Content uploaded by Claudia Riccioni
Author content
All content in this area was uploaded by Claudia Riccioni on Feb 14, 2020
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=umyc20
Mycologia
ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: https://www.tandfonline.com/loi/umyc20
Ribosomal DNA polymorphisms reveal genetic
structure and a phylogeographic pattern in the
Burgundy truffle Tuber aestivum Vittad.
Claudia Riccioni, Andrea Rubini, Aziz Türkoğlu, Beatrice Belfiori & Francesco
Paolocci
To cite this article: Claudia Riccioni, Andrea Rubini, Aziz Türkoğlu, Beatrice Belfiori &
Francesco Paolocci (2019): Ribosomal DNA polymorphisms reveal genetic structure and
a phylogeographic pattern in the Burgundy truffle Tuber�aestivum Vittad., Mycologia, DOI:
10.1080/00275514.2018.1543508
To link to this article: https://doi.org/10.1080/00275514.2018.1543508
View supplementary material
Published online: 24 Jan 2019.
Submit your article to this journal
Article views: 58
View Crossmark data
Ribosomal DNA polymorphisms reveal genetic structure and a phylogeographic
pattern in the Burgundy truffle Tuber aestivum Vittad.
Claudia Riccioni
a
, Andrea Rubini
a
, Aziz Türkoğlu
b
, Beatrice Belfiori
a
, and Francesco Paolocci
a
a
Institute of Biosciences and Bioresources Perugia Division, National Research Council, Via Madonna Alta n. 130, 06128 Perugia, Italy;
b
School
of Environmental and Forest Sciences, Box 352100 University of Washington, Seattle, Washington 98195-2100
ABSTRACT
Ectomycorrhizal ascomycetes belonging to the genus Tuber produce edible fruiting bodies known as
truffles. Tuber aestivum, in particular, is a fungus appreciated worldwide and has a natural distribution
throughout Europe. Most of the molecular studies conducted on this species have been focused on
the question as to whether or not T. aestivum and the morphologically similar T. uncinatum are
conspecific. Conversely, only a handful of studies have assessed the level and distribution of genetic
diversity and occurrence of phylogeographic patterns in this species. Here, we analyzed the genetic
diversity of T. aestivum over a wide geographic range, performing an extensive sampling of specimens
from Turkey, which is novel, to the best of our knowledge. We compared the internal transcribed
spacer (ITS) profiles of 45 samples collected in different Turkish areas with those of 144 samples from
all over Europe. We identified 63 haplotypes, 32 of which were exclusively present in Turkey. The
majority of these haplotyes were also population specific. Haplotype network analysis and statistical
tests highlighted the presence of a genetic structure and phylogeographic pattern, with three spatially
distinct genetic clusters (northeastern Europe, southern Europe, and Turkey), with Turkey representing
a diversity hotspot. Based on these results, we hypothesize the presence of glacial refugia for
T. aestivum in Turkey, whereas European populations likely experienced a population bottleneck.
The possible occurrence of cryptic species among Turkish T. aestivum samples also emerged. Our
results are of practical relevance for the marketing of T. aestivum truffles and mycorrhizal seedlings
and the preservation of the biodiversity of this species.
ARTICLE HISTORY
Received 4 October 2017
Accepted 19 October 2018
KEYWORDS
Ectomycorrhizal fungi;
genetic pattern; internal
transcribed spacer;
phylogeography; truffles;
Turkey
INTRODUCTION
Macrofungi are fungi producing visible fruiting bodies
and are characterized by a saprotrophic, parasitic, or
symbiotic lifestyle. Plant-fungus symbioses play key
ecological roles in agroforest ecosystems (Smith and
Read 2010). Some fungi, belonging to the Ascomycota
and Basidiomycota phyla, establish symbiotic associa-
tions with the lateral roots of many tree and shrub
species by developing structures known as ectomycor-
rhizae. The ectomycorrhizal associations are beneficial
to both partners: the fungi use the carbon compounds
photosynthesized by the host plants while providing
their hosts with nutrients, water, and protection against
biotic and abiotic stresses. Besides their ecological role,
ectomycorrhizal ascomycetes belonging to the order
Pezizales genus Tuber produce hypogeous fruiting
bodies, which are the true truffles. These are edible
macrofungi that in some Tuber species hold distinctive
aromatic properties, which make them appreciated and
marketed worldwide as food delicacies. Truffles live in
native forests throughout the Northern Hemisphere
(Bonito et al. 2013), with patterns of geographic dis-
tribution depending on the species. The most valuable
white and black truffle species, T. magnatum and
T. melanosporum, respectively, have very limited geo-
graphic ranges: T. magnatum is only harvested in Italy
and in some Balkan areas, although records from a few
other countries have been reported (Tabouret 2011;
Riccioni et al. 2016); T. melanosporum grows sponta-
neously in Italy, France, and Spain (Riccioni et al. 2008)
but has been introduced in several countries around the
world (Chen et al. 2016). Conversely, other truffle spe-
cies display a wider distributional range and among
them is T. aestivum, one of the most economically
important truffles, better known as the Burgundy truf-
fle. This species is widespread in nearly all European
countries, as well as in North Africa (Chevalier et al.
1979) and in Turkey (Türkoğlu et al. 2015). In sharp
CONTACT Francesco Paolocci francesco.paolocci@ibbr.cnr.it
Claudia Riccioni and Andrea Rubini contributed equally to this work.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/umyc.
Supplemental data for this article can be accessed on the publisher’s Web site.
MYCOLOGIA
https://doi.org/10.1080/00275514.2018.1543508
© 2019 The Mycological Society of America
Published online 24 Jan 2019
contrast to other Tuber species, T. aestivum is success-
fully cultivated in diverse environments and countries,
both within and outside its natural geographic range
(Chevalier 2008; Chevalier and Sourzat 2012; Stobbe
et al. 2013; Morcillo et al. 2015), due to its adaptation
to a large spectrum of climatic and pedological
conditions.
Due to the presence of truffles having different mor-
phological characteristics (i.e., spore ornaments), bou-
quet, ripening period, and ecological requirements, in
the late 19th century Chatin (1887) split T. aestivum
into two distinct species: T. aestivum and T. uncinatum,
the former representing truffles that ripen in summer,
the latter in the fall. However, in the last few years,
phylogenetic studies carried out by different research
groups have all come to the conclusion that the pheno-
typic differences between T. uncinatum and T. aestivum
are not enough to justify the separation into two dis-
tinct species and recommend the use of T. aestivum
Vittad. as the scientific name (Paolocci et al. 2004;
Wedén et al. 2004,2005; Chevalier and Sourzat 2012;
Molinier et al. 2013).
Assessing the extent and distribution of genetic
diversity of fungal species, on both large scale and
small scale, is crucial to understanding their biology
and demographic history and to guide biodiversity
conservation programs (Douhan et al. 2011).
Concerning truffles, large-scale genetic diversity studies
have been carried out on T. magnatum (Rubini et al.
