FEMS Microbiology Letters 180 (1999) 147^155

Phylogenetic relationships between European and Chinese tru¥es

based on parsimony and distance analysis of ITS sequences
Christophe Roux *, Nathalie Sëjalon-Delmas, Monique Martins,
Agne©s Parguey-Leduc, Robert Dargent, Guillaume Bëcard
Equipe de Mycologie vëgëtale, UMR 5546, Universitë Paul Sabatier, Poªle de Biotechnologie vëgëtale, Chemin de Borde-Rouge, BP 17,
Castanet-Tolosan 31326, France

Received 3 August 1999; received in revised form 9 September 1999; accepted 13 September 1999

Abstract

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of
the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared
polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum,
T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum
and T. himalayense, were grouped in an independent clade. The Përigord black truffle T. melanosporum and the Chinese black
truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance
analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal
transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to
distinct taxa. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.

Keywords : Species delimitation; Internal transcribed spacer; Tuber

1. Introduction ent order: the Tuberales [5,6]. Dissing and

Traditional systematics has given the broad lines
of the natural classi¢cation of tru¥es [1^4]. How-
ever, the small number of micro- and macro-mor-
phological characters used has led to controversy
on the position of these fungi at the supra- and in-
frageneric level. Analysis of structural and ultra-
structural characters placed tru¥es in an independ-
* Corresponding author. Tel.: +33 562 193 504;
Fax: +33 562 193 502; E-mail: roux@cict.fr
Schumacher [6] underlined the absence of signi¢cant
di¡erences between epigeous and hypogeous taxa
upon which the de¢nition of Tuberales versus Pezi-
zales was based. These authors suggested that tru¥es
be placed in a family (Tuberaceae) in the order Pe-
zizales. More recently, molecular phylogenetic anal-
ysis based on the comparison of 18S and/or 28S
rDNA genes supported the latter position [7], but
some problems of systematics remain at the infrage-
neric level. For example, the separation of the taxa
Tuber aestivum Vitt and Tuber uncinatum Ch. [7,8],
or Tuber rufum Pico and Tuber ferrugineum Vitt. [9],

