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Truffle trouble: what happened to the
Tuberales?
Article in Mycological Research · October 2007
DOI: 10.1016/j.mycres.2007.08.004 · Source: PubMed
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mycological research 111 (2007) 1075–1099
journal homepage: www.elsevier.com/locate/mycres
Truffle trouble: what happened to the Tuberales?
Thomas LÆSSØEa,*, Karen HANSENb,y
a
Department of Biology, Copenhagen University, DK-1353 Copenhagen K, Denmark
Harvard University Herbaria – Farlow Herbarium of Cryptogamic Botany, Cambridge, MA 02138, USA
b
article info
abstract
Article history:
An overview of truffles (now considered to belong in the Pezizales, but formerly treated in
Received 10 February 2006
the Tuberales) is presented, including a discussion on morphological and biological traits
Received in revised form
characterizing this form group. Accepted genera are listed and discussed according to a sys-
27 April 2007
tem based on molecular results combined with morphological characters. Phylogenetic
Accepted 9 August 2007
analyses of LSU rDNA sequences from 55 hypogeous and 139 epigeous taxa of Pezizales
Published online 25 August 2007
were performed to examine their relationships. Parsimony, ML, and Bayesian analyses of
Corresponding Editor: Scott LaGreca
these sequences indicate that the truffles studied represent at least 15 independent lineages within the Pezizales. Sequences from hypogeous representatives referred to the fol-
Keywords:
lowing families and genera were analysed: Discinaceae–Morchellaceae (Fischerula, Hydnotrya,
Ascomycota
Leucangium), Helvellaceae (Balsamia and Barssia), Pezizaceae (Amylascus, Cazia, Eremiomyces,
Helvellaceae
Hydnotryopsis, Kaliharituber, Mattirolomyces, Pachyphloeus, Peziza, Ruhlandiella, Stephensia,
Hypogeous
Terfezia, and Tirmania), Pyronemataceae (Genea, Geopora, Paurocotylis, and Stephensia) and
Pezizaceae
Tuberaceae (Choiromyces, Dingleya, Labyrinthomyces, Reddellomyces, and Tuber). The different
Pezizales
types of hypogeous ascomata were found within most major evolutionary lines often nest-
Pyronemataceae
ing close to apothecial species. Although the Pezizaceae traditionally have been defined
mainly on the presence of amyloid reactions of the ascus wall several truffles appear to
have lost this character. The value of the number of nuclei in mature ascospores as a delimiting family character is evaluated and found to be more variable than generally assumed.
ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
Fungi pursuing the truffle strategy by producing underground
sporocarps have long been recognized as a polyphyletic group
with representatives in the former Zygomycota now Glomeromycota (Endogone, Glomus a.o.), Ascomycota, and Basidiomycota.
Those with asci were at one time all placed in the Tuberales
(e.g. Tulasne & Tulasne 1851; Fischer 1897; Knapp 1950;
Hawker 1954; Eckblad 1968; Korf 1973a). Nannfeldt (1946)
wrote: ‘The question is raised whether Tuberineae is monophyletic or whether it is composed of different operculates
that have evoluted convergently into hypogeous forms.’
Malençon (1938) also advanced ideas about the evolution of
truffles and their transformation from epigeous apothecial
species to hypogeous truffles, but, as pointed out by Burdsall
(1968), his system relied too heavily on macroscopic features.
Korf (1973b) discussed the evolution of convoluted pezizalean
forms, both above and below ground, and although he accepted the Tuberales, he indicated that at least some of the
taxa were derived along various evolutionary lines within
the Pezizales. He considered Tuberales to be a biological unit
rather than a phylogenetic one. Trappe (1971) published a similar statement, and finally Trappe (1979), proposed that the
order be abandoned, with one major part being moved to the
* Corresponding author.
E-mail address: thomasl@bi.ku.dk
y
Present Address: Swedish Museum of Natural History, Cryptogamic Botany, Box 50007, SE-10405 Stockholm, Sweden.
0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2007.08.004
1076
Pezizales and just Elaphomyces to the Elaphomycetales. Fischer
(1897) had earlier referred Elaphomyces to the ‘Plectascineae’
but alongside the Terfeziaceae. Later (Fischer 1938), Terfeziaceae
reappeared within the Tuberales. Trappe (1979) kept some hypogeous lines, as families, within his Pezizales, but other hypogeous taxa were placed alongside epigeous species in various
mixed families. Burdsall (1968) had already convincingly
merged one tuberalean genus (Geopora) with the pezizalean
genus Sepultaria. Eckblad (1968) gave many clear arguments
for not accepting the Tuberales but, nevertheless, concluded
the opposite. In the first Outline of the Ascomycetes (Eriksson
1982) Tuberales (with Geneaceae, Terfeziaceae, and Tuberaceae)
were relegated to synonymy of Pezizales. Ainsworth & Bisby’s
Dictionary of the Fungi (Hawksworth 1983) likewise abandoned
the use of Tuberales and listed the order under Pezizales (and
Elaphomycetales). Trappe’s hypothesis was tested in a longlasting study of the ultrastructure of pezizalean taxa guided
by Kimbrough and summarized in Kimbrough (1994), that
for example, led to the placement of Hydnobolites in the Pezizaceae, based on both cytological and ultrastructural features of
asci and ascospores. Also the placement of Barssia in the Helvellaceae followed from these studies. The most important
character used was the morphology of the complicated septal
pore-apparatus at the base of the asci (Kimbrough 1994). Another prominent feature, the number of nuclei in the mature
spores that originated in Berthet’s (1963) studies on epigeous
Pezizales, was also taken into account when trying to delimit
natural groups of truffles (e.g. Berthet 1982; Donadini 1986a,
b). With the onset of the molecular taxonomy era, these early
hypotheses have gradually been confirmed and expanded
upon, or in some cases, corrected (e.g. O’Donnell et al. 1997;
Norman & Egger 1999; Percudani et al. 1999; Hansen et al.
2005; Perry et al. 2007). In a comprehensive treatment of European (mainly Italian) truffles Montecchi & Sarasini (2000) refer
former Tuberales taxa to Elaphomycetales, with just Elaphomyces, and Pezizales with seven families: Pezizaceae (four genera),
Pyronemataceae (four genera), Geneaceae (two genera), Helvellaceae (three genera), Balsamiaceae (two genera), Terfeziaceae
(four genera) and Tuberaceae with two genera. Although, they
cite recent molecular results, they have chosen a conservative
approach by following the systems proposed in Trappe (1979)
and Pegler et al. (1993). One group of researchers (PargueyLeduc et al. 1987b, 1990; Janex-Favre & Parguey-Leduc 2003)
proposed to accept Tuberales based mainly on the genera Tuber
and Terfezia that were considered closely related, mostly
based on a perceived different development of asci and ascospores. van Brummelen (1994) gave a summary of the arguments put forward up to that time. Eriksson (2006b),
influenced by data published by e.g. de Hoog et al. (2005), discussed what to do nomenclatorily if Pezizales are restricted
to Pezizaceae. Although Tuberales are a possible choice, he proposed to find another name. Currently, however, there is no
supported molecular phylogenetic evidence that suggests
Pezizaceae are not part of the Pezizales (the Pezizaceae are supported as monophyletic by a BS value of 100 %, but the relationships among the included families in e.g. de Hoog et al.
(2005) are without support).
The purpose of this paper is to review morphological and biological traits, and the systematics of the passively dispersed,
more or less hypogeous Pezizales. Using all currently available
T. Læssøe, K. Hansen
LSU sequences from pezizalean truffles, in analyses with
a broad sample of epigeous pezizalean taxa, we will further investigate the phylogenetic relationships and evolution of these
truffle fungi. Ascomycetous truffles, which are now considered
to be non-pezizalean (Elaphomyces, Eurotiomycetes), are not
treated in detail. The taxonomic position of all accepted taxa
at and above generic level are given and compared with previous classifications. The accepted classification is based on molecular phylogenetic analyses and morphological characters.
A truffle definition
Ascomycete truffles can be defined as producing sporocarps
below or at ground level and with a simultaneous loss of active
spore dispersal. In several genera, for example Geopora and
Helvella, species with intermediate characters can be found.
Also Sarcosphaera coronaria is an example of a fungus that
has nearly become a truffle. It forms apothecia below ground
and often opens by a rather small aperture, but as the spores
are actively ejected it can still be classified as a ‘‘cup fungus’’.
The genus Caulocarpa was based on such hypogeous Sarcosphaera ascomata (Trappe 1975c). Although some species tend
to produce sporocarps in or on the litter, we still group them
with the truffles as long as they have lost active spore dispersal. Glaziella and Paurocotylis are good examples.
Morphological features of pezizalean truffles
The ascomata are typically fleshy but can be quite hard and
cartilaginous. An outer rind (peridium) is often present and
can be almost woody and sculptured. Even at maturity the
spores do not become powdery, except in a few genera (e.g.
Carbomyces) that are adapted to extreme xeric conditions.
There is a continuous variation from truffles with a single cavity lined with a hymenium, often with a single opening, to
truffles with intricate foldings or with pockets of asci in
a firm gleba. Weber et al. (1997) defined three different types
of hypogeous ascomata within the Pezizales: ptychothecia
with persistent, recognizable hymenia and variously folded
or even solid ascomata; stereothecia without hymenia and
solid ascomata; and exothecia with external hymenia. None
of these ascoma types can accommodate Paurocotylis and
Glaziella. These genera produce ascomata that are hollow,
without paraphyses, and furthermore, are unusual in being
fully exposed at maturity. Hansen et al. (2001) reviewed the
morphological features of the truffles considered to belong
to the Pezizaceae. Those pezizalean species that have been
studied in ontogenic detail, such as Tuber and Terfezia species
(Janex-Favre & Parguey-Leduc 2003), start out as apothecial
before folding occurs. The asci can at one end of the variation
resemble those of operculate species being cylindrical with
spores in one row or at the other end be completely globose
with or without a pedicel and with a variable number of often
very large spores. The ascospores vary in colour from hyaline
to almost black, and in surface features from smooth and
thin-walled to very thick-walled with intricate ornamentation. The ascus walls can be more or less layered and amyloid
or inamyloid. The Pezizaceae are characterized by amyloid asci,
but this feature appears to have been lost in many pezizaceous truffles (Hansen et al. 2001, 2005).
What happened to the Tuberales?
Truffle identification and nomenclature
Castellano et al. (1989) have published a slightly dated key to
the spores of genera found in north temperate forests. An
updated key, taking further characters into use, can be found
on the Internet (http://natruffling.org/ascokey.htm), and an
earlier printed version was published by Trappe & Castellano
(1992). Trappe’s (1979) synoptical key is still useful. In Europe
two main illustrated accounts with keys are current
(Montecchi & Sarasini 2000; Pegler et al. 1993). Other important
contributions include Lange (1956), Lawrynowicz (1988), and
Montecchi & Lazzari (1993).
The names of pezizalean truffles are given sanctioned status if included in Fries (1821–1832) and should be used when
available for a given taxon. In practice, however, another tradition has evolved, where Vittadini’s (1831) much more accurate work on European truffles has been used as the de facto
starting point for especially Tuber nomenclature. As Trappe
(2001) has pointed out, it will be necessary to propose these
Vittadini names for conservation over the sanctioned Friesian
names in order not to disrupt the very long usage of these
names for such economically important organisms.
Distribution, diversity, and dispersal
Although, false truffles (hypogeous Basidiomycota) have been
collected in extreme arctic environments, the true truffles
would appear to have a more limited distribution, with a clear
peak in diversity in temperate–subtropical, often rather dry
climates. Although a high number of publications are dedicated to truffles, a reasonable picture of the diversity and distribution of the group has still not been achieved. Castellano
et al. (2004) from one long Australian study suggest a figure
of 600 species (although mainly of false truffles), most of
which remain to be described. A part of this project was described in Claridge et al. (2000). Only ten of these species belong to the Ascomycota, and two apparently to undescribed
genera. [See also the extensive review of Australian and New
Zealand sequestrate fungi by Bougher & Lebel (2001).] Only
Europe and parts of North America can be claimed to be
reasonably well covered with respect to hypogeous fungi
(Castellano et al. 2004). Distributions of European taxa are
dealt with in Lawrynowicz (1991). Parts of Asia would seem
to be equally rich in truffles. Africa and South America are apparently especially poor in hypogeous ascomycetes but be
aware of the likely differences in sampling efforts in various
regions. Verbeken & Walleyn (2003) in a checklist of subsaharan sequestrate fungi only reported one pezizalean species,
Terfezia decaryi from Madagascar. In addition, three species are
known from the southern dry lands of continental Africa,
including the Kalahari (Marasas & Trappe 1973; Ferdman
et al. 2005). Two were separated as new genera (Ferdman
et al. 2005). The third, Terfezia austroafricana, was listed as
a member of Terfezia subgen Mattirolomyces and may require
a new combination, as Mattirolomyces has been raised to generic rank. Although too little is known, it is fairly clear that
many localized endemics are to be found among pezizalean
truffles.
It has been hypothesized that all, or nearly all, truffles are
passively dispersed with animal vectors, but there is very little
1077
experimental evidence to support this assertion. Various
small mammals, including Australian marsupials (e.g.
