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Phylogenetic overview of the genus Genea (Pezizales, Ascomycota)
with an emphasis on European taxa
Pablo Alvarado
1
ALVALAB, La Rochela 47, E-39012 Santander, Spain
Julio Cabero
C/ Barrio del Carmen 6, E-49800 Toro, Zamora, Spain
Gabriel Moreno
Departamento de Ciencias de la Vida (Botanica),
Universidad de Alcalá, Facultad de Biología,
E-28871 Alcalá de Henares, Spain
Zoltán Bratek
Department of Plant Physiology and Molecular Plant Biology,
ELTE University, Budapest, Hungary
Nicolas van Vooren
36 rue de la Garde, F-69005 Lyon, France
Vasileios Kaounas
Sokratous 40, Artemis Attika, TK-19016, Greece
Giorgos Konstantinidis
Ag. Kosma 25, 51100 Grevena, Greece
Carlo Agnello
Via Antonio Gramsci 11 –I-72023 Mesagne, Italy
Zsolt Merényi
Department of Plant Physiology and Molecular Plant Biology,
ELTE University, Budapest, Hungary
Matthew E. Smith
Department of Plant Pathology, University of Florida,
Gainesville, Florida 32611
Alfredo Vizzini
Dipartimento di Scienze della Vita e Biologia dei Sistemi,
Università di Torino, Viale P.A. Mattioli 25, I-10125,
Torino, Italy
James M. Trappe
Department of Forest Ecosystems and Society,
Oregon State University, Corvallis, Oregon 97331
Abstract: We constructed a comprehensive phylogeny
of the genus Genea, with new molecular data from sam-
ples collected in several countries in temperate and
Mediterranean Europe, as well as North America.
Type specimens and authentic material of most species
were examined to support identifications. The molecu-
lar identity of the most common species in Genea was
compared with nuc rDNA internal transcribed spacer
(ITS), D1-D2 domains of 28S nuc rDNA (28S rDNA)
and translation elongation factor 1-agene (TEF 1) pro-
files of 10 recently proposed taxa, G. brunneocarpa,G.
compressa,G. dentata,G. fageticola,G. lobulata,G. oxygala,
G. pinicola,G. pseudobalsleyi,G. pseudoverrucosa and G.
tuberculata, supporting their status as distinct species.
Genea mexicana and G. thaxteri on the one hand and
G. sphaerica and G. lespiaultii on the other are closely
related. Multiple lineages were recorded for G. verru-
cosa and G. fragrans, but we found no morphological
traits to discriminate among them, so we tentatively
interpreted them as cryptic species. A key to species
of the genus Genea is provided to facilitate identifica-
tion. We provide macroscopic images of fresh speci-
mens and of representative spores of most species.
Finally, we conducted a molecular analysis of the diver-
gence time for Genea and discuss the implications of
our results.
Key words: hypogeous, Mediterranean, phylogeny,
Pyronemataceae, taxonomy, truffle fungi
INTRODUCTION
Carlo Vittadini (1831) proposed the hypogeous asco-
mycete genus Genea Vittad. dedicated to zoologist Dr
Joseph Gené. It is one of the more common truffle
genera in the Mediterranean basin, although it
receives considerably less attention than the esteemed
true truffles in Tuber P. Micheli ex F.H. Wigg. Genea
accommodates species with odoriferous hypogeous
ascomata that are more or less lobed and have a basal
tuft of hyphae that attaches the ascomata to the sub-
strate. The inamyloid, uniseriate, cylindrical asci typi-
cally contain eight verrucose spores and are arranged
in an organized hymenium (ptycothecium) and pro-
tected by an epithecium that usually resembles the
peridium. Trappe (1979) considered Genea as the
type genus of the family Geneaceae, but it later was
transferred to the Pyronemataceae on the basis of
both morphological and molecular evidence (Pfister
1984, Smith et al. 2006, Perry et al. 2007, Hansen et al.
2013). There are approximately 35 validly described
species currently included in Genea, and several vari-
eties justified by different spore dimensions (Gross
1992, 1996).
The type species, Genea verrucosa Vittad., was
described with hollow ascomata covered with minute
black warts on the surfaces of both the peridium and
epithecium (Vittadini 1831). Originally it was reported
to have spherical spores, but they later were described
from authentic material as ellipsoidal, verrucose and
Submitted 23 Jul 2015; accepted for publication 11 Nov 2015.
1
Corresponding author. E-mail: pablo.alvarado@gmail.com
Mycologia, 108(2), 2016, pp. 441–456. DOI: 10.3852/15-199
#2016 by The Mycological Society of America, Lawrence, KS 66044-8897
441
,32 mm diam by Berkeley and Broome (1846) and
Tulasne and Tulasne (1851). Mattirolo (1900a)
described the ascospores of this species as ellipsoidal,
27–30 mm long 621 mm wide, and ornamented with
small, regularly spaced, conical or hemisphaerical pro-
tuberances. He also discussed the great color variability
of G. verrucosa. He described it as ranging from tan to
dark brown to rarely black and suggested that color
was influenced by developmental stage of the ascomata
as well as collection site. Finally, he reported that most
original specimens of G. verrucosa from Vittadini were
covered with pyramidal warts similar to those of Tuber
aestivum.
