No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Population genetics of the westernmost distribution of the glaciations-surviving black truffle Tuber melanosporum
Abstract
The black truffle (Tuber melanosporum Vittad.) is an important natural resource due to its relevance as a delicacy in gastronomy. Different aspects of this hypogeous fungus species have been studied, including population genetics of French and Italian distribution ranges. Although those studies include some Spanish populations, this is the first time that the genetic diversity and genetic structure of the wide geographical range of the natural Spanish populations have been analysed. To achieve this goal, 23 natural populations were sampled across the Spanish geographical distribution. ISSR technique demonstrated its reliability and capability to detect high levels of polymorphism in the species. Studied populations showed high levels of genetic diversity (h
N
= 0.393, h
S
= 0.678, Hs = 0.418), indicating a non threatened genetic conservation status. These high levels may be a consequence of the wide distribution range of the species, of its spore dispersion by animals, and by its evolutionary history. AMOVA analysis showed a high degree of genetic structure among populations (47.89 %) and other partitions as geographical ranges. Bayesian genetic structure analyses differentiated two main Spanish groups separated by the Iberian Mountain System, and showed the genetic uniqueness of some populations. Our results suggest the survival of some of these populations during the last glaciation, the Spanish southern distribution range perhaps surviving as had occurred in France and Italy, but it is also likely that specific northern areas may have acted as a refugia for the later dispersion to other calcareous areas in the Iberian Peninsula and probably France.
Supplementary resource (1)
Data
April 2014
... Following this study, population genetics studies have been carried out in several truffle species, utilizing different types of gene makers. In brief, the genetic markers used include random amplified polymorphic DNA (RAPD) [8], simple sequence repeat (SSR, microsatellite) [9][10][11][12][13][14][15][16], amplified fragment length polymorphism (AFLP) [10], inter-simple sequence repeat (ISSR) [17], mating type loci [11][12][13][14][15] and single-nucleotide polymorphisms (SNPs) from different DNA fragments, such as internal transcribed spacers (ITS) [18][19][20][21], beta-tubulin gene [20] and SNPs at the genomic scale [22]. The major concerns of these studies are (1) species delimitation of several species complex, e.g., T. indicum [3,20,21], T. aestivum [23], T. borchii [24] and T. brumale [25], (2) life cycle of true truffles [9,10,[13][14][15], and (3) genetic structure of several truffle species [16][17][18][19][25][26][27]. ...
... In brief, the genetic markers used include random amplified polymorphic DNA (RAPD) [8], simple sequence repeat (SSR, microsatellite) [9][10][11][12][13][14][15][16], amplified fragment length polymorphism (AFLP) [10], inter-simple sequence repeat (ISSR) [17], mating type loci [11][12][13][14][15] and single-nucleotide polymorphisms (SNPs) from different DNA fragments, such as internal transcribed spacers (ITS) [18][19][20][21], beta-tubulin gene [20] and SNPs at the genomic scale [22]. The major concerns of these studies are (1) species delimitation of several species complex, e.g., T. indicum [3,20,21], T. aestivum [23], T. borchii [24] and T. brumale [25], (2) life cycle of true truffles [9,10,[13][14][15], and (3) genetic structure of several truffle species [16][17][18][19][25][26][27]. ...
... In T. melanosporum, analyses of Italian and French samples, using either SNPs from ITS or SSR genotyping, identified higher levels of genetic diversity in southernmost populations, supporting the Italian Peninsula as its refugium during the last glaciation [10,19]. For samples from Spain, high levels of genetic diversity could be detected in southern, central, and northern populations using ISSR genotyping, suggesting that the Iberian Peninsula may represent another refugium of this species [17]. Unfortunately, different genetic markers were used, and so those results cannot be combined to reveal the genetic structure of T. melanosporum, covering its full distribution range. ...
True truffle (Tuber spp.) is one group of ascomycetes with great economic importance. During the last 30 years, numerous fine-scale population genetics studies were conducted on different truffle species, aiming to answer several key questions regarding their life cycles; these questions are important for their cultivation. It is now evident that truffles are heterothallic, but with a prevalent haploid lifestyle. Strains forming ectomycorrhizas and germinating ascospores act as maternal and paternal partners respectively. At the same time, a number of large-scale studies were carried out, highlighting the influences of the last glaciation and river isolations on the genetic structure of truffles. A retreat to southern refugia during glaciation, and a northward expansion post glaciation, were revealed in all studied European truffles. The Mediterranean Sea, acting as a barrier, has led to the existence of several refugia in different peninsulas for a single species. Similarly, large rivers in southwestern China act as physical barriers to gene flow for truffles in this region. Further studies can pay special attention to population genetics of species with a wide distribution range, such as T. himalayense, and the correlation between truffle genetic structure and the community composition of truffle-associated bacteria.
... In Tuber melanosporum, the genetic diversity and phylogeography have been investigated with different molecular markers such as randomly amplified polymorphism DNA, microsatellites, SNPs in the internal transcribed spacer (ITS) of ribosomal DNA and inter-simple sequence repeats (ISSR; Garc ıa-Cunchillos et al. 2014). These studies pointed to an important effect of the last glaciations (from 120 000 to 11 000 years ago; Van Andel & Tzedakis 1996) on the truffle population structure. ...
... For example, two putative post-glacial recolonization routes were hypothesized using 10 SNPs in ITSs . Through the use of microsatellites and ISSR fingerprinting, it has also been suggested that glacial refuges exist in Italy and Spain Garc ıa-Cunchillos et al. 2014). ...
... The positive Tajima's D values can result not only from balancing selection, but also from population structure and moderately intense bottlenecks (i.e. a reduction in the size of the population; Biswas & Akey 2006). In addition to significant population structure effects Garc ıa-Cunchillos et al. 2014), a population bottleneck due to the last glaciation has also been proposed for T. melanosporum . The six T. melanosporum geographical accessions were harvested from different populations, as demonstrated by the phylogeographical analysis (see below). ...
Accès restreint aux membres de l'Université de Lorraine jusqu'au 2015-12-31
... Electronic supplementary material The online version of this article (doi:10.1007/s00572-015-0649-0) contains supplementary material, which is available to authorized users. ectomycorrhizal species such as Laccaria amethystina (Hortal et al. 2012), Rhizopogon roseolus (Okuda et al. 2013), Tuber brumale (Merényi et al. 2014), Tuber magnatum (Rubini et al. 2005) and Tuber melanosporum (Murat et al. 2004;Riccioni et al. 2008;García-Cunchillos et al. 2014). On the other hand, the absence of any genetic differentiation among populations was shown in Pisolithus microcarpus (Hitchcock et al. 2011) and Suillus spraguei (Rivera et al. 2014). ...
... Among Tuber spp. these analyses have mainly been carried out in T. melanosporum, T. magnatum and T. brumale (Murat et al. 2004(Murat et al. , 2011(Murat et al. , 2013Rubini et al. 2004;Riccioni et al. 2008;Merényi et al. 2014;García-Cunchillos et al. 2014). For example, a strong genetic structure with significant fixation indices was observed among T. melanosporum populations at a European scale (F st =0.20 and F st =0.177; Murat et al. 2004;Riccioni et al. 2008). ...
... The absence of a recent bottleneck suggested that T. aestivum is not yet an endangered species, not even in Mediterranean regions since genetic group 3 is limited to southern populations (see above). A similar result was found for T. melanosporum in the Iberian Peninsula indicating a nonthreatened genetic conservation status (García-Cunchillos et al. 2014). However, even under conservative scenarios, future climate changes are likely to cause further increases in mean temperature (about 2-4°C globally) with significant drought in some regions (Seager et al. 2007) as well as increases in frequency and severity of extreme droughts, extreme heat levels and heat waves (IPCC 2013;Sterl et al. 2008). ...
Tuber species are ectomycorrhizal ascomycetes establishing relationships with different host trees and forming hypogeous fruiting bodies known as truffles. Among Tuber species, Tuber aestivum Vittad. has a wide distributional range being found naturally all over Europe. Here, we performed large-scale population genetic analyses in T. aestivum to (i) investigate its genetic diversity at the European scale, (ii) characterize its genetic structure and test for the presence of ecotypes and (iii) shed light into its demographic history. To reach these goals, 230 ascocarps from different populations were genotyped using 15 polymorphic simple sequence repeat markers. We identified 181 multilocus genotypes and four genetic groups which did not show a clear geographical separation; although, one of them was present exclusively in Southeast France, Italy and Spain. Fixation index values between pairs of genetic groups were generally high and ranged from 0.29 to 0.45. A significant deficit of heterozygosity indicated a population expansion instead of a recent population bottleneck, suggesting that T. aestivum is not endangered in Europe, not even in Mediterranean regions. Our study based on a large-scale population genetic analysis suggests that genetically distinct populations and likely ecotypes within T. aestivum are present. In turn, this study paves the way to future investigations aimed at addressing the biological and/or ecological factors that have concurred in shaping the population genetic structure of this species. Present results should also have implications for the truffle market since defining genetic markers are now possible at least for some specific T. aestivum genetic groups.
