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.
The growth of trees in forests and woodlands is promoted by root colonization by ectomycorrhizal fungi, which assist the trees in nutrient uptake. Johnson et al. (2012) highlighted that intraspecific fungal diversity can also influence plant diversity, as well as ecosystem function. Most ectomycorrhizal fungi belong to filamentous basidiomycetes and ascomycetes. Filamentous fungi can propagate asexually, through mycelial growth or conidia formation, and sexually, through the production of meiospores. The spatial genetic structure (SGS), that is, the distribution of genotypes in populations, results from a combination of their propagation modes and demographic processes. SGS can be assessed by the analysis of the size and persistence of genotypes (genets; Douhan et al., 2011). On a limited spatial scale, the formation of local pedigree structures resulting from limited gene dispersal is the predominant cause of SGS (Vekemans & Hardy, 2004). In this case, the similarity between neighbouring individuals is higher than that among more distant individuals, and the isolation-by-distance theory predicts the expected pattern of SGS (Vekemans & Hardy, 2004). In some fungal species, vegetative mycelial growth can reach several hundred metres, producing large genets (e.g. Armillaria mellea, Xerocomus chrysenteron; Fiore-Donno & Martin, 2001; Douhan et al., 2011; Travadon et al., 2012). Other species produce numerous small genets of a few metres, and genets can even produce a single fruiting body (e.g. Laccaria amethystina; Gherbi et al., 1999; Hortal et al., 2012). In some cases, the size of the genets correlates with ecological strategies, as was reviewed by Douhan et al. (2011). Late successional fungi tend to form large genets resulting from mycelial vegetative expansion, whereas early successional or pioneer fungi form numerous and small genets resulting from sexual reproduction. However, Fiore-Donno & Martin (2001) showed that species forming small (L. amethystina) and large (X. chrysenteron) genets could share the same site. Moreover, the same species could form both persistent genets derived from vegetative development and small genets exhibiting significant turnover each year (e.g. Hebeloma cylindrosporum and Suillus pungens; Bonello et al., 1998; Gryta et al., 2000). Investigating the SGS can therefore provide information about the ecological strategies (e.g. reproduction and dissemination mode) of a species. The understanding of and ability to promote sexual reproduction are among the most important topics concerning edible mycorrhizal fungi that have high economic values, such as Cantharellus spp., Tuber spp., Boletus spp. and Tricholoma matsutake (Yun & Hall, 2004). The balance between vegetative and sexual dissemination of Tuber spp. is poorly understood, although this information is critical for enhancing fructification in truffle orchards.
True truffles belong to the ascomycete genus Tuber, which is estimated to comprise > 200 species (Bonito et al., 2010, 2013). Some of these species produce edible fruiting bodies with distinctive olfactory bouquets. To complete their life cycle, these fungi establish symbiosis with the roots of several tree and shrub species by promoting the formation of ectomycorrhizas (ECMs). The production of truffles has dramatically decreased during the last century as a result of rural desertification, a reduction in truffle ground area and climate changes, among other causes (Büntgen et al., 2012; Olivier et al., 2012). To preserve and increase truffle production, the life cycle, reproductive mode and ecology of truffles need to be elucidated. The life cycle of Tuber spp. has not yet been reproduced in vitro (Rubini et al., 2007). However, population-genetics analyses coupled to methodological progress in the isolation and typing of single mycorrhizal root tips, asci and ascospores have clarified the basic aspects of the life cycle and reproductive biology of the two most economically important truffle species worldwide, Tuber magnatum and T. melanosporum (Rubini et al., 2005; Paolocci et al., 2006; Riccioni et al., 2008). Each truffle ECM results from host-plant colonization by a single, haploid mycelium. Evidence of recombination events coupled with the analysis of the mating type (MAT) locus, the genomic region that governs the fungal sexual cycle (Fraser & Heitman, 2003), has revealed that T. melanosporum is a heterothallic species (Riccioni et al., 2008; Martin et al., 2010; Rubini et al., 2011a,b). All known heterothallic ascomycetes have a single MAT