNA to delimit the species Tuber bonitoi, T. brunneum, T. pseudose- /rufum, /excavatum, /aestivum, /maculatum and /gennadii clades, parans and T. tequilanum to the list of truffles belonging to the /pu- which appear to be more commonly associated with angiosperm berulum group that includes Tuber separans, T. anniae, T. hosts, while species in the /japanicum and /gibbosum clades appear californicum, T. menseri ad. int., T. liui, T. zhongdianense, T. asa, T. la- to prefer hosts in the Pinaceae. In contrast, species in the /puberu- tisporum, T. sphaerosporum, T. dryophylum, T. puberulum, T. oligosper- lum clade tend to have a wider range host association (PAYEN et al., mum and T. borchii, along with many species yet to be described or 2014). Host-specificity, for species such as the black truffleT. mela- 371 372

Table 1 – Comparative morphology of the novel Tuber species described vs. T. separans (holotype)

Species and holo- Peridium surface Peridium color and Pellis and cell size Subpellis Dermatocystidia Spore size (range) Spore shape Number Geography of type collection # thickness without reticula- spore/asci the holotype tion

T. separans Smooth to slightly Light yellow brown Pseudoparenchyma, Prosenchyma, No recordered 31–50 × 24–37 μm Broadly ellipsoid 1–3 (–4) California, USA H.E. Parks 1052 rimose-papilose ± 250 μm cells 6–30 μm cells 3–5 μm Holotype

T. bonitoi Smooth to puberu- Yellow with pink Pseudoparenchyma, Prosenchyma 35–88 × 5–-6 μm 22–50 × 22–45 μm Broadly ellipsoid, 1–3 (–4) Veracruz, MX JT 32421 (OSU) lent dots 200–550 μm 150–300 μm cells 5– 75–400 μm, cells subglobose 53 μm 5–10 μm some globose

T. brunneum Smooth to finely Brown with white Pseudoparenchyma Prosenchyma 37–67 × 2–5 μm 23–53 × 20–40 μm Broadly ellipsoid, 1–4 (–5) Jalisco, MX JT 33835 (OSU) puberulent furros 180–400 μm 50–300 μm, cells 5– 75–210 μm, cells globose to sub- 30 μm 2–8 μm globose

T. pseudoseparans Smooth to finely White cream to light Pseudoparenchyma Prosenchyma 15–80 × 4–10 μm 46–65 × 34–46 μm Broadly ellipsoid 1–4 (–5) Jalisco, MX JT 33778 (OSU) puberulent brown 125–190 μm 75–125 μm, cells 5– 30–100 μm, cells to broadly fusi- 38 μm 3–10 μm form

T. tequilanum Smooth to puberu- White, grey to Pseudoparenchyma Prosenchyma 25–75 × 4–7 μm 42–55 × 30–38 μm Broadly fusiform 1–4 (–5) Jalisco, MX JT 33796 (OSU) lent cream 120–230 μm 50–70 μm, cells 4– 80-140 μm, cells 20 μm 2.5–5 μm Table 2 – Accession and voucher numbers of novel taxa

Taxon Genbank numbers Voucher number T. bonitoi KT897472 JT 32421 (OSU), holotype KT897473 913a (ITCV) T. brunneum KT897474 JT 33835 (OSU), holotype KT897475 JT 33836 (OSU) KT897476 JT 33820 (OSU) KT897477 JT 33827 (OSU) KT897478 JT 33830 (OSU) KT897479 JT 33837 (OSU) T. pseudoseparans KT897480 JT 33778 (OSU), holotype KT897481 JT 33774 (OSU) T. tequilanum KT897482 JT 33796 (OSU), holotype KT897483 JT 33794 (OSU) KT897484 JT 33803 (OSU) KT897485 JT 33755 (OSU) KT897486 JT 33790 (OSU) KT897487 JT 33852 (OSU)