... In Tuber melanosporum, the genetic diversity and phylogeography have been investigated with different molecular markers such as randomly amplified polymorphism DNA, microsatellites, SNPs in the internal transcribed spacer (ITS) of ribosomal DNA and inter-simple sequence repeats (ISSR; Bertault et al. 1998;Murat et al. 2004;Riccioni et al. 2008;Garc ıa-Cunchillos et al. 2014). These studies pointed to an important effect of the last glaciations (from 120 000 to 11 000 years ago; Van Andel & Tzedakis 1996) on the truffle population structure. ...
... For example, two putative post-glacial recolonization routes were hypothesized using 10 SNPs in ITSs (Murat et al. 2004). Through the use of microsatellites and ISSR fingerprinting, it has also been suggested that glacial refuges exist in Italy and Spain (Riccioni et al. 2008;Garc ıa-Cunchillos et al. 2014). ...
... The positive Tajima's D values can result not only from balancing selection, but also from population structure and moderately intense bottlenecks (i.e. a reduction in the size of the population; Biswas & Akey 2006). In addition to significant population structure effects (Murat et al. 2004;Riccioni et al. 2008;Garc ıa-Cunchillos et al. 2014), a population bottleneck due to the last glaciation has also been proposed for T. melanosporum (Bertault et al. 1998). The six T. melanosporum geographical accessions were harvested from different populations, as demonstrated by the phylogeographical analysis (see below). ...
The Périgord black truffle (Tuber melanosporum Vittad.), considered a gastronomic delicacy worldwide, is an ectomycorrhizal filamentous fungus that is ecologically important in Mediterranean French, Italian, and Spanish woodlands. In this study, we developed a novel resource of single nucleotide polymorphisms (SNPs) for T. melanosporum using Illumina high-throughput re-sequencing. The genome from six T. melanosporum geographic accessions was sequenced to a depth of ~20×. These geographic accessions were selected from different populations within the northern and southern regions of the geographical species distribution. Approximately 80% of the reads for each of the six re-sequenced geographic accessions mapped against the reference T. melanosporum genome assembly, estimating the core genome size of this organism to be ~110 Mbp. A total of 442,326 SNPs corresponding to 3,540 SNPs/Mbps were identified as being included in all seven genomes. The SNPs occurred more frequently in repeated sequences (85%), although 4,501 SNPs were also identified in the coding regions of 2,587 genes. Using the ratio of non-synonymous mutations per non-synonymous site (pN) to synonymous mutations per synonymous site (pS) and Tajima's D index scanning the whole genome, we were able to identify genomic regions and genes potentially subjected to positive or purifying selection. The SNPs identified represent a valuable resource for future population genetics and genomics studies. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... This value is a reflection of allele diversity and frequency among the varieties. The high values of PIC parameter exhibited a high polymorphism and variety; therefore, high PIC markers are useful to separate genotypes with close relationship [8]. ...
... Since truffles grow underground, they spread spores in the way different from other mushrooms. Truffles attract 'fungivores' by their unique aroma, to that enjoy snacking on them [8], include mammals like mice, squirrels and rabbit, pigs and bears. The most of the flesh is digested, when consumed; the spores pass without scathed by animal's body. ...
Tuber species are edible fungi and plant-symbiotic microorganisms that form a beneficial relationship with the roots of certain trees and plants (ectomycorrhizae). After interaction with a plant host, tuber species produced hypogeous fruit bodies of great economic value known as forest truffles. There are different species of truffles, but based on species and place of origin varied their quality and market price. Truffle identification is based on morphological analysis maybe fail to distinguish them due to highly susceptible to environmental conditions. But using molecular markers to identify truffles can be more accurate, less expensive and reliable monitoring. In this context, twelve inter-simple sequence repeats (ISSR) primers were chosen for amplifying the genetic materials of black and brown truffles. In this study, a total of 57 polymorphic bands were amplified (an average of 5.18 bands). The Polymorphism Information Content (PIC) value and gene diversity (H) was with an average 0.37 and 0.50, respectively. During the ISSR screening good amplification products were obtained from primers based on GA, (AG) G, (AG)T, and GAC repeats. The population analysis result revealed that there are three main clusters A, B and C. Four strains Ardabil, Khalkhal, Zanjan and Urmia were identified to be in the group A cluster. The strains of at second and third groups were black and brown truffles respectively. The results indicated that truffles had two separate speciation events (DK = 2). According to DK = 2, the samples of Ardabil, Khalkhal, Zanjan and Urmia grouped in the same group and rest of truffles in other groups.
... Wild populations of T. melanosporum were sampled from thirteen regions in the northern Pre-Pyrenees mountains, in the Iberian System and Sub-betic mountain as described by García-Cunchillos, Sánchez, Barriuso, and Pérez-Collazos (2014). All truffle samples were lyophilized, ground sieved to obtained particle size lower than 0.5 mm and kept at −80°C until further use. ...
... Other geographical impediments such as the sub-betic mountain system in the South divided the populations furthermore into other genetic sub-groups (SE1 and SE2). In the North, the sub-division was not so drastic (NE1 and NE2) but the discontinuity of the limestone soils between locations could act as isolation barrier for their distribution (García-Cunchillos et al., 2014). Moreover, the Spanish forests naturally producing black truffle can be divided in three clusters by clear climatic gradients: warm and dry (cluster 1); cold and wet (cluster 2) and cold and dry (cluster 3) (Garcia-Barreda, Sánchez, Marco, & Serrano-Notivoli, 2019). ...
Truffles, besides the appreciated aromatic compounds, contain other molecules with interesting bioactive properties. A screening of fungal sterols and β-glucans within different truffle species and locations was carried out. These compounds were extracted with pressurized liquids (PLE) generating enriched fractions. Extraction efficiency was studied with a full-factorial experimental design (Response surface methodology, RSM), using water and ethanol as extraction solvents. Polysaccharides from truffle powder (TP) and the optimal PLE extract (EP) obtained were precipitated and analysed by NMR and GC–MS. THP-1 cell cultures were utilized to test immunomodulatory properties. With the optimal PLE conditions (16.7 MPa, 180 °C, 30 min) 64 and 22.5% yields were obtained respect, with water and ethanol, generating fractions containing respect, 9.1% β-glucan and 4.5% ergosterol. NMR analyses detected (1 → 3)-β-glucan structures in truffle. The EP induced a reduction of 40% IL-1β and 60% IL-6 pro-inflammatory cytokines secretion suggesting potential immunomodulatory activity.
... For example, within the black truffle clade, T. melanosporum survived in refugia located in the Iberian and Italian peninsulas, as inferred by ITS, ISSR and SSR markers [3,5,44]. Conversely the surviving pattern of T. brumale aggr., resulting from the phylogeny of ITS, LSU and PKC loci, was more complex: Within the T. brumale clade A, populations of haplotype I survived the last glaciation in Western Europe, those of haploytpe II in Eastern Europe whereas those of clade B in the Carpathian basin and Balkan region [6]. This latter clade was later proposed as representing a cryptic species, T. cryptobrumale [45]. ...
... Diversity 2020, 12,44 ...
The ectomycorrhizal fungus Tuber magnatum produces the white truffle appreciated worldwide for its unique aroma. With respect to other Tuber spp. of economic interest, T. magnatum presents a narrower geographical range. This species has, in fact, long been considered endemic to Italy. However, over the last few decades several reports have documented the presence of white truffles in different Mediterranean countries and in particular in various areas of southeast Europe. In this study, samples from several Pannonian and Balkan countries such as Hungary, Serbia, Romania, Bulgaria and Greece have been collected and genotyped with microsatellite markers and the data merged with those available for Italian populations. Our objectives were to test whether Italian and southeast European populations are differentiated and to evaluate the genetic diversity of T. magnatum all over its distributional range. We show the genetic structure of T. magnatum populations with the differentiation of four main groups: northern Italy, central-northern Italy, southern Italy and the Balkan/Pannonian region. The present study allowed us to refine the evolutionary history of T. magnatum and track the possible post-glacial expansion route of this species. The assessment of T. magnatum's genetic structure is not only of scientific relevance, but it is also important for the conservation and market traceability of this prestigious fungus.
... For instance, European populations of Tuber aestivum revealed four genetic clusters inconsistent with geography; the absence of SGS over up to 2400 km could be explained by human dispersal via inoculated plant material (Molinier et al. 2016). Regional-scale substructure of T. melanosporum populations may also be related to active human inoculation and trade of plants (García-Cunchillos et al. 2014). Interestingly, Taschen et al. (2016) revealed no loss of natural regional SGS in inoculated plantations. ...
... As the distribution of T. melanosporum haplotypes is consistent with that of oak trees, this species may have followed the two postglacial re-colonisation routes of oaks, through the Rhone valley and through southern France to the Atlantic coast (Murat et al. 2004;Bertault et al. 2001;Payen et al. 2015). Host-associated glacial refugia for T. melanosporum were also found in Iberian Peninsula (García-Cunchillos et al. 2014). ...
... Figura 1. Mapa de pH de los suelos de la España peninsular (Rodríguez et al., 2009). El área de distribución natural de Tuber melanosporum (García-Cunchillos et al., 2014), aparece sombreada, y la localización de la zona de plantación marcada en rojo. Figure 1. ...
... Soil pH map of peninsular Spain (Rodríguez et al., 2009). The area of natural distribution for Tuber melanosporum (García-Cunchillos et al., 2014) is shaded, and the location of the plantation is marked in red. ...
