Research Article |
Corresponding author: Roberto Garibay-Orijel ( rgaribay@ib.unam.mx ) Academic editor: Kevin D. Hyde
© 2018 Carolina Piña Páez, Gregory M. Bonito, Gonzalo Guevara-Guerrero, Michael A. Castellano, Roberto Garibay-Orijel, James M. Trappe, Rafael Peña Rámirez.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Piña Páez C, Bonito GM, Guevara-Guerrero G, Castellano MA, Garibay-Orijel R, Trappe JM, Rámirez RP (2018) Description and distribution of Tuber incognitum sp. nov. and Tuber anniae in the Transmexican Volcanic Belt. MycoKeys 41: 17-27. https://doi.org/10.3897/mycokeys.41.28130
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The genus Tuber is a lineage of diverse ectomycorrhizal, hypogeous, sequestrate ascomycete fungi that are native to temperate forests in the Northern Hemisphere. Recently, many new species of Tuber have been described in North America and Asia, based on morphological characteristics and molecular data. Here we describe and illustrate a new species, Tuber incognitum, based upon phylogenetic analysis and morphological description. We also present a new record for Tuber anniae in México. These two Tuber species are distributed in the Transmexican Volcanic Belt in the states of México, Michoacán, Guanajuato, Querétaro and Tlaxcala at altitudes between 2,000 and 3,200 meters. These species are associated with Pinus (T. anniae) and Quercus forests (T. incognitum).
Sequestrate fungi, truffles, Ascomycota , Systematics, new species
Fungal species, within the genus Tuber, produce hypogeous, sequestrate ascomata, that are more commonly known as truffles. These fungi are ectomycorrhizal (EcM) symbionts of angiosperm or gymnosperm hosts, including many species of trees as well as orchids. Plant hosts provide their EcM symbionts with carbohydrates in exchange for greater access to water and nutrients (
Ascomata were collected from the states of Guanajuato, México, Michoacán, Querétaro, Tlaxcala and were deposited in herbaria at Oregon State University (OSC), Herbario Nacional de México (MEXU) and Herbario José Castillo Tovar (ITCV). Macroscopic characters were recorded from fresh specimens and microscopic characters were described from both sections of fresh specimens and dried specimens mounted in 5% potassium hydroxide (KOH) following protocols from
DNA was extracted from ascomata of collections OSC157842 and OSC150066 using a CTAB chloroform extract protocol and ITS rDNA was amplified and sequenced as previously described (
DNA sequences were manually trimmed and edited with Sequencher 4.0 (Gene Codes Corp., Ann Arbor, Michigan). ITS sequences were queried against the NCBI public database GenBank by use of the BLASTn algorithm to retrieve similar sequences (
Taxon | GenBank | Voucher | Origin | Reference |
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T. incognitum Piña Páez. Bonito, Guevara & Castellano | GQ221447 | OSC 150066 | Ascoma | This paper |
KJ595013 | MEXU 26218 | Ascoma | This paper | |
KJ595014 | MEXU 25995 | Ascoma | This paper | |
MH174661 | – | EcM | This paper | |
MH447961 | ITCV 1695 | Ascoma | This paper | |
T. anniae W. Colgan & Trappe | MH174660 | OSC 157842 | Ascoma | This paper |
T. bonitoi G. Guevara & Trappe | KC152256 | MEXU 26541 | Ascoma |
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Tuber sp. 3 | KJ152267 | MEXU 26504 | Ascoma | This paper |
MÉXICO, State of Querétaro, Huimilpan, San Pedro, under Quercus crassifolia Humb. and Bonpl., Quercus spp., hypogeous, gregarious, 24 September 1996, M.A. Castellano (Holotype: OSC 150066), GB GQ221447. State of Michoacán, Zinapécuaro, el Jaral, under Quercus polymorpha Schltdl. and Cham., hypogeous, solitary or in groups of two, 2380 m alt., 19°46'48"N, -100°47'24"W, 4 September 2008, R. Garibay-Orijel (Paratype: MEXU 25995), GB KJ595014. State of México, Temascaltepec, under Quercus spp., hypogeous, solitary, 2011 m alt., 19°04'12"N, -100°01'48"W, 8 July 2009, R. Garibay-Orijel (Paratype: MEXU 26218), GB KJ595013.
Tuber incognitum is distinctive in the structure of its peridium (two-layered) and spore size (25–55 × 20–44 μm), which separates it from the rest of the species within the Puberulum clade reported from México.
