Phytotaxa 635 (3): 206–216 ISSN 1179-3155 (print edition)

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Article PHYTOTAXA
Copyright © 2024 Magnolia Press ISSN 1179-3163 (online edition)

https://doi.org/10.11646/phytotaxa.635.3.2

Tuber itzcuinzapotl sp. nov. (Tuberaceae, Pezizomycetes), the first edible truffle
reported from Mexico with traditional biocultural importance
JAVIER ISAAC DE LA FUENTE1,4, WENDY ROSALES-ROSALES2,5, CÉSAR RAMIRO MARTÍNEZ-
GONZÁLEZ3,6, MAGDALENA MARTÍNEZ-REYES1,7, ANDREA CAROLINA ELIZONDO-SALAS2,8 & JESÚS
PÉREZ-MORENO1,9*
1
Colegio de Postgraduados, Campus Montecillo, Edafología, Km 36.5, 56230, Montecillo, Texcoco, Estado de México, Mexico.
2
Tecnológico Nacional de México. Instituto Tecnológico Superior de Zongolica, Km 4 Carretera a la Compañía S/N, 95000, Tepetitla-
napa, Zongolica, Veracruz, Mexico.
3
Tecnológico Nacional de México. Instituto Tecnológico de Ciudad Victoria, Tecnológico Nacional de México, Herbario Micológico
José Castillo, Boulevard Emilio Portes Gil No. 1301, 87010 Ciudad Victoria, Tamaulipas, Mexico.
4 �
[email protected]; https://orcid.org/0000-0003-4667-1574
5 �
[email protected]; https://orcid.org/0009-0001-8475-412X
6 �
[email protected]; https://orcid.org/0000-0002-0256-0840
7 �
[email protected]; https://orcid.org/0000-0003-2352-917X
8 �
[email protected]; https://orcid.org/0000-0002-4201-8845
9 �
[email protected]; https://orcid.org/0000-0001-5216-8313
*Author for correspondence

Abstract

Tuber itzcuinzapotl is described as a new species to science. This species is characterized by its pale brown ascomata, finely
granular peridium, pale brown to gray gleba, and 22–52 × 15–40 µm alveolate ascospores. The new species is putatively
associated with Pinus patula in conifer mixed forests in the state of Veracruz, located in eastern Mexico. Phylogenetic
analysis based on the nrITS region places the new species in the Maculatum clade, closely related, but morphologically
distinctive, to T. miquihuanense and T. mexiusanum. The new species consumed by the Nahua people, traditionally named
“Itzcuinzapotl”, constitutes the first edible truffle with biocultural importance in Mexico. Macro- and micromorphological
characterization, results of phylogenetic analysis, and photographs are presented. Ethnomycological aspects related to the
species are also briefly discussed.

Key words: 1 new species, biocultural erosion, ethnomycology, hypogeous fungi, Mexican truffles, mycorrhizal fungi,
traditional knowledge

Introduction

Tuber P. Micheli ex F.H. Wigg is a genus of the order Pezizales whose species, commonly known as truffles, are
characterized by their hypogeous habits with echinulate or alveolate ascospores, and by presenting characteristic
aromas that allow them to be dispersed by some invertebrates and vertebrates (Montecci & Sarasini 2000; Trappe et
al. 2009). The genus Tuber includes ectomycorrhizal fungi associated with gymnosperms and angiosperms, mainly
Carya, Pinus, Quercus, and even some orchid species. Ecologically, they play a role of paramount relevance in the
nutrient cycling in forests (Bonito et al. 2012; Castellano et al. 1989; Guevara-Guerrero et al. 2013a). Currently, more
than 230 species grouped into 11 clades around the world are known (Bonito et al. 2013). Due to their peculiar aroma
and flavor, some species are highly appreciated in gourmet gastronomy and are traded internationally at annual-in
season retail market higher than USD 150 million (Payen et al. 2014; Pérez-Moreno et al. 2021b). Tuber species can
be either collected from natural forests or from artificial plantations e.g., T. melanosporum Vittad. is known as the
Périgord black truffle (Guevara-Guerrero et al. 2022).
Currently, in Mexico, 25 species belonging to the genus Tuber have been recorded, mainly from northeastern
temperate forests and the Neovolcanic axis, most of them without commercial value (Guevara-Guerrero et al. 2013a;
García-Jiménez et al. 2021). Despite the fact that Mexico constitutes the second biocultural center of edible wild