... For instance, European populations of Tuber aestivum revealed four genetic clusters inconsistent with geography; the absence of SGS over up to 2400 km could be explained by human dispersal via inoculated plant material (Molinier et al. 2016). Regional-scale substructure of T. melanosporum populations may also be related to active human inoculation and trade of plants (García-Cunchillos et al. 2014). Interestingly, Taschen et al. (2016) revealed no loss of natural regional SGS in inoculated plantations. ...
... As the distribution of T. melanosporum haplotypes is consistent with that of oak trees, this species may have followed the two postglacial re-colonisation routes of oaks, through the Rhone valley and through southern France to the Atlantic coast (Murat et al. 2004;Bertault et al. 2001;Payen et al. 2015). Host-associated glacial refugia for T. melanosporum were also found in Iberian Peninsula (García-Cunchillos et al. 2014). ...
Studies on ectomycorrhizal fungi (EcMF) populations, their genetic diversity, dynamics and the ecological drivers of their structure have long struggled with characterisation of fungal infraspecific variability, until development of molecular tools. Population dynamics have enhanced understanding of basic biological features of EcMF at population level, such as mating systems, local dispersal patterns and relationships between soil mycelial colonisation and fructification. Investigating population structure and dynamics also allowed the transfer of ecological colonisation strategies derived from plant ecology to EcMF species, although local environmental drivers and individual variations can shape populations beyond such ecological strategies. Characterisation of genetic diversity of EcMF populations from contrasted habitats has then been used to explore the role of environmental drivers, for instance soil parameters, in shaping the genetic structure and adaptive responses of populations. Detection of genetic structure of populations also proved relevant to explore host specialisation versus generalism in the ectomycorrhizal symbiosis. Extended up to the breadth of species’ range, investigation through the prism of landscape genetics and demographic reconstruction helped deciphering ancient and modern environmental drivers of population diversity, indicating the value of EcMF for testing biogeographic hypotheses. Present development of high-throughput sequencing methods is now allowing to explore the evolutionary mechanisms and traits beyond EcMF population history and response to environmental variation.
... For instance, European populations of Tuber aestivum revealed four genetic clusters inconsistent with geography; the absence of SGS over up to 2400 km could be explained by human dispersal via inoculated plant material (Molinier et al. 2016). Regional-scale substructure of T. melanosporum populations may also be related to active human inoculation and trade of plants (García-Cunchillos et al. 2014). Interestingly, Taschen et al. (2016) revealed no loss of natural regional SGS in inoculated plantations. ...
... As the distribution of T. melanosporum haplotypes is consistent with that of oak trees, this species may have followed the two postglacial re-colonisation routes of oaks, through the Rhone valley and through southern France to the Atlantic coast (Murat et al. 2004;Bertault et al. 2001;Payen et al. 2015). Host-associated glacial refugia for T. melanosporum were also found in Iberian Peninsula (García-Cunchillos et al. 2014). ...
... Tuber melanosporum biogeography was investigated with different molecular markers such as randomly amplified polymorphism DNA (RAPD), microsatellites, ITS sequencing and inter-simple sequence repeats (ISSR) (Bertault et al. 1998;Murat et al. 2004;Riccioni et al. 2008;García-Cunchillos et al. 2014). These studies highlighted an important genetic structure of T. melanosporum in natural populations mainly resulting from the effects of the last glaciation. ...
... Two putative postglacial recolonisation routes have been hypothesised for France: one through the west and another through the east (Murat et al. 2004). The existence of glacial refuges was suggested in Italy and Spain (Murat et al. 2004;Riccioni et al. 2008;García-Cunchillos et al. 2014). Recently, Payen and colleagues (2015) estimated that the 60,507 SNPs present in the genomic regions free of selection pressure accumulated between 100,000 and 150,000 years ago. ...
The European species Tuber melanosporum and the Asian species T. indicum are phylogenetically and morphologically very close. T. indicum is formed by a complex of cryptic species or ecotypes spread from India to Japan, while T. melanosporum is a well-defined species localised in South of Europe. T. melanosporum has been harvested and consumed in Europe for several centuries and is cultivated in orchards since the XIXe century. On the contrary, T. indicum, naturally occurring in primary or secondary Asian forests, is rarely consumed by local populations. Since the 1990s, T. indicum has been traded from China in the European market to compensate the decline of T. melanosporum production. After a peak of production of about 1000 t in 2000, production and exportation of T. indicum have considerably decreased in the last years. This decline could be explained by the destruction of its natural habitat due to the systematic digging of forest soils. T. indicum ectomycorrhizas were detected in Italy in 2008 and in 2011 in the USA. Moreover, T. indicum is able to fruit in the USA. For the moment we do not know if both species are able to inbreed nor if T. indicum can successfully compete with T. melanosporum and replace it in European truffle grounds.
... Most fungi that are pathogenic to humans reside naturally in the environment with occasional transmission to humans or other animals. Many fungi, especially the dimorphic fungi, display a limited and distinct phylogeographic distribution, suggesting that growth and persistence in the environment is linked to specific biogeographic and ecological factors [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. For example, Coccidioides immitis is localized primarily to the San Joaquin Valley of California, while Coccidioides posadasii has a wider biogeographical distribution including southwestern United States, southern California, Mexico, and South America [2]. ...
... Like Blastomyces spp., microsatellite and/or sequence typing of other fungi has revealed that most fungi exhibit genetically divergent populations localized to specific geographic regions [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Two notable exceptions are Aspergillus fumigatus and Penicillium chrysogenum which show world-wide dispersal with no correlation between genotype and geographic location [59][60][61]. ...
Blastomyces dermatitidis and Blastomyces gilchristii are dimorphic fungal pathogens that cause serious pulmonary and systemic infections in humans. Although their natural habitat is in the environment, little is known about their specific ecologic niche(s). Here, we analyzed 25 microsatellite loci from 169 strains collected from various regions throughout their known endemic range in North America, representing the largest and most geographically diverse collection of isolates studied to date. Genetic analysis of multilocus microsatellite data divided the strains into four populations of B. dermatitidis and four populations of B. gilchristii. B. dermatitidis isolates were recovered from areas throughout North America, while the B. gilchristii strains were restricted to Canada and some northern US states. Furthermore, the populations of both species were associated with major freshwater drainage basins. The four B. dermatitidis populations were partitioned among (1) the Nelson River drainage basin, (2) the St. Lawrence River and northeast Atlantic Ocean Seaboard drainage basins, (3) the Mississippi River System drainage basin, and (4) the Gulf of Mexico Seaboard and southeast Atlantic Ocean Seaboard drainage basins. A similar partitioning of the B. gilchristii populations was observed among the more northerly drainage basins only. These associations suggest that the ecologic niche where the sexual reproduction, growth, and dispersal of B. dermatitidis and B. gilchristii occur is intimately linked to freshwater systems. For most populations, sexual reproduction was rare enough to produce significant linkage disequilibrium among loci but frequent enough that mating-type idiomorphic ratios were not skewed from 1:1. Furthermore, the evolutionary divergence of B. dermatitidis and B. gilchristii was estimated at 1.9 MYA during the Pleistocene epoch. We suggest that repeated glaciations during the Pleistocene period and resulting biotic refugia may have provided the impetus for speciation as theorized for other species associated with temperate freshwater systems.
... However, these truffles were harvested on the same day, with the same dog and same orchard. Therefore, the differences might be due to environmental or genetical factors [37,38]. ...
Nowadays, the truffle aroma attribute is not included as a quality parameter in the current recommendation that explains the truffle quality (UNECE standard 53 FFV3) and establishes the truffle commercial categories. However, the aroma is the main reason why truffles are worldwide appreciated. Indeed, more than 30 aromatic molecules compose it, and this is the reason why the human evaluation and identification of these odorants, without previous training, is quite subjective. Analytical techniques such as gas chromatography techniques, however, can establish an aromatic profile and detect potential aromatic markers. In this study, 16 tasting experts were trained to make more objective the truffle aroma evaluation and odorants identification. For this, a comparison between solid-phase microextraction gas chromatography coupled with mass spectrometry (SPME-GC-MS) and sensory expert evaluation was carried out in six sessions during different harvesting times in the black truffle season (December, January, and February). Both techniques were able to separate truffles depending on the harvesting time. Also, a list of volatile organic compounds related to the aromatic attributes was reported. This information will help to provide a more objective T. melanosporum truffle sensory evaluation.
... Wild populations in these areas would likely be highly vulnerable to climate change. Conservation strategies should take into account the existing genetic diversity (García-Cunchillos et al., 2014). ...
Black truffle is a highly appreciated edible fungus that grows wild in southwestern Europe, although its cultivation has recently spread to other continents. In Spain the widespread exploitation of wild truffles began only after 1950, whereas plantations play a relevant role in truffle production from the late 1990s. However, most plantations continue to apply empiric practices not taking into account local environmental conditions. The identification of environmental factors driving black truffle distribution and yield could help to optimise cultivation practices, but no agro-climatic zoning is currently available for this fungus in Spain. This study char-acterises the climate of Spanish forests naturally producing black truffle, defines an agro-climatic zoning for the fungus and examines the climatic patterns across its spatial distribution. The examined forests presented climatic ranges coherent with the available experts' surveys, except for an extended low end in annual precipitation. The clustering identified three agro-climatic zones, with dry environments tending to be dominant. The principal components analysis indicated that the examined forests tended to cluster along water availability and temperature gradients. Only in one of the zones mean precipitations during the vegetative period were similar to those characterising optimum years for black truffle fruiting, thus suggesting that plantations could benefit from practices increasing soil water content. Similarly, the results suggested that in two of the zones plantations could benefit from practices increasing soil temperature in winter and early spring. The study provides a basis for large-scale planning of truffle cultivation and identification of research priorities in Spain.