2004,2005) and T. melanosporum (Bertault et al. 1998;
Murat et al. 2004,2011; Riccioni et al. 2008;
García-Cunchillos et al. 2014). Geographically struc-
tured populations and phylogeographic signals were
identified in both species. Moreover, these studies sug-
gested that both T. magnatum and T. melanosporum
experienced a population bottleneck during the last ice
age and that their refugia were located in the southern-
most areas of their distributional ranges. Using differ-
ent markers, such as random amplification of
polymorphic DNA (RAPD), internal transcribed
spacers (ITS) of rDNA, amplified fragment length poly-
morphism (AFLP), and simple sequence repeats (SSRs),
several authors also reported the existence of genetic
diversity among populations of T. aestivum
(Gandeboeuf et al. 1997; Mello et al. 2002; Wedén
et al. 2004; Splivallo et al. 2012b; Molinier et al.
2016b). However, these studies were centered only on
the European populations.
Interestingly, in accordance with its wider habitat
range, T. aestivum displays a higher rate of genetic
and morphological diversity with respect to other
Tuber species of economic relevance (Rubini et al.
2005 and references therein; Molinier et al. 2016b).
More in depth, in a global survey conducted on Tuber
species, T. aestivum was shown to be the most poly-
morphic, since its ITS variation was up to 3.7% (Bonito
et al. 2010). More recently, four genetic groups have
been recognized in the European populations of
T. aestivum, by using SSRs, and the presence of an
ecotype, potentially adapted to the climate of southern
Europe, has been envisaged (Molinier et al. 2016b). On
a smaller spatial scale, some studies have focused on the
spatial and temporal genetic structure of T. aestivum in
both natural and man-made truffle orchards, as well as
on the potential link between the genetic diversity of
the fruiting body and the aromatic profiles (Splivallo
et al. 2012b; Molinier et al. 2015,2016a). Here, we
extended the sampling to include the natural popula-
tions of T. aestivum of Turkey, with the aim of evaluat-
ing the extent and distribution of genetic diversity and
of disclosing the phylogeographic patterns in this spe-
cies on a larger spatial scale compared with previous
studies. To this end, we characterized the ITS profiles
of the 45 Turkish specimens from 11 populations and
compared their haplotypes with those of 144 previously
characterized specimens from all over Europe.
MATERIALS AND METHODS
Sample source, DNA isolation, PCR, and sequencing.
—At
otalof45T. aestivum ascocarps were collected in
the years 2010–2014 from 11 natural areas in Turkey
(TABLE 1;FIG. 1; SUPPLEMENTARY TABLE 1). The
Aegean region (populations 1–7 and 11) was the most
sampled, followed by the Marmara (population 9),
Mediterranean (population 8), and Black Sea
(population 10) regions. Dried herbarium specimens as
well as fresh specimens were used. Soon after collection,
the peridium was removed from the fresh ascocarps and
the inner part of the gleba was cut into slices, frozen in
liquid nitrogen, and stored at −70 C until DNA
extraction.
All ascocarps were morphologically checked accord-
ing to Chevalier et al. (1979). Spore examination was
performed to exclude the presence of similar species
such as Tuber mesentericum. Genomic DNA was iso-
lated from 0.3 g of gleba. The internal transcribed
spacer (ITS) of the rDNA region was amplified by
polymerase chain reaction (PCR), as previously
described by Paolocci et al. (2004)(). Dimethyl sulfox-
ide (DMSO) 10% (v/v) was added to the PCR reaction
mixtures to amplify most of the DNA, due to the
presence of a G/C-rich region in the T. aestivum ITS
(Paolocci et al. 2004). In addition, DNA amplification
from certain herbarium specimens was obtained only
after adding bovine serum albumin (BSA; 7 mg/ml;
2RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
Sigma-Aldrich, Steinheim, Germany) to the reaction
mixture (Paolocci et al. 1999). The amplified fragments
were purified using the EuroGold Cycle-Pure kit
(Euroclone, Milan, Italy ).
ITS sequencing was carried out using the primers ITS1,
ITS4,5.8sf,and5.8sb(Rubinietal.1998) and the BigDye
Terminator Cycle Sequencing Kit (version 3.1; Applied
Biosystems, Foster City, CA, USA), according to the sup-
plier’s instruction manual. DMSO 10% (v/v) was also
added to the sequencing reaction mixtures. An ABI 3130
Genetic Analyzer (Applied Biosystems) was used for capil-
lary electrophoresis. Electropherograms were analyzed
with FinchTV 1.3.1 (Geospiza, Seattle, Washington;
http://www.geospiza.com). A BLASTn analysis (Altschul
et al. 1990) was carried out for each sequence to confirm
the species. The ITS sequences were deposited in GenBank
under the following accession numbers: KU664080 to
KU664124 (SUPPLEMENTARY TABLE 1).
Sequence mining from GenBank.—In order to obtain
aT. aestivum sampling representative of the entire
species distributional range, 144 ITS sequences from
specimens collected throughout Europe, and referenced
in previous papers (Mello et al. 2002;Paoloccietal.
2004; Wedén et al. 2005; Gryndler et al. 2011), were
downloaded from GenBank (SUPPLEMENTARY
TABLE 1). Thus, we obtained a data set consisting of
189 T. aestivum specimens, by combining the 45 ex novo
sequenced samples from Turkey with the GenBank
samples. These were grouped in 36 populations
according to their place of origin (TABLE 1;
SUPPLEMENTARY TABLE 1). Assembly, editing, and
alignment of the ITS sequences were performed by the
software Geneious 4.8.5 (Biomatters, Auckland, New
Zealand; http://www.geneious.com).
Data analyses.—The number of ITS haplotypes and
their frequency in the entire collection and at the
population/area level were calculated using the
software Arlequin 3.5.1.2 (Excoffier and Lischer 2010).
To evaluate the representativeness of our sampling,
a rarefaction analysis was performed by plotting the
Table 1. Geographic information and diversity of the T. aestivum populations.
Population Origin Country Latitude Longitude N nH nHG
1 Bozkurt Turkey 37.82 29.61 11 9 0.82
2 Acipayam Turkey 37.42 29.35 3 1 0.33
3 Honaz Turkey 37.76 29.27 9 9 1.00
4 Cal Turkey 38.08 29.4 9 4 0.44
5 Fethiye Turkey 36.58 29.25 1 1 1.00
6 Burdur Turkey 37.42 30.67 1 1 1.00
7 Izmir Turkey 38.2 26.84 1 1 1.00
8 Hatay Turkey 36.2 36.16 1 1 1.00
9 Çatalca Turkey 41.14 28.46 1 1 1.00
10 Ordu Turkey 41.02 37.5 1 1 1.00
11 Muğla Turkey 37.21 28.36 7 4 0.57
Turkey 45 32 0.71
12 Emilia Romagna Italy 44.6 11.22 3 2 0.67
13 Umbria Italy 42.94 12.62 9 5 0.56
14 Abruzzo Italy 42.34 13.45 9 6 0.67
15 Molise Italy 41.56 14.66 2 2 1.00
16 Liguria Italy 44.32 8.4 3 3 1.00
17 Lombardia Italy 45.48 9.84 9 2 0.22
18 Piemonte Italy 44.8 9.29 11 6 0.55
19 Marche Italy 43.63 12.76 2 1 0.50
20 Italy Italy 41.87 12.57 2 2 1.00
21 Teruel Spain 40.34 -1.11 1 1 1.00
22 Castilla Spain 41.75 -4.79 2 2 1.00
23 Dordogne France 45.1 0.75 2 1 0.50
24 Bouches du Rhone-Drome France 44.36 4.91 5 4 0.80
Southern Europe 60 24 0.4
25 Yonne-Haute Marne France 48.8 6.09 8 4 0.50
26 Oxford England 51.75 -1.26 1 1 1.00
27 Gotland Sweden 57.65 18.71 35 4 0.11
28 Oland Sweden 56.66 16.67 2 1 0.50
29 Aarhus Denmark 56.16 10.2 1 1 1.00
30 NPR Karlstejn Czech Republic 49.93 14.18 9 3 0.33
31 Damborice-Klinek Czech Republic 49.03 16.91 1 1 1.00
32 Teplice Czech Republic 50.64 13.83 3 1 0.33
33 Chodec Czech Republic 50.41 14.51 1 1 1.00
34 Pest-Baranya Hungary 47.16 20.18 3 3 1.00
35 TribečSlovakia 48.68 18.39 12 4 0.33
36 Povazsky Inovec Slovakia 48.77 18.03 8 3 0.38
Northern Europe 84 13 0.15
Total 189 63 0.33
Note. N = number of samples; nH = number of haplotypes; nHG = ratio nH/N.