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 7 4 - 7

FEMSLE 9058 28-10-99

148 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155
or Tuber brumale Vitt. var brumale Vitt. and T. bru-
male Vitt. var. moschatum F. de B. [7,9^11] is still
controversial.
The di¤culty in di¡erentiating some species and/or
varieties in the Tuber taxa can be due to the overall
similarity of morphological characters and the fact
that descriptions have been somewhat approximate.
The separation of T. aestivum and T. uncinatum into
two species, for example, is the result of contradic-
tory taxonomical interpretations. These two species
formerly separated by their morphological and eco-
logical di¡erences [8,12^14] are now, after biochem-
ical and molecular analyses, considered as a complex
of strains in which intermediate forms make up a
continuum between the two extreme forms of the
species [8,9,15]. This heterogeneity within one species
underlines the need to integrate population dynamics
analyses in phylogenetic studies. However, in system-
atics, morphological description referred to only one
holotypus, and molecular phylogenetic analyses are
usually based on a unique sequence for each species.
The interspeci¢c phylogeny of organisms is con-
fronted with the problem of species delimitation. In
the case of the genus Tuber, phylogenetic approaches
based on isozyme polymorphism [10,16] and RAPD
patterns [9,17] used several samples for each species
to estimate the intraspeci¢c variation of the charac-
ters. In spite of their interest, these analyses are
sometimes contradictory as the characters considered
were the result of a mix of inter- and intraspeci¢c
variations or could be not correlated at all [18^20].
The problem of species delimitation was intensi¢ed
with the importation into Europe of black tru¥es
from China. Macroscopic characters of the fruit
bodies and microscopic observation of ascospores
do not allow them to be easily distinguished from
the famous black tru¥e Tuber melanosporum, espe-
cially when the ascocarps are immature [21]. Com-
parison of T. melanosporum and Tuber indicum with
RAPD markers led to the conclusion that the two
taxa are distinct [9]. However this work also reported
a high polymorphism among Chinese tru¥es, like-
wise observed with microsatellite and internal tran-
scribed spacers (ITS) sequence analyses [22,23]. It
was unclear whether this polymorphism was related
to geographic variations as proposed by Paolocci et
al. [23] and Longato and Bonfante [22], or if it re-
£ected a misidenti¢cation of several taxa taken to be
FEMSLE 9058 28-10-99
T. indicum. Several black tru¥es have been described
in Asia (T. indicum Cooke and Massee, Tuber hima-
layense BC Zang and Winter, Tuber pseudohima-
layense [24], Tuber sinense K Tao and Liu) and the
distinction between them is sometimes tricky.
In this study, a phylogenetic investigation of the
genus Tuber was completed by a subspeci¢c popula-
tion analysis in order to limit incorrect phylogenetic
interpretations due to intraspeci¢c variations. Parsi-
mony inferences were applied on the ITS1-5.8S-ITS2
sequences. ITS regions have already been proven to
be useful in the phylogeny of closely related species
[18], although intraspeci¢c ITS polymorphism was
shown in several fungi [25^28]. ITS polymorphism
of several samples of T. melanosporum and of some
Chinese tru¥es collected in di¡erent regions was
then analysed by polymerase chain reaction-restric-
tion fragment length polymorphism (PCR-RFLP).
2. Materials and methods
2.1. Fungal isolates and sequences
The samples used in our assays, their geographic
origin and the GenBank accession numbers are listed
in Table 1. The tru¥es from the di¡erent geographic
areas were identi¢ed in this laboratory.
2.2. DNA extraction and PCR ampli¢cation
DNA extraction from ascocarps was performed on
200 mg of gleba. The samples were ground in sand
with 1 ml of extraction bu¡er (0.2 M Tris-HCl pH 8,
0.02 M EDTA pH 8, 1.4 M NaCl, 2% CTAB, 0.2%
L-mercaptoethanol) [29]. After centrifugation, 20 Wg
of proteinase K and 10 Wg of RNAse A were added
to the supernatant and left for 5 min at 62³C. After
centrifugation, 10 min at 8000 RPM, 2 ml of 5 M
potassium acetate (pH 6) were mixed to the super-
natant. After 10 min at 4³C, proteins were pelleted
by centrifugation, 15 min at 8000 RPM. The super-
natant was gently mixed with half a volume of
chloroform and centrifuged for 15 min at low speed.
The supernatant was then mixed with 0.7 vol. of
isopropanol, 10 min at 4³C. DNA was pelleted by
centrifugation (15 min, 8000 RPM) and washed
twice with 70% ethanol. The air-dried pellet was re-
 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155 149

suspended in 100 Wl of TE bu¡er (10 mM Tris-HCl
and 1 mM EDTA pH 8). The purity and quantity of
the DNA were checked by spectrophotometry at
260, 280 and 320 nm.
The ITS 1F and ITS 4 primers used to amplify
the ITS sequence have been described by Gardes
and Bruns [29]. The ampli¢cations used 25 Wl
DNA template solution in 50 Wl of reaction mixture.
The ¢nal solution contained 25 WM each of dATP,
dCTP, dGTP and dTTP (Euromedex, France),
0.5 WM of each primer and one unit of thermostable
polymerase (Red Hot, AB-Fischer Scienti¢c,
France). The cycling conditions consisted of an ini-
tial denaturation step at 94³C for 5 min, 1 min at
52³C and 1 min at 72³C (30 cycles). The ampli¢ed
DNA was visualized by electrophoresis on 1.5%
agarose gels.
For sequencing, the PCR products were puri¢ed
using the QIAquick kit (QIAGEN S.A., France) ac-
cording to the manufacturer's protocol. The DNA
templates were mixed with PRISM dye deoxy termi-
nator cycle sequencing kit. Samples were loaded on a
4% polyacrylamide, 48 cm gel, using an Applied Bio-
systems automated sequencer (model 373 Perkin El-

Table 1
Origin of the fungal material and sequences used in this study
Species Isolate Origin GenBank accession number
T. melanosporum Vitt. Tm3 Lot (France)
Tm5 Huesca (Spain)
Tm6 Lot (France)
Tm12 Lot (France)
Tm13 Vaucluse (France) AF132501*
Tm14 Vaucluse (France)
TmM2 unknown (EMBL) Y09790
TmA1 L'Aquila (Italy) U89359