... 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. , 2005 and T. melanosporum (Bertault et al. 1998;Murat et al. 2004Murat et al. , 2011Riccioni et al. 2008;García-Cunchillos et al. 2014). Geographically structured populations and phylogeographic signals were identified in both species. ...
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.
... Spatial autocorrelation analysis from the mammal-dispersed truffle Tuber magnatum determined that populations separated by greater than 450 km are genetically more distant than would be expected by chance (Rubini, Paolocci, Riccioni, Vendramin, & Arcioni, 2005). A similar analysis of another mammaldispersed truffle, T. melanosporum, identified subpopulations clustering together that were separated by more than 250 km, which is thought to be the result of spore dispersal by large mammals such as wild deer and boars, which can move between 10 to 200 km per day (García-Cunchillos, Sánchez, José Barriuso, & Pérez-Collazos, 2014). However, in Guyanagaster, allopatric speciation appears to occur at these scales (Koch et al., 2017). ...
Understanding how species accomplish dispersal of their propagules can shed light on how they are adapted for their ecosystem. Guyanagaster necrorhizus is a sequestrate fungus, meaning its dispersal propagules, or spores, are entirely enclosed within a fruiting body, termed a sporocarp. This fungus is most closely related to Armillaria and its allies. While Armillaria species form mushrooms and have forcibly discharged spores, G. necrorhizus spores have lost this ability, and by necessity, must be passively dispersed. However, G. necrorhizus does not possess characteristics of other sequestrate fungi with known dispersal mechanisms. Repeated observations of termites feeding on G. necrorhizus sporocarps, and spores subsequently adhering to their exoskeletons, led to the hypothesis that termites disperse G. necrorhizus spores. To test this hypothesis, we used microsatellite markers and population genetics analyses to understand patterns of clonality and population structure of G. necrorhizus. While Armillaria individuals can spread vegetatively over large areas, high genotypic diversity in G. necrorhizus populations suggests spores are the primary mode of dispersal, consistent with termite dispersal. Spatial genetic structure analyses show that G. necrorhizus sporocarps within 238 m of each other are more closely related than would be expected by chance and conservative estimates from population assignment tests suggest gene flow no longer occurs between sporocarps separated by two km. These distances are consistent with previous studies analyzing foraging distances of the termites found associated with G. necrorhizus sporocarps. Termites have rarely been recorded to specifically target fungal sporocarps, making this a potentially novel fungal‐insect interaction.
This article is protected by copyright. All rights reserved.
... Recent studies have revealed new information contributing to the assessment of the phylogenetics and phylogeography of Tuber species with respect to their origin, evolution and biogeographic history (Bonito et al., 2013;García-Cunchillos, 2014;Merényi et al., 2014;Payen et al., 2014). The genus Tuber is regarded as monophyletic with a common ancestor, which was most likely an aboveground cup fungus, although there is still much uncertainty about whether the origin of Tuber is European or Asian (Bonito et al., 2013;Jeandroz et al., 2008). ...
Tuber indicum is an ectomycorrhizal ascomycete that produces edible ascocarps. Based on a number of specimens with known exact origin, we investigate the speciation of the Tuber indicum complex in southwest China. Internal transcribed spacer (ITS) and simple sequence repeat (SSR) markers were used in the study. Phylogeography and population genetics analyses were combined to detect 31 wild populations of the T. indicum complex. Two distinct lineages, Tuber cf. indicum and Tuber cf. himalayense, were identified in the T. indicum complex that exhibited significant phylogeographic structures and genetic differentiation. The characteristics of haplotypes distributing along the river demonstrate that the diffusion and modern distribution pattern of species was influenced by river expansion. These findings are critical for the protection of the diversity of truffles in this region.
... In the hypothesis, the truffle populations in southern areas in Italy and Spanish acted as epibiotic species during glaciation and they spread northward along with their host trees as the ice receded (Bertault et al. 1998). The research results of García-Cunchillos et al. (2014) also supported this hypothesis. In this study, the Spanish populations of T. melanosporum were found to have higher genetic diversity than the Italian and French populations, with the species separated into two groups by the Iberian Mountain System (Table 1). ...
Macrofungi refers to all fungi that produce visible fruiting bodies. These fungi are evolutionarily and ecologically very divergent. Evolutionarily, they belong to two main phyla, Ascomycota and Basidiomycota, and many of them have relatives that cannot form visible fruiting bodies. Ecologically, macrofungi can be associated with dead organic matter, plants, and animals. Here we review our current understanding of population structure and biogeography of macrofungi associated with animals. Their interactions, functions, and patterns of coevolution are described and discussed. Our focus is on studies using molecular markers. Our analyses suggest that the types of fungi-animal associations play an important role in the structure of these animal-associated fungal populations.
... Understanding and conserving the natural diversity within truffle populations is important to future truffle culture. García-Cunchillos et al. (2014) observe a high level of genetic diversity between two main groups separated by the Iberian mountain system when examining natural Spanish populations of T. melanosporum. They discuss the possibility that Spanish populations of T. melanosporum acted as a refuge during glaciation periods and later migrated to southwest France during post glaciation. ...
Background
Recent climatic history has strongly impacted plant populations, but little is known about its effect on microbes. Alders, which host few and specific symbionts, have high genetic diversity in glacial refugia. Here, we tested the prediction that communities of root symbionts survived in refugia with their host populations. We expected to detect endemic symbionts and a higher species richness in refugia as compared to recolonized areas.
Methods
We sampled ectomycorrhizal (EM) root tips and the nitrogen-fixing actinomycete Frankia communities in eight sites colonized by Alnus glutinosa subsp. barbata close to the Caucasus in Georgia. Three sites were located in the Colchis, one major Eurasian climatic refugia for Arcto-Tertiary flora and alders, and five sites were located in the recolonized zone. Endemic symbionts and plant ITS variants were detected by comparing sequences to published data from Europe and another Tertiary refugium, the Hyrcanian forest. Species richness and community structure were compared between sites from refugia and recolonized areas for each symbionts.
Results
For both symbionts, most MOTUs present in Georgia had been found previously elsewhere in Europe. Three endemic Frankia strains were detected in the Colchis vs two in the recolonized zone, and the five endemic EM fungi were detected only in the recolonized zone. Frankia species richness was higher in the Colchis while the contrary was observed for EM fungi. Moreover, the genetic diversity of one alder specialist Alnicola xanthophylla was particularly high in the recolonized zone. The EM communities occurring in the Colchis and the Hyrcanian forests shared closely related endemic species.
Discussion
The Colchis did not have the highest alpha diversity and more endemic species, suggesting that our hypothesis based on alder biogeography may not apply to alder’s symbionts. Our study in the Caucasus brings new clues to understand symbioses biogeography and their survival in Tertiary and ice-age refugia, and reveals that isolated host populations could be of interest for symbiont diversity conservation.
Truffles are ectomycorrhizal filamentous fungi belonging to genus Tuber naturally found across a range of climates in the Northern Hemisphere. Some truffle species such as Périgord black truffle (T. melanosporum) and the white truffle (T. magnatum) in Europe, the black Chinese truffle (T. indicum) in Asia, and the pecan truffle (T. lyonii) in North America are particularly famous due to the organoleptic qualities of their fruiting bodies and are among the world's most precious culinary delicacies. In this chapter, we summarize the recent studies pertaining to the phylogeography and biogeographic history of the genus Tuber, genomic analysis of the black truffle Tuber melanosporum, and comparative genomics towards understanding the life cycle, ecology, and evolution of Tuber species. Such understanding is critical to the anticipated future of truffle production in light of global change.
Arlequin ver 3.0 is a software package integrating several basic and advanced methods for population genetics data analysis, like the computation of standard genetic diversity indices, the estimation of allele and haplotype frequencies, tests of departure from linkage equilibrium, departure from selective neutrality and demographic equilibrium, estimation or parameters from past population expansions, and thorough analyses of population subdivision under the AMOVA framework. Arlequin 3 introduces a completely new graphical interface written in C++, a more robust semantic analysis of input files, and two new methods: a Bayesian estimation of gametic phase from multi-locus genotypes, and an estimation of the parameters of an instantaneous spatial expansion from DNA sequence polymorphism. Arlequin can handle several data types like DNA sequences, microsatellite data, or standard multilocus genotypes. A Windows version of the software is freely available on http://cmpg.unibe.ch/software/arlequin3.