Claridge & May 1994), and voles and chipmunks in North
America, collect and often hoard ascomata and by this activity
are thought to play an active dispersal role (e.g. Fogel & Trappe
1978; Maser et al. 1978). The stomach contents of voles and
chipmunks have been found to contain over 70 % truffles. So
far it has not been shown that pezizalean truffle spores can
germinate after gut passage but in all likelihood they can.
The volatile compounds truffles exude when ripe clearly substantiate the claim that these mammals are the key dispersal
vectors. Also larger mammals such as boar and deer are well
known for their ability to locate and digest truffles, and presumably, also act in a beneficial way to the truffles by their
dispersal abilities. The volatile compounds may resemble
pheromones (Claus et al. 1981) and can also be used in species
recognition (e.g. Marin et al. 1984; Pacioni et al. 1990). Trappe
(1977) and Trappe et al. (2001) have speculated that the ectomycorrhizal truffle partners migrated along with the rodent
dispersers and the truffles themselves, many populations
later becoming isolated as a result of continental drift. Many
invertebrates (Diptera etc) also actively seek out truffles, but
although a more parasitic aspect to this relationship can be
postulated, additional dispersal ability cannot be ruled out.
Even birds have been claimed to actively seek out truffles
and possibly act as dispersal vectors (Alsheikh & Trappe
1983b; Castellano et al. 2004). One example concerns the desert
truffle Phaeangium (or Picoa) lefebvrei, which is believed to be
dispersed by various species of desert-adapted larks, but
also by cream-coloured courser and hoopoe. Another case
deals with Paurocotylis pila, which at maturity has epigeous,
orange–red fruit bodies coinciding with the fall of likewise
bright-coloured Podocarpus fruits, known to be bird dispersed.
Whether birds may also be involved in the dispersal of introduced British populations of Paurocotylis is not known.
Material and methods
Taxon sampling and alignment
To summarize and determine the phylogenetic placement of
hypogeous taxa within Pezizales, LSU rDNA sequences from
48 hypogeous species (represented by 55 specimens) and 134
epigeous pezizalean species (represented by 141 specimens)
were compiled for analyses (for sequence accession numbers,
see online Supplementary Data Table 1). Sequences were selected to represent all sub-lineages within Pezizales based primarily on Hansen et al. (2001, 2005), O’Donnell et al. (1997), and
Perry et al. (2007). Nucleotide sequences were aligned by hand
using the software program Se-Al v. 2.0a11 (Rambaut 1996
Se-Al: Sequence Alignment Editor; available at http://evolve.
zoo.ox.ac.uk/). The LSU rDNA contains highly divergent regions across all of the Pezizales. Therefore, three subset
alignments were constructed, each representing one of three
distinct lineages identified within the Pezizales (Fig 1) (Landvik
et al. 1997; Hansen & Pfister 2007). The three alignments include representative taxa from the families Pezizaceae (lineage
A; Fig 2); Caloscyphaceae, Discinaceae, Helvellaceae, Morchellaceae,
Rhizinaceae, and Tuberaceae (lineage B; Fig 3); and
1078
T. Læssøe, K. Hansen
Pyronemataceae
Ascodesmidaceae
C
Glaziellaceae
Sarcoscyphaceae
Sarcosomataceae
Morchellaceae-Discinaceae
B
Helvellaceae-Tuberaceae
Rhizinaceae
Caloscyphaceae
Pezizaceae
A
Ascobolaceae
Fig 1 – Schematic tree giving an overview of the three major
clades (A–C) identified within Pezizales using SSU rDNA
sequences (after Landvik et al. 1997). Truffles have evolved
within the families highlighted in bold. Families listed for
each clade follow Eriksson (2006a). The Morchellaceae–
Discinaceae and Helvellaceae–Tuberaceae lineages are
according to O’Donnell et al. (1997).
Ascodesmidaceae and Pyronemataceae (lineage C; Fig 4). Members
of the Sarcoscyphaceae and Sarcosomataceae were not included,
because no truffle taxa were affiliated with these families.
The final datasets included 68 epigeous species (from 72 specimens) and 17 hypogeous species (20 specimens) (lineage A); 22
epigeous species (one specimen each) and 22 hypogeous species (23 specimens) (lineage B); and 44 epigeous species (47
specimens) and nine hypogeous species (12 specimens) (lineage C). Based on phylogenetic analyses of higher-level relationships (e.g. Landvik 1996; Hansen & Pfister 2007; Perry et al.
2007), Neolecta vitellina was used as an outgroup for lineage A
(with the ingroup also including taxa from the lineages B and
C); two species of Peziza and Iodophanus for lineage B; and Ascobolus and Peziza for lineage C. Alignments are available from
TreeBASE (http://www.treebase.org) as accessions M3364 (lineage A), M3363 (lineage B), and M3362 (lineage C).
Phylogenetic analyses
Analyses of the LSU were performed using PAUP version
4.0b10 for Unix (Swofford 2002) and MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001) on G5 Macintosh computers. MP, parsimony BS (PB), and Bayesian analyses were performed as in
Hansen et al. (2005), except Bayesian MCMC were run for 5M
generations. The GTR þ I þ G model of sequence evolution
was selected for each dataset using MrModeltest v. 2.2
(Nylander 2004). In Bayesian analyses, the first 1500 trees
were deleted as the ‘burn-in’ period of the chain for the lineage
A dataset, and Bayesian PP are based on the last 48,500 trees
sampled. For the lineages B and C, the last 46,700 and 49,000
trees were used, respectively. Clades represented by
PB 75 % and/or PP 95 % are considered to be significantly
supported.
Based upon the results of the phylogenetic analyses, topologically constraint MP and ML analyses were used to evaluate
how many times hypogeous taxa have been derived from
epigeous apothecia-forming taxa, with loss of forcible spore
discharge. Constraint topologies were manually specified in
PAUP. The MP analyses were performed under the constraints,
using the same settings as specified above (Hansen et al. 2005).
The ML analyses consisted of heuristic searches with ten random addition sequence replicates, tree bisection–reconnection (TBR) branch swapping and starting trees obtained via
stepwise addition. The ML GTR þ I þ G model parameters
used, were fixed to values estimated from one of the unconstrained MP trees (from the original MP analyses). The
Kishino–Hasegawa test (Kishino & Hasegawa 1989) and the
Shimodiara–Hasegawa tests (Shimodaira & Hasegawa 1999)
were used to compare constrained and unconstrained topologies in PAUP version 4.0b10.
Results
Phylogenetic relationships of truffles within lineage A
The LSU dataset of lineage A included 973 characters with 338
being parsimony informative. Parsimony analyses resulted in
1391 equally MPTs (1327 steps, CI ¼ 0.333, RI ¼ 0.678). The
Pezizaceae are highly supported as monophyletic (PB 99 %, PP
100 %), with Ascobolaceae as the sister group (PB 97 %, PP 100,
Fig 2). The strict consensus tree of all MPTs is highly resolved,
but the deep level relationships are not well supported. Fourteen fine-scale lineages that correspond to the lineages resolved in Hansen et al. (2005) are recovered by all analyses.
The 17 truffle species (11 genera) sampled are nested within
five or six of the 14 lineages; Eremiomyces echinulatus is resolved separately with Peziza vacini in the MP analysis (Fig 2),
but is placed in the Plicaria–Hapsidomyces lineage, along with
Peziza phyllogena in ML and Bayesian analyses. The truffle
Amylacus tasmanicus forms a highly supported sister taxon
(PB/PP 100 %), to a highly supported clade of three species of
the truffle genus Pachyphloeus, the anamorph Glischroderma
sp. and the apothecial Scabropezia (PB 98 %, PP 100 %). The
two species of the truffle genus Hydnotryopsis form a strongly
supported group with Sarcosphaera (PB/PP 100 %). The three
specimens of Sarcosphaera coronaria (from North America and
Denmark) exhibit quite large sequence variation, but form
a monophyletic group (PP 95 %). The placement of Mattirolomyces is uncertain; it is deeply nested within the Peziza s. str. lineage in the strict consensus tree of all MP trees, but is grouping
with Iodophanus, as a sister group to the Peziza s. str. lineage in
ML and Bayesian analyses (none of these positions are with
80
100
97
Pezizaceae
Neolecta vitellina
100
Byssonectria terrestris
98
Melastiza contorta
100
100
Otidea onotica
98
Otidea umbrina
100
100
Smardaea amethystina
Greletia reticulosperma
100
Morchella elata
Ascobolus
carbonarius
100
Ascobolus denudatus
100
81
Ascobolus crenulatus
Marcelleina pseudoanthracina
Marcelleina persoonii
100
76
Peziza gerardii (2)
100
71
100
PachyphloeusPeziza gerardii (1)
100
Iodophanus hyperboreus
Amylascus
100
Iodophanus carneus
Amylascus tasmanicus
Scabropezia flavovirens
100
Scabropezia scabrosa
100
100/89 89
Pachyphloeus melanoxanthus
100
Glischroderma sp.
98
100
Pachyphloeus citrinus (1)
98 100/99 Pachyphloeus citrinus (2)
Pachyphloeus virescens
100
Boudiera tracheia
100
Boudiera dennisii
100
Pachyella babingtonii
77
Pachyella punctispora
100
Pachyella violaceonigra
100
Pachyella habrospora
100
Peziza natrophila
100
Peziza quelepidotia
Peziza apiculata
100
Peziza succosa
98
100
Peziza succosella
78
Peziza michelii
Peziza sp.
95
Sarcosphaera coronaria (1)
Sarcosphaera coronaria (2)
Sarcosphaera* 100 95
Sarcosphaera coronaria (3)
Hydnotryopsis
Hydnotryopsis sp.
100
100
Hydnotryopsis setchellii
99
Mattirolomyces terfezioides
Peziza howsei
*
96
Peziza proteana, petersii
90
Peziza exogelatinosa
92
Peziza proteana f. sparassioides
Peziza lobulata
Peziza subviolacea
Peziza s. str.
Peziza ampelina
Peziza subcitrina
96/72
Peziza echinispora
*
Peziza varia
98 Peziza arvernensis
97 78 Peziza ampliata
93
Peziza vesiculosa
100 Iodowynnea auriformis (2)
100
Iodowynnea
auriformis (1)
Iodowynnea-Kaliharituber
*
75
Kalaharituber pfeilii
*
Peziza luteoloflavida
100
Peziza obtusapiculata
100
100/95 Peziza polaripapulata
Peziza retrocurvata
Ruhlandiella berolinensis
Peziza whitei
99/76 Peziza limnaea
Peziza
* Pezizabadia
alaskana
*
97/
* Peziza badiofusca
*
Peziza saniosa
Peziza depressa
Peziza griseorosea
Peziza depressa-Ruhlandiella
Peziza atrovinosa
Peziza ellipsospora
100
100 Terfezia boudieri (1)
100 94 Terfezia boudieri (2)
Terfezia claveryi (1)
91
100/94 Terfezia claveryi (2)
Cazia flexiascus
Tirmania pinoyi
100/98
Tirmania nivea
infolded or chambered
Peziza ostracoderma
ptycothecium
*
Peziza phyllogena (1, 2)
Hapsidomyces venezuelensis
solid ptycothecium
Plicaria carbonaria
98/74 * Plicaria trachycarpa
stereothecia
Plicaria endocarpoides
Peziza vacini
exothecium
Eremiomyces echinulatus
100
99
100/98
Peziza bananicola
Peziza subisabellina (2)
Peziza subisabellina (1)
10 changes
Fig 2 – Phylogenetic relationships among epigeous and hypogeous taxa in Pezizaceae (lineage A), derived from parsimony
analyses of LSU rDNA sequences. One of 1391 most parsimonious trees. Terminal taxa represent individual specimens
(from Hansen et al. 2001, 2005; Ferdman et al. 2005; Norman & Egger 1999). Neolecta vitellina was used to root the
phylogeny. Hypogeous lineages are shown in bold. Numbers above branches represent PP (95 %). Numbers below
branches represent PB support (70 %). Symbols by taxon names indicate specific fruiting body types of truffles.
Fine-scale lineages, as defined in Hansen et al. (2005), that include truffles are indicated for discussion in the text.