MYCOLOGIA 3
number of haplotypes as a function of sample size using
the software EstimateS 9.1. A phylogenetic tree was
inferred with the software MEGA6 (Tamura et al.
2013) by using the neighbor joining method and the
composite maximum likelihood distance. The
alignment gaps were considered using the pairwise
deletion option. Bootstrap analysis was performed
with 100 replicates. The alignments were submitted to
TreeBASE (S22696). The analysis of the haplotype
network was performed with the software Haploview
(http://www.cibiv.at/~%20greg/haploviewer) using the
maximum parsimony (MP) method implemented in
the DNAPARS module of the PHYLIP package 3.696
(Felsenstein 1989).
The clustering of samples was examined by the soft-
ware BAPS (Bayesian Analysis of Population Structure)
4.14 (Corander et al. 2003,2008). The genetic divergence
between the groups identified by BAPS was estimated
using MEGA6 (Tamura et al. 2013) as an average pro-
portion (p) of nucleotide sites at which two sequences
being compared are different (p-distance).
To check for deviations from neutrality, i.e., to see
whether DNA sequences evolve in manners
inconsistent with the neutral theory of molecular evo-
lution, we performed Tajima’s D and Fu and Li’sD*
and F* neutrality tests by using DnaSP 5.1 (Librado and
Rozas 2009). A significant deviation from genetic neu-
trality could be interpreted as a result of a recent popu-
lation expansion or bottleneck (Tajima 1989). The
mismatch distribution analysis, i.e., the distribution of
pairwise differences in our set of sequences, was per-
formed as an additional test for demographic expansion
using Arlequin 3.5.1.2. The graph resulting from this
analysis is usually multimodal in samples drawn from
populations at equilibrium, whereas it is unimodal in
populations that have been through a recent demo-
graphic expansion.
The hierarchical analysis of molecular variance
(AMOVA) was performed using Arlequin 3.5.1.2. The
spatial structure of populations was inferred using the
software Spatial Analysis of Molecular Variance
(SAMOVA) 2 (Dupanloup et al. 2002;http://cmpg.
unibe.ch/software/samova2/). SAMOVA defines groups
of populations that are geographically homogeneous
and maximally differentiated from each other on the
basis of Fct statistics. Fct indicates the proportion of
Figure 1. Geographic map showing the locations of the populations of T. aestivum under study.
4RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
molecular variance among groups of populations. The
pairwise nucleotide difference was used as a measure of
distance, and the significance of the Fct index was
evaluated through a permutation test (1000 permuta-
tions). The haploid genetic distance among populations
was calculated after conversion of the DNA sequence
data into numeric codes using GenAlEx 6.501 (Peakall
and Smouse 2012). To evaluate the presence of
a pattern of isolation by distance (Rousset 1997), we
performed the Mantel correlation test between genetic
distance and the natural logarithm of geographic dis-
tances (km) among populations using the software
GenAlEx and 1000 random permutations.
RESULTS
Successful PCR amplification and sequencing of the ITS
region was obtained for all 45 samples from Turkey.
The nucleotide alignment between these sequences and
those downloaded from GenBank was 542 bp long and
contained a total of 108 variable sites (single-nucleotide
polymorphisms [SNPs] and indels).
By considering these polymorphisms, 63 haplotypes,
namely, H1 to H63, were detected within the 189
T. aestivum samples under study (SUPPLEMENTARY
TABLE 1; SUPPLEMENTARY FIG. 1). An even higher
number of haplotypes should be expected by increasing
the sample size, as is shown by the rarefaction curve
(SUPPLEMENTARY FIG. 2).
Out of the 63 haplotypes identified, 32 exclusively
belonged to the 11 Turkish collection sites
(SUPPLEMENTARY TABLES 1 and 2). In addition,
with the only exception of H9, which was shared
between two nearby populations (Honaz and
Bozkurt), all the Turkish haplotypes were also specific
to single populations (SUPPLEMENTARY TABLE 2).
As shown in TABLE 1, the ratio between number of
haplotypes and sample size (nHG) in Turkey was about
2- and 5-fold higher than that of southern and northern
Europe, respectively. This ratio increased from north to
south if the entire population set was subdivided into
four groups according to latitude (TABLE 2).
The phylogenetic tree of the haplotypes highlighted
the presence of three main clusters (FIG. 2). Cluster
I grouped most of the European haplotypes (25), since
only 5 were the Turkish haplotypes, whereas most (25)
of the Turkish haplotypes belonged to cluster II, which
only included 6 haplotypes from Europe. A third, small
cluster (III) only included 2 haplotypes from Turkey.
We performed a network analysis that also included
geographic information and haplotype frequencies in
order to infer relationships among the haplotypes
(FIG. 3). The existence of three main clusters of hap-
lotypes, corresponding to three major geographic
areas, emerged from this analysis: southern Europe
(cluster A), northeastern Europe (cluster B), and
Turkey (cluster C). Only a few haplotypes, all belong-
ing to the Turkish samples, were not included in these
clusters. The three most frequent haplotypes (H33,
H36, and H57) were largely shared between
European populations, with H36 mainly found in
Italy (cluster A). Conversely, H33 and H57 were
mainly found in northern and eastern Europe, respec-
tively (cluster B), and were very similar to each other,
only differing by one SNP. Many rare haplotypes,
whichonlydifferedbyonetothreepolymorphisms
compared with the most common haplotypes, were
also present (FIG. 3).
In contrast to what emerged in the European popu-
lations, where some haplotypes dominated whole
regions, Turkey presented a high diversity and was
characterized by many exclusive haplotypes. These hap-
lotypes were very different not only from the European
ones but also between each other, both at intra- and
interpopulation levels (FIG. 3; SUPPLEMENTARY
FIG. 1). The most frequent Turkish haplotype, although
only shared by four individuals from Cal, was H19
(FIG. 3; SUPPLEMENTARY TABLE 2). A few haplo-
types (4), which only differed from H19 by one to two
polymorphisms, were detected in the same population
of Cal and in the nearby population of Honaz. It is
worth mentioning that two Turkish haplotypes, H1 and
H4, that were harbored by samples 1 and 5 from
Bozkurt, respectively, showed an exceptionally high
phylogenetic distance from all others to the extent
that they were grouped in a cluster of their own
(FIGS. 2 and 3). Also, H1 and H4 were placed in
a separate clade with an intermediate position between
the European/Turkish T. aestivum and the Chinese
specimens in the phylogenetic tree built including
taxa from China, namely, Tuber sinoaestivum, and
T. aestivum sensu lato (Zhang et al. 2012; Zambonelli
et al. 2012) (SUPPLEMENTARY FIG. 6).