T. indicum Cooke and Massee Ti16 Huidong (China)

Ti17 Huili (China) AF132502*
Ti22 Huidong (China)
Ti25 Huidong (China)
Ti26 Miyi (China)
Ti27 Huili (China)
Ti80 unknown U89360
Ti20 unknown U89362

T. himalayense Zhang and Minter Th15 unknown

Th20 Huidong (China) AF132503*
T. pseudohimalayense? Ti38 U89361
Tuber mesentericum Vitt. AF132508*
Tuber panniferum Tul. AF132507*
T. borchii Vitt. AF132505*
T. ferrugineum Vitt. AF132506*
T. uncinatum Ch. AF132509*
T. brumale Vitt. AF132504*
T. brumale f moschatum AF001010
T. magnatum AF003913
T. puberulum AF003918
Tuber dryophilum AF003917
T. maculatum AF003919
T. aestivum U95175
T. excavatum AF073509
P. vulgaris U66008
*Sequences obtained in this study. The others were obtained from GenBank or EMBL.

FEMSLE 9058 28-10-99

150 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155

Fig. 1. Cladogram obtained with 18 aligned ITS1-5.8S-ITS2 sequences of di¡erent species of tru¥es. The ITS sequence of P. vulgaris (As-
comycotina, Discomycetes, Pezizales) is added as an outgroup. The consensus tree resulted from 500 bootstrap replicates on 329 sites (117
informative), while 406 steps were required for maximum parsimony. Partial ITS sequences of T. aestivum and T. excavatum were added
in independent analyses (*: based on the comparison of ITS1-5.8S sequences. **: based on the comparison of 5.8S-ITS2 sequences) and
the resulting groups are noted in the tree.

mer/Applied Biosystems Division, Foster City, CA,
USA)
2.3. DNA restriction
Ten microlitre of ampli¢ed DNA was digested for
RFLP analysis using the conditions de¢ned by the
manufacturer. The solutions were incubated for 3 h
at the recommended optimal temperature. For each
PCR product, 5 restriction enzymes were used:
MseI, NlaIV, RsaI, HinfI and DdeI (MBI Fermen-
tas-Euromedex, France). These enzymes were chosen
to cut inside the most variable regions of the ITS1
and ITS2 sequences as revealed by restriction maps.
The restriction digests were then electrophoresed on
3% agarose gels (Euromedex, France).