Vittadini (1831) also described G. papillosa Vittad. as
forming smaller, more reddish ascomata covered with
papillae. Mattirolo (1900a) considered G. papillosa a
variant of G. verrucosa differing only in the reddish tones
of the peridium and proposed the variety G. verrucosa
var. badia Mattir. Zobel (Corda 1854) proposed G. kun-
zeana Zobel, because it was brown and had subglobose
and lobed but not wrinkled ascoma. However, Mattirolo
(1900b) synonymized G. kunzeana with G. verrucosa var.
badia and G. papillosa because these taxa have similar
spore sizes and overall peridium color. Several authors
(Fischer 1897, Hawker 1954, Ceruti 1960) also consid-
ered G. perlata Corda (Corda 1854) a likely synonym
of G. verrucosa, because the former differs only by
having an irregular ascoma, a feature observed also
in G. verrucosa by other authors (Tulasne and Tulasne
1851, Mattirolo 1900b). Moreno-Arroyo et al. (1998b)
described another species putatively related to G. verru-
cosa from southern Spain, G. subbaetica Moreno-Arroyo,
Gómez & Calonge. The spores are similar to those
of G. verrucosa,butG. subbaetica has smaller cells in
the outer peridial layer and is collected in early
winter, while G. verrucosa is usually found in spring or
early summer.
Wallroth (1833) described some specimens of a hypo-
geous fungus in Germany as Hydnocaryon fragrans Wallr.
This was considered a synonym of G. verrucosa by Die-
trich and Klotzsch (1839) but soon recognized as an
independent taxon by Berkeley and Broome (1846) as
G. klotzschii Berk. & Broome. They incorrectly gave the
species epithet klotzschii priority over fragrans, later cor-
rected as G. fragrans (Wallr.) Paoletti by Saccardo
(1889). This species was distinguished by subglobose
spores that are larger than those of G. verrucosa (Berke-
ley and Broome 1846, Corda 1854, Fischer 1897), and
ornamented with block-like or pyramidal warts (Hawker
1954). Another species with fairly large spores, Genea
vagans Mattir., was proposed by Mattirolo (1900a).
This species forms small, round ascomata with a black
verrucose peridium. The spores are ellipsoidal, 35 6
27 mm, and ornamented with big conical warts that
tend to coalesce at their bases (Ceruti 1960). Genea
vagans is reportedly found in autumn under Abies,
Fagus, and putatively Castanea (Mattirolo 1900b). Vele-
novský (1922) proposed G. neuwirthi Velen. a unique
species based on its smooth brownish peridium. This
species is reported to have ascomata with a single hollow
cavity, spores 35–40 mm and ornamented with promi-
nent thick warts that have blunt, rounded edges.
Berkeley and Broome (1846) also published a
description of some G. papillosa specimens from the
UK that were quite different from those originally
described by Vittadini (1831). These later were recog-
nized as a new species by Tulasne and Tulasne (1851)
under the name G. hispidula Berk. ex Tul. & C. Tul.
Tulasne and Tulasne (1851) describe G. hispidula as
having small, globose toflattened ascomata with a single
inner cavity, a basal tuft of reddish mycelium, a peri-
dium covered by pyramidal warts that are smaller than
those of G. verrucosa, and an epithecium with minute
blackish warts. Spores of G. hispidula are described as
ellipsoidal, 38–42 632 mm, and ornamented with
crowded, obtuse to roundish warts. Genea hispidula is
thought to be a symbiont of Fagus and Castanea trees
in France, Britain and Germany (Tulasne and Tulasne
1851, Fischer 1897, Hawker 1954). Corda (1854)
proposed a second species with hairy peridium from
the Czech Republic, G. pulchra Corda. This species dif-
fered from G. hispidula because of its smaller spores
(20–28 615–24 mm), spore ornamentation composed
of minute or subacute warts and a pseudoparenchymatic
peridium with scattered lacunae (locules inside the
ascomata that are lined with a palisade of hymenium).
Another species was described by Tulasne and
Tulasne (1851) from France as G. sphaerica Tul. & C.
Tul. and later reported from several European coun-
tries (Hesse 1891, Vacek 1951, Hawker 1954). As sug-
gested by the name this species forms almost
perfectly sphaerical, medium-sized ascomata that are
blackish and in which the peridium and epithecium
are covered with minute polygonal warts. Internally it
has labyrinth-like chambers and a characteristically
white trama. Spores are ellipsoidal, ornamented with
obtuse roundish warts and are larger than those of
G. verrucosa. Mattirolo (1903) described G. sphaerica f.
sporis spinuloso-tuberculatis, a Mediterranean variety of
G. sphaerica with spores ornamented with spiney-tuber-
culate warts. A more or less similar feature also was
observed by Moreno-Arroyo et al. (1998a) in their
G. sphaerica f. lobulata Mor.-Arr., J. Gómez & Calonge
described from southern Spain as a lobed variety of
G. sphaerica. The species G. lespiaultii Corda (Corda
1854) is macroscopically similar to G. sphaerica (Mattir-
olo 1903) but has spores with unusual, irregularly flat
warts.
Although Genea is a common and frequently col-
lected genus of truffles across both Europe and North
442 MYCOLOGIA
America (Montecchi and Sarasini 2000; Gori 2005;
Smith et al. 2006, 2007; Trappe et al. 2007, 2009; Gue-
vara-Guerrero et al. 2012), there has been no modern
synthesis based on both morphological and molecular
data. The only available molecular phylogenies thus far
were based on partial 28S rDNA and included only
Genea from the western USA (Smith et al 2006, Perry
et al. 2007). Ten new Genea species were proposed to
accommodate several groups of specimens with shared
morphological and ecological features collected from
European countries (Alvarado et al. 2014). Here we
compare these recently described taxa with previously
described species based on molecular data to support
their taxonomic status. The purpose of this work is to
delimit the phylogenetic species within Genea, charac-
terize the common taxa in Europe and determine
the phylogenetic relationships within the genus.