1080
T. Læssøe, K. Hansen
Peziza vesiculosa
Peziza depressa
Iodophanus carneus
Morchella conica
100
100
Morchella esculenta
Verpa bohemica
Morchellaceae
Verpa conica
Disciotis venosa
Fischerula subcaulis
Leucangium carthusianum
100
Pseudorhizina californica
98
Discina macrospora
Gyromitra melaleucoides
Discinaceae
Hydnotrya cerebriformis
99/73
Hydnotrya cubispora
Balsamia magnata
100
91
Barssia oregonensis
Underwoodia columnaris
Wynnella silvicola
Helvella crispa
100
* Helvella rivularis
* Helvella pezizoides
93
Helvella aff. cupuliformis
* Helvella albella
100
Helvella atra
96
Helvella lacunosa
Tuber californicum
98/78
Tuber puberulum (1)
100/89
Tuber puberulum (2)
90 Tuber oligospermum
Tuber borchii
77
Tuber maculatum
98
Tuber canaliculatum
Tuber gibbosum
100
100
99
Tuber aff. gibbosum
Tuber rufum var. rufum
Tuber melanosporum
100
Tuber excavatum
84
Dingleya verrucosa
100
Reddellomyces donkii
95
Labyrinthomyces varius
Choiromyces venosus
Choiromyces alveolatus
100/70
Rhizina undulata
Rhizinaceae
Caloscypha fulgens
Caloscyphaceae
infolded or chambered
ptycothecium
100/97
100
100
solid ptycothecium
stereothecia
10 changes
Helvellaceae
Tuberaceae
Fig 3 – Phylogenetic relationships among epigeous and hypogeous taxa within the families Morchellaceae, Discinaceae,
Helvellaceae and Tuberaceae (lineage B), derived from parsimony analyses of LSU rDNA sequences. One of three most
parsimonious trees. Terminal taxa represent individual specimens (primarily from O’Donnell et al. 1997). Peziza vesiculosa,
P. depressa and Iodophanus carneus were used to root the phylogeny. Hypogeous lineages are shown in bold. Numbers
above branches represent PP (95 %). Numbers below branches represent PB support (70 %). Symbols by taxon names
indicate specific fruiting body types of truffles.
significant support). Kaliharituber is suggested as closely related to Iodowynnea (PB 75 %). The truffle genera Cazia, Ruhlandiella, Terfezia, and Tirmania, and two truffle species of Peziza,
P. ellipsospora and P. whitei, are resolved among apotheciaforming Peziza species in the P. depressa–Ruhlandiella lineage.
This lineage, excluding Ruhlandiella, is supported by 100 %
PP, but is with only 53 % PB.
At least nine independent origins of hypogeous forms are
supported by the LSU gene trees (Fig 2). Constrained MP and
ML analyses forcing the two species of Hydnotryopsis to be
monophyletic could not be rejected (Table 1). Likewise, forced
monophyly of the hypogeous taxa within the P. depressa–
Ruhlandiella lineage (not including Eremiomyces), did not yield
trees that were significantly longer than the unconstrained
MP trees. However, under this constraint the ML tree was
significantly worse than the unconstrained optimal ML tree
(Table 1). Trees rejected by MP and ML include the following
monophyly constraints: truffles in the P. depressa lineage
including Eremiomyces (with or without Ruhlandiella), Amylascus–Pachyphloeus, Pachyphloeus, Mattirolomyces with Terfezia,
and Kaliharituber with Terfezia (Table 1). The most conservative
conclusion is thus, that forcible spore discharge has been
lost only once within each of the lineages Sarcosphaera–
Hydnotryopsis and P. depressa–Ruhlandiella, once in Eremiomyces,
Kaliharituber, Mattirolomyces, and Amylascus, and three times in
Pachyphloeus (assuming that active spore discharge, once lost,
can not be regained).
Phylogenetic relationships of truffles within lineage B
Parsimony analyses of lineage B yielded three equally MPTs
(1235 steps, CI ¼ 0.423, RI ¼ 0.639) produced from 699 characters, of which 233 were parsimony informative. The strict consensus tree of the three MPTs is highly resolved, but support
for the families are lacking, except for Tuberaceae (PB 84 %,
PP 100 %, Fig 3). The trees recovered by MP, Bayesian, and
What happened to the Tuberales?
100
100
100
98
Pyronemataceae
1081
Ascobolus carbonarius
ptycothecia, infolded or
Peziza vesiculosa
chambered
Smardaea amethystina
ascoma without hymenium,
Greletia reticulosperma
Otidea onotica
chambered
100
99 98
Otidea concinna
10 changes
Otidea alutacea
Humaria hemisphaerica (1, 2)
100/ Genea hispidula (1)
100 Genea hispidula (2)
100
Genea sp.
100 100/
100 Genea harknessii (1)
100/83
100 Genea harknessii (2)
73
Genea arenaria
99/86
Genea verrucosa
Parascutellinia carneosanguinea
Wilcoxina mikolae
100/81 Wilcoxina rehmii
Pyronema confluens
Melastiza cornubiensis
Aleuria aurantia
Byssonectria terrestris
Cheilymenia vitellina
100/
100 Geopora cooperi
96/74 99 Geopora cooperi f. gilkeyae
Geopora sp. 2
100
100 100 Geopora sp. 3
99 Geopora cf. cervina
96/
100
Geopora sp. 1
100/98 Geopora arenicola (1, 2)
*
Miladina lecithina
96
100
Ramsbottomia asperior
100/
Scutellinia scutellata
Trichophaea woolhopeia
Sphaerosporella brunnea
*
*
Anthracobia macrocystis
Sowerbyella imperialis
Tarzetta pusilla
100
Tarzetta catinus
100
100
Stephensia shanorii
99
100
Stephensia bombycina
98 74 Geopyxis carbonaria (1, 2)
100
Paurocotylis pila
Geopyxis sp.
Lasiobolus
cuniculi
100
Lasiobolus ciliatus
100 100
Ascodesmidaceae
92 100 Ascodesmis nigricans
100 Eleutherascus lectardii
100
Pulvinula constellatio
100
100
Pulvinula convexella
Pseudombrophila merdaria
Pseudombrophila theioleuca
100/100
Octospora axillaris
96
100
Neottiella rutilans
76
Lamprospora cf. ascoboloides
100
100
Lamprospora dictydiola
Fig 4 – Phylogenetic relationships among epigeous and hypogeous taxa in Pyronemataceae (lineage C), derived from
parsimony analyses of LSU rDNA data consisting of 894 aligned nucleotides for 56 taxa. One of three most parsimonious
trees. Terminal taxa represent individual specimens. Hypogeous lineages are shown in bold. Numbers above branches
represent PP (95 %). Numbers below branches represent PB support (70 %). Symbols by taxon names indicate specific
fruiting body types of truffles.
ML analyses did not possess any supported conflict. Bayesian
analyses support Helvellaceae (PP 99 %) excluding Underwoodia
columnaris, which is unresolved. A Morchellaceae–Discinaceae
(PP 100 %) and a Helvellaceae–Tuberaceae lineage are resolved
by MP and ML analyses (Fig 3), in accordance with O’Donnell
et al. (1997), who used both SSU and LSU. The truffles Leucangium and Fischerula subcaulis are variously placed within the
Morchellaceae–Discinaceae lineage, and their exact position is
unknown. Two species of Hydnotrya, H. cerebriformis and
H. cubispora, form a monophyletic group (PB 73 %, PP 99 %)
nested within the Discinaceae in all analyses. Balsamia, B. magnata and B. oregonensis, is likewise monophyletic (PB 91 %, PP
100 %) and forms a sister group to a highly supported clade
of apothecial Helvella species and Wynella silvicola in all analyses. The 11 species of Tuber included form a monophyletic
group (PB 69 %, PP 100 %), as a sister group to a clade of four
additional truffle genera, Dingleya, Reddellomyces, Labyrinthomyces, and Choiromyces s. str.
The most parsimonious interpretation of the LSU phylogeny suggests that the truffle form originated four times
within lineage B (Fig 3). Nevertheless, constraint MP and ML
analyses forcing Fischerula, Leucangium, and Hydnotrya into a
monophyletic group could not be rejected (Table 1). This suggests that forcible spore discharge has been lost at least three
times within lineage B, once in the Morchellaceae–Discinaceae
lineage, once in Helvellaceae, and in Tuberaceae.
Phylogenetic relationships of truffles within lineage C
Parsimony analyses of lineage C yielded three equally MPTs
(1679 steps, CI ¼ 0.424, RI ¼ 0.637) from 894 total characters,
of which 302 were parsimony informative. The strict
1082
T. Læssøe, K. Hansen
Table 1 – Evaluation of different constrained tree topologies in MP and ML analyses, compared with the MPTs and the
optimal MLT, respectively, using the Kishino–Hasegawa test for MP and the Shimodiara–Hasegawa test for ML ( p < 0.05)
Tree
MP
a
Lineage A, unconstrained MPT
Lineage A, unconstrained optimal MLT
Hydnotryopsis monophyletic
Truffles in ‘P. depressa lineage’ monophyletic
(including Ruhlandiella and Eremiomyces)
Truffles in ‘P. depressa lineage’ monophyletic
(not including Ruhlandiella,
but including Eremiomyces)
Truffles in ‘P. depressa lineage’ monophyletic
(not including Eremiomyces,
but including Ruhlandiella)
Amylascus and Pachyphloeus monophyletic
Pachyphloeus monophyletic
Mattirolomyces with Terfezia
Kaliharituber with Terfezia
Lineage B, unconstrained MPT
Lineage B, unconstrained optimal MLT
Fischerula–Leucangium with Hydnotrya
Lineage C, unconstrained MPT
Lineage C, unconstrained MLT
Stephensia with Paurocotylis
Stephensia monophyletic
ML
Tree lenght
Significantly
worse?
Ln likelihood
Difference
in LnL
P-value
Significantly
worse?
2078
–
2082 (þ4)
2095 (þ17)
Best
–
No
Yes
–
11183.03738
11189.40900
11224.21753
–
–
6.37162
41.18015
–
–
0.083
0.006*
–
Best
No
Yes
2093 (þ15)
Yes
11213.75681
30.71942
0.004*
Yes
2085 (þ7)
No
11201.55494
18.51755
0.020*
Yes
2098
2090
2094
2098
1235
–
1239
1679
–
1694
1692
Yes
Yes
Yes
Yes
Best
–
No
Best
–
Yes
Yes
11219.85875
11214.23004
11215.54524
11218.85867
–
6540.88728
6550.10480
–
8958.87503
8991.20102
8992.05211
36.82137
31.19266
32.50785
35.82129
–
–
9.21752
–
–
32.32599
33.17708
0.041*
0.005*
0.009*
0.051
–
–
0.077
–
–
0.001*
0.001*
Yes
Yes
Yes
No
–
Best
No
–
Best
Yes
Yes
(þ16)
(þ12)
(þ12)
(þ20)
(þ4)
(þ15)
(þ13)
a Difference in length between MPTs and constrained trees in parentheses.
consensus tree of the three MPTs is nearly completely resolved, but as for the lineages A and B the deep level relationships are poorly supported. Pyronemataceae are suggested to be
paraphyletic, because Ascodesmidaceae are nested within it.
Ascodesmidaceae are highly supported as monophyletic
(Fig 4). Twelve clades of pyronemataceous taxa are recovered
by all analyses, which correspond to those identified by Perry
et al. (2007) who used a much larger taxon sampling. The nine
truffle species included are nested within three, moderate to
highly supported clades with apothecial pyronemataceous
taxa (Fig 4). The five species of the hypogeous genus Genea
form a monophyletic group (PB 86 %, PP 99 %), as a sister group
to the epigeous Humaria hemisphaerica (PB/PP 100 %). The truffle Geopora cooperi, forms a highly supported monophyletic
group with five epigeous species of Geopora (PB/PP 100 %).
Geopora is suggested to be a sister group to a clade of the
apothecial Ramsbottomia, Scutellinia, and Miladina (PP 100 %).
The truffles Stephensia and Paurocotylis pila form a highly supported group with apothecial Tarzetta and Geopyxis (PB 99 %, PP
100 %). Stephensia is suggested to be non-monophyletic;
Stephensia bombycina form a well-supported group with
Geopyxis carbonaria (PB 74 %, PP 100 %), with Paurocotylis pila
(PP 98 %), Geopyxis sp. (PP 100 %), and Stephensia shanorii as successive sister taxa.
The most parsimonious interpretation of the LSU phylogeny suggests that forcible spore discharge has been lost at
least five times within the Pyronemataceae (in Genea, Geopora
cooperi (not completely), Paurocotylis and twice in Stephensia).
The constraint analyses forcing Stephensia to be monophyletic,
or Stephensia and Paurocotylis to be monophyletic were rejected
(Table 1).
Evolution of ascomata types
At least five different forms of ascomata exist within Pezizales.
Epigeous apothecia of various shapes with forcible spore discharge are the most common form and occur in each of the
A, B, and C lineages. This is likely the ancestral form, and
the molecular data suggest that the apothecia-forming
Pezizales have given rise to at least four different types of
hypogeous ascomata without forcible spore discharge ( pro
parte sensu Weber et al. 1997): ptychothecia [hollow to folded
with internal hymenia, in Pezizaceae, Discinaceae, Helvellaceae,
Tuberaceae and Pyronemataceae (Figs 2–4)]; stereothecia [solid
without hymenia, in Pezizaceae, Discinaceae–Morchellaceae,
and Tuberaceae (Figs 2 and 3)]; exothecia [external hymenia,
Ruhlandiella (Pezizaceae, Fig 2)]; and an unnamed type found
in Glaziella and Paurocotylis (Glaziellaceae and Pyronemataceae,
Fig 4; recalls bladder-shaped ptychothecia, but without organized hymenia). The molecular data suggests that ptychothecia and stereothecia have evolved multiple times in different
lineages within Pezizales.
Taxonomy
Taxonomic implications: an overview of accepted
hypogeous Pezizales taxa
Lineage A
The Ascobolaceae have no confirmed hypogeous representatives but various truffle taxa have at times been placed in
What happened to the Tuberales?
the family, e.g. Sphaerosoma and Ruhlandiella (as Muciturbo) (e.g.
Castellano et al. 2004). See Figs 2 and 5A-D.