A phylogenetic link was suggested between Turkey
and southern Europe, since five of the Turkish haplo-
types were very close to the highly frequent H36 and
vice versa six haplotypes from Italy, France, and Spain
were close to the Turkish haplotypes (FIGS. 2 and 3).
Table 2. Relative numbers of haplotypes at different latitudinal
ranges.
Latitudinal range (°N) N nH nH/N
58–51 (populations 26–29) 39 6 0.15
50–47 (populations 25, 30–36) 45 10 0.22
45–42 (populations 12–14, 16–19, 23, 24) 53 20 0.38
41–36 (populations 1–11, 15, 20–22) 52 39 0.75
Note. N = number of samples; nH = number of haplotypes; nHG = ratio nH/N.
MYCOLOGIA 5
Figure 2. Phylogenetic tree of the haplotypes observed. Numbers near the branches indicate the bootstrap values (percentage over
100 replicates). Haplotypes are indicated as H1 to H63 as in SUPPLEMENTARY FIG. 1. Populations where each haplotype is present
are indicated in brackets and numbered as in TABLE 1.
6RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
The Bayesian clustering performed with BAPS
detected one minor and three major clusters showing
a clear link with the already cited geographic areas
(FIG. 4A). In fact, cluster A grouped most of the
Turkish samples, together with the same few specimens
that were grouped by phylogenetic analyses from other
geographic areas (Italy, France, and Spain); cluster
B grouped the only two Turkish samples from
Bozkurt (population 1), confirming their divergence
from all other haplotypes (FIGS. 2,3, and 4A); cluster
C mainly included Italian samples and cluster D, north-
ern and eastern European samples. Moreover, the four
clusters perfectly matched the pattern of the haplotype
network (FIG. 4B).
To test for departure from neutral selection, we
performed the Tajima’s D and Fu and Li’s D* and F*
neutrality tests for northeastern Europe, southern
Europe, and Turkey, together with the mismatch dis-
tribution analysis (SUPPLEMENTARY FIG. 3).
Negative values were observed for all neutrality tests,
although only D* and F* were significant for southern
Europe (SUPPLEMENTARY FIG. 3B). In the northern
and eastern European populations, the mismatch dis-
tribution analysis showed that sequence pairs differing
for low numbers of nucleotide sites (0 and 1) had
a particularly high frequency, whereas in the southern
European and Turkish populations sequences differing
for a low (0, 1) and a higher (5, 10) number of nucleo-
tide sites had similar frequencies.
The analysis of molecular variance (AMOVA)
revealed a strong genetic differentiation between popu-
lations (Fst = 0.44; P< 0.001). We performed the
SAMOVA analysis to define groups of populations
that were geographically homogeneous and maximally
differentiated from each other. Quite similar Fct values
were obtained when the number of groups of popula-
tions (K) ranged from 3 to 8 (SUPPLEMENTARY FIG.
4A). When K = 3 was considered, we identified groups
of populations basically corresponding to the geo-
graphic areas of northeastern Europe, southern
Europe, and Turkey. With the increase in K, this geo-
graphic pattern was basically confirmed and the ten-
dency to split Turkish populations into further groups
emerged (SUPPLEMENTARY FIG. 4B). The Mantel
test showed that among-population differentiation sig-
nificantly increased with geographic distance (R
2
=
0.109; P< 0.01), revealing a pattern of isolation by
distance (SUPPLEMENTARY FIG. 5).
In order to evaluate the presence of cryptic species
among T. aestivum samples, we calculated the diver-
gence levels in terms of sequence similarity between the
four BAPS clusters. The average nucleotide distances
ranged from 0.81% to 2.14% between clusters A–D, A–
C, and D–C and from 3.9% to 4.44% between clusters
A–B, D–B, and C–B(TABLE 3), suggesting the possible
presence of cryptic species within cluster B.
DISCUSSION
In the present study, we carried out the first ITS
sequence analysis of populations of T. aestivum from
Turkey, which, to the best of our knowledge, represents
one of the southernmost areas of the distributional
range of this truffle species. We further compared the
ITS profiles of the Turkish specimens with ITS profiles
of those collected from other regions of Europe, with
the aim of performing a large-scale population genetics
study and of assessing the possible presence of phylo-
geographic signals. Although it is highly unlikely that
our data set reflects the whole genetic diversity of the
species, the acquired data were enough to demonstrate
the presence of a strong genetic structure among popu-
lations of T. aestivum, with those from Turkey display-
ing a higher rate of genetic diversity and private
haplotypes compared with those from Europe.
A phylogeographic pattern with three main spatially
distinct genetic clusters (located in northeastern
Europe, southern Europe, and Turkey) and the possible
existence of cryptic species also emerged from our
data set.
Our study sheds light on the historical population
dynamics of this species. These results are also of prac-
tical relevance for the marketing of T. aestivum truffles
and mycorrhizal seedlings and for the preservation of
the biodiversity of this species.
ITS analysis suggests that geographic and genetic
barriers shaped the spatial structure of
T. aestivum.—Considering the polymorphisms of the
ITS region, 63 ITS haplotypes were identified among
the 189 T. aestivum samples analyzed. The level of
T. aestivum genetic diversity, which emerged from
our data set, was much higher than that of other
Tuber species, as previously reported in analogous
large-scale studies. In fact, in two independent
screenings of 188 and 205 T. melanosporum European
samples, only 10 and 13 ITS haplotypes were identified,
respectively (Murat et al. 2004; Riccioni et al. 2008).
Likewise, only 13 ITS haplotypes were found among the
136 samples of T. brumale analyzed by Merényi and
colleagues (2014).
The phylogenetic analysis performed to evaluate the
relationship between haplotypes showed a clear ten-
dency of the Turkish and European haplotypes to be
different from each other. The network analysis
MYCOLOGIA 7
conducted was even more effective in highlighting the
presence of a phylogeographic structure, which also
showed the tendency of northeastern and southern
European samples to cluster in two distinctive groups.
This pattern was still consistent even when the Turkish
populations were excluded from the analysis (data not
shown). The finding of two genetic clusters in Europe
calls for additional studies aimed at verifying whether
or not samples of the two clusters differ in morphology,
ripening time, and/or habitat requirement.
Bayesian analysis and SAMOVA confirmed the pre-
sence of three main genetic clusters, corresponding to
the three major phylogeographic areas under study, and
of a fourth cluster, which only included two samples
from Turkey.
High levels of genetic structure are usually associated
with limited gene flow between populations as
a consequence of either reproductive barriers or
geographic isolation (Slatkin 1987). Reproductive isola-
tion plays an important role in sympatric speciation
processes (Giraud et al. 2008). Our analysis suggests
that geographic isolation likely played a major role in
limiting gene flow among populations of T. aestivum,
as the isolation by distance test showed a significant
correlation between genetic and geographic distances
among the populations.