locus with two alternative genes (MAT1-1-1 and MAT1-2-1; Metzenberg & Glass, 1990; Debuchy et al., 2010). In heterothallic fungi, same-clone mating is prevented because only haploid cells that carry different alleles at the mating type locus/loci can fuse (Murtagh et al., 2000; Kronstad, 2007; Paoletti et al., 2007; Billiard et al., 2012). Furthermore, because the gleba of the ascocarps, composed of homokaryotic tissue of uniparental origin, collected from a natural T. melanosporum site, exhibited the same multilocus profiles as the nearby ECMs, it has been postulated that the haploid mycelium forming the ECMs serves as the maternal partner in the cross and that it must have come into contact with an opposite mating type strain that serves as the paternal partner (Rubini et al., 2011a). We note, however, that the paternal reproductive structure in Tuber spp. has not yet been identified and that the dikaryotic structure derived from the fertilization process is temporally limited to the early stages of fruit body development, as this structure is undetectable at maturity (Paolocci et al., 2006; Rubini et al., 2011b). In light of heterothallism of T.melanosporum, light of heterothallism investigating the mating type distribution in truffle grounds is of relevance. To this end, Rubini et al. (2011a) reported the biased distribution of the two mating types in artificially inoculated seedlings, as well as in host plants from a natural T. melanosporum site. Similarly, Zampieri et al. (2012) identified only one mating type in the soil harvested from under a single tree, and Linde & Selmes (2012) found that only half of the investigated trees in Australian truffle orchards harboured ECMs of both mating types in their roots. Simple sequence repeat (SSR)-based studies have been used to investigate T. melanosporum population genetics over its natural distributional range (Italy, France and Spain) and have highlighted the absence of isolation by distance at this scale (Bertault et al., 1998). However, a strong genetic structure with significant fixation indices was observed (FST = 0.20 and FST = 0.177) (Murat et al., 2004; Riccioni et al., 2008). Interestingly, on a smaller scale, Bertault et al. (2001) observed isolation by distance in a truffle field, but this study did not investigate the contributions of vegetative vs sexual dissemination. The genetic structure of T. melanosporum populations at a small scale has not been adequately investigated. Therefore, in the present study, the spatial genetic structure of T. melanosporum was investigated in order to: characterize, at the small-scale level, the spatial and temporal distributions of T. melanosporum genets and assess the occurrence of SGS; assess the importance of vegetative vs sexual recruitment in this species; and test whether the T. melanosporum genets have a random distribution according to their mating type locus. To achieve these goals, we applied a finely mapped sampling strategy in two productive T. melanosporum orchards, one located in northern France and the other in central Italy. We investigated the genetic profiles of ECMs using both SSR markers and mating-type genes and analysed the distribution of T. melanosporum mycelia of the two mating types in the soil. Furthermore, the genetic profiles of the fruit bodies were compared with those of the underlying ECMs in a 2 yr study in the French truffle orchard.
Materials and Methods
Two independent experiments were performed at two different sites, one located in Montemartano (Umbria, Italy) and the other in Rollainville (Lorraine, France). Both truffle orchards were set within the natural range of this species. A detailed description of the Montemartano and Rollainville plantations is available in the Supporting Information (Methods S1). The two experiments were designed independently such that the sampling and genotyping strategies differed.
Sampling of soil, ectomycorrhizal roots and ascocarps
In Montemartano, 19 soil cores were collected in June 2010 under four 15-yr-old Quercus pubescens known to produce ascocarps (numbers 212, 213, 214 and 253; Fig. 1). Thirty-six root samples were obtained in October 2010 from under the same trees by collecting up to 10 evenly distributed root samples from around each tree. The soil and root samples were collected from within the brulé (i.e. the area around the tree without vegetation) using a soil corer at a c. 15–20 cm depth (Fig. 1). The root samples were stored in sterile plastic bags for no longer than 3 d at 4°C, soaked in tap water and sieved to separate the root fragments and ECMs from the soil.