MATERIALS AND METHODS
Fungal samples.—More than one hundred Genea specimens
have been studied through morphological or molecular
methods or both. European collections are preserved in the
herbaria of the Universidad de Alcalá (AH), Hungarian Nat-
ural History Museum and Budapest (BP), whereas North
American taxa are deposited at the University of Florida
(FLAS). Original codes from personal herbaria also are pro-
vided (SUPPLEMENTARY TABLE I).
Morphological studies.—Samples were morphologically identi-
fied with the aid of available literature (Gilkey 1954, Hawker
1954, Ceruti 1960, Montecchi and Sarasini 2000). Newly col-
lected specimens also were compared with authentic mate-
rial of common European Genea species stored at the
University of Turin (TO) as part of Mattirolo’s collections.
These were sent to Mattirolo from the personal herbaria of
the species authors (Mattirolo 1900b). Authentic specimens
included samples of G. verrucosa,G. papillosa,G. sphaerica,G.
sphaerica f. sporis spinuloso-reticulatis and G. vagans. The type
specimen of G. hispidula and G. klotzschii were obtained
from K (Royal Botanical Gardens, Kew, UK), and compared
with new specimens. Authentic specimens of American spe-
cies also were compared with the newly analysed samples.
Original material came from TO, BERN (University of
Bern, Switzerland) and OSC (Oregon State University,
USA). Finally, a comprehensive descriptive work of authentic
material of common European and American Genea species
by J.M. Trappe (unpubl) was used as a guide for comparisons
and identifications.
Microscopic images were taken with an Olympus BH2-
BHS 100W camera with Apo optical. Helicon Focus software
(Helicon Soft. Ltd.) was used to blend focused areas of multi-
ple images. Spores were measured with Piximètre software
(Alain Henriot) and Mycometre 2.01 (Georges Fannechère).
Microscopic study was performed in water, lactophenol cot-
ton blue, Trypan blue or Congo red-floxine. Scanning elec-
tron microscopy (SEM) was performed at University of
Alcalá (Spain) with a Zeiss DSM-950. Ascospore sizes exclude
spore ornamentation and both ascospore sizes and spore
ornament sizes are presented as height 6width.
DNA processing and phylogenetic analyses.—DNA extraction
and PCR amplification were performed as described by
Alvarado et al. (2012). Primers ITS1F and ITS4 (White et al.
1990, Gardes and Bruns 1993) were used for the ITS region;
primers LR0R and LR5 (Vilgalys and Hester, 1990) were used
for the 28S rDNA ribosomal region (28S rDNA) and EF1-
983F and EF1-1567R (Rehner and Buckley 2005) for the
translation elongation factor 1a(TEF1) gene, for both PCR
and sequencing. Sequences were edited for errors in MEGA
5 (Tamura et al. 2011).
Sequence alignment and phylogenetic analyses.—Sequences were
aligned with the most similar sequences in GenBank identi-
fied through BLASTn searches (Altschul et al. 1997). Two
alignments were constructed: 192 ITS sequences and a com-
bined 28S rDNA-TEF1 alignment including sequences from
65 specimens. Homologous sequences of Genea were
retrieved from Smith et al. (2006), Tedersoo et al. (2006),
Perry et al. (2007), Guevara-Guerrero et al. (2012), Hansen
et al. (2013) and Osmundson et al. (2013). Sequences first
were aligned in MEGA 5 (Tamura et al. 2011) with Clustal
W (Higgins et al. 1994) and edited manually. The ITS align-
ment contained insertions in some species, but these regions
were excluded from the final anlyses. Alignments are avail-
able in TreeBase (ID18402).
Phylogenetic analyses follow those of Alvarado et al. (2012).
Aligned loci were subjected to MrModeltest 2.3 (Nylander
2004) in PAUP*4.0b10. Bayesian analysis was performed with
optimal models in MrBayes 3.1 (Ronquist and Huelsenbeck
2003) with ITS1-5.8S-ITS2 data partitioned, two simultaneous
runs, six chains, temperature set to 0.2, and sampling every
100th generation until convergence parameters were met
after about 5 550 000 (ITS) and 1 390 000 generations (28S
rDNA –TEF1). The first 25% trees were discarded as burn-
in. Last a full query for the best-scoring maximum likelihood
tree was performed in RAxML (Stamatakis 2006) using the
standard search algorithm (ITS1-5.8S-ITS2 data partitioned,
2000 bootstrap replications). Support values were considered
significant when bootstrap (BP) values were above 70% and
posterior probability (PP) values were above 0.95.
Estimation of divergence times.—Genea divergence times were
estimated with BEAST 1.8 (Drummond et al. 2012). Because
no fossil records or previous studies of dating time are avail-
able for Pyronemataceae, we employed a secondary calibra-
tion approach according to Prieto and Wedin (2013) and
Bonito et al. (2013). Molecular clock analysis of the ITS
region was rejected because ITS was too divergent. Thus
only the 28S rDNA dataset was analyzed. The jModel‐
test (Posada 2008) identified GTR+G(Rodríguez et al. 1990)
as the best fit to our data. We created two monophyletic
taxon sets; the first contains all Pezizomycetes taxa except
Orbilia sp.; the second contains two Tuberaceae sequences
(T. aestivum and Choiromyces meandriformis). Analyses were
run under an uncorrelated lognormal relaxed molecular
clock, setting the tree prior to the birth-death speciation
ALVARADO ET AL.: GENUS GENEA 443
FIG. 1. 50% majority rule consensus ITS rDNA phylogram of the genus Genea obtained in MrBayes from 20 550 sampled
trees. Nodes supported by .0.95 Bayesian PP and .70% ML BP are shown with boldface bars.