Pezizaceae Dumort. 1829 (syn. Terfeziaceae E. Fisch. 1897)
The family Terfeziaceae as defined by Zhang (1992a, 1992b)
are included in this family, but was accepted in the latest
Dictionary of the Fungi (Kirk et al. 2001). Recent molecular results
(e.g. Norman & Egger 1999; Hansen et al. 2005) clearly demonstrate it should be relegated to synonymy of the Pezizaceae (see
review in Hansen & Trappe 2002). Thirteen out of 25 genera in
the Pezizaceae (Eriksson 2006a) are exclusively truffle or trufflelike taxa, but several truffle species have also been described
in Peziza. Hansen et al. (2001) gave a review of the genera.
The genus Peziza was found to be non-monophyletic and all
other pezizaceous genera nested within it (Hansen et al.
2001, 2005), and a revised generic arrangement is under way
(Hansen & Pfister, in preparation.). Two lineages discovered
comprise most of the Peziza species, the Peziza s. str. and the
P. depressa–Ruhlandiella lineages, the latter including several
truffles (Cazia, Peziza ellipsospora, P. whitei, Ruhlandiella, Terfezia
and Tirmania; Fig 2). The P. depressa–Ruhlandiella lineage was
highly supported in combined analyses of LSU, b-tubulin,
and RPB2 (Hansen et al. 2005). Only one truffle genus, Mattirolomyces, clusters in Peziza s. str. in MP analyses, but without
support (Fig 2). Three types of hypogeous ascomata exist
within the family (Fig 2). The Amylascus–Pachyphloeus and
the P. depressa–Ruhlandiella lineages produce both ptychothecia and stereothecia. The cardinal feature of Pezizaceae, the
amyloid reaction of the ascus wall, has been lost in several
of the hypogeous taxa (e.g. Cazia and Terfezia).
1083
Amylascus Trappe 1971
Type: Amylacus herbertianus.
The type species of Amylacus has not been sampled for molecular phylogenetic study, but the genus is most likely monophyletic [A. tasmanicus has even been considered a synonym
of A. herbertianus (Beaton & Weste 1982)] and is suggested to
be closely related to Scabropezia and Pachyphloeus. Amylascus
was originally placed in the Terfeziaceae or Geneaceae (Trappe
1971, 1975a), but later, based on the thick-walled, amyloid
asci, was placed in the Pezizaceae (Trappe 1979). Trappe
(1975a) and Beaton & Weste (1982) monographed the genus.
Amylascus includes only the two mentioned species, both
recorded only in Australia.
Cazia Trappe 1989
Type: Cazia flexiascus.
Originally, and at times by some subsequent authors,
placed in the Helvellaceae (Trappe 1989), but Kirk et al. (2001)
place it in the Terfeziaceae. O’Donnell et al. (1997) were the first
to place Cazia in the Pezizaceae. As can be seen from Fig 2, it is
nested within the P. depressa–Ruhlandiella lineage containing
both epigeous and hypogeous taxa. Cazia quercicola Fogel &
States (2002) is only the second recognized species.
Eremiomyces Trappe & Kagan-Zur 2005
Type: Eremiomyces echinulatus (syn. Choiromyces echinulatus).
Ferdman et al. (2005) found this species to cluster within the
Pezizaceae (with Terfezia and Tirmania species) and not with the
type of Choiromyces, which has affinities with the Tuberaceae.
Fig 5 – Fruiting body forms in lineage A (Pezizaceae). (A-B) Sarcosphaera coronaria (A) Closed apothecia, JHP-95.074 (C). (B) Open
apothecia. (C) Hydnobolites cerebriformis, ptychothecia. (D) Terfezia leptoderma, stereothecia. Photos: J.H. Petersen (A),
K. Hansen (B), J. Nitare (C), J. Santos (D).
1084
Besides the molecular results the highly inflated exipular cells
also suggest this fungus belongs to Pezizaceae rather than
Tuberaceae. The exact placement of Eremiomyces within Pezizaceae is not resolved in our analyses, but it is likely among members of the P. depressa–Ruhlandiella, Plicaria–Hapsidomyces or P.
phyllogena lineages (the inclusive clade A in Hansen et al. 2005).
Hydnobolites Tul. & C. Tul. 1843 (Fig 5C)
Type: Hydnobolites cerebriformis.
This genus apparently has only two accepted species,
H. cerebriformis from Europe and H. californicus from North
America. The type species has saccate, amyloid [when pretreated in potassium hydroxide (KOH)] asci formed in poorly
defined hymenia, without well-differentiated paraphyses, in
brain-like, pale ascomata. The spores are globose with a reticulate and spinulose ornament. The genus was previously placed
in the Tuberaceae (Gilkey 1955; Korf 1973a; Castellano et al. 2004)
or in the Terfeziaceae (Hawker 1954; Trappe 1971, 1979). Trappe
(1979) regarded Hydnobolites to be close to Pachyphloeus and
Terfezia (Fig 5D). Kimbrough et al. (1991) suggested a placement
in the Pezizaceae based on ultrastructural observations of septal
pores. They also found the asci to be weakly amyloid after treatment in 2 % KOH. No molecular data are available for Hydnobolites, and the placement is mainly based on the amyloid asci and
the suggested close relationship to Pachyphloeus and Terfezia.
Hydnotryopsis Gilkey 1916
Type: Hydnotryopsis setchellii.
Gilkey (1954) later abandoned the genus and placed it in
Choiromyces. In agreement with Hansen et al. (2005), Hydnotryopsis setchelli and an unnamed Hydnotryopsis are suggested
as closely related to the near hypogeous Sarcosphaera coronaria
(Figs 2 and 5A-B). The constraint analyses forcing the two Hydnotryopsis species to be monopyletic could not be rejected
(Table 1). Hydnotryopsis was placed in the Pezizaceae by Fischer
(1938), and based on the diffusely amyloid asci, followed by
e.g. Trappe (1975c, 1979). The solid ascomata have a peridium
of globose cells, and asci and paraphyses in a hymenial
configuration.
Kalaharituber Trappe & Kagan-Zur 2005
Type: Kalaharituber pfeilii (syn. Terfezia pfeilii).
Ferdman et al. (2005) demonstrated (using ITS and LSU) the
non-monophyletic nature of Terfezia and erected Kalaharituber
for a southern African desert truffle originally described as
T. pfeilii (basionym in error given as Tuber pfeilii). No epigeous
representatives were included in their analyses. Our analyses
indicate a relationship with the epigeous Iodowynnea (PB 75 %,
Fig 2). Taylor et al. (1995) discussed the biology of K. pfeilii and
suggested it could be mycorrhizal with species of Acacia, although a strong association with grasses was noted.
Mattirolomyces E. Fisch. 1938
Type: Choiromyces terfezioides (syn. Mattirolomyces terfezioides, Terfezia terfezioides).
This genus was reinstated by Percudani et al. (1999) and accepted as such by Diéz et al. (2002) and Ferdman et al. (2005), after having been sunk under Terfezia, where it still recides in e.g.
Montecchi & Sarasini (2000). Unlike species of Terfezia, M. terfezioides occurs in woodland or in ruderal habitats rather than in
T. Læssøe, K. Hansen
deserts (e.g. Montecchi & Sarasini 2000). Kovács et al. (2003)
reviewed the mycorrhizae studies on Mattirolomyces and similar taxa and concluded that there is no clear evidence for a mycorrhizal function, and an ectomycorrhizal anatomy does not
develop (with Robinia or Helianthemum ovatum) but, instead, an
anatomy referred to as ‘terfezioid’. Kovács et al. (2007) maintained that the trophic strategy of this fungus remains ambiguous. It forms sclerotia in the same manner as certain species
of Morchella. Although the position of Mattirolomyces is uncertain in our analyses, constraint analyses forcing Mattirolomyces
to group with Terfezia were rejected (Table 1). Healy (2003) described an additional American species, but based on molecular data (R. Healy, K. Hansen and G. Kovács, unpublished
results) this species is not a member of Mattirolomyces.
Mycoclelandia Trappe & Beaton 1984 (syn. Clelandia)
Type: Clelandia arenacea (syn. M. arenacea).
Beaton & Weste (1982) revised the two known species (as
Clelandia) and Trappe & Beaton (1984) replaced the invalid
homonym Clelandia for Mycoclelandia. The asci stain strongly
or diffused blue in iodine solutions. There are no sequences
available, but based on the known morphological features
the genus clearly belongs in the Pezizaceae.
Pachyphloeus Tul & C. Tul. 1844 (syn. Pachyphlodes, Cryptica)
Type: Pachyphloeus melanoxanthus.
The ascomata typically have an apical depression or pore
connecting to a few open veins. The peridium is verrucose
and contains globose cells. Trappe (1979) placed the genus in
the Terfeziaceae and gave the above synonymy (Trappe
1975c). It had mainly been treated within the Tuberaceae (e.g.
Knapp 1951; Korf 1973a). Amyloid asci occur in some species
of Pachyphloeus (e.g. the type species), and based on this and
anatomical features the genus was placed in the Pezizaceae
(Dissing & Korf 1980). This has been confirmed by molecular
data (Norman & Egger 1999; Percudani et al. 1999; Hansen
et al. 2005). Phylogenetic analyses of LSU suggest that the
type species is more closely related to species of Scabropezia
than to other species of Pachyphloeus (PB 89 %, PP 100 %, Fig 2).
Also, constraint analyses forcing the three included Pachyphloeus spp. to be monophyletic were rejected (Table 1). This
suggests that Scabropezia may be a synonym of Pachyphloeus.
Peziza Fr. 1822 (syn. Hydnoplicata)
Type (lectotype): Peziza vesiculosa.
Several hypogeous species, with passive spore dispersal,
have been accepted in the otherwise epigeous, apothecial
genus Peziza. Trappe (1979) noted six hypogeous species in
Peziza and recently Peziza infossa (syn. P. quercicola) was added
(Fogel & States 2002, 2003). Although this latter species is described as having operculate asci, no active spore discharge
had been observed. Peziza has been demonstrated several
times, using molecular phylogenetics, to be non-monophyletic (see above under Pezizaceae). The two pezizas with passive spore dispersal, P. whitei and P. ellipsospora, included in
the molecular analyses, are nested within the P. depressa–Ruhlandiella lineage (Hansen et al. 2001, 2005) (Fig 2). In this lineage,
these two taxa represent a less derived truffle form; both
produce infolded ptycothecia, with a single opening, cylindrical asci with eight ascospores in a single row and paraphyses
What happened to the Tuberales?
placed in hymenia. Both species have retained the amyloid reaction of the asci. More derived truffle forms in this lineage
(Terfezia and Tirmania) produce compact ascomata (stereothecia), with elongate-clavate to sub-globose asci (5-8 spored),
randomly arranged in fertile areas, separated by sterile veins.
Tirmania has amyloid asci, whereas this reaction is lost in
Terfezia. The relationships among the taxa in the P. depressa–
Ruhlandiella lineage are not unambiguously resolved, and a hypothesis about the evolution of these forms must await
further molecular studies using more variable gene regions
and a larger taxon sampling. Trappe & Claridge (2006), nevertheless, resurrected Hydnoplicata for P. whitei based on the molecular results by Hansen et al. (2001). However, depending on
the delineation within this lineage (see also Hansen et al. 2005
and Fig 2), other possible and older generic names could be
Terfezia or Tirmania. Hydnoplicata was based on H. whitei later
transferred to Peziza (Trappe 1975c), and this was again
confirmed by the molecular phylogenetic study by Hansen
et al. (2001, 2005). Beaton & Weste (1982) gave an account of
P. whitei. Also Korf (1973b) discussed this species (as P. jactata).
Ruhlandiella Henn. 1903 emend. Dissing & Korf 1980 (syn.
Tremellodiscus C.G. Lloyd, ?Muciturbo P.H.B. Talbot 1989)
Type: Ruhlandiella berolinensis.
Ruhlandiella is more or less epigeous but with passive spore
dispersal and with a somewhat convoluted ascoma, where the
hymenium covers the surface (exothecial) rather than being
disposed internally. The paraphyses have characteristic gelatinous sheaths. Dissing & Korf (1980) placed this genus in the
Pezizaceae with a proposed relationship to the genera Sphaerozone, Boudiera, and Plicaria. Molecular results place it in the
P. depressa–Ruhlandiella lineage (Hansen et al. 2005) (Fig 2).
Muciturbo was accepted and listed in the Ascobolaceae by
Castellano et al. (2004), but Galán & Moreno (1998) and Hansen
(2000) suggest it as a synonym of Ruhlandiella based on a detailed comparative study of the proposed distinguishing characters. Dissing & Korf (1980) also noted that another Hennings
genus, Exogone, could represent an additional generic synonym and an additional species. Ruhlandiella (as Muciturbo)
has been associated with a Chromelosporium anamorph
(Warcup & Talbot 1989) in accordance with other connections
in this clade (Hansen et al. 2005). Warcup & Talbot (1989)
reported the spores of Muciturbo species to be uninucleate.
Sphaerozone Zobel 1854 (syn. Sphaerosoma subgen. Tulasnia)
Type: Sphaerozone ostiolatum (syn. S. tulasnei).