nosporum, could be caused by ectomycorrhiza-specific genes, which cificity, and long-distance dispersal. Molecular Ecology, 19 (22): help to modulate plant-fungal interactions (MARTIN & BONITO, 2012). 4994-5008. The genus Tuber is characterized by high levels of continental en- BONITO G., SMITH M.E., NOWAK M, HEALY R.A, GUEVARA G, CÁZARES E. KINO- demism e.g. T. gibbosum in North America, T. gennadii and T. multi- SHITA A., NOUHRA E.R., DOMINGUEZ L. S., TEDERSOO L., MURAT C., WANG Y., maculatum in Europe, T. japonicum in Asia (PAYEN et al., 2014). ARROYO MORENO B., PFISTER D. H., NARA K., ZAMBONELLI A., TRAPPE J. M. Putatively, this pattern could be the result of dispersal limitation. & VILGALYS R. 2013. — Historical Biogeography and Diversification While the /puberulum clade is geographically widespread, the spe- of Truffles in the Tuberaceae and Their Newly Identified Southern cies described here appears to be recently diverged along the sou- Hemisphere Sister Lineage. PLoS ONE, 8(1): e52765. doi: 10.1371/ thern range of North American ‘Tuber separans complex’, differing journal.pone.0052765 phylogenetically and morphologically from related species in more CASTELLANO M.A., TRAPPE J.M., MASER Z. & MASER C. 1989. — Key to spores northern latitudes. Further research on these taxa may provide clea- of the genera of hypogeous fungi of north temperate forest with spe- rer understanding of the genetic and environmental factors related cial reference to animal mycophagy. Eureka, Mad. River Press, 186 p. to speciation in truffles. CÁZARES E., GARCIA J., CASTILLO J. & TRAPPE J.M. 1992. — Hypogeous fungi from northern Mexico. Mycologia, 84 (3): 341-359. CHEN J. & LIU P. 2007. — Tuber latisporum sp. nov. and related taxa Acknowledgements based on morphology and DNA sequence data. Mycologia, 99 (3): 475-481. Gonzalo Guevara thanks CONACYT, DGEST (Dirección General de EDGAR R.C. 2004. — Muscle: A multiple sequence alignment method Educación Superior Tecnológica) and PROMEP (Programa para el with reduced time and space complexity. BMC Bioinformatics, 5: Mejoramiento del Profesorado) for funding this research. Participa- 1-19. tion by Efrén Cázares and James Trappe was supported by the U.S. FAN L., CHENG-LIN H. & YU L. 2012. — Tuber microverrucosum and T. hui- Forest Service, Pacific Northwest Research Station. We thank Jesús zeanum―Two new species from China with reticulate ascospores. García from Tecnológico Nacional de México for comments and sug- Mycotaxon, 122: 161-169. gestions. These herbaria generously accessioned and curated our GARDES M. & BRUNS T.D. 1993. — ITS primers with enhanced specificity collections: Duke University, Durham, North Carolina (DUKE); Insti- for basidiomycetes - application to the identification of mycorrhi- tuto Tecnológico de Ciudad Victoria, México (ITCV); Oregon State zae and rusts. Molecular Ecology, 2 (2): 113-118. University, Corvallis (OSC). Gregory Bonito acknowledges NSF pro- GILKEY H.M. 1939. — Tuberales of North America. Oregon State Mo- ject 0641297, Michigan State University and AgBioResearch for sup- nographs Studies in Botany, 1: 1-63. port. GILKEY H.M. 1954. — Tuberales. North America Flora 2: 1-36. GUEVARA G., BONITO G., CÁZARES E., RODRIGUEZ J.A. & VILGALYS R. 2008. — Tuber regimontanum, new species of truffle from Mexico.Revista References Mexicana de Micología, 26: 17-20. GUEVARA G., BONITO G. & CÁZARES E. 2013. — Revisión del género Tuber BIDARTONDO M.I., BURGHARDT B., GEBAUER G., BRUNS T.D. & READ D.J. (Tuberaceae: Pezizales) de México. Revista Mexicana de Biodiversi- 