206 Accepted by Samantha C. Karunarathna: 21 Jan. 2024; published: 26 Jan. 2024

Licensed under Creative Commons Attribution-N.C. 4.0 International https://creativecommons.org/licenses/by-nc/4.0/
edible fungi in the world, only behind China (Pérez-Moreno et al. 2021b), in the country there is no commercial trade
of Tuber species. However, recently the synthesis of the black truffle (T. melanosporum) with native oaks has been
successfully achieved, which opens the possibility of developing an industry with high economic relevance (Guevara-
Guerrero et al. 2022). Additionally, some species that grow in natural forests have potential economic importance,
e.g., T. regiomontanum G. Guevara, Bonito & Julio Rodr., T. canaliculatum Gilkey, T. bonitoi G. Guevara & Trappe
and T. lyonii Butters. This latter species, reported from northeastern Mexico (Guevara-Guerrero et al. 2013a), has an
international price of up to USD 400 per kilogram (Healy et al. 2016).
Mexico is one of the diversification centers of the Quercus and Pinus genera, which have been reported to
establish ectomycorrhizal symbiosis with species of the genus Tuber, and therefore the potential number of truffle
species may be higher than those reported so far (García-Jiménez 2014). In the present study, a new edible Tuber
species, grew under Pinus patula Schiede ex Schltdl. & Cham trees in coniferous mixed forests in eastern Mexico, with
traditional biocultural importance among Nahua people, is described. Morphological characterization, photographs,
and phylogenetic analysis based on the ITS region are presented.

Material and methods

Collection site and sample collection

The study area is the Mexcala locality, municipality of Soledad Atzompa, Veracruz state located in eastern Mexico. The
area is dominated by Pinus patula plantations, and natural forests of P. patula, P. ayacahuite Ehrenb. Ex Schltdl., Abies
hickelli Flous & Gaussen, Arbutus xalapensis Kunth and Cupressus spp. The collection of the specimens was carried
out following the protocols of Castellano et al. (1989) in the company of local people, two of them being coauthors of
this contribution. The collected specimens were photographed and dried at 40°C (Hu et al. 2022). Temporal slides were
made using 5% KOH and Melzer’s reagent to observe and measure the peridium hyphae, asci, and ascospores. The
reviewed material is deposited in the herbarium of the Instituto Tecnológico Superior de Zongolica (ZON), Veracruz
state.

Extraction, amplification, and sequencing of DNA

Genomic DNA was obtained with the CTAB method (Martínez-González et al. 2017) using 2-3 mg of dried glebal
tissue. The PCR amplification included 35 cycles with an annealing temperature of 54 ºC and was carried out with
the ITS5 and ITS4 primers (White et al. 1990) for the ITS nrDNA region. The PCR products were verified by agarose
gel electrophoresis. The gels were run for 1 h at 95 V cm⁻³ in 1.5% agarose and 1× TAE buffer (Tris Acetate-EDTA).
The gel was stained with GelRed (Biotium, USA) and the bands were visualized in an Infinity 3000 transilluminator
(Vilber Lourmat, Germany). The amplified products were purified with the ExoSAP Purification kit (Affymetrix,
USA), following the manufacturer’s instructions. They were quantified and prepared for the sequence reaction using
a BigDye Terminator v. 3.1 (Applied Biosystems, USA). These products were sequenced in both directions with an
Applied Biosystems model 3730XL (Applied BioSystems, USA), at the Instituto de Biología of the Universidad
Nacional Autónoma de México (UNAM).

Sequence assembly

The sequences of both strands of each of the genes were analyzed, edited, and assembled using BioEdit v.7.0.1 (Hall
2004) to generate a consensus sequence. These consensus sequences were compared with those deposited in the
GenBank of the National Center for Biotechnology Information (NCBI), using the tool BLAST 2.2.19 (Zhang et al.
2000).

Phylogenetic analyses

To study phylogenetic relationships, our newly produced sequences of two individuals of the studied species were
added to reference sequences of nrITS deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/genbank/). The
nrITS dataset included sequences from 54 specimens representing 36 taxa. The nrITS region was aligned using the

Tuber itzcuinzapotl, A New Species from Mexico Phytotaxa 635 (3) © 2024 Magnolia Press • 207
online version of MAFFT v. 7 (Katoh et al. 2002; Katoh & Standley 2013). The alignment was revised in PhyDE v.
10.0 (Müller et al. 2005), followed by minor manual adjustments to ensure character homology between taxa. The
matrix was composed of 54 individuals (685 characters). The data were analyzed using maximum parsimony (MP),
maximum likelihood (ML), and Bayesian inference (BI). Maximum parsimony analyses were carried out in PAUP*
4.0b10 (Swofford 2002) using the heuristic search mode, 1000 random starting replicates, and TBR branch swapping,
with MULTREES and Collapse on. Bootstrap values were estimated using 1000 bootstrap replicates under the heuristic
search mode, each with 100 random starting replicates. Maximum likelihood analyses were carried out in IQ-tree ver.
1.6.12 (Nguyen et al. 2015) with a GTR + G model of nucleotide substitution. To assess Branch support, 1000 rapid
bootstrap replicates were run with the GTRGAMMA model. Bayesian inference was carried out in MrBayes v.3.2.7
x64 (Ronquist et al. 2012) with four chains and the best evolutionary model for alignment was sought using Partition
Finder (Lanfear et al. 2014; 2017), the best-fit model selected for these three partitions of nrITS sequences was
GTR+G for ITS1, JC for 5.8s, and HKY+G for ITS2. The information block for the matrix includes two simultaneous
runs, four Montecarlo chains, temperature set to 0.2, and sampling 10 million generations (standard deviation ≤0.1)
with trees sampled every 1000 generations. The first 25% of samples were discarded as burn-in, and convergence was
evaluated by examining the standard deviation of Split frequencies among runs and by plotting the log-likelihood
values from each run using TRACER v.1.7.1 (Rambaut et al. 2018). The remaining trees were used to calculate a 50%
majority-rule consensus topology and posterior probabilities (PP). Trees were visualized and optimized in FigTree
v.1.4.4 (Rambaut 2018).