This is a monotypic genus with exothecial, more or less
spherical, and to some extent convoluted ascomata, and
amyloid asci. These characters, on current evidence place
the genus within the Pezizaceae. The asci are more or less
as in typical members of the family but indehiscent, and
the paraphyses are likewise typical. The exposed hymenium
also suggests a fairly recent radiation from actively dispersed
ancestors. Beaton & Weste (1978) overlooked the amyloid asci
in the type species. The non-amyloid species, Sphaerozone
echinulatum and S. ellipsosporum, should not be accepted in
the genus, as also stated in Beaton & Weste (1982), and
were duly transferred to Gymnohydnotrya (Zhang & Minter
1989b). Dissing & Korf (1980) first noted the amyloidity of
the asci in the type species and also clarified the
1085
nomenclatural confusion surrounding the names Sphaerozone and Sphaerosoma. There is a certain resemblance to the
genus Ruhlandiella. All known collections are from the
vicinity of ectotrophic plants, so it is most likely ectomycorrhizal. Montecchi & Sarasini (2000) illustrate and
describe the genus but also cited Sphaerosoma as a synonym
(see this).
Terfezia (Tul. & C. Tul.) Tul. & C. Tul. 1851 (Fig 5D)
Type (lectotype): Terfezia leonis (syn. Terfezia arenaria).
Two species of Terfezia, T. boudieri and T. claveryi, are
deeply nested within the P. depressa–Ruhlandiella lineage
(Hansen et al. 2005) (Fig 2). The Terfeziaceae were based on
the lack of structure in the arrangement of the asci that
led early workers (e.g. Fischer 1897) to consider Terfezia outside the Tuberales. Vizzini (2003) considered the Tuberaceae
and Terfeziaceae to exhibit extreme convergent morphology
and also noted that the relationship of these families have
been especially controversial. Trappe (1971) accepted the
family Terfeziaceae within the Tuberales and later in the
Pezizales (Trappe 1979). Trappe & Sandberg (1977) studied
the Japanese/North American non-desert species T. gigantea
in detail and described a rather complicated ascospore wall
with minute spines, while Janex-Favre & Parguey-Leduc
(1985) and Janex-Favre et al. (1988) studied the ascus structure and ascospores in T. claveryi and T. leptoderma and found
similarities to Tuber. Janex-Favre & Parguey-Leduc (2003)
again studied the ascomata and concluded that Tuber and
Terfezia should be retained within the Tuberales. Norman &
Egger (1999) and Percudani et al. (1999) found evidence for
a position within the Pezizaceae (see also Kalaharituber and
Mattirolomyces). Diéz et al. (2002), in a recent ITS study,
hypothesized a single origin of the so-called desert truffles,
Tirmania and Terfezia, but included only hypogeous taxa
and no other truffle taxa from the P. depressa–Ruhlandiella
lineage. Trappe (1971) characterized the genus Terfezia as
the most heterogenous genus in the Terfeziaceae. For a review
of the mycorrhizal biology of the genus see Kovács et al.
(2003).
Tirmania Chatin 1892 [date disputed: 1890 sec Trappe; 1891
sec Hansen et al. 2001]
Type: Tirmania africana (syn. T. nivea).
The amyloid reaction of the asci combined with a double
ascospore wall, with the outer smooth and the inner with a reticulate-roughened wall, characterize the genus according to
Alsheikh & Trappe (1983a). The two species accepted by these
authors associate with species of Helianthemum, but the exact
nature of this association is disputed (Kovács et al. 2003). They
apparently disperse by wind after drying in situ, rather than
relying on an animal vector. The species are prized as food
items, and Rayss (1959, as cited in Alsheikh & Trappe 1983a)
suggested that the manna that fed the Israelites could have
been Tirmania truffles. Moreno et al. (2000) reported smooth
spores in T. nivea and a fine net-like ornament on T. pinoyi
spores. See also the descriptions in Malençon (1973) and in
Montecchi & Sarasini (2000). Diéz et al. (2002) studied a number
of desert truffles by molecular phylogenetic analyses, and
concluded that the sampled species formed a monophyletic
group. Trappe (1979) transferred the genus to the Pezizaceae
1086
based on the amyloid asci. Our analyses place Tirmania in the
P. depressa–Ruhlandiella lineage.
Lineage B
The monotypic, parasitic Rhizinaceae and Caloscyphaceae have
no known hypogeous representatives. Truffles forming
ptychothecia and stereothecia are identified in both the
Morchellaceae–Discinaceae and Helvellaceae–Tuberaceae lineages
(Figs 3, 6A-H). The family Tuberaceae is unique in its high diversity of strictly hypogeous taxa.
Morchellaceae–Discinaceae
O’Donnell et al. (1997) placed Leucangium and Fischerula as
incertae sedis due to suspected long-branch attraction between
these taxa. Additional sampling of hypogeous taxa in this
group, including the type species of Fischerula, could possibly
help resolve this problem.
Fischerula Mattir. 1928 (Fig 6A)
Type: Fischerula macrospora.
Mattirolo (1928) and Knapp (1951) separated Fischerula from
Tuber based on the peculiar spore ornamentation and the
more or less fusiform asci. Trappe (1975b, 1979) placed Fischerula in the Helvellaceae, a placement that can be rejected as long
as the two known species are considered congeneric. The
ascoma of the American taxon, F. subcaulis, has a stipe-like
extension, as the name indicates, which is absent on the
European taxon.
Leucangium Quél. 1883
Type: Leucangium ophthalmosporum (syn. L. carthusianum).
Li (1997) studied the ultrastructure of Leucangium carthusianum, often treated within Picoa, and found it to be close to species in Morchellaceae and Helvellaceae. The structure of the
excipulum also indicated such a relationship. Li found the ascospores to be multi-nucleate, which would point towards the
Morchellaceae rather than the Helvellaceae. Likewise, O’Donnell
et al. (1997) found that L. carthusianum clustered in the neighbourhood of the Morchellaceae, while the type of Picoa clustered
with Otidea (Pyronemataceae) (data not shown in O’Donnell et al.
1997). L. carthusianum has apiculate-fusiform ascospores in
saccate asci. Palfner & Agerer (1998b) described the ectomycorrhizae of this species.
Discinaceae Benedix 1961 (syn. Hydnotryaceae M. Lange 1956)
Besides the epigeous taxa Discina, Pseudorhizina and Gyromitra, this family also includes the hypogeous taxon Hydnotrya
(O’Donnell et al. 1997) (Figs 3, and 6C-D). The family name Hydnotryaceae has been used to replace Pseudotuberaceae (nom.
inval., Art. 36.1) (e.g. Burdsall 1968), but is itself invalid (no
Latin nor any other kind of diagnosis, e.g. Art. 36.1).
Hydnotrya Berk. & Broome 1846 (syn. Geoporella, Gyrocratera)
(Figs 6C-D)
Type: Hydnotrya tulasnei.
Knapp (1950, 1952) discussed the genus, including the synonym Geoporella, and gave a fairly detailed description, whilst
a thorough key with a few misplaced taxa can be found in
Gilkey (1954). Trappe (1975c) dealt with the generic names
Geoporella and Gyrocratera. Trappe (1979), Donadini (1986b)
T. Læssøe, K. Hansen
and later Abbott & Currah (1997) accepted the genus in the
Helvellaceae, which cannot be confirmed by the molecular
data. Donadini (1986a) reported 4-nucleate spores. The morphological variation within the genus spans more or less hollow ascomata with cylindrical asci to nearly solid ascomata
(Figs 6C-D) with clavate-saccate asci. There is likewise a great
variation in spore shape and ornamentation. Whether the variation in spore characters should be given taxonomic importance in generic assignment awaits further molecular data.
Zhang (1991b) demonstrated that there is a conspicuous, but
non-functional, opening in the ascus apex of H. cerebriformis.
Gymnohydnotrya B.C. Zhang & Minter 1989
Type: Gymnohydnotrya australiana.
Zhang & Minter (1989b) accepted three Australian species
and placed the genus in the Helvellaceae based on the four nuclei in the spores. The main diagnostic feature was the lack of
a peridium, an external, and in the type species also internal
hymenium, and the non-pigmented spores with an unusual
and intricate ornamentation (a complex reticulum) as
revealed by SEM. Vizzini (2003) lists this genus in the Discinaceae based on the similarity to Hydnotrya and the 4-nucleate
ascospores. There are no published LSU sequences available
for phylogenetic analysis. Two of the species had previously
been placed in Sphaerozone (Beaton & Weste 1978).
Helvellaceae Fr. 1823 (syn. Balsamiaceae E. Fisch. 1897)
The Balsamiaceae, a family accepted in an emended version
by Trappe (1979) and by e.g. Pegler et al. (1993), were considered a synonym of the Helvellaceae by van Brummelen (in
Dissing & Schumacher 1994) and in an emended version by
O’Donnell et al. (1997), a conclusion that was followed by
e.g. Eriksson & Winka (1998) and Hansen & Knudsen (2000).
Analyses of LSU identified a Balsamia–Barssia lineage (PB 91 %
and PP 100 %) as a poorly supported sister group to a
Helvella–Wynnella lineage (Fig 3). However, this relationship
was highly supported in combined analyses of LSU and SSU
(PB 100 %, O’Donnell et al. 1997; Hansen & Pfister 2007).
Balsamia Vittad. 1831 (syn. Pseudobalsamia E. Fisch.) (Fig 6E)
Type (lecto): Balsamia vulgaris.
Knapp (1950) gave a detailed account of this genus, which he
placed in ‘section B’ of his own (invalid; Art. 36.1) family
Pseudotuberaceae, but he later (Knapp 1952) placed it in his
‘Eu-tuberaceae’, based on further developmental studies; a
conclusion also reached by Hawker (1954). Donadini (1986b) observed four nuclei in mature spores and proposed a placement
in the Helvellaceae. The asci can be more or less organized in
a palisade-like structure. Morphologically, Balsamia species
are typical truffles with closed fruit bodies with a veined interior and a coarse peridium (Fig 6E). The asci are sac-like with
clustered spores. The spore morphology is simple as in many
species of Helvella. Species delimitation has been a subject of
discussion with Szemere (1965) taking a very broad view.
Trappe (1975c) agreed that Pseudobalsamia should be placed in
synonymy with Balsamia. Palfner & Agerer (1998a) described
the ectomycorrhiza formed between B. alba and Pseudotsuga.
Barssia Gilkey 1925
Type: Barssia oregonensis.
What happened to the Tuberales?
1087
Fig 6 – Fruiting body forms in lineage B (Morchellaceae-Discinaceae-Helvellaceae-Tuberaceae). (A) Fischerula macrospora,
solid ptychothecium. (B) Choiromyces venosus, solid ptychothecium. (C) Hydnotrya tulasnei, ptychothecia, JV87-356 (C).
(D) Hydnotrya michaelis, ptychothecia, JHP-00.018 (C). (E) Balsamia polysperma, ptychothecia, JV97-080 (C). (F) Helvella astieri,
ptychothecia, (C-65663). (G) Tuber aestivum, stereothecia, JHP-00.395. (H) Tuber rufum, stereothecia, JV93-321(C). Photos:
J. Santos (A), J.H. Petersen (B, D, G detail), J. Vesterholt (C, E, H), T. Læssøe (F), C. Lange (G).
1088
Kimbrough et al. (1996) studied the ultrastructure of the type
species. Trappe (1979) included another monotypic genus, Phymatomyces, in Barssia, but as the type has been lost, this
Japanese taxon should be re-investigated. Barssia ascomata
have a smoother surface compared to species of Balsamia.
Molecular results indicate a very close relationship between
Barssia and Balsamia, so that it may be a sound move to
synonymize these genera, but more taxa, including the type
of Balsamia, should be sampled before such a decision is made.
Helvella L. 1753: Fr.
Helvella astieri Korf & Donadini (Fig 6F) (Korf 1973b) is the
only known truffle within the genus. No molecular data are
available for H. astieri, but its placement in Helvella is convincing on morphological grounds. It has closed semi-hypogeous
fruit bodies and apparently passive spore dispersal, but an
operculum is still present. The species is very rarely recorded,
but is known from France and Denmark (Hansen & Knudsen
2000). The similarity of H. astieri and species of Hydnotrya
was used in placing Hydnotrya in the Helvellaceae (Trappe
1979; Pegler et al. 1993). Trappe (1979): ‘Korf in effect emended
the family (Helvellaceae) to include astipitate, infolded and
chambered ascomata by the description of Helvella astieri
Korf & Donadini. This species is essentially a Hydnotrya with
operculate asci and hyaline spores’. This view was strongly
opposed by Donadini (1986a), as he found spores, paraphyses
and excipulum exactly as in Helvella.
Insufficient data [placed here in Eriksson (2006a)]:
Picoa Vittad. 1831
Type: Picoa juniperi.
This genus was placed in the Balsamicaeae by e.g. Trappe
(1979) and likewise in Montecchi & Sarasini (2000). Some species have asci arranged in a clear palisade, whereas in others
the asci are more dispersed. The genus can be difficult to differentiate from Balsamia based on the characters employed
e.g. by Montecchi & Sarasini (2000). Preliminary LSU rDNA sequence data of P. juniperi, suggest it is more closely related to
Otidea (unpublished data in O’Donnell et al. 1997) than to the
taxa in clade B (as sampled by O’Donnell et al. 1997).