Conversely, at a small-scale level, reproductive genetic
barriers might be evoked to explain the presence of
strongly differentiated samples within a given population
such as the case of samples 1 and 5 from Bozkurt. These
samples were in fact not only differentiated from all
other samples but also from the other nine samples
belonging to the same population and formed
a distinct genetic group (cluster B of BAPS analysis).
The average distance of cluster B from the others was
much higher than the average distances between all
Figure 3. Haplotype network. Circles are shaded according to the percentage of samples belonging to the different populations
numbered according to TABLE 1. The sizes of the circles are proportional to the haplotype frequency. Haplotypes are indicated as H1
to H63. Points along the lines indicate intermediate mutational steps between haplotypes.
8RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
Figure 4. A. Genetic structure as estimated by BAPS software for K = 4. Each individual is represented by a vertical line shaded
according to one of the four K groups. Numbers and black marks indicate the different populations. B. Haplotype network with
overlapped BAPS clusters.
MYCOLOGIA 9
other clusters. The divergence values of cluster B was
higher than the 3% threshold commonly used in fungal
community studies to delineate species boundaries
(Smith et al. 2007; Peay et al. 2008;Hughesetal.2009)
and the 4% threshold suggested by Bonito et al. (2010)
for Tuber species. According to our phylogenetic analy-
sis, samples of cluster B represent an intermediate taxon
between T. aestivum and the related Chinese taxa
T. aestivum sensu lato and T. sinoaestivum. To establish
whether or not this intermediate taxon represents
a cryptic species, further morphological and molecular
analyses, based on additional genetic markers and
a more representative number of fruit bodies from
Bozkurt as well as from other Eastern countries, such
as Iran and Azerbaijan, will be needed.
The study of the mating type (MAT) locus would be
particularly informative to disclose inter- and intraspe-
cific genetic barriers in T. aestivum. This locus has been
identified in Tuber species of economic relevance,
including T. aestivum (Martin et al. 2010,2012;
Rubini et al. 2011; Belfiori et al. 2013,2016; Payen
et al. 2014). As an example, in T. indicum, the sequen-
cing of this locus revealed an extensive polymorphism
and rearrangements between samples belonging to dif-
ferent ITS clades, suggesting the presence of cryptic
species (Belfiori et al. 2013).
Genetic diversity and phylogeography suggest
different demographic histories of T. aestivum in
Europe and Turkey.—About 50% (32 out of 63) of the
total number of haplotypes detected in this study were
from Turkey. Interestingly, almost all of these haplotypes
(31) were not shared among the Turkish populations.
This scenario changed when European samples were
considered: three haplotypes were largely shared
between populations; two (H33 and H57) only differed
byoneSNP,andthereweremanyrarehaplotypesthat
only differed from the three most common by one to
three polymorphisms (FIG. 3). Such a star-shaped
network of the European haplotypes is consistent with
the hypothesis of a recent population expansion. In fact,
the few highly frequent haplotypes may represent
ancestral haplotypes from which many rare ones likely
derived in recent times (McCormack et al. 2008; Merényi
et al. 2014).ThenegativevaluesofTajima’sDandFuand
Li’s D* and F* neutrality tests also point to a possible
population expansion following a recent population
bottleneck. The mismatch distribution analysis, in
addition, highlighted a different trend between the
populations from northeastern Europe and those from
Turkey/southern Europe, since a large prevalence of
haplotypes with few pairwise differences only emerged
intheformergroup.Also,thehaplotypediversitywas
shown to increase from north to south. These results can
be explained by the fact that T. aestivum experienced
a population bottleneck, during the last glacial age,
which likely was more severe in northern and eastern
Europe than in southern Europe and Turkey. These
latter areas were probably refugia of the species, due to
the favorable climatic conditions. In contrast, according to
Molinier and colleagues (2016b), T. aestivum has not
experienced a recent population bottleneck, although it
must be underlined that no Turkish specimens were
considered in their investigation. The occurrence of
a population bottleneck and the presence of refugia in
the southernmost distributional ranges were already
envisaged for other Tuber species, such as
T. melanosporum and T. magnatum (Bertault et al. 1998;
Murat et al. 2004;Rubinietal.2005;Riccionietal.2008).
Since Tuber species are mycorrhizal fungi, their
postglacial population dynamics should track those of
their host plants. In this respect, we note that Turkey
was the refugium for several plant species (Médail and
Diadema 2009) and, among them, for T. aestivum host
species, such as Quercus cerris (Brewer et al. 2002;
Bagnoli et al. 2016) and Pinus sylvestris (Cheddadi
et al. 2006; Naydenov et al. 2007).
The very high level of genetic diversity and haplo-
type endemism of T. aestivum in Turkey may also be
explained by the presence of geographic barriers to
gene flow, such as mountain chains and the Central
Anatolian Lake System, whose formation can be traced
back to the Neogene (Bilgin 2011). The limited truffle
knowledge and tradition in Turkey, where truffle culti-
vation and marketing are still at their infancy, might
have also concurred to preserve such a local truffle
biodiversity. Although there are only a few molecular
investigations on fungal biodiversity in Turkey, the
presence of diversity hot spots in this area was already
hypothesized for other mushrooms belonging to the
genus Morchella (Taşkınet al. 2012).
The haplotype network highlighted a clear-cut
separation between European and Turkish strains in
different clusters, with at least eight mutational steps
from each other (FIG. 3). Nevertheless, a few Turkish
strains clustered within the “European lineage”and vice
versa some strains from southern Europe were detected
Table 3. Divergence between the groups identified by BAPS.
BAPS group Pairwise p-distance
A-D 1.99
A-C 2.14
A-B 4.44
D-C 0.81
D-B 3.90
C-B 4.04
10 RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
in the Turkish lineage, although without any haplotype
sharing. We also note that the ITS sequences of a few
T. aestivum samples from Iran recently published by
Jamali (2017) show the same haplotype, H36, of some
European samples. These pieces of evidence suggest an
effect of either sequence homoplasy or the recent
human-mediated introduction of allocthonous strains
in non-native areas. Indeed, both T. aestivum ascocarps
and mycorrhizal seedlings have been intensively mar-
keted for decades, and T. aestivum is considered to be
one of the few Tuber species that has become a non-
native colonizer of regions distant from its habitat of
origin (Vellinga et al. 2009; Bonito et al. 2010). Most of
the Turkish samples were collected from natural Pinus
forests, with the exception of samples from the Black
Sea region where extensive cultivations of hazelnut
trees are present. Interestingly, some of the Turkish
samples of T. aestivum, holding haplotypes similar to
those of the European samples, came from the latter
region. It is therefore conceivable that non-native
T. aestivum strains were introduced in Turkey as
a consequence of the cultivation of hazelnut trees.
However, additional Turkish samples from this area
should be analyzed to test and confirm this hypothesis.
Potential impact of the current findings on truffle
marketing and biodiversity conservation.—The
rising truffle demand in a globalized market and the
decrease in harvesting of the premium black and white
truffles, coupled with the wider ecological range and
higher cultivation potential of T. aestivum compared
with other Tuber species, are reasons that have
increased the scientific and commercial interest for
T aestivum over the last few years (Stobbe et al. 2013).