444 MYCOLOGIA
process. We set a normal prior distribution for the node of
Pezizomycetes group (mean 5317 Mya, SD 537.0 Mya),
as well as for the node of Tuberaceae (mean 5156.9 Mya,
SD 59.0 Mya). A time-calibrated maximum clade credibility
(MCC) tree was reconstructed by running the MCMC simula-
tion for 10 million generations, sampling trees every 1000
generations and discarding the first 50% sampled trees as
burn-in. The effective sample sizes were checked with the
program TRACER 1.5 (Rambaut and Drummond 2009).
RESULTS
The phylogenetic clades that were recovered based on
ITS rDNA (FIG. 1) and 28S rDNA - TEF1 analysis (FIG.
2) matched the morphological species concept of cir-
cumscribed taxa in the genus Genea. Morphologically
(FIGS.3–6) LM and SEM microscopy confirmed the
spore descriptions that have been reported for
recently described species (Alvarado et al. 2014). Sam-
ples from different species and countries (Greece,
Spain and Germany) were colonized by a basidiomy-
cete with a 100% ITS match to Infundibulicybe geotropa
(Bull.) Harmaja (ALV4344, GenBank KT122792,
KT122793). Specimens of G. hispidula,G. arenaria,
G. fragrans,G. balsleyi and G. harknessii matched
sequences with these names already in public data-
bases. Several taxa formed monophyletic groups with
significantly distinct subclades. The high genetic dis-
tances of some subclades suggest the presence of cryp-
tic species within the morphological species concepts,
e.g. in G. verrucosa and G. fragrans. Sequences of
G. sphaerica and G. lespiaultii belong to a single mono-
phyletic group and differ by only 6/570 bp in ITS,
mainly because of two repetitive insertions in G. sphaer-
ica. 28S rDNA sequences were almost identical
between G. lespiaultii and G. sphaerica (FIG. 2), with
only scattered point mutations, while these species dif-
fered in only one nucleotide in the TEF 1 gene.
Many unidentified light brown European specimens
as well as several identified as G. verrucosa f. badia had
ITS sequences clustered with the North American
taxa G. thaxteri Gilkey and G. mexicana Guevara, Göker
& Stielow (Cázares et al. 1992, Guevara-Guerrero et al.
2012), indicating that this clade is found in Europe
and North America. Three European collections
were tentatively identified as the North American
taxon G. anthracina based on morphology (Stewart
and Heblack 1979) and comparison with ITS rDNA
data obtained from G. anthracina isotype (OSC
39,482). The sequence (KT950256) was clean and
had a clear relationship with those obtained from Eur-
opean specimens, although it probably represents an
independent lineage. The only specimen identified as
G. brachytheca revealed a significant genetic relation-
ship with G. hispidula (FIGS. 1, 2) but is here maintained
as an independent taxon until further analysis. We also
generated the first sequences from specimens of
G. vagans (Mattirolo 1900b, Ceruti 1960) suggesting
that this taxon is monophyletic. In contrast, specimens
identified as G. harknessii were not monophyletic based
on ITS, indicating that more work is needed to clarify
the identity of this taxon (FIG. 1).
FIG. 2. 50% majority rule consensus 28S rDNA-TEF 1
phylogram of the genus Genea obtained in MrBayes from
10 425 sampled trees. Nodes supported by .0.95 Bayesian
PP and .70% ML BP are shown with boldface bars and
major clades are labeled A through G. Nodes supported by
ITS inference, but not in 28S rDNA-TEF1 analysis are
annotated shown as labels with the actual Bayesian posterior
probabilities (left) and ML bootstrap proportions (right).
ALVARADO ET AL.: GENUS GENEA 445
FIG. 3. Ascomata of Genea.a.G. brunneocarpa (JC35). b. G. brunneocarpa (VK2922). c. G. compressa (JC28). d. G. fageticola
(H139). e. G. pseudoverrucosa (GK5286). f. G. oxygala (AVM1745). g. G. tuberculata (JC32). h. G. pinicola (VK2865). i. Genea cf.
subbaetica (VK1342). j. G. pseudobalsleyi (CA1). k. G. vagans (AH42939). l. Genea cf. anthracina (GH20100829). m. G. lobulata
(VK2238). Bars: a 0.5 cm, b 1 cm, c–e 0.5 cm, f–h 1 cm, i–k 0.5 cm, l 0.25 cm, m. 1 cm.
446 MYCOLOGIA
FIG. 4. Ascomata of Genea.a.G. hispidula (JC23). b. G. hispidula (GH20050713). c. G. hispidula (NVP). d. G. arenaria
(RM1140). e. G. arenaria (NVH). f. G. arenaria (JC8). g. G. thaxteri (JC24). h. G. thaxteri (JC26). i. G. thaxteri (JC27). j. G. thaxteri
(RM2231). k. G. thaxteri (CA4). l. G. fragrans (JC21). m. G. fragrans (GK4401). Bars: a, b 0.25 cm, c 0.5 cm, d, e 0.25 cm, f 0.5 cm,
g 0.25 cm, h–k 0.5 cm, l, m 0.25 cm.
ALVARADO ET AL.: GENUS GENEA 447
FIG.5.a–k. Ascomata of Genea:a.G. sphaerica (GH20100613). b. G. lespiaultii (NVJ). c. G. verrucosa (AH42941). d. G. verrucosa
(AH42936). e. G. verrucosa (PSS3704). f. G. verrucosa (JC1). g. G. verrucosa (RM2117). h. G. verrucosa (RM2131). i. G. verrucosa
(CA6). j. G. verrucosa (PSS3701). k. G. verrucosa (NVM). l–m. Spores of G. verrucosa:l.G. verrucosa spores (from Vittadini 1831), m.