Tuberaceae Dumort. 1822
Only hypogeous taxa cluster alongside the likewise hypogeous genus Tuber. Ascomata produced by the Dingleya–
Choiromyces lineage show a persistent hymenium (chambered
to completely compressed ptychothecia), whereas ascomata
produced by Tuber spp. have lost the hymenium (stereothecia). Tuber is the most speciose genus of ascomycetous truffles, and it is known from many areas around the world,
including North America, Central America, Europe, and Asia,
but apparently not from subsaharan Africa and South America. It has been introduced to Australia (Bougher & Lebel
2001). Dingleya, Reddellomyces, and Labyrinthomyces clearly
have a centre of diversity in Australia and New Zealand.
Choiromyces Vittad. 1831 (syn. e.g. Piersonia) (Fig 6B)
Type: Choiromyces meandriformis (syn. C. venosus)
Although often placed in the Helvellaceae (e.g. in Pegler et al.
1993) current molecular phylogenies place the type species as
T. Læssøe, K. Hansen
a sister to Tuber, making it possible to include it in the Tuberaceae (O’Donnell et al. 1997). Gilkey (1955) and also Korf
(1973a) suggested this placement, whereas Hawker (1954)
and others (e.g. Trappe 1979) placed the genus in the Terfeziaceae based on structural studies. Zhang & Minter (1989a) studied C. gangliformis (considered by some, e.g. Montecchi &
Sarasini (2000), as a possible synonym of C. meandriformis) in
detail and found four nuclei in the spores, which could
indicate the Helvellaceae. However, 4-nucleate spores are also
commonly found in Tuber. Zhang & Minter (1989a) found
multi-layered ascus walls in taxa belonging to Choiromyces as
opposed to taxa of f.ex. Terfezia. This complex wall system
would appear to characterize taxa in the Tuberaceae. They
also emphasized the strange, pitted spore ornamentation.
Dingleya Trappe 1979, emend. Trappe, Castellano &
Malajczuk 1992
Type: Dingleya verrucosa.
The genus was described from New Zealand and stated to
differ from Hydnotrya species by having a more solid, but
apparently still chambered gleba and a verrucose peridium.
Later, the affinities were considered to be with Reddellomyces
and Labyrinthomyces (Trappe et al. 1992), which our analyses
confirm (Fig 3). Trappe et al. (1992) recognized six species. It
is not unlikely that in a future revision the three genera will
be lumped.
Labyrinthomyces Boedijn 1939, emend. Trappe, Castellano &
Malajczuk 1992
Type: Labyrinthomyces varius.
Trappe et al. (1992) accepted this genus within the Pyronemataceae s.l. (as tribe Otideae or undescribed tribe), but the
type species is highly supported within Tuberaceae in molecular phylogenies (O’Donnell et al. 1997) (Fig 3). There is a strong
relation to Reddellomyces and Dingleya (PB 95 %, PP 100 %).
Zhang & Minter (1988), Beaton & Weste (1977), and Malençon
(1973) also discussed the status of this genus, but their concept
included Dingleya and Reddellomyces, whereas Trappe et al.
(1992) restricted the genus to the type species.
Paradoxa Mattir. 1935
Type: Paradoxa monospora.
Knapp (1951) discussed this genus and declared ‘Stellung
dieses Genus ist noch unsicher’. Vizzini (2003) indicated
that it nests within the genus Tuber (data not shown). It is
normally included in the Tuberaceae (e.g. Montecchi & Sarasini 2000; Castellano et al. 2004). As the name indicates this
Italian truffle has 1-spored asci, and the globose spores
have a low, net-like ornament. The ascoma surface is fibrillose from closely packed hyphae. We accept it ad interim
within the Tuberaceae.
Reddellomyces Trappe, Castellano & Malajczuk 1992 (syn.
Labyrinthomyces subgen. Simplex)
Type: Reddellomyces westraliensis.
Trappe et al. (1992) separated this taxon from Labyrinthomyces and Dingleya based on a smooth and glabrous peridium and
asci with 1–5 spores. They accepted four species. Our analyses
of existing sequences indicate a close relationship between
Labyrinthomyces, Dingleya, and Reddellomyces, a group of taxa
What happened to the Tuberales?
that Malençon (1973) treated in an expanded version of
Labyrinthomyces. Trappe et al. (1992) considered these taxa to
belong to the Pyronemataceae (but in different tribes), but as
can be seen from Fig 3, they clearly are close to Tuber, and
maybe they should be united under Labyrinthomyces.
Tuber F.H. Wigg. 1780: Fr. (syn. Aschion, Ensaluta, Oogaster,
Lespiaultinia, Delastreopsis, Terfeziopsis, Mukagomyces)
(Figs 6G-H)
Type: Tuber gulosorum. [This name is currently not understood and is open to interpretation, but most likely represents
T. aestivum Vittad. 1831 (Fig 6G). Various other typifications are
given in the literature, including Index Fungorum, which lists
T. aestivum. A conservation procedure will probably be needed
to solve this problem, as Trappe (2001) points out the sanctioned T. albidum also represents T. aestivum].
The apothecial nature of the primordial Tuber ascomata has
long been known (e.g. Parguey-Leduc et al. 1990; Janex-Favre &
Parguey-Leduc 2002, 2003) and in some species this can even
be hinted at in mature specimens. Parguey-Leduc et al. (1987a,
1987b) studied asci and spores of T. melanosporum in ultrastructural detail. Li & Kimbrough (1995) studied ultrastructural
characters and supported a placement within Pezizales. The
characters found were so divergent that they suggested that
Tuber could be polyphyletic. A Geniculodendron-like anamorph
has been reported from Tuber dryophilum (Urban et al. 2002). It is
a big genus with 63 species according to Kirk et al. (2001). The genus forms a rather diverse group with a well-supported separate
position within the present phylogenetic analysis (Fig 3). The
synonymy cited above is according to Trappe (1975c, 1979).
Janex-Favre & Parguey-Leduc (2002) apparently recognized the
genus Delastreopsis. The multinuclear condition of the mature
spores is a well-known character in some species of Tuber (e.g.
Donadini 1987). Mello et al. (2005) investigated the white (Piedmont) truffle (T. magnatum) in detail and discussed various explanations for the nuclear condition. Vizzini (2003) indicated that
most species have four nuclei in the majority of the ascospores,
whereas a few species have less or more nuclei in the spores. Recently, Wedén et al. (2005) tested whether the height of the spore
ornament can be used (as has been claimed) to distinguish two
disputed truffles T. aestivum and T. uncinatum. All samples
formed a single fully supported group and the names should
be treated as synonyms, thus confirming the conclusion reached
by some early workers (e.g. Hawker 1954). Kovács & Jakucs (2006)
published a detailed phylogenetic and anatomical paper on what
they termed the white truffles. Papers describing Tuber mycorrhizae include Blaschke (1987), Rauscher et al. (1995) and Zambonelli et al. (1993, 1999). Chevalier & Frochot (1997) published
a whole book on the Burgundy truffle, a name traditionally attached to T. uncinatum, now considered a synonym of T. aestivum
(Wedén et al. 2005). The review of Tuber by Ceruti et al. (2003)
should also be consulted. Roux et al. (1999) compared some Chinese and European truffles based on molecular studies. A suite of
new species is currently being discovered and described in China
(e.g. He et al. 2004). Trappe et al. (1996) provided a key to Tuber species with a spiny-reticulate spore ornament. There is an ongoing
project to stabilize the use of Tuber names (e.g. Mello et al. 2000).
Insufficient data [In Eriksson (2006a) placed in Pezizales incertae
sedis]:
1089
Loculotuber Trappe, Parladé & I.F. Alvarez 1993
Type: Loculotuber gennadii.
The authors (Alvarez et al. 1993) stated this monotypic
genus to differ from Tuber in having glebal locules and stipitate asci. The spores tend to become citriform. They speculated that the genus formed an intermediate between an
unknown epigeous member of the Pezizales and the genus
Tuber. Castellano et al. (2004) listed this genus in the Tuberaceae.
Lineage C
The presumably strictly saprotrophic families Ascodesmidaceae, Sarcoscyphaceae and Sarcosomataceae have no known
hypogeous representatives. Glaziellaceae are suggested to belong to this clade (Hansen & Pfister 2007; Perry et al. 2007)
(Figs 4 and 7A-H).
Glaziellaceae J.L. Gibson 1986
Glaziella Berk. 1880
Type: Glaziella vesiculosa Berk (syn. G. aurantiaca).
This genus is unusual in several respects. It fruits more or
less on top of the soil and is completely hollow with a rather
thin rind that contains the monosporic asci, the spore being
enormous. The only species Glaziella aurantiaca (Fig 7A) has
been interpreted in many ways, including a placement in
Xylaria (Sordariomycetes, Ascomycota), in the Zygomycota and finally in the Pezizales. An early molecular study (Landvik &
Eriksson 1994b) suggested a relationship with members of
the Pyronemataceae. Later Landvik et al. (1997) expanded on
this and found further evidence, but still based on a very limited taxon sampling, for a relationship (low support) with e.g.
Pulvinula and the likewise semi-hypogeous genus Paurocotylis.
They ad interim accepted Glaziellaceae but not Glaziellales. Harrington et al. (1999) found support for inclusion in the Pezizales,
but did not resolve a position within, although their results
could indicate a closer relationship with the Sarcoscyphaceae
rather than with the Pyronemataceae. They erroneously cited
the origin of the specimen as Sweden. Castellano et al. (2004)
maintained a placement in the Glaziellales. Perry et al. (2007)
had Glaziellaceae in a sister position to Pyronemataceae but
with low statistical support. Eriksson (2006a) accepts the family in Pezizales. At least some collections of this pantropical
taxon are from decidedly ectotrophic communities, but the
exact nature of its biology is not known.
Pyronemataceae Schröter 1894 (syn. Geneaceae)
This family has been defined as having 1-nucleate ascospores and non-amyloid asci. It has relatively few hypogeous
members with Genea as the most prominent genus. Geopora
is represented with just one species that only pro parte qualifies
as a truffle (active spore dispersal not completely lost). Furthermore, with the exception of Paurocotylis, the truffles
formed in Pyronemataceae all still possess a hymenium; no
stereothecia are found. Epigeous members have both saprotrophic and mycorrhizal representatives, but Paurocotylis
would seem to be the only saprotrophic hypogeous member.
Although Geneaceae have gained wide acceptance, it can be
concluded both by morphological studies by e.g. Pfister
(1984) and Zhang (1992a), and molecular studies (Perry et al.
2007) (Fig 4), that it is part of Pyronemataceae as currently
circumscribed.
1090
T. Læssøe, K. Hansen
Fig 7 – Fruiting body forms in lineage C (Glaziellaceae-Pyronemataceae). (A) Glaziella aurantiaca, unnamed ascoma type,
TL-6168 (C). (B) Genea fragrans, ptychothecia, JV99-373 (C). (C) Humaria hemisphaerica, apothecia, JHP-03.144. (D) Genabea
cerebriformis, ptychothecia. (E) Geopora cooperi, ptychothecia. (F) Geopora arenicola, apothecia, JHP-93.114 (C). (G) Hydnocystis
clausa, ptychothecia, PH00-192 (C). (H) Stephensia bombycina, ptychothecia. Photos: T. Læssøe (A), J. Vesterholt (B, G), J.H.
Petersen (C, F), M. Tabarés (D), J. Nitare (E, H).
What happened to the Tuberales?
Genea Vittad. 1831 (syn. Hydnocaryon)
Type (lectotype): Genea verrucosa.
In Genea the ascomata have a more or less obvious opening,
and can be unfolded to strongly folded (ptychothecia; Fig 7B).
The asci are arranged in hymenia, but active spore dispersal
has been completely lost. The tips of the paraphyses have
fused to form an epithecium that protects the hymenium (Gilkey 1954). The more or less hyaline spores have a very prominent ornamentation. Trappe (1979) accepted 29 species. Li &
Kimbrough (1994) studied the ultrastructure that compared
with members of the Pyronemataceae s.l. (as Otideaceae). Phylogenetic analyses of LSU rDNA support the placement in Pyronemataceae, and suggest that Genea is closely related to
Humaria hemisphaerica (Figs 7B-C) (Perry et al. 2007) (PB and
PP 100 %, Figs 4). Pfister (1984) proposed to place G. hispidula
in Humaria based on analysis of excipular structures. Like species of Genea, H. hemisphaerica has also been shown to be ectomycorrhizal (Tedersoo et al. 2006).
Smith et al. (2006) studied the phylogeny, morphology,
and taxonomy of a group of Quercus-associated species and
listed some minor differences between Genea and the closely
related Genabea (Fig 7D) and Gilkeya. They added a couple of
new species.
Genabea Tul. & C. Tul. 1844 (syn. Myrmecocystis, Pseudogenea)
Type: Genabea fragilis.
The genus was accepted by Trappe (1975c) and again by
Smith et al. (2006). It differs from Genea in having clavate to ellipsoid asci in hymenia enclosed in pockets, and in having
echinulate spores rather than verrucose. Index Fungorum lists
five binomials, two based on European material, two on North
American, and one on Tasmanian. Zhang (1991a) placed
Genabea in synonymy with Genea, which Korf (1973a) also had
suggested. Smith et al. (2006) only dealt with one species, G. cerebriformis (Fig 7D), that clustered separately from the included
Genea species based on LSU data. However, the type species of
Genabea has not been sampled for molecular phylogenetic
studies, which are needed in order to fully test the delimitation
of Genabea, Myrmecocystis (type: M. cerebriformis) and Genea.