As an example, in Italy, where 70–80% of the national
truffle production reaches the processing industry,
about 65% of the processed truffles are T. aestivum
(Pampanini and Martino 2006).
The pronounced genetic variability among popula-
tions of different geographic regions, which has
emerged from the present study, opens the way to
further efforts to trace natural populations of
T. aestivum according to their geographic origin. This
goal is of particular interest for the socioeconomic
development not only of areas traditionally known for
the presence of T. aestivum, but also of those in coun-
tries, such as Turkey, having a productive capacity that
is not yet fully exploited. Such genetically distinct local
productions might gain further value if specific aro-
matic profiles also could be assigned to them
(Molinier et al. 2015). Ecological implications also
emerge from our results. For instance, priority should
be given to the protection of truffle resources from the
areas, such as Turkey, that have a relatively short har-
vesting history, harbor autochthonous strains, and have
limited to null tradition concerning truffle cultivation.
For the biodiversity conservation of this species in these
areas, in fact, the use of native strains as spore inocula
to produce mycorrhizal plants to promote T. aestivum
cultivation should be recommended. Ecological threats
due to the undiscriminated use of allochtonous strains
are unpredictable in terms of biodiversity erosion as
well as adaptability of the newly introduced strains to
new environmental conditions. Finally, although the
effects of climate change on productivity of natural
and man-made truffle orchards is a controversial
topic (Büntgen et al. 2012; Splivallo et al. 2012a), com-
parative genetics and genomics studies on T. aestivum
strains adapted to different ecological conditions may
allow us in the very near future to identify traits related
to tolerance and strains with higher adaptability to the
ongoing climatic changes. The present study suggests
that populations and strains of T. aestivum from
Turkey are of outstanding relevance to approaching
such a goal.
FUNDING
Dr. Aziz Türkoğlu received funding from the Scientific and
Technological Research Council of Turkey (project number
T-BAG-111T530). This study was also supported by a CNR-
Tubitak bilateral project 2014–2015 (number TBAG-
113Z893) entitled “Gaining insight into genetic diversity
and sexual propagation patterns of truffles belonging to the
T. aestivum/T. uncinatum species complex.”
ORCID
Claudia Riccioni http://orcid.org/0000-0003-1069-8943
Andrea Rubini http://orcid.org/0000-0003-3988-6294
Beatrice Belfiori http://orcid.org/0000-0002-5366-8579
Francesco Paolocci http://orcid.org/0000-0002-9394-876X
LITERATURE CITED
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990.
Basic local alignment search tool. Journal of Molecular
Biology 215:403–410, doi:10.1016/S0022-2836(05)80360-2
Bagnoli F, Tsuda Y, Fineschi S, Bruschi P, Magri D, Zhelev P,
Paule L, Simeone MC, González-Martínez SC,
Vendramin GG. 2016. Combining molecular and fossil
data to infer demographic history of Quercus cerris:
insights on European eastern glacial refugia. Journal of
Biogeography 43:679–690, doi:10.1111/jbi.12673
Belfiori B, Riccioni C, Paolocci F, Rubini A. 2013. Mating
type locus of Chinese black truffles reveals heterothallism
and the presence of cryptic species within the Tuber indi-
cum species complex. PLoS ONE 8:e82353.
MYCOLOGIA 11
Belfiori B, Riccioni C, Paolocci F, Rubini A. 2016.
Characterization of the reproductive mode and life cycle
of the whitish truffle T. borchii. Mycorrhiza 26:515–527,
doi:10.1007/s00572-016-0689-0
Bertault G, Raymond M, Berthomieu A, Callot G,
Fernandez D. 1998. Trifling variation in truffles. Nature
394:734, doi:10.1038/29428
Bilgin R. 2011. Back to the suture: the distribution of intraspe-
cific genetic diversity in and around Anatolia. International
Journal of Molecular Sciences 12:4080–4103, doi:10.3390/
ijms12064080
Bonito G, Smith ME, Nowak M, Healy RA, Guevara G,
Cázares E, Kinoshita A, Nouhra E, Domínguez LS,
Tedersoo L, Murat C, Wang Y, Moreno BA, Pfister DH,
Nara K, Zambonelli A, Trappe JM, Vilgalys R. 2013.
Historical biogeography and diversification of truffles in
the Tuberaceae and their newly identified Southern
Hemisphere sister lineage. PLoS ONE 8:e52765,
doi:10.1371/journal.pone.0052765
Bonito GM, Gryganskyi AP, Trappe JM, Vilgalys R. 2010.
A global meta-analysis of Tuber ITS rDNA sequences:
species diversity, host associations and long-distance
dispersal. Molecular Ecology 19:4994–5008, doi:10.1111/
j.1365-294X.2010.04855.x
Brewer S, Cheddadi R, De Beaulieu JL, Reille M. 2002. The
spread of deciduous Quercus throughout Europe since the
last glacial period. Forest Ecology and Management
156:27–48, doi:10.1016/S0378-1127(01)00646-6
Büntgen U, Egli S, Camarero JJ, Fischer EM, Stobbe U,
Kauserud H, Tegel W, Sproll L, Stenseth NC. 2012.
Drought-induced decline in Mediterranean truffle
harvest. Nature Climate Change 2:827–829, doi:10.1038/
nclimate1733
Chatin MA. 1887. Une nouvelle espécé de truffe (Tuber
uncinatum). Bulletin de la Société Botanique de France
34:246–248.
Cheddadi R, Vendramin GG, Litt T, François L, Kagey-ama
M, Lorentz S, Laurent JM, De Beaulieu JL, Sadori L, Jost A,
Lunt D. 2006. Imprints of glacial refugia in the modern
genetic diversity of Pinus sylvestris. Global Ecology and
Biogeography 15:271–282, doi:10.1111/j.1466-8238.2006.
00226.x
Chen J, Murat C, Oviatt P, Wang Y, Le Tacon F. 2016. The
black truffle Tuber melanosporum and Tuber indicum. In:
Zambonelli A, Iotti M, Murat C, eds. True truffle (Tuber
spp.) in the world. Soil ecology, systematic and biochem-
istry. Switzerland: Springer International Publishing. p.
19–32.
Chevalier G. 2008. Truffes et trufficulture en Europe. In: Atti
del terzo congresso internazionale di Spoleto sul tartufo,
Spoleto, Italy, 25th–28th November 2008. Perugia, Italy:
Ed. Comunità Montana dei Monti Martani Serano e
Subasio. p. 65–72.
Chevalier G, Desmas C, Frochot H, Riousset L. 1979.L’espèce
Tuber aestivum Vitt. I. Definition. Mushroom Science X
(Part 1):957–975.
Chevalier G, Sourzat P. 2012. Soils and techniques for culti-
vating Tuber melanosporum and Tuber aestivum in
Europe. In: Zambonelli A, Bonito GM, eds. Edible ecto-
mycorrhizal mushrooms. Springer Berlin and Heidelberg,
Germany: Springer. p. 163–189.
Corander J, Marttinen P, Sirén J, Tang J. 2008. Enhanced
Bayesian modelling in BAPS software for learning genetic
structures of populations. BMC Bioinformatics 9:539,
doi:10.1186/1471-2105-9-539
Corander J, Waldmann P, Sillanpää MJ. 2003. Bayesian ana-
lysis of genetic differentiation between populations.