Truffle (Tuber melanosporum Vitt.) culture is an agroforestry sector in Chile of increasing interest due to the high prices that truffles fetch in the national market and the recent evidence that its commercial production is possible in Chilean climatic and soil conditions. In this study, the efficiency of three methods of DNA extraction from a mix of 5 g of soil and roots from both nursery and field plants of Quercus ilex L. mycorrhized with T. melanosporum were evaluated, and a simple and reproducible protocol was established. Detection of T. melanosporum was performed by the technique of cleaved amplified polymorphic sequence (CAPS) from amplicons generated with the primers ADL1 (5́-GTAACGATAAAGGCCATCTATAGG-3́) and ADL3 (5́-CGTTTTTCCTGAACTCTTCATCAC-3 '), where a restriction fragment of 160 bp specific for T. melanosporum was generated, which allows the discrimination of this species from the rest of the species belonging to the Tuber sp. genus. Direct detection of T. melanosporum in one step was also obtained by polymerase chain reaction (PCR) from total DNA isolated from mycorrhized roots and with the primers ITSML (5́-TGGCCATGTGTCAGATTTAGTA-3 ́) and ITSLNG (5 ́-TGATATGCTTAAGTTCAGCGGG-3́), generating a single amplicon of 440 bp. The molecular detection of T. melanosporum by the methods presented here will allow the rapid and accurate detection of mycorrhization of trees, both under nursery and field conditions. This technology will also provide more security to farmers by controlling the quality of the mycorrhized trees they will plant and also by following the mycorrhization status of established orchards.
Truffle (Tuber melanosporum Vitt.) culture is an agroforestry sector in Chile of increasing interest due to the high prices that truffles fetch in the national market and the recent evidence that its commercial production is possible in Chilean climatic and soil conditions. In this study, the efficiency of three methods of DNA extraction from a mix of 5 g of soil and roots from both nursery and field plants of Quercus ilex L. mycorrhized with T. melanosporum were evaluated, and a simple and reproducible protocol was established. Detection of T. melanosporum was performed by the technique of cleaved amplified polymorphic sequence (CAPS) from amplicons generated with the primers ADL1 (5´-GTAACGATAAAGGCCATCTATAGG-3´) and ADL3 (5´-CGTTTTTCCTGAACTCTTCATCAC-3`), where a restriction fragment of 160 bp specific for T. melanosporum was generated, which allows the discrimination of this species from the rest of the species belonging to the Tuber sp. genus. Direct detection of T. melanosporum in one step was also obtained by polymerase chain reaction (PCR) from total DNA isolated from mycorrhized roots and with the primers ITSML (5´-TGGCCATGTGTCAGATTTAGTA-3´) and ITSLNG (5´-TGATATGCTTAAGTTCAGCGGG-3´), generating a single amplicon of 440 bp. The molecular detection of T. melanosporum by the methods presented here will allow the rapid and accurate detection of mycorrhization of trees, both under nursery and field conditions. This technology will also provide more security to farmers by controlling the quality of the mycorrhized trees they will plant and also by following the mycorrhization status of established orchards.
The genetic structure of ectomycorrhizal ( ECM ) fungal populations results from both vegetative and sexual propagation. In this study, we have analysed the spatial genetic structure of Tuber melanosporum populations, a heterothallic ascomycete that produces edible fruit bodies.
Ectomycorrhizas from oaks and hazels from two orchards were mapped and genotyped using simple sequence repeat markers and the mating type locus. The distribution of the two T. melanosporum mating types was also monitored in the soil. In one orchard, the genetic profiles of the ascocarps were compared with those of the underlying mycorrhizas.
A pronounced spatial genetic structure was found. The maximum genet sizes were 2.35 and 4.70 m in the two orchards, with most manifesting a size < 1 m. Few genets persisted throughout two seasons. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types.
Our findings suggest that competition occurs between genets and provide basic information on T. melanosporum propagation patterns that are relevant for the management of productive truffle orchards.
Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae - the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.
Of the ten species of European truffle (fungi of the genus Tuber, phylum Ascomycota), some have economic value because of their organoleptic properties (taste and perfume), in particular the black truffle (Tuber melanosporum Vitt.) and the summer and burgundy truffles¹,². The black truffle is mainly found in Spain, France and Italy (Fig. 1a), and it shows variation in several traits, including in its famous organoleptic properties, across this geographical range. Here we show that this variation probably results from environmental, rather than genetic, influences.
To select candidate populations of wild species to be given priority for conservation, genetic criteria gained from the study of molecular markers may be useful. Traditionally, diversity measures such as expected heterozygosity or percentage of polymorphic loci have been considered. For conservation we propose instead that priority should be given to measures of allelic richness. To standardize the results of allelic richness across populations, we used the technique of rarefaction. This technique allows evaluation of the expected number of different alleles among equal-sized samples drawn from several different populations. We also show how the contribution of each population to total diversity can be partitioned into two components. The first is related to the level of diversity of the population and the second to its divergence from the other populations. For conservation purposes the uniqueness of a population - in terms of its allelic composition - may be at least as important as its diversity level. These new descriptors are illustrated by means of isozyme and chloroplast DNA data obtained for an endangered tree species, the argan tree of Morocco (Argania spinosa (L.) Skeels). With these analyses the conservation value of the argan tree populations, especially those of two isolates present in the north of the country, can be better appreciated. The methods proposed to identify priority areas for conservation of the genetic resources of the argan tree are compared to those sometimes advocated in the case of reserve design, where one of the goals is to maximize species richness.
The geographic distribution throughout Europe of each of 32 chloroplast DNA variants belonging to eight white oak species sampled from 2613 populations is presented. Clear-cut geographic patterns were revealed by the survey. These distributions, together with the available palynological information, were used to infer colonisation routes out of the glacial period refugia. In western Europe in particular, movements out of the Iberian and the Italian Peninsulas can be clearly identified. Separate refugia are also present in eastern Balkans, whereas further west in this peninsula similarities with Italy were evident. Movements resulting in the exchange of haplotypes between refugia both during the present interglacial and probably also during earlier glacial cycles were therefore inferred. The consequences of these past exchanges is that phylogenetically divergent haplotypes have sometimes followed very similar colonisation routes, limiting somewhat the phylogeographic structure. Cases of geographic disjunction in the present-day distribution of haplotypes are also apparent and could have been induced by the existence of rapid climatic changes at the end of the glacial period (specifically the Younger Dryas cold period), which resulted in range restriction following an early warm period during which oak first expanded from its primary refugia. This cold phase was followed by a new period of expansion at the outset of the Holocene, involving in some cases 'secondary' refugia. It is expected that these short climate oscillations would have led to a partial reshuffling of haplotype distribution. Early association between haplotypes and oak species are also suggested by the data, although extensive introgression among species has ultimately largely blurred the pattern. This implies that colonisation routes may have been initially constrained by the ecological characteristics of the species hosting each chloroplast variant. We suggest for instance that two oak species distributed in the north of the Iberian Peninsula (Quercus petraea and Q. pubescens) are recent post-glacial immigrants there. When considered together, conclusions on the location of glacial period refugia and the colonisation routes derived from molecular information and fossil pollen data appear to be both largely compatible and complementary. # 2002 Elsevier Science B.V. All rights reserved.
Eleven enzymes from 139 specimens of thirteen Italian populations of Tuber magnatum were analysed with multilocus horizonfal starch gel electrophoresis. The research was carried out to obtain insight into the genetic variability of this species across its geographic range. All the gene-enzyme systems scored appear to be fixed in homozygosity in accordance with what is hitherto known in the genus Tuber; nine seemed monomorphic, whereas two, that is MDH-1 and ME-2, showed three alleles each. These results indicated a self-reproductive system and the low genetic variability is in agreement with the restricted endemism of the white truffle. The distribution of the electrophoretic types is discussed as a basis for further molecular applications.
Tuber melanosporum is a truffle native to Europe and is cultivated in countries such as Australia for the gastronomic market, where production
yields are often lower than expected. We assessed the genetic diversity of T. melanosporum with six microsatellite loci to assess the effect of genetic drift on truffle yield in Australia. Genetic diversity as assessed
on 210 ascocarps revealed a higher allelic diversity compared to previous studies from Europe, suggesting a possible genetic
expansion and/or multiple and diverse source populations for inoculum. The results also suggest that the single sequence repeat
diversity of locus ME2 is adaptive and that, for example, the probability of replication errors is increased for this locus.
Loss of genetic diversity in Australian populations is therefore not a likely factor in limiting ascocarp production. A survey
of nursery seedlings and trees inoculated with T. melanosporum revealed that <70% of seedlings and host trees were colonized with T. melanosporum and that some trees had been contaminated by Tuber brumale, presumably during the inoculation process. Mating type (MAT1-1-1 and MAT1-2-1) analyses on seedling and four- to ten-year-old
host trees found that 100% of seedlings but only approximately half of host trees had both mating types present. Furthermore,
MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in established trees, suggesting a competitive advantage for
MAT1-1-1 strains. This study clearly shows that there are more factors involved in ascocarp production than just the presence
of both mating types on host trees.
Quantification of extraradical mycelium of black truffle (Tuber melanosporum) has been carried out in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites, within the natural distribution area of the black truffles in Navarre (northern Spain). Specific primers and a Taqman® probe were designed to perform real-time PCR with DNA extracted from soil samples. Amplification of T. melanosporum DNA was obtained from 131 out of the 160 soil samples. The detection limit of the technique was 1.48 μg mycelium/g of soil. The extraradical mycelium biomass detected in the soil from the natural truffle ground was significantly greater (up to ten times higher) than the mycelium biomass detected in any of the orchards. Soil from productive, nonirrigated orchards in the Tierra Estella site contained significantly more extraradical mycelium than the rest of orchards irrigated, productive of T. brumale, or nonproductive. The comparison of soil mycelium biomass in nonirrigated evergreen oak orchards in both sites showed significantly more mycelium biomass in the Tierra Estella site. This study is the first attempt to quantify extraradical mycelium of T. melanosporum in the soil using Taqman® probes. The obtained quantitative results are of special interest to evaluate the fungal response to cultural treatments and to monitor the dynamics of the extraradical mycelium of T. melanosporum in the soil.