In the Rollainville plantation, two 15-yr-old hazels (Corylus avellana; F10 and F11) and one oak (Quercus petraea; E10) tree were selected for their high truffle production, based on the previous year's harvest (Fig. 2). A grid of 1 m × 1 m squares was set up with camping pickets and light cords. During the 2010–11 and 2011–12 periods of truffle production (winter), 41 ascocarps corresponding to all the production beneath these three trees were located using a well-trained dog. The ascocarps were carefully retrieved from the soil using a small spade and precisely mapped on the grid to within 5 cm (Fig. 3c,d). To confirm the results observed with the F10/F11/E10 samplings, 18 ascocarps, corresponding to all the production, were harvested from under three additional hazel trees (A11, B11 and D11; Fig. 2) during the 2011–12 winter and positioned on a 1 m × 1 m square grid (Fig. 3a,b). In the spring of 2011, 42 root samples and 48 soil cores were collected from under the F10/F11/E10 trees in the first 10 cm of soil and mapped using the same grid that was used to position the ascocarps (Fig. S1a). The fine tree roots were carefully retrieved from the soil and washed in water under a dissecting microscope. Three ECMs per sample core were genotyped, as reported in the following section. All plant debris was discarded from the soil, and the soil samples were kept at −20°C.
Genotyping of ECMs, ascocarps and soil DNA
From each root sample, the T. melanosporum ECMs were identified as described by Zambonelli et al. (1993) and Rauscher et al. (1995) and stored individually in microcentrifuge tubes at −80°C for molecular analyses. The numbers and positions of the T. melanosporum ECMs found are presented in Table S1. Genomic DNA was isolated from single mycorrhizal root tips using the method described by Paolocci et al. (1999) from Montemartano samples and using the DNeasy Plant Mini Kit (Qiagen), following the manufacturer's instructions, for samples from Rollainville. T. melanosporum ECMs were also checked using species-specific primers designed on the internal transcribed spacer (ITS) of the nuclear ribosomal-DNA (Rubini et al., 1998; Paolocci et al., 1999). DNA from soil samples collected in Montemartano was isolated using a PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Solana Beech, CA, USA), according to the manufacturer's instructions. DNA from soil samples collected in Rollainville was extracted using the Fast DNA Spin kit for soil (MP Biomedicals, Illkirch, France), according to Luis et al. (2004). The presence of fungal species in the soil samples was determined by amplifying DNA with the universal fungal primer pairs ITS1/ITS4 (White et al., 1990) or ITSf1/IT2, as described by Belfiori et al. (2012) and Zampieri et al. (2010), respectively. The presence of T. melanosporum hyphae was assessed by PCR amplification using the T. melanosporum-specific ITS primers described earlier. The mating types of the T. melanosporum ECMs and T. melanosporum hyphae present in the soil samples were analysed using specific primers for either the MAT1-2-1 or the MAT1-1-1 gene using the PCR conditions previously described by Rubini et al. (2011b). Hereafter, following Rubini et al. (2011b), the mating types are termed MAT1-2 and MAT1-1. To confirm the identity of the amplicons, the PCR products resulting from the amplification of the ECM and soil samples with T. melanosporum-specific ITS, MAT1-2 and MAT1-1 primer pairs were sequenced, as previously described (Rubini et al., 2011a).
Simple sequence repeat genotyping of T. melanosporum ECMs from Montemartano was performed using primer pairs corresponding to 12 polymorphic SSR markers (08ssrme02, 08ssrme03, 08ssrme09, 08ssrme14, 08ssrme15, 08ssrme17, 08ssrme18, 08ssrme27, 08ssrme29, 08ssrme30, 08ssrme49, and 09ssrme01; from Murat et al., 2011 and C. Riccioni et al., unpublished). The SSR genotyping conditions for these markers are described in Methods S1. The SSR genotyping of T. melanosporum ECMs and ascocarps from Rollainville was performed using primer pairs corresponding to 10 SSR markers (Tm16_ATA12, Tm241_TAA17, Tm2_TAT15, Tm98_TAT15, Tm112_TAT19, Tm9_ATCA12, Tm1_ATTG18, Tm75_GAAA14, Tm22_CCTCAT17 and Tm269_TGTTGC15), as described in Murat et al. (2011).