Results

Molecular analyses

We successfully amplified and sequenced the nrITS region from two specimens of the studied species. After
incorporation of additional sequences downloaded from GenBank (Table 1), the aligned nrITS dataset included 706
characters (including gaps), of which 264 conserved sites, 435 variable sites, and 349 parsimony informative sites.
The three phylogenetic analyses, MP, ML, and BI, of the nrITS dataset recovered similar topologies (Fig. 1). No
significant conflict (bootstrap value70%) was detected among the topologies obtained via the separate phylogenetic
analyses. The parsimony analysis of the alignment found 851 trees of 245 steps (CI=0.2541, HI=0.1041, RI=0.4485,
RC=0.6877). The best RAxML tree with a final likelihood value of –40578.924927 is presented. The matrix had
1001 distinct alignment patterns, with 5.65% undetermined characters or gaps. Estimated base frequencies were as
follows: A= 0.102548, C= 0.207510, G= 0.104213, T= 0.296420; substitution rates AC= 1.080347, AG= 1.507910,
AT= 1.151281, CG= 1.008021, CT= 5.407841, GT= 1.000000; gamma distribution shape parameter α= 0.003014.
In the Bayesian analysis, the standard deviation between the chains stabilized at 0.00002 after 4 million generations.
No significant changes in tree topology trace or cumulative Split frequencies of selected nodes were observed after
about 0.25 million generations, which were discarded as 25% burn-in. In the present study, a new species based on
morphological characters and phylogenetic analysis of nrITS (GenBank accession number OR429351- OR429352)
sequences is described (Fig. 1). The analysis of nrITS produced a phylogenetic tree where Tuber itzcuinzapotl is shown
as a monophyletic group (BS = 100%, BS = 100%, BI p = 1), related to T. mexiusanum Guevara, Bonito & Cázares
and T. miquihuanense G. Guevara, Bonito & Cázares.

TABLE 1. Species, voucher numbers, countries and corresponding GenBank accessions of the Tuber specimens used for
the phylogenetic analysis.
Species Voucher Country ITS
Tuber alboumbilicum YAAS L2324 China KJ742702
Tuber asa M1828 Italy HM485341
Tuber aztecorum GG1109 Mexico KY271790
Tuber aztecorum GG993 Mexico KY271791
Tuber beyerlei OSC 130875 USA NG042577
Tuber bomiense SKM106 China KC517481
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TABLE 1. (Continued)
Species Voucher Country ITS
Tuber bonitoi JT32421 Mexico KT897472
Tuber bonitoi ITCV913a Mexico KT897473
Tuber borchii GB62 Italy HM485342
Tuber brunneum JT33835 Mexico KT897474
Tuber brunneum JT33836 Mexico KT897475
Tuber brunneum JT33830 Mexico KT897478
Tuber californicum JT28058 USA HM485346
Tuber castilloi ITCV 149 Mexico NR119865
Tuber cistophilum AH39275 Spain JN392231
Tuber dryophilum GB37 Italy HM485354
Tuber foetidum ZB516 Hungary JQ288907
Tuber huizeanum FAN186 China JQ910651
Tuber itzcuinzapotl 52-ZON Holotype Mexico OR429351
Tuber itzcuinzapotl 53 ZON Mexico OR429352
Tuber latisporum HKAS 44315 China NR119620
Tuber lauryi OSC 130885 USA MT156528
Tuber linsdalei L63 USA HM485370
Tuber lijiangense HKAS52005 China KF805727
Tuber maculatum FLAS: MES-885 USA MT156493
Tuber maculatum GO-2008-144 Mexico KJ595014
Tuber maculatum Db-A Poland MH040280
Tuber mixtecorum 21880 ITCV Mexico MW209737
Tuber mexiusanum ITCV 3785 Mexico HM485412
Tuber mexiusanum ITCV 181 Mexico HM485411
Tuber microsphaerosporum BJTCFan152 China KF805726
Tuber microverrucosum LY-2012a China JN870099
Tuber miquihuanense ITCV 885 Mexico HM485414
Tuber panzhihuanense DXJ260 China JQ978644
Tuber pseudomagnatum BJTC FAN 403 China OM265246
Tuber pseudomagnatum BJTC FAN 532 China OM265248
Tuber pseudomagnatum BJTC FAN 391 China OM365244
Tuber pseudoseparans JT33778 Mexico KT897480
Tuber pseudoseparans JT33774 Mexico KT897481
Tuber pseudosphaerosporum BJTCFan250 China KF744063
Tuber rapaeodorum CMI-UNIBO 2483 Italy DQ011849
Tuber separans JT7697 USA HM485385
Tuber shearii OSC51052 USA HM485389
Tuber sinosphaerosporum BJTC: FAN135 China JX092086
Tuber sp. 14 KA-2010 Japan AB553464
Tuber sp. 14 GB-2010 Mexico GQ221447
Tuber sp. JT8755 USA HM485416
Tuber sp. 48D Mexico JF419253
Tuber sp. RH974 USA JF419256
Tuber tequilanum JT33796 Mexico KT897482
Tuber tequilanum JT33755 Mexico KT897485
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Tuber itzcuinzapotl, A New Species from Mexico Phytotaxa 635 (3) © 2024 Magnolia Press • 209
TABLE 1. (Continued)
Species Voucher Country ITS
Tuber tequilanum JT33790 Mexico KT897486
Tuber vesicoperidium I155 China JQ690071
Tuber zhongdianense wang0299 China DQ898187