Genetics 163:367–374.
Douhan GW, Vincenot L, Gryta H, Selosse MA. 2011.
Population genetics of ectomycorrhizal fungi: from current
knowledge to emerging directions. Fungal Biology
115:569–597, doi:10.1016/j.funbio.2011.03.005
Dupanloup I, Schneider S, Excoffier L. 2002. A simulated
annealing approach to define the genetic structure of
populations. Molecular Ecology 11:2571–2581, doi:10.1046/
j.1365-294X.2002.01650.x
Excoffier L, Lischer HE. 2010. Arlequin suite ver 3.5: a new
series of programs to perform population genetics analyses
under Linux and Windows. Molecular Ecology Resources
10:564–567, doi:10.1111/j.1755-0998.2010.02847.x
Felsenstein J. 1989. PHYLIP 3.2 manual. Berkeley, California:
University of California Herbarium.
Gandeboeuf D, Dupré C, Chevalier G, Roeckel-Drevet P,
Nicolas P. 1997. Grouping and identification of Tuber
species using RAPD markers. Canadian Journal of
Botany 75:36–45, doi:10.1139/b97-005
García-Cunchillos I, Sánchez S, Barriuso JJ, Pérez-Collazos E.
2014. Population genetics of the westernmost distribution
of the glaciations-surviving black truffle Tuber
melanosporum. Mycorrhiza 24:89–100, doi:10.1007/
s00572-013-0540-9
Giraud T, Refrégier G, Le Gac M, de Vienne DM, Hood ME.
2008. Speciation in fungi. Fungal Genetics and Biology
45:791–802, doi:10.1016/j.fgb.2008.02.001
Gryndler M, Hršelová H, Soukupová L, Streiblová E, Valda S,
Borovička J, Gryndlerová H, GažoJ,MikoM.2011. Detection
of summer truffle (Tuber aestivum Vittad.) in ectomycorrhizae
and in soil using specific primers. FEMS Microbiology Letters
318:84–91, doi:10.1111/j.1574-6968.2011.02243.x
Hughes KW, Petersen RH, Lickey EB. 2009. Using hetero-
zygosity to estimate a percentage DNA sequence similarity
for environmental species’delimitation across basidiomy-
cete fungi. New Phytologist 182:795–798, doi:10.1111/
j.1469-8137.2009.02802.x
Jamali S. 2017. First report of identification and molecular
characterization of Tuber aestivum in Iran. Agroforestry
Systems 91:335–343, doi:10.1007/s10457-016-9932-0
Librado P, Rozas J. 2009.DnaSPv5:asoftwareforcomprehen-
sive analysis of DNA polymorphism data. Bioinformatics
25:1451–1452, doi: 10.1093/bioinformatics/btp187
Martin F, Kohler A, Murat C, Balestrini R, Coutinho PM,
Jaillon O, Montanini B, Morin Emmanuelle, Noel B,
Percudani R, Porcel B, Rubini A, Amicucci A, Amselem
A, Anthouard V, Arcioni S, Artiguenave F, Aury JM,
Ballario P, Bolchi A, Brenna A, Brun A, Buée M,
Cantarel B, Chevalier G, Couloux A, Da Silva C,
Denoeud F, Duplessis S, Ghignone S, Hilselberger B, Iotti
M, Mello A, Miranda M, Pacioni G, Quesneville H,
Riccioni C, Ruotolo R, Splivallo R, Tisserant E, Stocchi
V, Zambonelli A, Zampieri E, Viscomi AR, Henrissat B,
Paolocci F, Bonfante P, Ottonello S, Wincker P. 2010.
Périgord black truffle genome uncovers evolutionary
12 RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
origins and mechanisms of symbiosis. Nature
464:1033–1038, doi:10.1038/nature08867
Martin F, Murat C, Paolocci F, Rubini A, Riccioni C,
Belfiori B, Arcioni S. 2012. Molecular method for the
identification of mating type genes of truffles species.
European Patent Application EP2426215; 2010.
McCormack JE, Bowen BS, Smith TB. 2008. Integrating
paleoecology and genetics of bird populations in two sky
island archipelagos. BMC Biology 6:28, doi:10.1186/1741-
7007-6-28
Médail F, Diadema K. 2009. Glacial refugia influence
plant diversity patterns in the Mediterranean Basin.
Journal of Biogeography 36:1333–1345, doi:10.1111/
j.1365-2699.2008.02051.x
Mello A, Cantisani A, Vizzini A, Bonfante P. 2002. Genetic
variability of Tuber uncinatum and its relatedness to other
black truffles. Environmental Microbiology 4:584–594,
doi:10.1046/j.1462-2920.2002.00343.x
Merényi Z, Varga T, Geml J, Orczán ÁK, Chevalier G,
Bratek Z. 2014. Phylogeny and phylogeography of the
Tuber brumale aggr. Mycorrhiza 24:101–113, doi:10.1007/
s00572-014-0566-7
Molinier V, Murat C, Baltensweiler A, Büntgen U, Martin F,
Meier B, Moser B, Sproll L, Stobbe U, Tegel W, Egli S,
Peter M. 2016a. Fine-scale genetic structure of natural
Tuber aestivum sites in southern Germany. Mycorrhiza
26:895–907, doi:10.1007/s00572-016-0719-y
Molinier V, Murat C, Frochot H, Wipf D, Splivallo R. 2015.
Fine-scale spatial genetic structure analysis of the black
truffle Tuber aestivum and its link to aroma variability.
Environmental Microbiology 17:3039–3050, doi:10.1111/
1462-2920.12910
Molinier V, Murat C, Peter M, Gollotte A, De la Varga E,
Meier B, Egli S, Belfiori B, Paolocci F, Wipf D. 2016b. SSR-
based identification of genetic groups within European
populations of Tuber aestivum Vittad. Mycorrhiza
26:99–110, doi:10.1007/s00572-015-0649-0.
Molinier V, Van Tuinen D, Chevalier G, Gollotte A, Wipf D,
Redecker D. 2013. A multigene phylogeny demonstrates
that Tuber aestivum and Tuber uncinatum are conspecific.
Organisms Diversity & Evolution 13:503–512, doi:10.1007/
s13127-013-0146-2
Morcillo M, Sanchez M, Vilanova X. 2015. Truffle farming
today—a comprehensive world guide. Barcelona, Spain:
Micologia Forestal & Aplicada. p. 270–272.
MuratC,DíezJ,LuisP,Delaruelle C, Dupré C, Chevalier G,
Bonfante P, Martin F. 2004. Polymorphism at the ribosomal
DNA ITS and its relation to postglacial re-colonization routes
of the Perigord truffle Tuber melanosporum. New Phytologist
164:401–411, doi:10.1111/j.1469-8137.2004.01189.x
Murat C, Riccioni C, Belfiori B, Cichocki N, Labbé J,
Morin E, Tisserant E, Paolocci F, Rubini A, Martin F.