Multilocus genotype (MLG) identification was performed with only the microsatellite data. MLGsim2.0 (Stenberg et al., 2003, http://www.rug.nl/fmns-research/theobio/_downloads/manual_MLGsim2.0.pdf) was used to identify the MLGs and to calculate the probability of independent occurrence (PGen) and, for those MLGs that were sampled more than once, the probability of arising by chance (PSex). The P-value for testing the significance of PSex for each MLG was estimated using 1000 simulations. When PSex values fell below a P-value of 0.05, it was concluded that identical genotypes originated from the same genet.
The clonal richness and the spatial component of clonal growth were estimated using the following indices calculated with GenClone ver. 2.0 (Arnaud-Haond & Belkhir, 2007; see Methods S1 for details): the clonal diversity index (G/N) (where G is the number of genets and N is the total number of ramets); the Simpson's diversity index modified for finite sample sizes (D*); the clonal evenness, evaluated as the Simpson evenness (ED*); the complement of the slope of the Pareto distribution (c Pareto) of clonal membership; the aggregation index (Ac); the Edge effect (Ee); and the clonal subrange, defined as the maximum spatial distance between two replicates of the same MLG corresponding to the size of the largest genet.
The spatial component of the fine-scale genetic structure was assessed using a spatial autocorrelation analysis of the kinship coefficient corresponding to an estimator of the genetic relatedness, as described in Methods S1. Geneland (ver. 3.1.4; Guillot et al., 2005) was used to infer the population structure and to assign individuals to genetic entities. For these analyses, a first dataset containing only the microsatellite markers and a second dataset containing the microsatellites and mating type loci were tested. The parameters used for the analysis are described in Methods S1.
Spatial distribution of T. melanosporum according to the mating type
In Montemartano, T. melanosporum ECMs were found in five of 10, four of 10, six of seven and eight of nine root samples taken from oak trees 212, 213, 214 and 253, respectively, for a total of 116 T. melanosporum ECMs (Fig. 1; Table S1). Their taxonomic attribution was confirmed by positive amplification with T. melanosporum ITS-specific primers (data not shown). Each single ECM manifested only one of the two MAT idiomorphs. Moreover, as shown in Table S1, all of the T. melanosporum ECMs collected on the same host plant shared the same mating type. The MAT1-2 idiomorph was found in all T. melanosporum ECMs collected from oak trees 212, 213 and 214, and the MAT1-1 idiomorph was detected in all T. melanosporum ECMs on tree 253 (Fig. 1). All soil DNA samples displayed either one or multiple bands when amplified using the universal ITS primers, and 18 out of the 19 samples collected yielded an ITS band of the expected size when amplified with T. melanosporum-specific ITS primers (Table S2). When PCR amplification was performed using either the MAT1-2 or MAT1-1 primer pairs, all soil samples in which T. melanosporum was detected produced a single amplicon, corresponding to either MAT1-2 or MAT1-1. The only exception was sample 213-7, which did not yield a MAT-specific band. The mating type found in the soil DNA samples (i.e. from hyphae) corresponded to that exhibited by the T. melanosporum mycorrhizal root tips sampled at a few cm apart, with the exception of one sample from tree 253 (253-5) (Fig. 1, Tables S1, S2).