2004. — Changing partners in the dark: Isotopic and molecular dad, 84: 39-49. evidence of ectomycorrhizal liaisons between forest orchids and HALÁSZ K., BRATEK Z., SZEG D., RUDNÓY S., RÁCZ I., LÁSZTITY D. & TRAPPE J.M. trees. Proceedings of the Royal Society of London, B, 271 (1550): 2005. — Tests of species concepts of the small, white, European 1799-1806. group of Tuber species based on morphology and rDNA se- BONITO G., TRAPPE J.M. & VILGALYS R. 2009. — North American Truffle in quences with special reference to Tuber rapaeodorum. Mycological the Tuberaceae: Molecular and Morphological Perspectives. Acta Progress, 4 (4): 291-298. Botanica Yunnanica, 16: 39-51. HALL I.R, BROWN G.T. & ZAMBONELLI A. 2007.— Taming the Truffle. The BONITO G., GRYGANSKYI A., TRAPPE J. & VILGALYS R. 2010. — A global meta- history, Lore, and Science of the Ultimate Mushroom. Portland Tim- analysis of Tuber ITS rDNA sequences: Species diversity, host spe- ber Press, 304 p. 373 HARKNESS H.W. 1899. — Californian hypogæous fungi. Proceedings of PALENZONA M., CHEVALIER G. & FONTANA A. 1972. — Sintesi micorrizica the California Academy of Sciences, 3rd ser. Botany, 1 (8): 241-292. tra i miceli in coltura di Tuber brumale, T. melanosporum, T. rufum HUELSENBECK J.P. & RONQUIST F. 2001. — MrBayes: Bayesian inference e semezali di conifere e latifoglie. Allionia, 18: 42-52. of phylogenetic trees. Bioinformatics, 17 (8): 754-755. PAOLOCCI F., RUBINI A., RICCIONI C. & ARCIONI S. 2006. — Reevaluation of IOTTI M., LANCELLOTTI E., HALL I. & ZAMBONELLI A. 2010. — The ectomy- the life cycle of Tuber magnatum. Applied and Environmental Mi- corrhizal community in natural Tuber borchii grounds. FEMS Mi- crobiology, 72 (4): 2390-2393. crobiology Ecology, 72 (2): 250-260. PAYEN T., MURAT C. & BONITO G. 2014. — Truffle phylogenomics: New JEANDROZ S., MURAT C., WANG Y.J., BONFANTE P. & LE TACON F. 2008. — Mo- insights into truffle evolution and truffle life cycle.In : MARTIN F. lecular phylogeny and historical biogeography of the genus (ed.). Advances in Botanical Research, 70: 211-234. Tuber, the ‘true truffles’. Journal of Biogeography, 35 (5): 815-829. PEGLER D.N., SPOONER B.M. & YOUNG T.W.K. 1993. — British truffles, a re- KOVÁCS G.M. & JAKUCS E. 2006. — Morphological and molecular com- vision of British hypogeous fungi. Kew, The Royal Botanic Gardens, parison of white truffle ectomycorrhizae.Mycorrhiza , 16 (8): 567- 216 p. + 26 pl. 574. STAMATAKIS A., HOOVER P. & ROUGEMONT J. 2008. — A rapid bootstrap al- KINOSHITA A., SASAKI H. & KAZUHIDE N. 2011. — Phylogeny and diversity gorithm for the RAxML web servers. Systematic Biology, 57 (5): of Japanese truffles (Tuber spp.) inferred from sequences of four 758-771. nuclear loci. Mycologia, 103 (4): 779-794. SWOFFORD D.L. 2002. — Paup* phylogenetic analysis using parsimony LALLI G., LEONARDI M., ODDIS M., PACIONI G., SALERNI E., IOTTI M. & ZAMBO- (*and other methods). Sunderland, Sinauer Associates. Version NELLI A. 2015. — Expanding the understanding of a forest ectomy- 4.0. corrhizal community by combining root tips and fruiting bodies: TRAPPE J.M. & CÁZARES E. & GUZMÁN G. 1971. — Notes on some hypo- a case study of Tuber magnatum stands. Turkish Journal of Botany, geous fungi from Mexico. Mycologia, 63 (2): 317-332. 