2011. Distribution and localization of microsatellites in
the Perigord black truffle genome and identification of
new molecular markers. Fungal Genetics and Biology
48:592–601, doi:10.1016/j.fgb.2010.10.007
Naydenov K, Senneville S, Beaulieu J, Tremblay F,
Bousquet J. 2007. Glacial vicariance in Eurasia: mitochon-
drial DNA evidence from Scots pine for a complex heritage
involving genetically distinct refugia at mid-northern
latitudes and in Asia Minor. BMC Evolutionary Biology
7:233, doi:10.1186/1471-2148-7-233
Pampanini R, Martino G. 2006.L’importanza del tartufo
nell’economia della montagna. Micologia Italiana 3:3–17
Paolocci F, Rubini A, Granetti B, Arcioni S. 1999. Rapid
molecular approach for a reliable identification of Tuber
spp. ectomycorrhizae. FEMS Microbiology Ecology
28:23–30, doi:10.1111/j.1574-6941.1999.tb00557.x
Paolocci F, Rubini A, Riccioni C, Topini F, Arcioni S. 2004.
Tuber aestivum and Tuber uncinatum. two morphotypes
or two species? Fems Microbiology Letters 235:109–115,
doi:10.1111/j.1574-6968.2004.tb09574.x
Payen T, Murat C, Bonito G. 2014. Truffle phylogenomics:
new insights into truffle evolution and truffle life cycle.
Advances in Botanical Research 70:211–234.
Peakall R, Smouse PE. 2012. GenAlEx 6.5: genetic analysis in
Excel. Population genetic software for teaching and
research—an update. Bioinformatics 28:2537–2539.
Peay KG, Kennedy PG, Bruns TD. 2008. Fungal community
ecology: a hybrid beast with a molecular master.
BioScience 58:799–810, doi:10.1641/B580907
Riccioni C, Rubini A, Belfiori B, Gregori G, Paolocci F. 2016.
Tuber magnatum: the special one. What makes it so dif-
ferent from the other Tuber spp.? In: Zambonelli A,
Iotti M, Murat C, eds. True truffle (Tuber spp.) in the
world. Soil ecology, systematic and biochemistry.
Switzerland: Springer International Publishing. p. 87–103
Riccioni C, Belfiori B, Rubini A, Passeri V, Arcioni S,
Paolocci F. 2008.Tuber melanosporum outcrosses: analysis
of the genetic diversity within and among its natural
populations under this new scenario. New Phytologist
180:466–478, doi:10.1111/j.1469-8137.2008.02560.x
Rousset F. 1997.Genetic differentiation and estimation of
gene flow from F-statistics under isolation by distance.
Genetics 145:1219–1228.
Rubini A, Belfiori B, Riccioni C, Tisserant E, Arcioni S,
Martin F, Paolocci F. 2011. Isolation and characteriza-
tion of MAT genes in the symbiotic ascomycete Tuber
melanosporum. New Phytologist 189:710–722,
doi:10.1111/j.1469-8137.2010.03492.x
Rubini A, Paolocci F, Granetti B, Arcioni S. 1998. Single step
molecular characterization of morphologically similar
black truffle species. FEMS Microbiology Letters
164:7–12, doi:10.1111/j.1574-6968.1998.tb13060.x
Rubini A, Paolocci F, Riccioni C, Vendramin GG, Arcioni S.
2005. Genetic and phylogeographic structures of the symbio-
tic fungus Tuber magnatum. Applied and Environmental
Microbiology 71:6584–6589, doi:10.1128/AEM.71.11.6584-
6589.2005
Rubini A, Topini F, Riccioni C, Paolocci F, Arcioni S. 2004.
Isolation and characterization of polymorphic microsatel-
lite loci in white truffle (Tuber magnatum). Molecular
Ecology Notes 4:116–118, doi:10.1111/j.1471-8286.2004.
00587.x
Slatkin M. 1987. Gene flow and the geographic structure of
natural populations. Science 236:787–792, doi:10.1126/
science.3576198
Smith ME, Douhan GW, Rizzo DM. 2007. Intra-specific and
intra-sporocarp ITS variation of ectomycorrhizal fungi as
assessed by rDNA sequencing of sporocarps and pooled
MYCOLOGIA 13
ectomycorrhizal roots from a Quercus woodland.
Mycorrhiza 18:15–22, doi:10.1007/s00572-007-0148-z
Smith SE, Read DJ. 2010. Mycorrhizal symbiosis. London:
Academic Press. 800 p.
Splivallo R, Rittersma R, Valdez N, Chevalier G, Molinier V,
Wipf D, Karlovsky P. 2012a. Is climate change altering the
geographic distribution of truffles?. Frontiers in Ecology
and the Environment 10:461–462, doi:10.1890/12.WB.020
Splivallo R, Valdez N, Kirchhoff N, Ona MC, Schmidt JP,
Feussner I, Karlovsky P. 2012b. Intraspecific genotypic
variability determines concentrations of key truffle vola-
tiles. New Phytologist 194:823–835, doi:10.1111/j.1469-
8137.2012.04077.x
Stobbe U, Egli S, Tegel W, Peter, M, Sproll L, Büntgen U.
2013. Potential and limitations of Burgundy truffle
cultivation. Applied Microbiology and Biotechnology
97:5215–5224, doi:10.1007/s00253-013-4956-0
Tabouret P. 2011. Exclusif! Description d’un site français pro-
ducteur de truffe blanche d’Italie. Le Trufficulteur 78:18.
Tajima F. 1989. Statistical method for testing the neutral
mutation hypothesis by DNA polymorphism. Genetics
123:585–595.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013.
MEGA6: Molecular Evolutionary Genetics Analysis
Version 6.0. Molecular Biology and Evolution
30:2725–2729, doi:10.1093/molbev/mst197
Taşkın H, Büyükalaca S, Hansen K, O’Donnell K. 2012.
Multilocus phylogenetic analysis of true morels
(Morchella) reveals high levels of endemics in Turkey
relative to other regions of Europe. Mycologia 104:446–
461, doi:10.3852/11-180
Türkoğlu A, Castellano MA, Trappe JM, Yaratanakul
Güngör M. 2015. Turkish truffles I: 18 new records for
Turkey. Turkish Journal of Botany 39:359–376,
doi:10.3906/bot-1406-42
Vellinga EC, Wolfe BE, Pringle A. 2009. Global patterns of
ectomycorrhizal introductions. New Phytologist
181:960–973, doi:10.1111/j.1469-8137.2008.02728.x
Wedén C, Danell E, Camacho FJ, Backlund A. 2004. The
population of the hypogeous fungus Tuber aestivum syn.
T. uncinatum on the island of Gotland. Mycorrhiza
14:19–23, doi:10.1007/s00572-003-0271-4
Wedén C, Danell E, Tibell L. 2005. Species recognition in the
truffle genus Tuber—the synonims Tuber aestivum and
Tuber uncinatum. Environmental Microbiology
7:1535–1546, doi:10.1111/j.1462-2920.2005.00837.x
Zambonelli A, Iotti M, Piattoni F. 2012. Chinese Tuber aes-
tivum sensu lato in Europe. The Open Mycology Journal
6:22–26.
Zhang JP, Liu PG, Chen J. 2012.Tuber sinoaestivum sp. nov.,
an edible truffle from southwestern China. Mycotaxon
122:73–82.
14 RICCIONI ET AL.: PHYLOGEOGRAPHIC PATTERN IN TUBER AESTIVUM