Incognitum is Latin for unknown. The name incognitum is not derived from its morphology, rather from the fact that it was overlooked for so long. The holotype was collected in 1996 and not described until now.
Ascomata 10–15 mm broad, subglobose to slightly irregular, white with light brown areas when dry, glabrous, with canals that continue with the veins into the gleba. Gleba pinkish to purplish pale-brown in youth, dark brown at maturity, marbled with white veins. Odour fruity, pleasant.
Peridium two-layered, when handled the upper layer is lost and only the inner layer is observable under the light microscope, 350–400 μm thick, pellis 175–240 μm thick, composed of isodiametric or angular cells, 6–15 μm broad, walls 1.75–2.0 µm thick, yellowish hyaline in KOH. Subpellis 110–140 µm thick, composed of septate, interwoven hyphae (textura intricata), 4.5–7.0 µm broad, thin walled < 1 μm thick, hyaline in KOH. Gleba composed of septate, interwoven hyphae (textura epidermoidea), 5.0–7.5 µm broad, thin walled < 1 μm thick, hyaline in KOH. Ascospores broadly ellipsoid; excluding their alveolate-reticulate ornamentation, in 1-spored asci 45–55 × 34–44 μm (Q = 1.3), 2-spored 37–43 × 29–34 μm (Q = 1.25–1.36), 3-spored 30–42 × 26–31 μm (Q = 1.2–1.37), 4-spored 28–33 × 24–28 μm (Q = 1.09–1.25) and 5-spored 25–28 × 20–28 μm (Q = 1.2–1.25), spore colour orange-yellow in KOH, the walls > 2 μm thick; reticulum with 3–8 alveolae across the spore surface; the alveolar walls 3.5–4.0 μm tall. Asci globose, subglobose to broadly ellipsoid, pyriform, 88–100 × 70–95 μm, pedicel lacking to prominent, hyaline in KOH, hyphae around the asci prostrated or interwoven, cylindrical, 3.5–6.0 μm broad at the septa, thin walled, hyaline in KOH.
Tuber incognitum (Holotype, OSC 150066). a Ascoma, surface and cross-section view bPeridium in cross-section c Light microscopy of spores in cross-sectional view, highlighting the spines and ornamentation d Light microscopy of spores in surface view, highlighting the surface and reticulum. Scale bars: 5 mm (a), 20 µm (b), 15 µm (c, d).
Only known from central and southwest México (Querétaro, Michoacán, State of México, Guanajuato and Hidalgo). Ascocarps always associated with Quercus species (Q. crassifolia, Q. polymorpha). An EcM association with Quercus has been verified (MH174661) and its DNA has been recovered only from soil in Quercus forest in Hidalgo, México.
MÉXICO, State of Guanajuato, Guanajuato, Las Palomas, under Quercus spp., hypogeous, in groups of two, 2534 m alt., 21°03'50"N, -101°13'23"W, 10 October 2016, R. Peña-Ramirez (ITCV 1695).
Tuber incognitum resembles Tuber pseudoseparans in the colour of the peridium and the lack of dermatocystidia but differs by the size of the spores (being smaller in T. incognitum, 31–50 × 24–37 μm vs. T. pseudoseparans, 46–65 × 34–46 μm) and in the thickness of the peridium (being thinner in T. pseudoseparans, by ± 250 μm). Tuber incognitum is similar to Tuber bonitoi in spore size and ornamentation, but differs by the presence of dermatocystidia, which are absent in T. incognitum and the thickness of the peridium, being thicker in T. bonitoi (200–500 μm). Tuber incognitum is similar to Tuber guzmanii in the peridial organisation, both species have a well differentiated two-layered peridium but differ in the thickness (being thinner in T. guzmanii, 100–160 μm) and spore ornamentation (alveolate reticulum, 2–4 μm tall) and the size of the spores (being larger in T. guzmanii, 27–68 × 30–50 μm). The collection from Guanajuato represents a young developmental stage of T. incognitum, this collection has a thinner peridium (130–345 μm) and smaller spores (1-spored asci 23–35 × 19–25 μm, Q = 1.09 1.59; 2-spored 18–29 × 17–22 μm, Q = 1.0–1.61; 3-spored 30–42 × 26–31 μm, Q = 1.11–1.2; 4-spored 23–27 × 19–25 μm, Q = 1.08–1.26). These differences represent morphological variation within the species and its identity was confirmed with molecular data.