448 MYCOLOGIA
Combined 28S rDNA - TEF1(FIG. 2) analysis sup-
ported the existence of several infrageneric lineages
(A–F). Most of these also were supported by ITS (FIG.
1), although ITS data split Genea-B clade into Genea-
B1 (G. fageticola,G. oxygala,G. tuberculata) and Genea-
B2 (G. brunneocarpa,G. dentata,G. pinicola). Similarly
ITS resolved Genea-C into Genea-C1 (G. lobulata)
and Genea-C2 (G. gardneri and Genea cf. subbaetica).
We suspect that G. bihymeniata falls in into Genea-C1
with G. lobulata, but no ITS data are available. The
American species G. cazaresii represented the most
basal lineage of Genea in both ITS and combined 28S-
TEF1 analyses, which we refer to as Genea-G. No evi-
dent apomorphic phenotypical trait could be identi-
fied in any of these clades, except for the occurrence
of peridia hairs in Genea-F lineage, although it appears
that these are sometimes absent in G. thaxteri.
The BEAST analysis to estimate divergence times
yielded sufficient effective sample sizes (.200) for
all relevant parameters, indicating adequate sampling
of the posterior distribution. The MCC tree (FIG.7)
was topologically congruent with the inferred com-
bined 28S rDNA - TEF1 tree (FIG. 2). Estimates of
divergence times and the chronogram are illustrated
(FIG. 7). The Genea lineage may have split from Gena-
bea and Gilkeya around 145.5 Mya (95% HPD: 86.7–
206.8 Mya). Similar to previous phylogenies based on
ITS (Erős-Honti et al. 2008, Guevara-Guerrero et al.
2012), the 28S rDNA data suggest that G. cazaresii
forms a lineage with Humaria and is distantly related
to the remaining Genea species. The split between
Genea and the G. cazaresii-Humaria group was estimated
at 84.1 Mya (95% HPD: 47.9–124.8 Mya). The first
divergence within Genea occured during the Creta‐
ceous, about 76.5 Mya (95% HPD: 42.9–112.7 Mya).
DISCUSSION
We examined authentic specimens of the common
European Genea species and compared them directly
with newly collected specimens of these taxa as well
as with the original protologs. When incorporated
with the results of molecular analyses these examina-
tions of old and new specimens made it possible to
identify the most useful diagnostic features within the
genus and resulted in the taxonomic key (see below).
The most controversial species concept is that of the
type taxon G. verrucosa, because of the conflict between
Vittadini’s original protolog and the observations
made on authentic material by all authors after him
regarding spore shape (Berkeley and Broome 1846,
Tulasne and Tulasne 1851, Mattirolo 1900a, Ceruti
1960, Montecchi and Sarasini 2000). Without a desig-
nated type specimen, the original published illustra-
tions (Vittadini 1831, T.II FIG. VII and T.V FIG. I),
probably composed of several collections, must be con-
sidered a lectotype. In T.V FIG.I a cross section of a G.
verrucosa hymenium is depicted (I-a–I-d) along with a
single, isolated, rounded spore (I-h, Q 51.04) and
an ascus containing eight spores (I-f, av. Q 51.20). Vit-
tadini’s original specimens in TO contained spores
(20–)21.5–30(–34) 6(17–)19–24(–26) mm, with Q 5
(1.15–)1.2–1.43(–1.5) (av. Q 51.29). The newly col-
lected specimens tentatively identified as G. verrucosa
had differently shaped spores, ranging from almost
spherical or subspherical to subellipsoidal or ellipsoi-
dal, in accordance with previous reports. Hence we
propose accepting also subspherical spores as typical
of this species. In the present work molecular data
revealed also the existence of at least four distinct
lineages that are refered to as “G. verrucosa complex”
because of our inability to designate a type lineage or
phenotypically discriminate among them. Both black-
ish and reddish specimens of G. verrucosa can be found
intermixed within this complex and neither form con-
stitutes a monophyletic lineage. We observed high ITS
divergence between these lineages (7.62% variable
sites on average between G. verrucosa subclades,
1.15% variable sites on average within each subclade),
suggesting that these probably represent cryptic spe-
cies. This phenomenon is similar to that found with
G. harknessii in California (Smith et al. 2006). It is pos-
sible that some of these cryptic species could be asso-
ciated with some European species names. One
example is G. perlata, which has spores similar to G. ver-
rucosa and has been considered a synonym (Fischer
1897, Hawker 1954, Ceruti 1960) but was differen-
tiated by a highly wrinkled black ascoma. Additional
studies are needed to determine which lineage within
the G. verrucosa complex is the true G. verrucosa. Once
this is completed an epitype could be designated to sta-
bilize the taxonomic status of G. verrucosa.
Samples of G. sphaerica from northern Europe with
rounded spore ornaments are not closely related to
Genea cf. sphaerica from Mediterranean Europe that
have pointed warts on their spores. Studies of
Tulasne’s authentic specimens of G. sphaerica at TO
match with the later descriptions of this species from
central Europe (Hesse 1891, Vacek 1951, Hawker
1954) and the newly collected specimens from these
areas. Spores are on average 28–31 621–24 mmexclud-
ing ornamentation, which is formed by hemispherical
r
G. verrucosa spores viewed with SEM (JC20). n–o. Ascomata of G. verrucosa:n.G. verrucosa (AM2452). o. G. verrucosa (VK2132).
Bars: a, b 0.5 cm, c 1 cm, d 0.5 cm, e, f 1 cm, g, h 0.5 cm, i 1 cm, j, k 0.5 cm, n, o 5 mm.