In Rollainville, 17 T. melanosporum ascocarps were sampled from under trees F10, F11 and E10 during the 2010–11 harvest season (Fig. 3d). The maternal tissue, or gleba, from the ascocarps harvested along the transect, corresponding to lines four to seven, always carried the MAT1-2 idiomorph, whereas the gleba from the ascocarps harvested along the transect, corresponding to lines eight to 10, always carried MAT1-1 (Fig. 3d; Table S1). During the 2011–12 harvest season, 24 ascocarps were sampled from under trees F10, F11 and E10. These ascocarps were mainly found in different coordinates from the 2010–11 season (Fig. 3c; Table S1). A similar spatial distribution of mating type loci was observed in both years, with the MAT1-2 idiomorph detected in the eastern part of the brulés and the MAT1-1 idiomorph detected in the western part (Fig. 3c). A total of 205 T. melanosporum root tips were identified in the 42 root samples collected in 2011 under trees F10, F11 and E10 (Fig. S1). T. melanosporum ECMs were absent in only two of the root samples located in squares G6 and G7. One hundred and eight out of 117 T. melanosporum ECMs selected were successfully genotyped with MAT-specific primers. For each T. melanosporum ECM, only one MAT-specific band was obtained, and a clear spatial segregation between the mycelia of different mating types was observed (Fig. 3d; Table S1). In spring 2011, soil samples were also collected to investigate the distribution of T. melanosporum mycelia with different mating types (Fig. S1b). At least one MAT gene was amplified in 45 out of the 48 soil DNA samples (Table S2). Mycelia harbouring the MAT1-2 idiomorph were identified in almost all soil samples, even next to sites in which the mycorrhizas and the maternal ascocarp tissues were formed by mycelia harbouring MAT1-1 (Table S2). By contrast, the presence of only MAT1-1 in soils was confined to squares E8 and F8 (Table S2).
During the 2011–12 season, six, seven and five ascocarps were harvested from under trees A11, D11 and B11, respectively (Fig. 3a,b). All ascocarps harvested from under trees A11 and D11 harboured MAT1-2 maternal tissue, but the ascocarps harvested from under B11 harboured MAT1-1 maternal tissue (Fig. 3a,b; Table S1).
Multilocus genotype identification and genet size in the two truffle orchards
At the Montemartano orchard, a single amplicon per SSR locus was obtained for each of the 116 ECMs genotyped. Of the 12 SSR loci, six (08ssrme03, 08ssrme27, 08ssrme30, 08ssrme49, 08ssrme18 and 09ssrme01) were monomorphic, whereas the remaining six loci showed two to five alleles (Table S1). By combining the SSR profiles, a total of eight MLGs were identified (Table S1). Notably, ECMs belonging to the same root system always exhibited identical MLGs. MLGsim analysis (Stenberg et al., 2003) confirmed that the samples of each MLG, with the exception of MLG VI, were likely to belong to the same genet (the probability of the MLG sampled belonging to the same genet (PSex) is < 0.05). As the five ramets bearing MLG VI showed a PSex value > 0.05, the hypothesis that they resulted from sexual reproduction could not be discarded (Table 1). Additionally, Ac was equal to 1, indicating that when the ECMs are close, that is, when they belong to the same root branch or are harvested from within a distance of a few centimetres, they have a high probability of belonging to the same MLG (Table 2). Only tree 214 hosted a single MLG (VIII). The other oak trees investigated hosted different T. melanosporum genets that carry the same mating type (Fig. 1; Table S1). For Rollainville samples, two SSR markers (out of the 10 SSRs tested) were monomorphic (Tm16_ATA12 and Tm269_TGTTGC15) when both the maternal ascocarp tissues and ECMs were analysed. The remaining eight SSRs identified two to four alleles (Table S1). Genotyping of 36 T. melanosporum ECMs harvested in squares A3, B4, D4, F4, B5, D6, C9, F9 and D10 failed. Consequently, these ECM samples were discarded from future analyses. The genotyping of 81 ECMs and 59 ascocarps identified a total of 28 MLGs (Tables 1, S1; Fig. 3). With the exception of R8, R9, R10, R14 and R21, the other MLGs were considered genets (PSex < 0.05; Table 1). The root tips harvested at the same position shared the same MLG, with the exception of one sample from square D8 under the F10/F11/E10 trees where two MLGs with the same mating type (MAT1-1) were found (Table S1). In both Montemartano and Rollainville, the value of Ac was high when only the ECMs were considered (1.0 and 0.98, respectively; Table 2). The Ac was lower when only the ascocarps were considered (0.46–0.49, respectively; Table 2). The mycelium forming mycorrhizas and the maternal tissue of the ascocarps shared the same genotype when harvested at the same location during the 2010–11 season (Fig. 3d), with the exception of one ascocarp in square E9 (Table S1). The MLGs of three mycelia forming ECMs were not recorded in the maternal tissue of the ascocarps. The MLG exhibited by the maternal tissue of a single ascocarp harvested during the 2010–11 season was not recorded among the sampled ECMs (R6; Fig. 3d). Of the 13 MLGs found under the F10/F11/E10 trees in the 2011–12 season, only three MLGs (R2, R3 and R7) were previously detected in the samples from the 2010–11 season (Fig. 3c,d). The maximum size of the genets was determined by calculating the clonal subrange that corresponds to the maximum distance between samples belonging to the same genet. In Montemartano, the clonal subrange was 2.35 m. For the samples harvested in Rollainville during the 2010–11 season, the clonal subrange was 4.52 m (when ascocarps and ECM were considered) and 4.70 m (for ascocarps only) during the 2011–12 season (Table 2). Eight out of the 13 genotypes identified in the ascocarps harvested at Rollainville during the 2011–12 season were sampled only once (Fig. 3).