Ascomata subglobose to slightly irregular, 10–12 mm broad, white, cream, light brown when dry. Peridium thin, < 0.2 mm, smooth to velvety, irregularly roughened, furrows with depressions continuing as canals into the gleba. Gleba solid, brown, marbled with white veins that emerge as depressions on the peridium. Odour and taste not recorded.
Peridium 85–140 μm thick; pellis a pseudoparenchyma, 40–65 μm thick, cells 6–18 μm broad, versiform, isodiametric, squared, rectangular or angular, hyaline to yellowish in KOH, thick walled (> 1.0 μm), dermatocystidia absent; subpellis 45–75 μm thick, of hyaline, septate, interwoven hyphae (textura epidermoidea), 4.0–5.5 µm broad, thin walled, < 1 μm thick. Gleba of hyaline, interwoven, sinuous hyphae, 5.0–7.5 µm broad, constrained at the septum, 3.0–4.5 μm broad at the septa, thin-walled (< 1.0 μm).
Ascospores subglobose; excluding their alveolate-reticulate ornamentation, 1-spored asci 40–50 × 30–46 μm (Q= 1.03–1.15), 2-spored 28–38 × 26–35 μm (Q = 1.05–1.13), 3-spored 26–33 × 24–30 μm (Q = 1.04–1.15), spore colour orange-yellowish in KOH; walls > 2 μm thick, yellow; reticulum with 5–6 aveolae across the spore surface; the alveolar walls 3–4.5 μm tall. Asci subglobose, 84–105 × 75–85 μm, pedicel lacking to prominent, walls with 2–3 layers, hyaline in KOH; hyphae around the asci interwoven, 3.5–5.5 μm broad at the septum, thin walled (< 1.0 μm), hyaline in KOH.
Tuber anniae (OSC 157842). a Ascoma, surface and cross-section view bPeridium in cross-section c Light microscopy of spores in cross-sectional view, highlighting the spines and ornamentation d Light microscopy of spores in surface view, highlighting the surface and reticulum. Scale bars: 5 mm (a), 15 µm (b, c, d).
MÉXICO, State of Tlaxcala, Huamantla, cañada central, La Malinche National Park, under Pinus leiophylla Schiede and Deppe and Abies religiosa (Kunth) Schltdl. and Cham., hypogeous, solitary, 3220 m alt., 19°14'7"N, -97°59'9"W, 23 September 2007, G.M. Bonito (OSC 157842), GB MH174660.
Tuber anniae is similar to Tuber pacificum Trappe, Castellano and Bushnell, however, the latter species has narrower, ellipsoid spores (23–15 × 16–35 μm) and a thicker peridium (250–400 μm) than the former. T. pacificum has also been found co-occurring with Pseudotsuga menziesii and Tsuga heterophylla (Raf.) Sarg. along costal Oregon, while T. anniae has been found co-occurring with P. leiophylla and A. religiosa.
Tuber anniae was first described by
Species in Puberulum clade can be found in North America, Europe and Asia and some regions of North Africa and South America (
Most likely tree based on maximum likelihood phylogenetic inference showing the placement of Tuber incognitum within the Puberulum clade. Bootstrap values ≥ 70% are labelled above nodes. Nodes with posterior probabilities ≥ 99% are blacked. Holotype collections are labelled. The phylogeny is rooted with species belonging to the Maculatum clade. Scale bar corresponds to the mean number of nucleotides substitutions per site.
The T. anniae species complex is recovered as a strongly supported clade. There is an internal structure in this clade, with different branch lengths and nested subclades, but additional markers are needed to resolve relationships within this species complex. The Mexican specimens’ group with those from Alaska (JX094351), form a nested clade that is closely related to a collection from Canada (EU554720). The members in the T. anniae species complex are closely related to T. pacificum from Oregon, USA. Given the relatively high ITS similarity, phylogenetic position and similar morphology to the T. anniae holotype collection, we have identified the Mexican collection as T. anniae, extending its known range and southernmost distribution of this species in North America.
This research was made possible through NSF award number 0641297, REVSYS: Phylogenetic and Revisionary Systematics of North American Truffles (Tuber). GB thanks AgBioResearch MICL02416 for research support. DNA sequencing was supported by the MEXBOL network project CONACYT 280896. CPP thanks the North American Truffling Society for research support, Daniel Luoma, Javier Tabima and Antonio Gómez who provided insight and expertise that greatly assisted the research. GG thanks CONACYT and TecNM for research support. We thank the herbaria of ITCV, MEXU and OSC for accessing their collections.