ALVARADO ET AL.: GENUS GENEA 449
FIG.6.a. G. brunneocarpa spores (JC6). b–cGenea spores viewed with SEM. b. G. brunneocarpa (JC6). c. G. dentata (NVR). d. G.
compressa spores (JC28). e–iGenea spores viewed with SEM. e. G. compressa (JC28). f. G. dentata (JC19). g. G. pseudobalsleyi (CA1).
h. G. fageticola (GK4129). i. G. fragrans (NVQ). j. G. pinicola spores (JC12). k–nGenea spores viewed with SEM. k. G. pseudoverrucosa
(GK5088). l. Genea subbaetica (BM7). m. G. lespiaultii (NVI). n. G. pinicola (JC12). o. G. oxygala spores (JC14). p–tGenea spores
450 MYCOLOGIA
warts 1–5mm diam. On the other hand newly collected
Mediterranean samples are frequently lobate with
spores 24–30 620–26 mm excluding ornamentation
that consists of spiny or sometimes conical warts
1–2.5 mm high and 1–2mm wide. These Mediterranean
collections fit the description of G. sphaerica f. lobulata
(Moreno-Arroyo et al. 1998a), which was elevated to
species rank by Alvarado et al. (2014) as G. lobulata.
They also may represent the unpublished G. sphaerica
“f. insolita”(Tulasne and Tulasne 1851) and G. sphae‐
rica “f. sporis spinuloso-reticulatis”(Mattirolo 1903),
although authentic material of the latter taxon
revealed a different spore ornamentation of slightly
larger conic or truncated digitated warts 2–4mm
high 61–3mm wide. Spores are also more ellipsoid
(24–26.5 619–21 mm). These characters are similar
to those of G. verrucosa. However, because the name
published by Mattirolo is invalid, we consider it doubt-
ful. Up to three distinct lineages were identified within
the G. lobulata complex, but we found no diagnostic
features that could be used to distinguish among
them, so these are interpreted as putative cryptic
r
viewed with SEM. p. G. oxygala (JC16), Genea spores q. G. tuberculata (JC32). r. G. vagans (JC17). s. G. sphaerica (NVF). t.
G. lobulata (BM1043). Bars: a 30 mm, b, c 5 mm, d 30 mm, e–h5mm, i 10 mm, j 20 mm, k 10 mm, l–n5mm, o 30 mm, p 5 mm, q 25 mm,
r–t5mm.
FIG. 7. Divergence time chronogram of Genea and related genera. Both mean of estimated divergence time and the 95%
highest posterior density bars are indicated at the nodes, based on BEAST analyses.
ALVARADO ET AL.: GENUS GENEA 451
species. Spore sizes of G. lobulata or G. sphaerica do not
match those reported for G. sphaerica var. lazzari (mean
24.5 621.5 mm), so the status of G. sphaerica var. lazzari
remains unresolved. Of interest, an ITS rDNA
sequence from Oregon (USA) identified as G. harknes-
sii (AY927851) is similar to our sequences of G. lobulata
(FIG. 1), indicating that this group is found on two
continents.
Our analyses based on ITS, 28S rDNA and TEF 1sug-
gest that G. sphaerica is closely related to G. lespiaultii,
and no significant value supported them as mutually
exclusive monophyletic groups. RNA polymerase II
second largest subunit (RPB2) data also was generated
from a single collection from each taxon (primers
bRPB2-6F and bRPB2-7R, Matheny 2005), revealing
4/365 differences (GenBank KT950257 and
KT950258). We conclude that these genetic markers
were insufficient to resolve a putatively recent evolu-
tionary split between these species. In addition, G.
lespiaultii is one of the most easily recognized species
of Genea because of its striking flat and irregular spore
ornamentation, preventing us from challenging its spe-
cies status.
Many European samples displayed a significant rela-
tionship with G. arenaria, from western North America,
although they formed distinct lineages (ca. 96% simi-
larity in ITS, 99.6% in 28S rDNA), interpreted as cryp-
tic species. Morphological features of European Genea
cf. arenaria match the holotype (Harkness 1899) and
the reexamination of authentic material from Hark-
ness sent by Lloyd to TO herbarium (Smith et al.
2006). This last specimen presents a dull brown verru-
cose peridium, with tomentum present at deep folds.
Spores measure 27–31 621–25 mm including orna-
mentation, which consists in small truncate papillae
or cones 1–2mm high.
A similar situation also can be seen in the clade con-
taining G. mexicana (Guevara-Guerrero et al. 2012), G.
thaxteri from eastern USA and several European collec-
tions of G. verrucosa f. badia, suggesting that these taxa
are closely related and perhaps synonyms. Morphologi-
cally G. mexicana has lobate ascomata with some peri-
dial trichomes while G. thaxteri is described as
globose-depressed, slightly lobed or without lobes and
without peridial hairs (Gilkey 1954). The inner cavity
was described as single in G. thaxteri but chambered
in G. mexicana. Spore ornamentation is formed of
mostly rounded or conical papillae in G. thaxteri
whereas G. mexicana is reported to have versiform con-
ical, truncate, bi- or trifurcate warts. A comparative
study of both types was conducted, revealing spore
size is almost identical (24.5–31.0 620.0–25.0 in G.
thaxteri vs. 25.5–31.0 618.0–22.0 in G. mexicana). Aver-
age Q was slightly lower in G. thaxteri (Q 51.27) than
G. mexicana (Q 51.42). Comparing these types with
other American G. thaxteri collections, some degree
of variability in spore size among genetically identical
specimens was observed, some specimens exceeding
the ranges mentioned (up to 36.5 mm). Another vari-
able feature is peridium hairiness. The observation of
scarce hairs in G. thaxteri type suggests this characteris-
tic is not a reliable diagnostic feature. European speci-
mens of this lineage were variable in color (ranging
from light yellow to tan or pale brown), shape (globose
to subglobose when young, to lobed or stellate when
mature) and in peridium wart morphology, which
can include minute papillae to small irregular warts
or small polygonal warts with a slightly darkened
apex. Spore size varied among specimens, some of
them matching the range reported by Gilkey (1939),
some others exceeding these values up to 35 mm. Due
to these similarities we hypothesize that both taxa
represent a single variable species. However, this
should be addressed in a specifically focused project
where additional collections and markers are studied.