In this study, sequence-related amplification polymorphism (SRAP) and inter-simple sequence repeat (ISSR) were analyzed for
accessing the genetic diversity of 37 Monascus isolates and 14 control strains. According to the dendrogram produced by SRAP data, all the tested strains were grouped into
four clusters at a 78% similarity level. Comparatively, 51 tested strains were divided into four major groups at a similarity
level of 74% based on the dendrogram generated via ISSR marker analysis. Based on the two sets of dendrograms, Monascus aurantiacus, M. purpureus, M. serorubescens, M. anka, and M. ruber were clustered in the same clade; M. albidus, M. fuliginosus, and M. barkeri were clustered with M. pilosus in a second clade; and M. lunisporas and M. argentinensis occurred together in a third cluster distinct from the other Monascus species. The cluster result produced by SRAP data shared great similarity with that by ISSR data with minor differences in
the subgroups, which is basically in agreement with morphological observations. In general, SRAP and ISSR are more simple,
rapid, and efficient, which may provide alternative molecular approaches to studying genetic diversity, classification, and
identification of Monascus strains.
KeywordsCluster analysis–Molecular marker–Polymorphism
Many molecular marker techniques are available today. PCR-based approaches are in demand because of their simplicity and requirement
for only small quantities of sample DNA. Nonanchored inter simple sequence repeats (ISSRs) are arbitrary multiloci markers
produced by PCR amplification with a microsatellite primer. They are advantageous because no prior genomic information is
required for their use. We found the technique stable across a wide range of PCR parameters. Polymorphisms were abundant among
7 dicot species tested with 2 tri-nucleotide and 2 tetra-nucleotide primers. Thus, nonanchored ISSR markers are a good choice
for DNA fingerprinting.
A study based on AFLP markers was conducted to characterise the present population genetic structure of Carpinus betulus in Europe and to formulate guidelines for the use of this species in plantations on a local scale in Flanders. High within-population diversity and little (but significant) genetic differentiation were detected at both Flemish and European scales. However, there was a pattern of isolation by distance only at the European scale. Within-population gene diversity, a new rarefaction-based measure of number of genotypes (band richness) and percentage of polymorphic loci are lower north of major mountain chains, suggesting that the mountain ranges formed a second bottleneck for the hornbeam during postglacial recolonisation. In Flanders, despite lower gene diversity, there were more polymorphic loci than in other European populations, a pattern that might have been caused by the mixing of material through planting, e.g. in hedges. In view of these findings, it is advised to create a single Flemish seed zone and to use preferentially reproductive material from this seed zone for new plantations in Flanders.
Aroma variability in truffles has been attributed to maturation (Tuber borchii), linked to environmental factors (Tuber magnatum), but the involvement of genetic factors has been ignored. We investigated aroma variability in Tuber uncinatum, a species with wide distribution. Our aim was to assess aroma variability at different spatial scales (i.e. trees, countries) and to quantify how aroma was affected by genotype, fruiting body maturity, and geographical origin.
A volatile fingerprinting method was used to analyze the aroma of 223 T. uncinatum fruiting bodies from seven European countries. Maturity was estimated from spore melanization. Genotypic fingerprinting was performed by amplified fragment length polymorphism (AFLP).
Discriminant analysis revealed that, regardless of the geographical origin of the truffles, most of the aroma variability was caused by eight-carbon-containing volatiles (C8-VOCs). In an orchard of T. uncinatum, truffles producing different concentrations of C8-VOCs clustered around distinct host trees. This clustering was not associated with maturity, but was associated with fungal genotype.
These results indicate that the variation in C8-VOCs in truffles is most likely under genetic control. They exemplify that understanding the factors behind aroma variability requires a holistic approach. Furthermore, they also raise new questions regarding the ecological role of 1-octen-3-ol in truffles.
Aspergillus terreus causes invasive aspergillosis (IA) in immunocompromised individuals and can be the leading cause of IA in certain medical centers. We examined a large isolate collection (n = 117) for the presence of cryptic A. terreus species and employed a genome scanning method, Inter-Simple Sequence Repeat (ISSR) PCR to determine A. terreus population structure.
Comparative sequence analyses of the calmodulin locus revealed the presence of the recently recognized species A. alabamensis (n = 4) in this collection. Maximum parsimony, Neighbor joining, and Bayesian clustering of the ISSR data from the 113 sequence-confirmed A. terreus isolates demonstrated that one clade was composed exclusively of isolates from Europe and another clade was enriched for isolates from the US.
This study provides evidence of a population structure linked to geographical origin in A. terreus.
Tuber melanosporum is an ectomycorrhizal ascomycete producing edible ascocarps. The prevalent view is that this species strictly selfs, since genetic analyses have never detected heterozygotic profiles in its putatively diploid/dikaryotic gleba. The selfing model has also forged the experimental approaches to assess the population genetic variability. Here, the hypothesis that T. melanosporum outcrosses was tested. To this end, SSR (simple sequence repeats) and ITS (internal transcribed spacer) markers were employed to fingerprint asci and the surrounding gleba within single ascocarps. The distribution of genetic variability was also investigated at different geographical levels using single (SSR and ITS) and multilocus (AFLP, amplified fragment length polymorphism) markers. It is shown that T. melanosporum outcrosses since asci display additional alleles besides those present in the surrounding, uniparental, gleba. Furthermore, SSR and AFLP data reveal a high rate of intrapopulation diversity within samples from the same ground and root apparatus and the highest rate of genetic variability within the southernmost populations of the distributional range. These data call for a profound re-examination of T. melanosporum mating system, life cycle and strategies for managing man-made plantations. They also strongly support the idea that the last glaciation restricted the species distribution to the Italian and Spanish peninsulas.
Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.
Leaf blight and purple seed, caused by the fungal pathogen Cercospora kikuchii (Matsumoto & Tomoyasu) M. W. Gardner are very important diseases of soybean (Glycine max L. Merr.) in Argentina. The aims of this work were: (a) to confirm and to assess the genetic variability among C. kikuchii isolates collected from different soybean growing areas in Santa Fe province using inter simple sequence repeats (ISSR) markers and sequence information from the internal transcribed spacer (ITS) region of rDNA and (b) to analyze the cercosporin production of the regional C. kikuchi isolates in order to assess whether there was any relationship between the molecular profiles and the toxin production. Isolates from different regions in Santa Fe province were studied. The sequence of the ITS regions showed high similarity (99-100%) to the GenBank sequences of C. kikuchii BRCK179 (accession number AY633838). The ISSR markers clustered all the isolates into many groups and cercosporin content was highly variable among isolates. No relationship was observed between ITS region, ISSR groups and origin or cercosporin content. The high degree of genetic variability and cercosporin production among isolates compared in this study characterizes a diverse population of C. kikuchii in the region.
The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis-'the symbiosis toolbox'-evolved along different ways in ascomycetes and basidiomycetes.
A new measure of the extent of population subdivision as inferred from allele frequencies at microsatellite loci is proposed and tested with computer simulations. This measure, called R(ST), is analogous to Wright's F(ST) in representing the proportion of variation between populations. It differs in taking explicit account of the mutation process at microsatellite loci, for which a generalized stepwise mutation model appears appropriate. Simulations of subdivided populations were carried out to test the performance of R(ST) and F(ST). It was found that, under the generalized stepwise mutation model, R(ST) provides relatively unbiased estimates of migration rates and times of population divergence while F(ST) tends to show too much population similarity, particularly when migration rates are low or divergence times are long [corrected].
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from http://www.stats.ox.ac.uk/~pritch/home.html.
Molecular markers derived from PCR amplification of genomic DNA are an important part of the toolkit of evolutionary geneticists. RAPDs, AFLPs, and ISSR polymorphisms allow analysis of species for which prior DNA sequence information is lacking, but dominance makes it impossible to apply standard techniques to calculate F-statistics. We describe a Bayesian method that allows direct estimates of Fst from dominant markers. In contrast to existing alternatives, we do not assume prior knowledge of the degree of within-population inbreeding. In particular, we do not assume that genotypes within populations are in Hardy-Winberg proportions. Our estimate of Fst incorporates uncertainty about the magnitude of within-population inbreeding. Simulations show that samples from even a relatively small number of loci and populations produce reliable estimates of Fst. Moreover, some information about the degree of within population inbreeding (Fis) is available from data sets with a large number of loci and populations. We illustrate the method with reanalysis of RAPD data from 14 populations of a North American orchid, Platanthera leucophaea.