Trappe (1975c) synonymized Myrmecocystis with Genabea.
Geopora Harkn. 1885 (syn. Sepultaria, Pseudohydnotrya)
Type: Geopora cooperi.
Burdsall (1965, 1968) studied this genus in detail and combined Sepultaria with Geopora after having found actively discharged spores in the type species of Geopora. Korf (1973b)
gave a detailed review. Biologically G. cooperi (Fig 7E) behaves
like an ordinary truffle but the operculum and the build up
of internal pressure within mature asci have not been lost.
Other species develop in the soil but open at the surface at
maturity (Fig 7F). Nannfeldt (1946) also gave a rarely cited, but
detailed summary (in Swedish) of the Geopora situation. He
regarded G. cooperi (as G. schackii) as a truffle based on biological arguments, such as passive animal dispersal, smell, etc.
Trappe (1975c) agreed on the above synonymy. Phylogenetic
analyses of LSU confirm the placement of G. cooperi among
epigeous Geopora spp. (PB and PP 100 %, Fig 4).
Gilkeya M.E. Sm., Trappe & Rizzo 2006
Type: Hydnocystis compacta (Gilkeya compacta).
1091
This genus was erected based on a separate, although unresolved, position of Hydnocystis compacta in a LSU analysis of
Genea (over six) and Genabea (one) species (H. compacta formed
a trichotomy with Genea and Genabea), in combination with a deviating reddish peridium colour compared with species of Genea
and Genabea. A similar molecular result was found by Perry et al.
(2007) with Gilkeya and Genabea as (unsupported) successive sister taxa to a highly supported Genea–Humaria hemisphaerica
clade. Further taxon sampling will hopefully resolve its position
in a clearer way. Gilkeya and Genabea differ from Genea in having
globose spores and the ascomata lack a basal tuft of mycelium.
Hydnocystis Tul. & C. Tul. 1844 (syn. Protogenea)
Type: Hydnocystis piligera.
Burdsall (1968) gave a detailed taxonomic and nomenclatural account of what he considered the only species of Hydnocystis, H. piligera. The genus is morphologically characterized
by its bladder-like hypogeous ascomata with a hairy, sandbinding outer surface and an irregular opening to the outside.
The spores are globose, eguttulate, and an epithecium is present. We accept its current position in the Pyronemataceae based
on morphological characters. No sequences are available.
Senn-Irlet & Aeberhard (2005) reviewed the genus in a
European context, and stated that the ectomycorrhizal status
of this fungus is uncertain. The placement of the species
H. clausa (Fig 7G) is disputed. Burdsall (1968) placed it in Geopora;
others have placed it with Hydnocystis (Montecchi & Sarasini
2000). Trappe (1975c) studied the type of Protogenea and proposed the above synonymy. Hydnocystis singeri from Argentina
was discussed in Burdsall (1968). It was not accepted in the genus, but compared with Labyrinthomyces and Phymatomyces. It
was thought to possibly represent a new genus. It is one of
very few ascomycetous truffles reported from South America.
Paurocotylis Berk. 1855
Type: Paurocotylis pila.
Patouillard (1903) was the first to recognize that the type
and only recognized species belongs to Ascomycota. The bright
red pigmentation points to a relationship with carotenoid
members of the Pyrenomataceae. Trappe (1979: 321) wrote ‘it
suggests an aleurioid fungus gone underground and fits nicely
in tribe Aleurieae sensu Korf’. Patouillard (1903) indicated a
position close to Hydnocystis, and noted that the remaining
taxa belong elsewhere. Paurocotylis pila forms a monophyletic
group with Stephensia, Geopyxis, and Tarzetta species (PB 99 %
and PP 100 %, Fig 4). Originally, the microscopical similarity between Paurocotylis and Stephensia was noted. The exact nature
of its ecology is far from understood. It is considered a native
of New Zealand and an introduction to the UK (Dennis 1975).
It is now fairly common in the northern parts of the UK, not
least in Orkney (Eggerling 2004), where it fruits during the wintertime in highly disturbed soils, in vegetable plots, along
roads, etc. As noted above, it has been suggested that the bright
red colour may attract birds (ground-dwelling species that fulfil
the small mammal niche in New Zealand) that may act as dispersal vectors in its natural setting (Castellano et al. 2004). Macromorphologically it resembles Glaziella (Fig 7A), which also
has hollow ascomata, that occur more or less on top of the
soil (see separate entry). Dennis (1975) noted that Paurocotylis
spores in mature ascomata are cream coloured and found in
1092
a powdery mass entangled with hyphae. Castellano et al. (2004)
list Paurocotylis as a saprotrophic fungus, and also Dennis (1975)
noted that no obvious mycorrhizal host was found in connection with the first UK find. However, the other members of
the clade, e.g. Geopyxis carbonaria (Vrålstad et al. 1998) and
Tarzetta (Tedersoo et al. 2006), have been shown to be
ectomycorrhizal.
Petchiomyces E. Fisch. & Mattir. 1938
Type: Hydnocystis twaitesii (syn. Petchiomyces twaitesii).
This genus was included in Geneaceae by Fischer (1938),
followed by Gilkey (1954). Burdsall (1968) studied the type of
the type species and concluded that it could not be placed in
Geopora based on the presence of an epithecium and ornamented spores. Gilkey (1939) described Petchiomyces kraspedostoma from California, the only additional species known
besides the type from Sri Lanka. P. kraspedostoma has an apical
opening with stiff, incurved hairs and smooth, ellipsoid
spores. The genus should be revised, but we ad interim accept
its position within the Pyronemataceae.
Phaeangium Pat. 1894
Type: Phaeangium lefebvrei.
This genus was sunk under Picoa by Maire (1906), but resurrected by Alsheikh & Trappe (1983b), a move not accepted by
e.g. Moreno et al. (2000). Gutierrez et al. (2003) described the
rather deviating mycorrhizae formed by Phaeangium lefebvrei
(as Picoa) with Helianthemum species. We ad interim accept
the genus (within Pyronemataceae).
Sphaerosoma Klotzsch 1839
Type: Sphaerosoma fuscescens.
Korf (1972) placed the genus in the Ascobolaceae following
previously published characters and was ad interim followed
by Trappe (1979). Gamundi (1976) could not find any amyloid reaction in the type material and considered it a likely member of
the Pyronemataceae (as Humariaceae tribe Otideae). Dissing & Korf
(1980) followed Gamundi but stated ‘studies on fresh material
are needed before the true systematic position of this genus
can be evaluated’. They felt, based on circumstantial evidence,
that Sphaerosoma fuscescens probably has forcible spore discharge. Montecchi & Sarasini (2000) cite Sphaerosoma as a synonym of the younger name Sphaerozone (Pezizaceae!), but it is
accepted in e.g. Vizzini (2003) in the Pyronemataceae and ad interim here. Kirk et al. (2001) stated the number of species as
three, but there are 11 names in Index Fungorum currently without other placement. A revision would seem to be required.
Stephensia Tul. & C. Tul. 1845 (syn. Densocarpa, Elderia)
Type: Stephensia bombycina (Fig 7H).
Knapp (1951) gave a description of the type species, whereas
Fontana & Giovannetti (1987) described its anamorph. Uecker
(1967) reported a similar anamorph for Stephensia shanori.
Trappe et al. (1997) published a key to the species. Our placement (Fig 4) is based on sequences obtained by Perry et al.
(2007). De Vito (2003) described a new species, S. colomboi, said
to differ from previously described species in being epigeous
on rotten wood. Based on the published picture the wood
more or less qualifies as soil, and some of the ascomata appear
to be at least partly immersed. Microscopically, S. colomboi is
T. Læssøe, K. Hansen
apparently very close to S. bombycina, but some minor macroscopical differences are noted.
Hypogeous pezizalean taxa currently not placed
within clade A–C
Carbomycetaceae Trappe 1971
Trappe (1971) erected this family as a segregate from Terfeziaceae. It was based on ‘brown-walled asci borne in fertile
pockets of large, inflated cells mixed with narrow, tubular
ascogenous hyphae, and in the fertile pockets being separated
by sterile veins of inflated cells only’. It never produces a hymenium in any kind of palisade. When dry the spore mass becomes pulverulent almost as in Elaphomyces. Eriksson (2006a)
accepts the family in the Pezizales.
Carbomyces Gilkey 1954
Type: Carbomyces emergens.
This interesting taxon, only known from three species in
southwestern North America, is currently under study by
K. Hansen using molecular techniques. According to Trappe
(1971) its mycorrhizal status has not been clarified. At maturity the ascomata are dispersed by the wind (Trappe 1979).
Zak & Whitford (1986) demonstrated the hypogeous nature
of immature Carbomyces emergens, and that rodents apparently eat the (immature?) ascomata.
Pezizalean truffles with unknown family placement
(based on Eriksson (2006a)
Delastria Tul. & C. Tul. 1843
Type (mono): Delastria rosea.
Not much is known about this southern European/
North African monotypic genus. Montecchi & Sarasini (2000)
include it in the Terfeziaceae (here considered a synonym of
the Pezizaceae), following Trappe (1979), and differentiate it
from the other accepted genera in this family by the evanescent peridium, the pinkish colour of the gleba, 2–4-spored
asci and a reticulate spore ornament. Castellano et al. (2004)
accepted the genus in the Tuberaceae. DNA studies are clearly
needed in order to clarify the position of this Tuber-like genus.
Unplaced Ascomycota truffles (Eriksson 2006a)
Diehliomyces Gilkey 1955
Type (mono): Diehliomyces microsporus.
This pest in mushroom beds (the ‘compost truffle’) is referred to as a ‘false truffle’ in Kirk et al. (2001), but its ascomycetous nature is not disputed, and it must be considered
a genuine although rather atypical truffle. Its position is unsettled, but it could belong in Pezizales and parallel the case
of Orbicula, another passively discharged, but epigeous fungus
that has led a tumultuous life, but now has found its place in
the Pezizales (Hansen et al. 2006). Both genera have had
Eurotiales/Onygenales proposed as proper placements, mainly
due to the production of small ascomata with small, globose
spores. Unlike almost all other truffles this species is clearly
not mycorrhizal. Diehl & Lambert (1930) introduced the
species as Pseudobalsamia microspora after having received material from an Ohio grower where the pest was ‘filling his beds
What happened to the Tuberales?
and completely stopping the production of mushrooms’. It
was later found in other American sites and later also in
Europe (e.g. Pegler et al. 1993). It resembles many typical
ascomycetous truffles in having a convoluted ascoma up to
3 cm diam with an outer rind. It may have one or several openings to the exterior. The asci are evanescent, long stipitate
with a sac-like, spore-containing part. Unlike typical pezizalean truffles, the spores are smooth and subglobose, 5–7 mm
diam, and form an ‘olivaceous sulphur-coloured dusty mass’
(Diehl & Lambert 1930; Gilkey 1955). Diehl & Lambert (1930)
also reported chlamydospores up to 13 mm diam, with a thick,
golden-brown wall. It was grown in artificial culture, where it
produced ascomata. These authors tentatively concluded that
the truffle could be considered a weed in mushroom beds
rather than a parasite of the mushrooms. Singer (1961) published a plate that clearly indicates the scale of a full-blown
‘infection’ in a mushroom bed. Hawker (1959) did some developmental studies on Diehliomyces and concluded that the ascomata were not truly folded as in a typical member of the
Tuberales, and she supported a transfer to the Eurotiales. She
found a completely irregular arrangement of the ascogenous
hyphae and asci, even at very early stages of development.
Currah (1985) excluded it from the Onygenales, where Benny &
Kimbrough (1980) had accepted it.
Excluded truffle taxa
Amylocarpus Curr. 1859
Type: Amylocarpus encephaloides.
This monotypic genus has passive spore dispersal but develops on intertidal wood and, although originally included in
the Tuberaceae, it cannot be considered a truffle in the sense of
this paper. Its current position is unsettled (e.g. Landvik et al.
1998). It is listed as Leotiomycetidae with unclear position in
Kirk et al. (2001) and as Helotiales incertae sedis in Eriksson (2006a).
General information
For general information on truffles refer, for example, to North
American Truffling Society (www.natruffling.org/) and e.g.
Bucquet-Grenet & Dubarry (2001). A very extensive bibliography on the genus Tuber can be found in Ceruti et al. (2003).
Also, Trappe & Maser (1977) and Trappe et al. (2001) should
be consulted. Recently a very illustrative guide to Andalucian
truffles directed at the general public has been published (Arroyo et al. 2005). Dannell (1996) published a useful popular review in Swedish.
1093
1994a; but see Landvik & Eriksson 1994b), but was later shown
to be closely related to Eurotiales and Onygenales (Landvik et al.