FEMSLE 9058 28-10-99

 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155
2.4. Sequence alignment, phylogenetic inference and
distance analysis
The sequences were aligned with the software Sea-
View [30] (algorithm ClustalW) and the phylogenetic
inferences performed with Phylo_win [30] (algorithm
DNApars from PHYLIP, v.3.5c, J. Felsenstein) on a
UNIX system. Bootstrap analyses were made of
500 replicates using the bootstrap and consensus op-
tions of Phylo_win. The ITS sequence of Pithya vul-
garis was used to root the tree. The data from re-
striction analyses were analysed using Restsite [31]
and Neighbor (option Neighbor-Joining algorithm)
from PHYLIP. As the restriction map for each spe-
cies was known, the results were analysed in terms of
presence or absence of site (s option of Restsite). The
dendogram was built with the software NJ Plot
available on PHYLIP.
3. Results
3.1. Phylogeny of Tuber taxa
ITS regions were ampli¢ed and sequenced. After
alignment of the sequences, a consensus tree was
built consecutively with a 406-step parsimony proce-
dure (Fig. 1). The ITS sequence of T. brumale being
longer due to a duplication of a part of the ITS1
region (880 versus 600^700 bp for the other Tuber
species), the alignment step was crucial to perform
any phylogenetic analysis. Of the 19 Tuber species
compared, 329 sites were selected giving 117 infor-
mative sites. Fig. 1 shows that most of the clades
were supported by high bootstrap values. The three
major clades (clades I, II, III) were obtained inde-
pendently of the number of species included in each
one (data not shown).
Since the T. aestivum and Tuber excavatum ITS
sequences obtained from GenBank were truncated,
their alignment with the full sequences of the other
species presented in Fig. 1 was not possible. Inter-
mediate steps were required to position these two
species on the ¢nal tree (Fig. 1). A ¢rst tree was built
after alignment with T. aestivum and ITS1-5.8S re-
gions of all the other species except T. excavatum, a
second one resulted in alignment of 5.8S-ITS2 re-
gions of all species plus T. excavatum but without
FEMSLE 9058 28-10-99
151
T. aestivum (data not shown). The position of these
species in the phylogeny estimated with the two trees
is reported in Fig. 1. T. excavatum appears related to
T. ferrugineum. However their relationships with
clades I or II is not clearly established (low bootstrap
value). T. aestivum constitutes a monophyletic group
with T. uncinatum and a bootstrap value of 100 val-
idates this clade.
Clade III in Fig. 1 is composed of morphologically
similar species (T. melanosporum, T. brumale and the
Chinese tru¥es). At least two sequences for each
species were used in this clade. Fig. 1 shows that
no di¡erences were found between the ITS sequences
of the two varieties of T. brumale described (var.
brumale and var. moschatum). T. melanosporum
forms an independent taxon distinct from the Chi-
nese tru¥es analysed. This distinction is strongly
supported by a bootstrap value of 100. Inside the
taxon with the Chinese tru¥es, T. indicum and
T. himalayense are separate. In addition, a third tax-
on appears (Ti38), close but distinct from the
T. himalayense clade.
3.2. Distance analysis of T. melanosporum and some
tru¥es from China
The unrooted dendogram in Fig. 3 was built with
the data obtained by the PCR-RFLP approach (Fig.
2). Among the ¢ve enzymes used (HinfI, MseI,
NlaIV, DdeI and RsaI), only one (MseI) presented
three intraspeci¢c polymorphic pro¢les for T. mela-
nosporum. Two enzymes, MseI and NlaIV, exhibited
Fig. 2. Electrophoregram obtained after restriction of the ITS se-
quence of T. melanosporum with the enzymes HinfI (lane 2),
MseI (lane 3), NlaIV (lane 4), DdeI (lane 5), RsaI (lane 6). Lanes
8^12 : restriction pro¢les of T. indicum obtained with Hinf1,
MseI, NlaIV, DdeI and RsaI, respectively. Lanes 1, 7 and 13:
100-bp ladder.
152 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155

Fig. 3. Distance analysis between several isolates of T. melanosporum, T. himalayense and T. indicum, based on PCR-RFLP pro¢les of
ITS regions. The unrooted tree was built with the Neighbor-Joining algorithm. The scale corresponds to the relative genetic distance.

¢ve di¡erent restriction patterns for T. indicum and
no polymorphism for T. himalayense. All the en-
zymes used showed a high interspeci¢c polymor-
phism. The genetic distances given on the dendo-
gram are relative and only relevant to the enzyme
system used in our analyses. In these conditions,
three groups are separate, with intraspeci¢c poly-
morphism appearing less than interspeci¢c polymor-
phism. These three separate groups correspond to
the three taxa analysed: T. melanosporum, T. indicum
and T. himalayense.
4. Discussion
The cladogram on Fig. 1 shows that three evolu-
tionary groups may be considered in the Tuber ge-
nus. The clades obtained are signi¢cant, being sup-
ported by high bootstrap values. In clade I, the
homology of T. uncinatum and T. aestivum argues
for the theory of a complex of species with ecological
and morphological varieties [8]. Clade II is composed
of the so-called white tru¥es. The species Tuber
borchii, Tuber maculatum and Tuber puberulum,
which make it up, have morphological similarities.
However, since Tuber magnatum, the famous Pied-
mont white tru¥e, is grouped with brown tru¥es in
clade I, the white-tru¥e group appears polyphyletic.
The presence of T. magnatum in the clade of
brown tru¥es, the proximity of T. excavatum and
T. ferrugineum, two species with alveolate and echi-
nulate ascospores, and the mix in clade III of asco-
spores that are echinulate (T. brumale, T. melanospo-
rum), more or less reticulate (T. indicum, T.
himalayense) and perhaps alveolate (Ti38), suggest
that ascospore ornamentation and colour of the as-
cocarp gleba and peridium are not phylogenetically
informative. The homoplasmy of the `ascospore or-
namentation' character is supported by observations
of spore ontogenesis showing that alveolate and echi-