Studies were made from 1965— 1970 within the Kampinos National Park forests near Warsaw and surrounding area, for the purpose of ascertaining the possibility of limiting the amount of damage caused by wild boar, Sus scrofa. Linnaeus, 1758 on commercial land. The purpose of managing the population was to bring about a greater num ber of older individuals. The population was regularly supplied with supplementary food, records were kept of the degree to which use was made of the food, feeding times, along with damage done by the ani mals to crops. A total of 507 individuals was marked, obtaining 2640 captures and 6180 separate observations. The supply of food brought about an increase in population numbers from about 30 to 250 animals (within an area of approx. 2.5 thousand ha of wooded land), followed by a decrease of about 50— 70 individuals. The population consisted of settled and migrating (ephemeral) individuals. Some of the individuals were found to migrate over a considerable distances (over 250 km). The number of females in the population exceeded the number of males, particularly in the older age classes. Increase in population numbers resulted in the animals feeding on supplementary food during the day time hours also. Consumption of supplementary food decreased damages in spite of increasing the population numbers. The wild boar caused 75% of all damage from May to August. It is suggested that supplemen tary food should be provided for wild boar during the period when maximum damage is done since, by appropriately controlling age structure, population density can be maintained on a level of 30—40 animals per 1000 ha of woodland.
Fritillaria imperialis grows at high altitudes of the alpine Zagros region of Iran and has a high medicinal and ornamental value and importance. In this study AFLP, ISSR and RAPD markers were used to detect intra- and inter-population genetic diversity of F. imperialis. 140 genotypes, belonging to seven populations, were studied using six random primers, six ISSR primers and five AFLP primer combinations, to investigate the genetic structure between populations. Percentage of polymorphic loci, estimated using Nei's genetic diversity index and Shannon's information index revealed low or moderate levels of genetic variations within each F. imperialis population. Similarly, the mean coefficient of gene differentiation between populations (Gst) for RAPD, ISSR and AFLP were 0.415, 0.47 and 0.63 respectively, indicating that 58.5%, 52.3% and 36.1% of genetic diversity resided within the population. AMOVA indicated that variation among populations was higher than those within F. imperialis populations. Gene flow was 0.70, 0.54 and 0.29 from RAPD, ISSR and AFLP marker analysis respectively indicating that there was limited gene flow among the populations. Results showed that AFLP is the best marker for investigating the genetic diversity and assessing differentiation between F. imperialis populations belonging to different geographical regions of Zagros, Iran. These results indicate that the conservation of existing natural population of F. imperialis is necessary by the development of different management practices such as: maintaining effective population sizes, construction of an in situ conservation area, ex situ conservation of seed and domestication of this wild plant species.
The colonization dynamics of the black truffle in an artificial field were assessed through analyses of microsatellite and RAPD markers. The truffle field was composed of three tree species and mycelial inoculum of three different origins, and was monitored for the first three years of truffle production. We found very low levels of genetic diversity. Isolation by distance was detected only at the between-tree level. This could be interpreted as local colonization around each tree facilitated by the presence of the tree root system. At the larger spatial scale of the European range, the absence of isolation by distance corroborates the hypothesis of an impact of glaciation on genetic variation, followed by rapid postglaciation demographic expansion. In addition, genetic variation of harvested truffles was explained by neither inoculation origin, nor tree species. Our study questions the real impact of man-made inoculation of tree root systems with fungal mycelia.Keywords: biogeography, microsatellite, population genetics, RAPD, truffle, Tuber melanosporum
Aim Various data sets and methods of analysis were combined to produce the first comprehensive molecular phylogeny of the genus Tuber and to analyse its biogeography .
Location Europe, North Africa, China, Asia, North America.
Methods Phylogenetic relationships among Tuber species were reconstructed based on a data set of internal‐transcribed spacer (ITS) sequences and various phylogenetic inference methods, specifically maximum parsimony, Bayesian analysis and neighbour joining. Tajima’s relative rate test showed that Tuber 18S rRNA, 5.8S rRNA, 5.8S‐ITS2 rRNA and β‐tubulin sequences evolved in a clock‐like manner. These genes, combined or not, were employed for molecular clock estimates after construction of linearized trees using mega 3.1. We reconstructed ancestral areas in the Northern Hemisphere by means of a dispersal–vicariance analysis ( diva 1.1) based on current distribution patterns of the genus Tuber determined from the literature.
Results The resulting molecular phylogeny divided the genus Tuber into five distinct clades, in agreement with our previously published studies. The Puberulum , Melanosporum and Rufum groups were diversified in terms of species and geographical distribution. In contrast, the Aestivum and Excavatum groups were less diversified and were located only in Europe or North Africa. Using a global molecular clock analysis, we estimated the divergence times for the origin of the genus and for the origin of several groups. diva inferred nine dispersal events and suggested that the ancestor of Tuber was originally present in Europe or was widespread in Eurasia. Equally optimal distributions were obtained for several nodes, suggesting different possible biogeographical patterns.
Main conclusions Our analyses identified several discrepancies with the classical taxonomy of the genus, and we propose a new phylogenetic classification. According to molecular clocks, the radiation of the genus Tuber could have started between 271 and 140 Ma. Used in combination with the results obtained from time divergence estimates, this allows us to propose two equally probable scenarios of intra‐ and inter‐continental diversification of the genus according to the geographic distribution of the most recent common ancestor in Europe or Eurasia. The biogeographical patterns imply intra‐continental dispersal events between Europe and Asia and inter‐continental dispersal events between North America and Europe or Asia, which are compatible with land connections during the Tertiary.
The Quaternary cold periods in Europe are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in both animals and plants. The phylogeographies of 10 taxa, including mammals (Ursus arctos, Sorex spp., Crocidura suaveolens, Arvicola spp.), amphibians (Triturus spp.), arthropods (Chorthippus parallelus), and plants (Abies alba, Picea abies, Fagus sylvatica, Quercus spp.), were analysed to elucidate general trends across Europe. Only a small degree of congruence was found amongst the phylogeographies of the 10 taxa, but the likely postglacial colonization routes exhibit some similarities. A Brooks parsimony analysis produced an unrooted area phylogram, showing that: (i) the northern regions were colonized generally from the Iberic and Balkanic refugia; and (ii) the Italian lineages were often isolated due to the presence of the Alpine barrier. The comparison of colonization routes highlighted four main suture-zones where lineages from the different refugia meet. Some of the intraspecific genetic distances among lineages indicated a prequaternary divergence that cannot be connected to any particular cold period, but are probably related mainly to the date of arrival of each taxon in the European continent. As a consequence, molecular genetics so far appears to be of limited use in dating Quaternary events.
Bayesian approaches have been widely applied to partitioning diversity within and among levels in many different multi-level modeling contexts. In spite of the structural similarities between these Bayesian models and hierarchical approaches to partitioning diversity in population genetics, population geneticists have not explored the use of hierarchical Bayesian models to provide estimates of Wright's F-statistics. In this paper I describe and illustrate the application of a simple multilocus, two-allele model sufficient for partitioning diversity within and among populations. Extenions of the model incorporate both fixed-effect and random-effect models of population sampling at multiple hierarchical levels with multiple alleles per locus. The Bayesian approach developed here is closely related to previously developed methods for likelihood analysis of the same problem. I illustrate the utility of the Bayesian approach with a reanalysis of previously published allozyme data from Argania spinosa.
5 These authors contributed equally to this work; 6 Present address: Institut für Botanik – Terrestrische Ökologie, Universität Leipzig – Johannisallee 21–23 D-04103 Leipzig, Germany Summary • Glaciations and postglacial migrations are major factors responsible for the present patterns of genetic variation we see in natural populations in Europe. For ectomyc-orrhizal fungi, escape from refugia can only follow range expansion by their specific hosts. • To infer phylogeographic relationships within Tuber melanosporum , sequences of internal transcribed spacers (ITS) and the 5.8S coding region of the ribosomal DNA repeat were obtained for 188 individuals sampled over the entire distribution of this species in France, and in north-western Italy and north-eastern Spain. • Ten distinct ITS haplotypes were distinguished, mapped and treated using F -and N ST -statistics and nested clade (NCA) analyses. They showed a significant genetic differentiation between regional populations. NCA revealed a geographical associ-ation of ITS haplotypes, an old fragmentation into two major groups of populations, which likely colonized regions on different sides of the French Central Massif. • This re-colonization pattern is reminiscent of the one observed for host trees of the Perigord truffle, such as oaks and hazelnut trees. This suggests that host postglacial expansion was one of the major factors that shaped the mycobiont population structure.
Krascheninnikovia ceratoides (L.) Gueldenst. (Chenopodiaceae) is an irano-turanian steppe plant with a striking geographical vicariance between the western Mediterranean and the central Asian regions. Its westernmost populations are located in the north-east part of the Iberian Peninsula, in the narrow middle Ebro and Alfambra valleys, where they are threatened and have been catalogued as 'vulnerable'. Genetic diversity and structure was studied in 150 individuals from five populations of these two valleys using inter-simple sequence repeats (ISSR). A total of 121 informative bands allowed the identification of 150 genotypes. Nei's genetic diversity found in all natural populations (h = 0.448) was higher than that of other geographical restricted species. Analysis of molecular variance and spatial correlations analyses showed a strong genetic differentiation among populations (35.88%) and among valleys (22.33%). The unweighted pair-group method of arithmetical averages and Neighbour-joining clusterings further demonstrated a substructure of two population cores within the Ebro valley. Our genetic data suggest that the high levels of genetic diversity and the strong genetic structure found among the northern Iberian populations and ranges of K. ceratoides might result from the summed effects of its tetraploidy, its outbreeding nature, and its success in colonizing newly altered areas. However, the ISSR data might also reflect the existence of an ancient wider distribution range of the plant in the Iberian Peninsula coupled with a more recent history of habitat fragmentation. Different management conservation guidelines are recommended for this plant after our genetic analysis: a microreserve of approximately 2 km2 is proposed for the highly threatened population of Osera (Ebro valley), whereas seed collection strategies and occasional population reinforcements are suggested for the remaining populations.