FIGURE 1. Maximum likelihood phylogeny based on the nrITS sequences data. Maximum parsimony and Bayesian analyses recovered
identical topologies with respect to the relationships among the main clades of the Tuber. For each node, the following values are provided:
maximum parsimony bootstrap (%) /maximum likelihood bootstrap (%) / and posterior confidence (p-value). The scale bar represents the
expected number of nucleotide substitutions per site. The new species Tuber itzcuinzapotl is shown in bold. Sequences obtained from this
study are in bold.

Taxonomy

Tuber itzcuinzapotl de la Fuente & Rosales-Rosales, sp. nov.

Mycobank number: MB 84987, GenBank: OR429351 and OR429351

210 • Phytotaxa 635 (3) © 2024 Magnolia Press DE LA FUENTE ET AL.

Etymology:—Refers to the name used by the Nahua people to designate this edibles species itzcuinzapotl (itzcuin=dog,
and zapotl=zapote, a native sweet fruit).
Ascomata 35 × 28 mm, subglobose irregular to lobate, pale brown, darkening when touched, finely verrucose or
granulose, 5−7 verrucae per mm, dry texture, without rhizomorphs at the base. Peridium thinner than 1 mm in width,
grey, brown, yellowish when dry. Gleba marbled, pale brown to greyish brown, dark brown when mature, with abundant
white veins, some coalescing in the peridium. Taste and smell fruity. Peridium 200−400 µm composed of two layers:
Epicutis 180−250 µm, composed of a pseudparenchymatous layer composed of hyaline, inamyloid, thin-walled, 5−20
µm subglobose to globose hyphae in diameter, with thick, brownish cell-walls, usually forming pyramid-like structures
of 150−200 × 60−180 µm, with scarce erect clavate hyphae of 11−30 × 6−10. Subcutis 80-150 µm width, composed of
strongly interwoven hyphae, prosenchymatous in some areas, 4−12 µm in diameter, tubulose, rarely subglobose, and
occasionally inflated near the septa, hyaline, inamyloid, thin-walled. Veins composed of strongly interwoven hyphae,
2−16 µm in diameter, hyaline, tubulose to inflate near the septa, hyaline, thin-walled, like those from the subcutis.
Asci 34−70 × 20−50 µm, globose to subglobose, hyaline, some with a globose pedicel reaching up to 10 µm, mostly
2-spored, monosporic, rarely 3 or 4-spored, thick-walled (4 µm in diameter). Ascospores ellipsoid, pale brown to light
brown, with angular alveoli of (2)5−10 × 3−5(7) µm, 5−8 sides, projecting up to 6 µm, thick-walled (up to 3 µm);
1-spored asci have ascospores of 40−52 × 23−30 µm; 2-spored asci have ascospores of 27−34 × 15−22 µm, 3-spored
asci have ascospores of 22−39 × 15−19 and 4-spored asci have ascospores of 25−42 × 13−30 µm.
Holotype:—MEXICO. Veracruz: Soledad Atzompa municipality, Mexcala town, 18° 41’ 46’’ N, 97° 10’ 00’’ W,
2400 m, 17 April 2023, Wendy Rosales-Rosales, (52-ZON, holotype designated here).
Habitat, habit, distribution:—Solitary. So far, it is only known from the type locality, growing under P. patula.
Diagnosis:—The new species differs from other species within the Maculatum clade by its pale brown ascomata,
finely granular peridium, composed of subglobose hyphae, forming pyramid-like structures, clavate terminal cells of
11−30 × 6−10 µm, pale brown to gray gleba, and 22−52 × 15−40 µm alveolate ascospores.

FIGURE 2. Tuber itzcuinzapotl (Holotype). A) Fresh ascomata. B) Dry ascomata. C) Peridium hyphal arrangement. D) Hyphae from
sterile veins. E) 3-spored ascus containing 2 mature ascospores showing alveolate ornamentation. Bar: A–B= 10 mm; C–E=20 µm.