FEMSLE 9058 28-10-99

 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155
nulate ornamentations have the same ultrastructural
origin [32,33]. Similar conclusions have been drawn
for other fungal groups [34]. However, this does not
decrease the value of these characters as determina-
tion keys.
The distance analysis performed with the PCR-
RFLP approach showed a polymorphism within
the ITS sequence of T. melanosporum. This is in
agreement with Henrion et al. [35] who observed
polymorphic sites in the ITS 2 region using AluI
and HinfI restriction enzymes. We obtained a similar
result with a di¡erent enzyme complex, supporting
the conclusion that a polymorphism higher than we
described in our distance analysis may be found in
the ITS region of T. melanosporum. Although slight,
the intraspeci¢c polymorphism observed of the usu-
ally constant ITS sequences is in contrast with the
very low diversity obtained with length polymor-
phism analyses using RAPD and mirosatellites
[9,36]. The use of a co-dominant technique like
PCR-RFLP to analyse variability should allow de-
tection of higher degrees of polymorphism than a
dominant technique. In the case of T. aestivum,
ITS polymorphism and the high variability of
RAPD patterns [9,15,23] could be related to the het-
erogeneity of the morphology of this complex taxon.
Such an interpretation cannot be made for T. mela-
nosporum. In addition, we did not ¢nd any correla-
tion between ITS polymorphism and the ascocarp
origins. This result has already been reported by
Bertault et al. [36].
The PCR-RFLP analyses were also applied to the
Chinese tru¥es in order to detect any intraspeci¢c
polymorphism. Of the two species analysed, T. hima-
layense and T. indicum, only the latter exhibited a
high polymorphism of its ITS sequence. As for
T. melanosporum, this polymorphism cannot be cor-
related to any geographical origin. In another ITS
sequence analysis, Paolocci et al. [37] reported an
even greater polymorphism than we observed. Mi-
crosatellite investigations also showed high polymor-
phism [22], whereas the RAPD approach only re-
vealed low genetic variability [9]. These di¡erent
analyses cannot be correlated considering the tech-
niques used. Two ITS sequences from GenBank in-
cluded in our analyses were obtained from ascocarps
(Ti80 and Ti20) determined as T. indicum by Paoloc-
FEMSLE 9058 28-10-99
153
ci et al. [37]. Ti80 appears to be included in the clade
with our T. indicum samples. However, Ti20 is in the
clade with T. himalayense samples, and we propose
that this isolate is actually T. himalayense, not
T. indicum. This result con¢rms the observations of
Paolocci et al. [37] on the morphology of the Ti20
tru¥e which di¡ered from the other T. indicum mor-
phology used in their study. This also explains the
very high divergence within the T. indicum species
reported by these authors and the absence of the
clear distinction they observed between T. melano-
sporum and T. indicum.
In spite of intraspeci¢c polymorphism of ITS se-
quences, the species T. melanosporum, T. indicum
and T. himalayense clearly formed three separate
groups (Fig. 3). The distance analysis supports the
results obtained with the phylogenetic tree (Fig. 1)
where the high bootstrap value for each node indi-
cates that the three taxa were unambiguously di¡er-
ent. In addition, sample Ti38 from Paolocci et al.
[37] was included in our parsimony analysis. We ob-
served that this sample was distinct from T. indicum
and close to T. himalayense. It would be interesting
to further investigate the delimitation of this taxon in
order to determine if it corresponds to a variety of
T. himalayense or to a di¡erent but closely related
species, like T. pseudohimalayense [24].
Our study has combined a molecular phylogenetic
investigation with a population analysis to clarify the
systematics of Tuber species. It illustrates that intra-
speci¢c analyses must be carried out to develop a
more reliable classi¢cation of the species based on
DNA comparison, especially with the ITS. Although
these sequences may be useful to di¡erentiate T. mel-
anosporum, T. indicum, T. himalayense and Ti38
[37,38], the polymorphism we observed must be tak-
en into account before primers are designed in re-
gions of the ITS where no variation occurs.
Acknowledgements
We are very grateful to Pebeyre S.A. (Cahors,
France) and to the Consiel Rëgional de Midi-Pyrë-
nëes (France) who supported this work, and to Dr
M. Kulifaj (Pebeyre S.A., Cahors, France) for pro-
viding the tru¥es used in this study.
154 C. Roux et al. / FEMS Microbiology Letters 180 (1999) 147^155
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