Migration of populations to and from glacial refugia is responsible for various cases of speciation and subspeciation in Europe. The pattern of distribution and the degree of diversification between lineages originated by isolation in different glacial refugia usually depends on ecological traits, especially to their dispersal ability. Zerynthia polyxena is a philopatric species, scattered in small populations and rarely colonizing mountain areas. These characteristics probably caused repeated isolation during the Quaternary and may have favoured diversification. Actually two studies, based on both morphological and genetic data, suggest the existence of two highly distinct lineages in Europe having in Northern Italy their contact zone. In this study, I applied geometric morphometrics to male genitalia and demonstrated that (i) two morphotypes exist in Europe approximately facing on the two sides of the Po River; (ii) the two lineages probably survived glaciations in Italy and the Balkan Peninsula, respectively; then the Balkans lineage expanded to Central and Eastern Europe; (iii) no hybrid populations seem to exist in the contact area and, in one locality at least, the two lineages live in sympatry without any evidence of intermediates. These results suggest that (i) two sister species of Zerynthia exist in Europe. Accordingly, Papilio cassandra Geyer, 1828 is reinstated, as Zerynthia cassandra stat. rev., as the species to which the Zerynthia from Italy South of the Po River belong. Male genitalia differences with Zerynthia polyxena are described.
The aims of this paper were to study the genetic diversity within and among 6 Tuber taxa, the phylogenetic relationships and the spatial distribution of truffles from the Basilicata region (southern Italy).
Molecular analyses, using the internal transcribed spacer region, microsatellites, minisatellites and random amplified polymorphic
loci revealed the highest expected intra-specific heterozygosity (He=0.30) in T. aestivum, followed by that of T. mesentericum (He=0.22). T. borchii (He=0.17), T. magnatum (He=0.16), T. aestivum fo. uncinatum (He=0.12) and T. brumale fo. moschatum (He=0.09) showed a lower average heterozygosity. The whole set of markers were demonstrated to be useful in clearly differentiating
Tuber taxa, separating them in two distinct groups and in five sub-clusters, each corresponding to one single taxon. The first
cluster (T. borchii, T. magnatum and T.␣brumale fo. moschatum) was more differentiated than the second one (T. aestivum, T.␣aestivum fo. uncinatum and T. mesentericum). T. brumale fo. moschatum was the most differentiated Tuber taxon. It was more related to the white truffles (the closely related T.␣borchii and T. magnatum) than to the remaining related black truffles T. aestivum and T.␣mesentericum. The amount of Tuber species richness was higher over the western side of Basilicata, in cold Lauretum and in Castanetum phytoclimatic Pavari’s zones. A spatial genetic structure was detected for T. aestivum, as shown by the significant correlation between geographic and genetic distances (r
s
=0.32; P<0.0001). Integration of molecular and geographic diversity patterns can allow the selection of sites for Tuber and Tuber-related biodiversity conservation.
KeywordsBasilicata region-Biodiversity-Ecogeographic distribution-Molecular markers-
Tuber taxa
It has been hypothesized that refugia of thermophilous tree species were located in Northern Italy very close to the Alps, though, this hypothesis has yet to be tested thoroughly. In contrast to Central and Southern Italy with its relative wealth of data, only a few fragmentary records are currently available from Northern Italy for the last Glacial (Würm, Weichselian). Our new study site Lago della Costa lies adjacent to the catchment of the megafans of the Alpine forelands and the braided rivers of the Northeastern Po Plain that have so far inhibited the recovery of continuous Glacial and Late-Glacial records. We analyze pollen, plant macrofossils, charcoal and ostracods to reconstruct the vegetation, fire and lake history for the period 33,000–16,000 cal. BP. We compare our data with Glacial records from Southern Europe to discuss similarities and dissimilarities between these potential refugial areas. A comparison with independent paleoclimatic proxies allows to assess potential linkages between environmental and climatic variability. New macrofossil and pollen data at Lago della Costa unambiguously document the local persistence of boreal tree taxa such as Larix decidua and Betula tree species around the study site during the last Glacial. The regular occurrence of pollen of temperate trees in the organic lake sediments (fine-detritus calcareous gyttja) suggests that temperate taxa such as Corylus avellana, Quercus deciduous, Tilia, Ulmus, Fraxinus excelsior, Carpinus, Abies alba and Fagus sylvatica, most likely survived the Last Glacial Maximum (LGM) at favorable sites in the Euganean Hills. The percentage values of temperate trees are comparable with those from Southern Europe (e.g. Monticchio in Southern Italy). We conclude that the Euganean Hills were one of the northernmost refugial areas of temperate taxa in Europe. However, the relative and absolute abundances of pollen of temperate trees are highly variable. Pollen-inferred declines of temperate tree communities (e.g. Quercetum mixtum) and low ostracod-inferred water levels at Lago della Costa correspond to the cold Heinrich events H-2 (LGM; 23,000–19,000 cal. BP) and H-3 (around 28,000 cal. BP), as recorded in the marine sediments of the North Atlantic. Similar patterns of significant temperate tree population collapses during cold Heinrich events are recorded at southern Mediterranean sites (e.g. Monticchio and the Alboran Sea). These findings suggest close linkages between Northern Atlantic and South-Central European climates during the past Glacial.
Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.
Genetic clustering algorithms require a certain amount of data to produce informative results. In the common situation that individuals are sampled at several locations, we show how sample group information can be used to achieve better results when the amount of data is limited. New models are developed for the structure program, both for the cases of admixture and no admixture. These models work by modifying the prior distribution for each individual's population assignment. The new prior distributions allow the proportion of individuals assigned to a particular cluster to vary by location. The models are tested on simulated data, and illustrated using microsatellite data from the CEPH Human Genome Diversity Panel. We demonstrate that the new models allow structure to be detected at lower levels of divergence, or with less data, than the original structure models or principal components methods, and that they are not biased towards detecting structure when it is not present. These models are implemented in a new version of structure which is freely available online at http://pritch.bsd.uchicago.edu/structure.html.
The level of genetic diversity and genetic structure in the Perigord black truffle (Tuber melanosporum Vittad.) has been debated for several years, mainly due to the lack of appropriate genetic markers. Microsatellites or simple sequence repeats (SSRs) are important for the genome organisation, phenotypic diversity and are one of the most popular molecular markers. In this study, we surveyed the T. melanosporum genome (1) to characterise its SSR pattern; (2) to compare it with SSR patterns found in 48 other fungal and three oomycetes genomes and (3) to identify new polymorphic SSR markers for population genetics. The T. melanosporum genome is rich in SSRs with 22,425 SSRs with mono-nucleotides being the most frequent motifs. SSRs were found in all genomic regions although they are more frequent in non-coding regions (introns and intergenic regions). Sixty out of 135 PCR-amplified mono-, di-, tri-, tetra, penta, and hexa-nucleotides were polymorphic (44%) within black truffle populations and 27 were randomly selected and analysed on 139 T. melanosporum isolates from France, Italy and Spain. The number of alleles varied from 2 to 18 and the expected heterozygosity from 0.124 to 0.815. One hundred and thirty-two different multilocus genotypes out of the 139 T. melanosporum isolates were identified and the genotypic diversity was high (0.999). Polymorphic SSRs were found in UTR regulatory regions of fruiting bodies and ectomycorrhiza regulated genes, suggesting that they may play a role in phenotypic variation. In conclusion, SSRs developed in this study were highly polymorphic and our results showed that T. melanosporum is a species with an important genetic diversity, which is in agreement with its recently uncovered heterothallic mating system.
• In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions. • In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation. • In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months. • The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.
The Tuber melanosporum genome has been analysed with the aim of identifying and characterizing the genes involved in the environmental stress response. A whole genome array (7496 genes/probe) was used to verify the fungal transcriptional profiling upon a cold temperature period (7 days at 4 °C). A total of 423 genes resulted to be differentially expressed in a significant manner (>2.5-fold; p-value<0.05) in the mycelia exposed to cold, compared to the control ones: 187 of these genes were up-regulated, while 236 were down-regulated. Sixty-six and fifty-one percent, respectively, of the up- or down-regulated transcripts had no KOG classification and were clustered as unclassified proteins, which was the most abundant category in the both up- and down-regulated genes. A gene subset, containing a range of biological functions, was chosen to validate the microarray experiment through quantitative real time PCR (qRT-PCR). The analysis confirmed the array data for 16 out of 22 of the considered genes, confirming that a cold temperature period influences the truffle global gene expression. The expressed genes, which mostly resulted to be genes for heat shock proteins (HSPs) and genes involved in cell wall and lipid metabolism, could be involved in mechanisms, which are responsible for fungal adaptation. Since truffle ascomata develop during the winter period, we hypothesize that these differentially expressed genes may help the truffle to adapt to low temperatures and/or perceive environmental signals that regulate the fructification.