Tuber itzcuinzapotl, A New Species from Mexico Phytotaxa 635 (3) © 2024 Magnolia Press • 211
Discussion

Analysis of the ITS region places the new species within the maculatum clade, which is characterized by having
species with granular or cottony peridium, clavate elements on the peridium, and honeycomb ascospores (Montecchi
& Sarasini 2000; Bonito et al. 2013). The species of this clade are located mainly in the northern hemisphere, being
present in the United States and Mexico (García-Jiménez et al. 2021). A synopsis of the diagnostic characteristics
of the Mexican Tuber species related to the new species described in this work, including their host and distribution
is presented in the Table 2. Even though the species in the maculatum clade are not highly appreciated due to their
inconspicuous taste and smell, some species such as T. miquinuanense could be commercialized (Guevara-Guerrero et
al. 2022). Most of the species within this clade are associated with trees of the genera Abies, Carya, Pinus and Quercus
(Sánchez-Ledezma et al. 2023). With the addition of the new species described in this study, a total of 26 species
belonging to the genus Tuber have been reported from Mexico, mostly distributed in northeastern Mexico (Figure 3).

TABLE 2. Diagnostic characteristics of the new species described in this work compared to related species.
Character T. aztecorum T. castilloi T. itzcuinzapotl T. miquihuanense
Ascomata
Size 5–23 × 4–16 × 3–11 11–25 × 10–17 35 × 28 30–20
Shape Subglobose, irregular, Subglobose to ovoid, Subglobose irregular to Subglobose to slightly
lobate or flattened or irregular lobate gibbose
globose
Texture Finely verrucose or Minutely verrucose to Finely verrucose or Flat-polygonal to pyramidal
granulose papillose granulose warts
Taste Not recorded Not recorded Fruity Not recorded
Smell Raw potato-like Not recorded Fruity Pleasant
Peridium
Width (μm) 110–350 80–150 200−400 110–360
Color Hyaline to reddish Hyaline to reddish Hyaline to reddish Yellowish to reddish brown
brown brown
Hyphal arrangement Pseudoparenchima Pseudoparenchima Pseudoparenchima Pseudoparenchima
Gleba
Appearance Marbled Marbled Marbled Marbled
Color White to greyish Light brown Pale brown to greyish Cream to light brown
brown, dark brown
when mature
Hyphal arrangement Interwoven Interwoven to periclinal, Interwoven Interwoven to periclinal
Asci
Size 62–95 × 57–77 90 × 50–65 34−70 × 20−50 65–90 × 51–70
Shape Globose, subglobose to Globose to subglobose Globose to subglobose, Globose to subglobose or
broadly or ovoid, some with a hyaline, some with a broadly ellipsoid, with a
ellipsoid short pedicel globose pedicel short pedicel when young
Number of spores 1 to 4 1 to 4 1 to 4 1 to 5
Ascospores
Shape Subglobose, globose to Subglobose to broadly Ellipsoid Globose to subglobose or
broadly ellipsoid ellipsoid broadly ellipsoid, with a
short pedicel when young
Size in spores per 1) 42–58 × 27–48; 2) 1) 27–63 × 20–40; 2) 1) 40−52 × 23−30; 2) 1) 40–50 × 30–39; 2) 29–40
asci (µm) * 25–52 × 23–40; 3) 27– 30–50 × 23–34; 27−34 × 15−22; 3) × 24–35; 3) 24–35 × 22–33;
40 × 20–30; 4) 23–38 × 3) 27–40 × 20–32; 4) 22−39 × 15−19; 4) 4)23–28 × 20–26; 5) 20–28
18–28 27–44 × 20–30 25−42 × 13−30 × 20–22
Changes of color None None None None
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TABLE 2. (Continued)
Character T. aztecorum T. castilloi T. itzcuinzapotl T. miquihuanense
Habit and habitat Hypogeous, gregarious Hypogeous, solitary to Hypogeous, solitary. Hypogeous, solitary to
in volcanic rock soil in scattered. scattered.
an Abies religiosa forest
Type of vegetation Abies religiosa forest Mixed forest with Pinus forest Pinus-Quercus and
Quercus spp. and Pinus mesophyll forests
spp.
Geographical State of Mexico, Nuevo León and Veracruz, Mexico. Tamaulipas, Mexico.
distribution Mexico. Tamaulipas, Mexico.
Reference Guevara-Guerrero et al. Guevara-Guerrero et al. This work Guevara-Guerrero et al.
2018 2013 2013
* the number before parenthesis indicates the number of spores per asci

FIGURE 3. Heatmap showing the distribution of the recorded Tuber species in Mexico.

Tuber itzcuinzapotl is mainly characterized by a light brown, verrucous-granular peridium, gray or pale brown
gleba, peridium formed by pseudoparenchymatous hyphae with 11−30 × 6−10 µm clavate elements and 22−52 ×
15−40 µm ascospores. Like the rest of the species of the maculatum clade, T. itzcuinzapotl has a pseudoparenchymatous
peridium with clavate terminal elements and alveolate ascospores. It resembles to T. miquihuanense, since they are
similar in the thickness and color of the peridium, including the size of the ascospores. However, these two species
phylogenetically belong to a different clade and morphologically they can be distinguished by its peridial hyphal
microscopic structure, being composed of erect hyphal chains in T. miquihuanense (Guevara-Guerrero et al. 2012) and
pyramidal-shaped hyphae in T. itzcuinzapotl. Another similar species is Tuber mexiusanum which phylogenetically is a
sister species of T. miquihuanense, and differs from T. itzcuinzapotl by having ascomata with a strong greenish reaction
when cut, strong acetone-like smell and larger terminal cells in the peridium (Guevara-Guerrero et al. 2013a).
Mexico constitutes one of the main biocultural centers of edible wild fungi in the world. In the country around 500
species are consumed by the 71 ethnic groups and by the rural communities which inhabit areas nearby the forested
areas. The country is ranked therefore as the second international genetic center of edible wild fungi, just after China,