Table 1. Probability of multilocus genotypes (PGen) and probability of their occurrence resulting from distinct sexual events (PSex) for all samples harvested in the Montemartano and Rollainville truffle orchards
The clonal recruitment is calculated as 1 – (G/N), where G is the number of genets and N is the total number of ramets .
The probability is indicated in brackets.
Both seasons ECM-ASCO
Both seasons ASCO
Clonal diversity and spatial genetic structure
Clonal diversity (G /N) was 0.07 in Montemartano and ranged between 0.1 and 0.5 in Rollainville. In this orchard, the highest value was obtained when the 2011–12 ascocarps were taken into consideration (Table 2). When the ECMs were used, the clonal diversity was lower because of the high number of ramets per genet. This sampling bias was confirmed by the edge effect that estimates the bias induced by the sampling scheme (Table 2). Accordingly, the average of clonality (clonal recruitment; Methods S1) was found to be 0.93 in Montemartano and ranged from 0.9 to 0.5 in Rollainville. In the latter orchard, the clonal recruitment was higher when the ECMs were considered (Table 2). The Simpson's diversity index and the evenness index ranged from 0.79 to 0.94 and from 0.84 to 0.95, respectively (Table 2). The c-Pareto values ranged from 1.26 to 1.74 (Table 2). These three indices were similar in both truffle orchards and varied slightly according the origin of the samples (ECMs and/or ascocarps). This variation can be explained by the fact that the ECMs were products of vegetative development of the mycelium, but the ascocarps resulted from sexual reproduction.
Overall, the SGS was significant at the 5% level, as indicated by the slope of the regression of the pairwise kinship coefficient (Fij) against the logarithm of the geographic distance (Figs 4, S3). High and significant SGS was found in the first 5 m, except when the sampling repetitions of the same genets were excluded in both Montemartano and Rollainville in the 2010–11 season. The Sp statistics was calculated with the b-log of the overall distance and the b-log of the first 5 m (Fig. 4 and Methods S1). In the first case, the Sp statistics ranged from 0.28 (Rollainville 2010–12 samples) to 6.13 (Rollainville 2010–11 samples) when all ramets were considered. When the first 5 m was considered, the Sp ranged from 0.37 (Rollainville, 2011–12 samples) to 19.25 (Montemartano). When considering only one sample per genet as defined by PSex (see earlier), the b-log and Sp statistic values were lower (Fig. S3). When Fij between all pairs of genets defined with PSex was plotted against the spatial distance, it emerged that the genets found under different trees could possess a high degree of kinship (Fig. 5). This was particularly true between genets II and VIII at Montemartano, isolated from under trees 212 and 214, which were almost 90 m apart.