Regarding G. fragrans (5G. klotzschii), both macro-
and microscopical features (black peridium generally
without pyramidal warts, large spores ornamented
with large truncated warts) can help discriminate this
species from G. verrucosa (Wallroth 1833, Dietrich
and Klotzsch 1839, Berkeley and Broome 1846, Corda
1854, Fischer 1897, Hawker 1954). Of the five authen-
tic specimens of G. klotzschii in Berkeley and Broome
herbarium in K(M), one is immature (K173360) and
two others are in poor condition (K173525,
K173526). K173368, from Leigh Woods, Bristol, UK,
matches the species concept of G. sphaerica. This is
probably the specimen mentioned by Hawker (1954)
and perhaps the same checked by Tulasne and
Tulasne (1851) and Fischer (1897), who reported
that spores did not exceed 32 mm. The remaining spe-
cimen in Berkeley’s and Broome’s herbarium is from
Stapleton, Bristol (K173527), and has spores 37–40 6
31–25 mm including ornamentation of large truncated
warts 3–4mm high. This is probably the one depicted
in Corda (1854) and matches other reports of this spe-
cies (Fischer 1897, Hawker 1954). The new species G.
fageticola (Alvarado et al. 2014) can be similar micro-
scopically to G. fragrans but has a more globose ascoma
without labyrinthic hymenium folds, a peridium
usually covered with small polygonal warts instead of
minute papillae, epithecium brownish instead of black-
ish and a large basal tuft of brownish or reddish
hyphae. Molecular data indicate that specimens mor-
phologically identified as G. fragrans belong to two or
three different clades. We could not identify any mor-
phological features to discriminate between these
lineages, although specimens were collected in differ-
ent habitats. Two G. fragrans lineages were found with
lowland trees such as Quercus or Carpinus, whereas
452 MYCOLOGIA
the other lineage seems associated with Fagus sylvatica.
Wallroth (1833) discovered this species under F. sylva-
tica at Straußberg (Thüringen, Germany), but later
reports of the same type collection mention an oak for-
est instead (Dietrich and Klotzsch 1839). Clearly addi-
tional studies are needed to resolve the species
concepts within this group.
We also report the first molecular data for G. vagans,
supporting its status as an independent species. Origi-
nal Italian collections by Mattirolo (1900) have been
described and illustrated by Ceruti (1960), and later
reports are known from Russia (Bucholtz 1901) and
Spain (Vidal 1997). Spore size documented by these
authors match our own observations on authentic
material from Mattirolo and those made on the new
specimens studied in the present work (32–41 627–
37 mm including ornamentation), although spore
ornamentation is slightly smaller (cones 3–6mm
wide 62–4mm high) in our recent collections.
This species resembles the recently proposed G. com-
pressa (Alvarado et al. 2014) but differs because of its
spore ornamentation (4–4.5 64mminG. compressa)
and the overall spore size (29.5–33 625.5–28 mmin
G. compressa).
Three European samples are related to the North
American species G. anthracina because their morphol-
ogy and genetic profile are an excellent match of the
isotype (Stewart and Heblack 1979). European speci-
mens are black, have a conspicuous basal tuft of
hyphae and are 0.8–1.3 cm diam. The ascomata are
covered with polygonal warts that extend through the
apical orifice to the internal epithecium, which lines
a single, regular inner chamber lacking peridium wall
projections. The peridium has scattered long hairs.
Asci are 190 626 mm on average whereas spores are
(23–)25–26(–27) 6(17.5–)18–20 (–21)mm (Qm 5
1.3) excluding ornamentation, which is formed by
small slightly scattered conical-truncate warts, 2.5 6
1.5–2.5 mm (width 6height). However, molecular dif-
ferences between the European specimens and the
American isotype prevented us from identifying them
all as G. anthracina before the potential intraspecific
variability of this species is explored with new Ameri-
can collections.
We also found three European collections that are
phylogenetically close to the North American taxon
G. gardneri Gilkey. Of interest, the European collec-
tions morphologically fit the description of G. subbae-
tica because of their long asci, small peridium cells,
fruiting season (winter but not spring) and the white
mycelium that is found in most specimens. In the pre-
sent work we tentatively used this epithet for these spe-
cimens, although the authentic molecular identity of
the original concept of G. subbaetica could not be
obtained from the type or other authentic material
(kindly loaned by Dr Baldomero Moreno-Arroyo) after
many attempts on different herbarium samples.
Finally, in the present study we attempted to esti-
mate the divergence time of the genus Genea and its
major lineages. We used a secondary calibration
approach according to the closest reliable divergence
times estimated by Prieto and Wedin (2013) for Pezi-
zomycetes based on fossils and by Bonito et al. (2013)
for the Tuberaceae sister taxa. In spite of the large
uncertainty it seems that the genus Genea might have
diverged in the late Cretaceous at the same time as
several infrageneric lineages of Tuber (Bonito et al
2013). A more accurate and reliable divergence time
estimation could be achieved only through a new ana-
lysis involving fossil records or some additional,
unlinked loci. In the future a worldwide sampling
(especially in Asia and South America) could facili-
tate the analysis of ancestral area reconstruction of
Genea.