Tuber itzcuinzapotl, A New Species from Mexico Phytotaxa 635 (3) © 2024 Magnolia Press • 213
where more than 1000 species of fungi are consumed (Pérez-Moreno et al. 2021a; 2021b). Paradoxically, most of the
species which are consumed in Mexico are epigeous and only a very reduced number of hypogeous fungi are consider
a food resource. Currently, only two species of the genera Melanogaster Corda and Gautieria Vittad. have been
reported as edible by different ethnic groups in Mexico (Cázares et al. 2008; Montoya et al. 2012). Due to the fact that
the country holds the largest diversity of oaks in the world, with more than 160 species, and 72 taxa of pines, which
are trees that establish ectomycorrhizal symbiosis with a high number of hypogeous fungi, is reasonably assume that
in general these group of fungi have been historically overlooked.
Tuber itzcuinzapotl is consumed in the central area of the Mexican state of Veracruz, located in eastern Mexico, by
members of the Nahua culture from the Sierra de Zongolica, where it is known as itzcuinzapotl (dog’s zapote). Trying
to trace the origin of these name of Nahuatl origin we learnt that according to local people, the name is related to the
fact that in the past local peasants were accompanied by dogs while conducting agricultural practices. And these dogs
use to dig up searching for this truffle; later on, at a time that is lost in time, people realize that they this truffle was
edible for humans as well, and they start enjoying their flavor. They named this truffle itzcuinzapotl, as it is called at the
present time. The word “Izcuintli”, comes from the Nahuatl, the language used by the Aztec people before the conquest
of Mexico by the Spaniards. It was used to designate a native hairless dog breed, often sacrificed in sacred rituals during
ancient times throughout Mexico, and then buried with their owners, believing they acted as guides to the soul on its
journey to the underworld. Meanwhile, the word “Zapotl” comes from the resemblance of the smell, color, taste and
shape of the new species of truffle with a local fruit known as “zapote”. Currently, peasants no longer use dogs when
they carry out their agricultural practices, and therefore the collection of Tuber itzcuinzapotl is carried out randomly
when they prepare the soil near forested areas for planting milpa (an ancient Mesoamerican agricultural system which
is used to produce maize, beans, and squash without employing artificial pesticides or chemical fertilizers). Nahua
people of this area enjoy eating this truffle, which is consumed raw when found in their mature stage. Its phenology is
included in the category known technically as “short at the middle of the rainy season” during the months of June to
August. Interestingly, the inhabitants do not include T. itzcuinzapotl in the category of fungi, but they consider it as a
fruit. The community of Mexcala, where the species was found, has 1,883 inhabitants (INEGI, 2020), among which
we only detected three women aged 50 to 70 years with a deep traditional knowledge of this fungus, who additionally
consume regularly T. itzcuinzapotl. Therefore, the accurate traditional knowledge related to the ecology, distribution,
phenology, and consumption is entirely associated with adult women, who are responsible for preparing the soil for
planting, indicating the huge cultural erosion and the high risk of disappearance of the traditional knowledge of this
species. Due to the great bio-cultural relevance of this truffle species, it would be very relevant to develop further
studies related to the determination of its nutrition contents and the identification of its potential bioactive compounds
with medicinal properties.
Therefore, this contribution reports the first edible truffle with traditional biocultural importance among ethnic
groups in Mexico. The contribution intends to incentivize the interest, development, and rescue of traditional knowledge
of the consumption of hypogeous fungi in Mexico. At the same time, it is intended to mitigate the mycocultural erosion
that has been happening in a short period of time, particularly in the last three decades, at a quick pace, as never
recorded before, not only in Mexico but on a global scale.

Acknowledgments

Javier de la Fuente thanks CONAHCYT for a doctoral scholarship. Wendy Rosales-Rosales and Carolina Elizondo
thank the Rosales-Rosales family and the Mexcala community for their support during fieldwork. This research was
funded by the project CONACYT-PRONACES-FOP07-2021-03-316198.

References

Bonito, G.M., Gryganskyi, A.P., Trappe, J.M. & Vilgalys, R. (2010) A global meta‐analysis of Tuber ITS rDNA sequences: species
diversity, host associations and long‐distance dispersal. Molecular Ecology 19 (22): 4994–5008.
https://doi.org/10.1111/j.1365-294X.2010.04855
Bonito, G., Smith, M.E., Nowak, M., Healy, R.A., Guevara, G., Cázares, E., Kinoshita, A., Nouhra, E.R., Domínguez, L.S., Tedersoo,
L., Murat, C., Wang, Y., Arroyo-Moreno, B., Pfister, D.H., Nara, K., Zambonelli, A., Trappe, J.M. & Vilgalys, R. (2013) Historical

214 • Phytotaxa 635 (3) © 2024 Magnolia Press DE LA FUENTE ET AL.

 Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage.
PLoS ONE 8 (1): e52765.
https://doi.org/10.1371/journal.pone.0052765
Castellano, M.A., Trappe, J.M., Maser, Z. & Maser, S. (19896) Key to spores of the genera of hypogeous fungi of North America, with
reference to animal mycophagy. Mad River Press, Eureka, 185 pp.
Cázares, E., Guevara, G., García, J. & Trappe, J.M. (2008) Melanogaster minysporus sp. nov., a new sequestrate member of the Boletales
from Mexico. Revista Mexicana de Micología 28: 67–69.
Cázares, E., García, J., Castillo, J. & Trappe, J.M. (1992) Hypogeous fungi from northern México. Mycologia 84: 341–359.
Contreras, L., Vázquez, A. & Ruan-Soto, F. (2018) Etnomicología y venta de hongos en un mercado del Noroeste del estado de Puebla,
México. Scientia Fungorum 47: 47–55.
De Avila, A., Welden, A. & Gúzman, G. (1980) Notes on the ethnomycology of Hueyapan, Morelos Mexico. Journal of Ethnopharmacology
2: 311–321.
García Jiménez, J. (2013) Diversidad de macromicetos en el Estado de Tamaulipas, México Diversity of macrofungi in the Tamaulipas
State, México. Doctoral dissertation, Universidad Autónoma de Nuevo León.
García-Jiménez, J., Ayala-Vásquez, O., Guevara-Guerrero, G., Garza-Ocañas, F. & de la Fuente, J.I. (2021) Tuber mixtecorum (Tuberaceae,
Pezizales) a new truffle in the Maculatum clade from Mexico. Phytotaxa 509: 113–120.
https://doi.org/10.11646/phytotaxa.509.1.6
Guevara, G., Bonito, G. & Cázares, E. (2013a) Revisión del género Tuber (Tuberaceae: Pezizales) de Mexico. Revista Mexicana de
Biodiversidad 84: 39–49.
https://doi.org/10.7550/rmb.31981
Guevara, G., Bonito, G., Trappe, J.A., Cázares, E., Williams, G., Healy, R., Schadt, C. & Vilgalys, R. (2012) New North American Tuber
spp. Mycologia 105: 194–209.
https://doi.org/10.3852/12-087
Guevara, G., Bonito, G., Trappe, J.M., Cázares, E., Williams, G., Healy, R.A., Schadt, C. & Vilgalys, R. (2013a) New North American
truffles (Tuber spp.) and their ectomycorrhizal associations. Mycologia 105: 194–209.
https://doi.org/10.3852/12-087
Guevara, G., Pacioni, G., Leonardi, M., Garza-Ocañas, F. & Gaitán-Hernández, R. (2022) Mycorrhizal synthesis of Périgord black truffle
(Tuber melanosporum) with mexican oak species. Microbiology and biotechnology letters 50: 40–50.
https://doi.org/10.48022/mbl.2109.09012
Hall, T. (2004) BioEdit v.7.0.1. Available at: Hall, Tom. “BioEdit version 7.0. 0.” Distributed by the author. Available from: https://www.
mbio.ncsu.edu/BioEdit/bioedit.html (accessed: 1 August 2023).
Healy, R., Bonito, G.M. & Smith, M.E. (2016) A Brief Overview of the Systematics, Taxonomy, and Ecology of the Tuber rufum Clade.
In: Zambonelli, A., Iotti, M., Murat, C. (Eds.) True Truffle (Tuber spp.) in the World. Soil Biology, vol 47. Springer, Cham. pp.
125–136.
https://doi.org/10.1007/978-3-319-31436-5_8
Hu, Y., Karunarathna, S.C., Li, H., Galappaththi, M.C., Zhao, C.L., Kakumyan, P. & Mortimer, P.E. (2022) The Impact of Drying
Temperature on Basidiospore Size. Diversity 14 (4): 239.
Instituto Nacional de Estadística y Geografía (2020) Censo de Población y Vivienda 2020. México. Available from: https://www.inegi.org.
mx/app/scitel/consultas/index# (Consulted 1 November 2023).
Katoh, K. & Standley, D.M. (2013) MAFFT Multiple Sequence Alignment Software version 7: improvements in performance and usability.
Molecular Biology and Evolution 30 (4): 772–780.
https://doi.org/10.1093/molbev/mst010
Katoh, K., Misawa, K, Kuma, K. & Miyata, T. (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier
transform. Nucleic Acids Research 30 (14): 3059–306.
https://doi.org/10.1093/nar/gkf436
Lanfear, R., Calcott, B., Kainer, D., Mayer, C. & Stamatakis, A. (2014) Selecting optimal partitioning schemes for phylogenomic datasets.
BMC Evolutionary Biology 14: 1–14.
https://doi.org/10.1186/1471-2148-14-82
Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T. & Calcott, B. (2017) PartitionFinder 2: New methods for selecting partitioned
models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34 (3): 772–773.
https://doi.org/10.1093/molbev/msw260
Martínez-González, C.R., Ramírez-Mendoza, R., Jiménez-Ramírez, J., Gallegos-Vázquez, C. & Luna-Vega, I. (2017) Improved method
for genomic DNA extraction for Opuntia Mill. (Cactaceae). Plant Methods 13: 1−10.
Montecchi, A. & Sarasini, M. (2000) Funghi ipogei d’Europa. Associazione Micologica Bresadola, Fondazione Centro Studi Micologici.