1996). The early study by Landvik & Eriksson (1994b) showed
that Glaziella, with the highly unusual ascomatal form, was
nested within Pezizales. Attempts to find out the exact relationship of Glaziella have since been carried out (see Glaziellaceae above). The molecular study by O’Donnell et al. (1997)
included a large number of truffles together with a large number of pezizalean epigeous taxa (from lineage B) and was the
first to discover multiple (at least five), independently derived,
hypogeous clades within Pezizales. It resulted in new family
assignments for several truffles and revealed a relationship
between Tuberaceae and Helvellaceae. Percudani et al. (1999)
focused on hypogeous Pezizales phylogeny and species
thought to belong to the Balsamiaceae, Terfeziaceae, and Tuberaceae. Unfortunately they included only few epigeous taxa,
which resulted in Cazia, Mattirolomyces, Pachyphloeus, and Terfezia (Terfeziaceae) erroneously formed a monophyletic group
within Pezizaceae. A study with a broader sampling of epigeous
pezizaceous species followed (Norman & Egger 1999) that
showed ‘Terfeziaceae’ are not monophyletic. The study of
epigeous-hypogeous relationships within Pezizaceae was
further expanded (Hansen et al. 2001) and gave support for at
least three independent origins of hypogeous forms within
the family. Most recently, Perry et al. (2007) focusing on Pyronemataceae, with a large taxon sampling, suggested that the truffle form has arisen at least five times independently within
that family. All of these studies used regions from the nuclear
ribosomal genes. One multi-locus study has emerged (Hansen
et al. 2005) substantiating the evolution of truffles within Pezizaceae using combined analyses of LSU rDNA and protein-coding genes, RNA polymerase II (RPB2), and b-tubulin.
Several further papers dealing with the phylogeny of truffles (e.g. Diéz et al. 2002; Ferdman et al. 2005) have unfortunately only included truffles in the analyses, which have
made it difficult to pinpoint epigeous relatives and fully
understand their relationships and taxonomy. Vizzini (2003)
gave the most recent review of ascomycetous truffles.
The 55 species of truffles included in the current review occur in 15 separate lineages within the Pezizales: in nine lineages within Pezizaceae (Fig 2), in three lineages within
Morchellaceae–Discinaceae–Helvellaceae–Tuberaceae (Fig 3) and
in three lineages within Pyronemataceae (Fig 4). The only strictly
hypogeous family known is currently Tuberaceae. Table 2 gives
an overview of recent classifications of pezizalean truffles and
an up-to-date classification based on both molecular and
morphological characters.
Discussion
Cytology
Phylogenetic relationships of truffles within Pezizales
The number of nuclei in mature ascospores within the Pezizales has long been considered a character of major importance in defining taxa (see e.g. Berthet 1964; Korf 1973a,
1973b and Zhang 1992a,b). It has been shown that the
hypogeous members of the Pezizales also tend to have the
same number of nuclei in the spores within a certain family
or genus. Tuber is an exception, as the spores can have from
one to 18 nuclei, although most species have four nuclei in
each spore (Vizzini 2003). The known numbers are summarized in Table 3. Zhang (1992a) found Genea (two species),
Within the last 13 y molecular phylogenetic studies have gradually confirmed and greatly expanded our knowledge on a repeated evolution of ascomycetous truffles across Pezizales.
The first study to address the controversial issue of the placement of Tuber was that of Landvik & Eriksson (1994a), who
confirmed the placement within Pezizales, as predicted by
Trappe (1979) and others. Elaphomyces was erroneously indicated to be nested within Pezizales (Landvik & Eriksson
1094
T. Læssøe, K. Hansen
Table 2 – Different recent classification schemes of
pezizalean truffles
Tuberales
(Korf 1973a)
Hypogeous
Pezizales
(Trappe 1979)
Elaphomycetaceae
Elaphomyces
Pezizaceae
Amylascus
Mycoclelandia
(as Clelandia)
Hydnotryopsis
Peziza spp.
Tirmania
Terfeziaceae
Carbomyces
Delastria
Mukagomyces
Paradoxa
Picoa
Terfezia
Tirmania
Terfeziaceae
Choiromyces
Delastria
Hydnobolites
Pachyphloeus
Terfezia
Tuberaceae
Barssia
Balsamia
Caulocarpa
Choiromyces
Elderia
Fischerula
Lespiaultinia
Labyrinthomyces
Hydnobolites
Hydnoplicata
Hydnotrya
Pachyphloeus
Phymatomyces
Piersonia
Protogenea
Pseudobalsamia
Stephensia
Tuber
Geneaceae
Genea
Hydnocystis
Petchiomyces
Suggested
classification of
hypogeous Pezizales
Pezizaceae
Amylascus
Cazia
Eremiomyces
Hydnobolites
Hydnotryopsis
Kalaharituber
Mattirolomyces
Mycoclelandia
Pachyphloeus
Peziza spp.
Ruhlandiella
Sphaerozone
Terfezia
Tirmania
Helvellaceae
Hydnotrya
Dingleya
Fischerula
Helvellaceae
Balsamia
Barssia
Helvella astieri
Balsamiaceae
Balsamia
Barssia
Picoa
Tuberaceae
Choiromyces
Dingleya
Labyrinthomyces
Paradoxa
Reddelomyces
Tuber
Tuberaceae
Paradoxa
Tuber
Pyronemataceae
Geopora cooperi
Hydnocystis
Labyrinthomyces
Paurocotylis
Petchiomyces
Sphaerozone
Stephensia
Geneaceae
Genea
Genabea
Morchellaceae/
Discinaceae
Gymnohydnotrya
Hydnotrya
Fischerula
Leucangium
Pyronemataceae
Genabea
Genea
Geopora cooperi
Gilkeya
Hydnocystis
Paurocotylis
Petchiomyces
Phaeangium ¼ Picoa?
Picoa
Sphaerosoma
Stephensia
Glaziellaceae
Glaziella
Carbomycetaceae
Carbomyces
Carbomycetaceae
Carbomyces
The adopted classification (right column) is based on recent molecular phylogenies combined with morphological characters.
Hydnobolites cerebriformis, Pachyphloeus citrinus, and Mattirolomyces terfezioides (as Terfezia), to be uni-nucleate. This
led Zhang to propose the synonymy of Geneaceae with
Pyronemataceae, and furthermore, restricted Terfeziaceae to
uninucleate taxa (now incorporated in the Pezizaceae). The Helvellaceae have been considered to be defined by tetra-nucleate
spores, but it is now evident that this number is a plesiomorphic character (also present in Discinaceae and some taxa of
Tuberaceae) and thus has very limited discriminative value.
The placement of f.ex Hydnotrya (Trappe 1979) and Choiromyces
(e.g. Pegler et al. 1993) in the Helvellaceae was argued along
those lines. However, molecular phylogenetic analyses of
SSU and LSU rDNA suggest that Hydnotrya belongs to Discinaceae and Choiromyces to Tuberaceae (O’Donnell et al. 1997) (Fig 3).
Ecological aspects of the truffle syndrome
Various evolutionary processes may be involved in the truffle
syndrome, but the most generally accepted is the avoidance
of desiccation (e.g. Thiers 1984). The high truffle diversity in
arid areas favours this hypothesis. Some truffles, like Tuber aestivum and T. melanosporum, clearly have an outer layer that renders protection, to both mechanical and desiccation stresses,
but many others have very delicate fruit bodies, often formed
in the upper soil layers, where desiccation pressures can exist,
although of a less harsh nature than above ground. Another
driving force could be protection against above-ground predation of immature ascomata. At maturity the production of pungent volatile compounds attracts predators of a kind the truffles
have co-evolved with, or at least adapted to, in order to facilitate
spore dispersal. Pacioni et al. (1990) speculated on other functions of the compounds, including microbial control of the micro-rhizosphere. Spores of hypogeous fungi probably persist for
longer in the soil than those of wind-dispersed relatives, which
presumably is of importance in respect to life in a xeric environment and as ectomycorrhiza formers (e.g. Miller et al. 1994).
It is generally assumed that most hypogeous fungi, including those in the Pezizales, form ectomycorrhiza. Direct proof of
this has not been established in all cases, but circumstantial
evidence clearly indicates the validity of this assumption
(e.g. Pacioni & Comandini 1999; Montecchi & Sarasini 2000).
Early on some of these relationships were considered parasitic, e.g. those with Cistaceae (Singer 1961). Awameh &
Alsheikh (1979) and Awameh et al. (1979) claimed that some
Terfezia and Tirmania spp. form ectomycorrhiza with Helianthemum, but Kovács et al. (2003) have pointed out some important morphological discrepancies compared to typical EM
structures casting doubt on these conclusions. Based on
morphotyping and sequencing of ectomycorrhizal root tips,
Tedersoo et al. (2006) identified 33 species of Pezizales to be
ectomycorrhizal, including species of Genea, Geopora, Helvella,
Hydnotrya, Pachyphloeus, Peziza, Sarcosphaera, and Tuber. They
hypothesized that the ectomycorrhizal lifestyle is a precondition for the switch to hypogeous fruiting. Most well-known
mycorrhizal trees would appear to be involved in associations
with pezizalean truffles, including various members of the
Fagaceae, Betulaceae, Pinaceae, and Myrtaceae. It is generally
assumed that truffles prefer warm, fairly dry climates and
calcareous soils, but this may be slightly overstated due to
the emphasis of requirements for the edible Tuber species.
Still, the overall species diversity appears to be highest in
alkaline soils in warm temperate to subtropical climates.
Desert areas around the world also have a special truffle
What happened to the Tuberales?
1095
Table 3 – A compilation of the known number of nuclei in mature ascospores in hypogeous Pezizales
Taxon
Nuclei/ascospore
Reference
Lineage A (Pezizaceae)
Hydnobolites cerebriformis
Mattirolomyces terfezioides, M. tiffanyae
Muciturbo (¼ Ruhlandiella?)
Pachyphloeus citrinus
Picoa juniperi
1 (to 4?)
1
1
1
1
4?
Zhang (1992a)
Zhang (1992a; Healy 2003)
Warcup & Talbot (1989)
Zhang (1992a)
Donadini (1986b)
Lineage B (Discinaceae-Tuberaceae)
Balsamia platyspora, B. vulgaris
Barssia oregonensis
Choiromyces gangliformis
Gymnohydnotrya australiana
Helvella astieri
Hydnotrya michaelis, H. tulasnei, H. cerebriformis
Leucangium carthusianum
Tuber rufum
Tuber aestivum, T. brumale, T. excavatum,
T. indicum, T. magnatum, T. mesentericum
Tuber maculatum
Tuber borchii, T. puberulum
Tuber melanosporum
1–17
4
4
4
4
4
4
4þ
1–2
2–4
Donadini (1986b)
Kimbrough et al. (1996)
Zhang & Minter (1989a)
Zhang & Minter (1989b)
Korf (1973b)
Berthet (1982); Donadini (1986a); Zhang (1991b)
Li (1997)
Vizzini (2003)
Vizzini (2003)
2–8
(2–)4–17
6–8
Vizzini (2003)
Donadini (1987), Vizzini (2003)
Parguey-Leduc et al. (1987a)
Lineage C (Pyronemataceae)
Genea klotzii, G. sinensis, G. variabilis, G. verrucosa
Geopora cooperi
Hydnocystis clausa, H. piligera
Stephensia shanori
1 (to 5?)
1
1
1
1 (to 5?)
Donadini (1986a); Zhang (1991a, 1992a)
Donadini (1987)
Donadini (1986a, 1987)
Uecker (1967)
funga, notably including Terfezia and Tirmania species.
Amongst pezizalean truffles only Paurocotylis is at present considered to be saprotrophic (Castellano et al. 2004) or suspected
to be so (Dennis 1975).
Conclusion
In conclusion, the trend that started with abandoning the
Tuberales, now robustly confirmed, has continued at the
family level where ‘pure’ hypogeous monophyla have been reduced to a single taxon, the Tuberaceae. At least 15 independent origins of hypogeous forms within the Pezizales are
supported by the LSU rDNA gene trees. Different types of
hypogeous ascomata forms, infolded or chambered
ptycothecia, solid ptycothecia and stereothecia, appear to
have evolved multiple times independently with the linages
A and B of Pezizales; within lineage C only infolded or chambered ptycothecia are present. No clear picture is shown by
the LSU phylogenies of the hypothesis that evolution from
an epigeous, actively dispersed form (apothecial) to a hypogeous, passively dispersed form (stereothecial), proceeds
through an intermediate semi-immersed form. Nevertheless,
several smaller clades include such forms and future studies,
including additional molecular data and taxa, providing
a more robust phylogeny, may likely show such a progression.
Much has been learnt on truffle biology, taxonomy, and phylogeny as the Tuberales were abandoned as an independent
order but all three fields are still very active research areas
where many exciting results will be forthcoming in the near
future.
Acknowledgements
Thanks to Jens H. Petersen for providing and editing photographs, and to Christian Lange, Jan Vesterholt, Johan Nitare,
Juan Santos, and Manuel Tabarés for allowing us to use their
pictures. Jim Trappe is thanked for help with references, advice on truffle matters in general and for supplying many of
the specimens used by K.H. for molecular work. Rosanne
Healy is thanked for providing specimens of Pachyphloeus.
Donald H. Pfister is thanked for discussions and support of
this work. The suggestions from two anonymous reviewers
are greatfully acknowledged. The research contributed by
K.H. was financially supported by an NSF grant to Donald
H. Pfister and K.H. (DEB-0315940). T.L. thanks David Hawksworth for initiating this review, and he and Scott LaGreca
are thanked for hosting ‘The New Bottles for Old Wine: fruit
body types, phylogeny and classication’ BMS annual conservation and taxonomy meeting.
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