39: 527-534. TRAPPE J.M., MOLINA R., LUOMA D.L., CÁZARES E., PILZ D., SMITH J.E., CASTEL- MADDISON W.P. & MADDISON D.R. 2009. — Mesquite: A modular system LANO M.A., MILLER L. & TRAPPE M.J. 2009. — Diversity, ecology and for evolutionary analysis. http://mesquiteproject.org. Version 2.6. conservation of the truffle fungi in forests of the Pacific northwest. MARTIN F. & BONITO G. 2012. — Ten years of genomics for ectomycor- US Dept. of Agriculture, Forest Service General Technical Report, rhizal fungi: what have we achieved and where are we heading. PNW-GTR-772. In: ZAMBONELLI A. & BONITO G.M. (eds.). Edible ectomycorrhizal mush- VARGA T., MERÉNYI Z., BRATEK Z. & SOLTI Á. 2014. — Mycorrhizal coloni- rooms. Berlin, Springer-Verlag: 381-401. zation by Tuber aestivum has a negative effect on the vitality of MASER C., CLARIDGE A.W. & TRAPPE J.M. 2008. — Trees, truffles, and beasts–how forests function. New Brunswick, Rutgers University oak and hazel seedlings. Acta Biologica Szegediensis, 58 (1): 49-53. Press, 281 p. VILGALYS R. & HESTER M. 1990. — Rapid genetic identification and map- ping of enzymatically amplified ribosomal DNA from several Cryp- MCGRAW R., DUNCAN N. & CÁZARES E. 2002. — Fungi and other items consumed by the blue-gray taildropper slug (Prophysaon coeru- tococcus species. Journal of Bacteriology, 172 (8): 4238-4246. leum) and the papilose taildropper slug (Prophysaon dubium). Ve- WALKER J.F., MILLER O.K. & HORTON J.L. 2005. — Hyperdiversity of ecto- liger, 45: 261-264. mycorrhizal fungus assemblages on oak seedlings in mixed fo- MELLO A., MURAT C. & BONFANTE P. 2006. — Truffles: Much more than a rests in the southern Appalachian Mountains. Molecular Ecology, prized and local fungal delicacy. FEMS Microbiology Letter, 260: 1- 14 (3): 829-838. 8. WANG Y., TAN Z.M., MURAT C., JEANDROZ S. & LE TACON F. 2007. — Mole- MELLO A., VIZZINI A., LONGATO S., ROLLO F., BONFANTE P. & T RAPPE J.M. cular taxonomy of Chinese truffle belonging to theTuber rufum 2000. — Tuber borchii versus Tuber maculatum: neotype studies and Tuber puberulum groups. Fungal Diversity, 24: 301-328. and DNA analyses. Mycologia, 92 (2): 326–331. WHITE T.M., BRUNS T., LEE S. & TAYLOR J. 1990. — Amplification and direct MILLER M.A., PFEIFFER W. & SCHWARTZ T. 2010. — Creating the CIPRES sequencing of fungal ribosomal RNA for phylogenetics. In: INNIS Science Gateway for inference of large phylogenetic trees. Pro- M.A., GELFAND D.H., SNINSKY J.J. & WHITE T.J. (eds). PCR protocols: A ceedings of the gateway Computing Environments Workshop guide to methods and applications. San Diego, Academic Press: (GCE): 14 Nov 2010, New Orleans, 1–8. http:// www.ngbw.org/ 315-321. ee/index.php/portal/cite_us. WURZBURGER N., BIDARTONDO M.I. & BLEDSOE C.S. 2001. — Characteriza- PACIONI G. & COMANDINI O. 1999. —Tuber. In: CAIRNEY J.W.G. & CHAMBERS tion of Pinus ectomycorrhizae from mixed conifer and pygmy fo- S.M. (eds.). Ectomycorrhizal Fungi: Key Genera in Profile. Berlin, rests using morphotyping and molecular methods. Canadian Springer-Verlag: 163-186. Journal of Botany, 79 (10): 1211-1216. ef

Gonzalo Guevara-Guerrero Gregory Bonito Instituto Tecnológico de Cd. Victoria Department of Plant, Soil and Microbial Sciences, Avenue Portes Gil 1301 Pte., Tamaulipas 87010 Michigan State University, East Lansing, MI 48825 Mexico USA [email protected] [email protected] Efrén Cázares-González Rosanne Healy Department of Forest Ecosystems and Society, Department of Plant Pathology Oregon State University, Corvallis, OR 97331 University of Florida, Gainesville, FL 32611 USA USA [email protected] [email protected] Rytas Vilgalys James Trappe Department of Biology, Duke University, Department of Forest Ecosystems and Society, Durham, NC 27708 Oregon State University, Corvallis, OR 97331 USA USA [email protected] [email protected] 374