Tuber itzcuinzapotl, A New Species from Mexico Phytotaxa 635 (3) © 2024 Magnolia Press • 215
 Vicenza. 714 pp.
Montoya, A., Hernández-Totomoch, O., Estrada-Torres, A. & Kong, A. (2003) Traditional knowledge about mushrooms in a Nahua
community in the state of Tlaxcala, Mexico. Mycologia 95 (5): 793–806.
Montoya, A., Kong, A., Estrada-Torres, A., Cifuentes, J. & Caballero, J. (2004) Useful wild fungi of La Malinche National Park, Mexico.
Fungal Diversity 17: 115–143.
Montoya, A., Torres-García, E., Kong, A., Estrada-Torres, A. & Caballero, J. (2012) Gender differences and regionalization of the cultural
significance of wild mushrooms around La Malinche volcano, Tlaxcala, Mexico. Mycologia 104 (4): 826–834.
https://doi.org/10.3852/11-347
Müller, K., Quandt, D., Müller, J. & Neinhuis, C. (2005) PhyDE®-Phylogenetic data editor, versión 10.0. Program distributed by the
authors. Available from: https://www.phyde.de (accessed: 16 February 2022).
Nguyen, L.T., Schmidt, H.A., Von Haeseler, A. & Minh, B.Q. (2015) IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating
Maximum-Likelihood Phylogenies. Molecular Biology and Evolution 32 (1): 268–274.
https://doi.org/10.1093/molbev/msu300
Payen, T., Murat, C. & Bonito, G. (2014) Truffle phylogenomics: New insights into truffle evolution and truffle life cycle. Advances in
Botanical Research 70: 211–234.
https://doi.org/10.1016/B978-0-12-397940-7.00007-0
Pérez-Moreno, J., Mortimer, P.E., Karunarathna, S.C. & Li, H. (2021b) Global perspectives on the ecological, cultural and socioeconomic
relevance of wild edible fungi. Studies in Fungi 6: 408–424.
https://doi.org/10.5943/sif/6/1/31
Pérez-Moreno, J., Guerin-Laguette, A., Rinaldi, A.C., Yu, F.G., Verbeken, A., Hernández-Santiago, F. & Martínez-Reyes, M. (2021a)
Edible mycorrhizal fungi of the world: What is their role in forest sustainability, food security, biocultural conservation and climate
change? Plants, People, Planet 3: 471–490.
https://doi.org/10.1002/ppp3.10199
Rambaut, A. (2018) FigTree version 1.4.4. Available from: http://tree.bio.ed.ac.uk/software/figtree/ (accessed: 1 August 2023).
Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. (2018) Posterior Summarization in Bayesian Phylogenetics Using
Tracer 1.7. Systematic biology 67 (5): 901–904.
https://doi.org/10.1093/sysbio/syy032
Reyes-López, R., Montoya, A., Kong, A., Cruz-Campuzano, E. & Caballero-Nieto, J. (2020) Folk classification of wild mushrooms from
San Isidro Buensuceso, Tlaxcala, Central Mexico. Journal of Ethnobiology and Ethnomedicine 16: 53.
https://doi.org/10.1186/s13002-020-00408-x
Ronquist, F., Teslenko, M., Van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P.
(2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across large model space. Systematic Biology 61:
539–542.
Sánchez-Ledezma, J.A., Guevara-Guerrero, G., Garibay-Origel, R., Ángeles-Argáiz, R., Ávila-Rodríguez, V., Arreola-Ávila, J.G.,
Carrasco-Hernández, V., Borja-De la Rosa, A. & Gonzáles-García, F. (2023) Tuber caryophilum, a new truffle species growing in
Carya illinoinensis orchards. Revista Mexicana de Biodiversidad 93: e934893.
https://doi.org/10.22201/ib.20078706e.2022.93.4893
Swofford, D.L. (2002) PAUP* Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland.
Trappe, J.M., Molina, R., Luoma, D.L., Cázares, E., Pilz, D., Smith, J.E., Castellano, M.A., Miller, L. & Trappe, J.M. (2009) Diversity,
ecology and conservation of the truffle fungi in forests of the pacific northwest. US Dept. of Agriculture, Forest Service General
Technical Report, PNW-GTR-772. Oregon. 194 pp.
White, T.J., Bruns, T.D., Lee, S. & Taylor, J.W. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J. & White, T.J. (Eds.) PCR protocols: a guide to methods and applications.
Academic, San Diego, pp. 315–322.
https://doi.org/10.1016/B978-0-12-372180-8.50042-1
Zhang, Z., Schwartz, S., Wagner, L. & Miller, W. (2000) A greedy algorithm for aligning DNA sequences. Journal of Computational
Biology 7: 203−214.

216 • Phytotaxa 635 (3) © 2024 Magnolia Press DE LA FUENTE ET AL.