KEY TO THE GENUS GENEA
Prolog: Truffle fungi have not been studied carefully
across the globe so the distribution of Genea remains incom-
plete, particularly in Asia where there are likely many unde-
scribed species. Most Genea species thus far appear to be
restricted to either Europe or North America. Based on these
putative distributions we have provided geographical infor-
mation in the key to make it more useful to future truffle tax-
onomists (NA 5North America, ENA 5eastern North
America, WNA 5western North America, EU - Europe. All
measures refer to average values).
1. Ascomata black or reddish black or with polygonal
warts, peridium not hairy ........................2
1. Ascomata yellowish, tan, brownish or reddish, or
hairy peridium ...............................18
2. Lacking wall projections, ascomata with a single reg-
ular inner cavity, not lobed (ENA) ................3
2. With wall projections or with multiple inner cavities,
lobed or not ..................................4
3. Peridium composed of a single layer, hemispherical
spore ornaments .................... G. anthracina
3. Peridium composed of two distinct layers, conical
spore ornaments ....................... G. balsleyi
4. Wall projections into inner cavity forming large ster-
ile whitish trama plates (EU) . . . ..................5
4. Wall projections into inner cavity, large whitish
trama plates absent ............................7
5. Spores ornamented with flat warts ........ G. lespiaultii
5. Spores ornamented with hemispherical or irregular
warts ........................................6
6. Spores ornamented with small irregular warts .G. lobulata
6. Spores ornamented with hemispherical warts G. sphaerica
7. Spores .32 mm long (ornamentation excluded) .....8
7. Spores ,32 mm long (ornamentation excluded) ....10
8. Spore covered with low semiglobose papillae (WNA) . . . .
........................G.gardneri
8. Spores ornamented with anvil-shaped warts ..........9
9. Ascomata lobed, small basal tuft of hyphae . . G. fragrans
ALVARADO ET AL.: GENUS GENEA 453
9. Ascomata globose, abundant basal tuft of hyphae ......
.......................G.fageticola
10. Paraphyses branched, hymenium interrupted,
American (WNA) ............................11
10. Paraphyses not branched, hymenium continuous,
European (EU) . ............................12
11. Spores with small irregular pointed warts ,3mmhigh...
.......................G.harknessii
11. Spores with rounded warts, hymenial layers some-
times fused ........................G. bihymeniata
12. Spores with truncated warts ....................13
12. Spores with irregular, pointed or conical warts .....14
13. Spore warts .2mm broad .........G. pseudoverrucosa
13. Spores warts ,2mm broad ..........G. pseudobalsleyi
14. Spores with irregular pointed warts ..............15
14. Spores with conical warts ......................17
15. Spore warts .3mm high, either fang-like or molar-
like .................................G. dentata
15. Spore warts ,3mm high ......................16
16. Spore warts irregular or pointed, fruiting in spring-
summer ............................G. verrucosa
16. Spore warts irregular, pointed or rounded, fruiting
in winter ...........................G. subbaetica
17. Spores ,32 mm long, including ornaments 4–4.5
64mm (wide 6high) ............... G. compressa
17. Spores .32 mm long, including ornaments 4–66
2–4mm (wide 6high) ..................G. vagans
18. Ascomata covered with hairs, at least at the apical
orifice .....................................19
18. Ascomata lacking hairs ........................24
19. Spores .30 mm long .........................20
19. Spores ,30 mm long .........................22
20. Spores .36 mm long, obtuse roundish warts (NA,
EU) ...............................G. hispidula
20. Spores sometimes .30 mm, versiform warts (NA) . . .21
21. Peridium with scarce hairs ...............G. thaxteri
21. Peridium hairy . . .....................G. mexicana
22. Truncated spore warts narrower than 2 mm (NA) .....
......................G.arenaria
22. Conical or rounded warts 2–4(5) mm broad (ENA) . .23
23. Conical warts, ascus wall thickened throughout .......
.....................G.brachytheca
23. Rounded warts, ascus wall thickened only at the base . .
......................G.asperula
24. Spores ,21 mm long, ornamentation 0.5 mm high
(WNA) ............................. G. cazaresii
24. Spores .21 mm long .........................25
25. Spores with medium truncated warts .............26
25. Spores with large hemispherical or small pointed
warts ......................................27
26. Associated with Pinus spp. (EU) ...........G. pinicola
26. Associated with Quercus spp. (EU) .....G. brunneocarpa
27. Spores with large hemispherical warts (EU) . G. oxygala
27. Spores with small pointed warts (EU) . . . G. tuberculata
ACKNOWLEDGMENTS
We thank Mr A. Priego and Mr J.A. Pérez of the Electron
Microscopy Service of the University of Alcalá de Henares
for their help with the SEM. We also thank Luis Monje and
Ángel Pueblas of the Department of Drawing and Scientific
Photography at the Alcalá University for their help with digi-
tal treatment of images; we also thank Dr J. Rejos, curator of
AH herbarium, for his assistance with specimens examined
in the present study. Financial support for ME Smith was pro-
vided by University of Florida’s Institute for Food and Agri-
cultural Sciences. We also thank P. Chautrand, A. García
Blanco, G. Hensel, R. Martínez, D. Mitchell, A. Montecchi,
B. Moreno-Arroyo, G. Pacioni, J.-B. Perez, C.M. Pérez del
Amo, P. Ribollet, M.A. Sanz, F. Sáinz, T. Sánchez and J.M.
Vidal for kindly sending us their collections and images.
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