To assess the presence of subpopulations within the truffle orchards, Geneland was used. Because of the low number of MLGs (12), Geneland did not generate significant results in Montemartano (data not shown). In Rollainville, when only the microsatellite data were considered, Geneland identified a single cluster; however, when the mating type data were merged with the SSR data, two clusters were identified (Fig. S2). Cluster 1 was composed of the MAT 1-1 MLG, whereas cluster 2 was composed of MLG harbouring the MAT 1-2 idiomorph.
A pronounced spatial genetic structure of T. melanosporum populations was identified within truffle orchards in two independent studies in which two tree species, oak and hazel, were sampled. Moreover, the present data show that T. melanosporum formed small genets and that most of the genets, because of turnover and/or fruiting dormancy, were not detected over two consecutive seasons. Interestingly, the spatial distribution of the T. melanosporum genotypes on its hosts was nonrandom, as indicated by the mating type locus. These data, coupled with the screening of soil and ascocarp samples, provide basic information about T. melanosporum vegetative and sexual propagation patterns.
T. melanosporum forms small-sized genets
Ectomycorrhizal populations result from a tradeoff between the asexual, vegetative extension of mycelium in the soil and the recruitment of dispersed meiotic spores (Douhan et al., 2011). Population-genetics analyses help mycologists to understand the patterns of the establishment and history of local ECM populations by inferring the number and size of genets within sites (Bonello et al., 1998; Gryta et al., 2000; Fiore-Donno & Martin, 2001; Hortal et al., 2012). The maximum distances between ramets of the same genet were 2.35 and 4.70 m in Montemartano and Rollainville, respectively. Interestingly, the same pattern was found under oaks and hazels at both sites. Most genets had a size < 1 m, and many were limited to one sampling core. In Rollainville, of the 10 and 13 genets found during the 2010–11 and 2011–12 seasons under trees F10, F11 and E10, only three genets were shared. These genets were the largest ones and produced the largest number of fruiting bodies (six for MLG R2). These results suggest the occurrence of a significant genet turnover, although we cannot exclude the possibility that genets of different sizes show a different fruiting behaviour. Because of this, future investigations are needed to test whether small-sized truffle genets are more prone to dormancy than large ones (i.e. number of yr without fruiting). It is also likely that the colonization pattern of T. melanosporum in the two sites resulted from a combination between the vegetative mycelial growth of a few genets that persisted for several years and the annual recruitment of new genets via meiospores. The mean size of the T. melanosporum genets was similar to that observed for early successional or pioneer fungi, such as Laccaria bicolor (maximum size = 3.3 m; Selosse et al., 1999) and Hebeloma cylindrosporum (maximum size = 7 m; Gryta et al., 2000). Douhan et al. (2011) reported that the genet size depends more on competition and the time elapsed since the last disturbance than on other factors such as forest age. The management of truffle orchards (e.g. tree pruning, tillage, grass cutting and searching for truffles with dogs) leads to the creation of open woodlands. T. melanosporum fruiting bodies are generally found in open woodlands, while the presence of competing fungi in closed canopies depresses its fructification (Hall et al., 2007). The disturbance resulting from the truffle orchard management might thus favour the persistence of different, small T. melanosporum genets under the trees, regardless of their age.
T. melanosporum populations are spatially structured
Positive Fij values were observed at short distances in both truffle orchards, indicating that neighbouring individuals had a higher degree of genetic relatedness than random pairs of individuals. The high Sp-statistical values indicate a pronounced SGS. These values, ranging from 0.28 to 5.22 when all ramets were considered, were higher than those found in plants (Sp varying from 0.000 31 to 0.263; Vekemans & Hardy, 2004) and pathogenic fungi (Sp = 0.022 for Armillaria mellea (Travadon et al., 2012); Sp = 0.062 for Cryphonectria parasitica (Dutech et al., 2008)). Because, to the best of our knowledge, these analyses have not yet been performed for other ectomycorrhizal species, no inferences on the possible relationship between the high Sp values and the ectomycorrhizal lifestyle can be drawn. The spatial autocorrelation suggests that the SGS was stronger within the first few metres. Isolation by distance was observed in both truffle grounds and was significant over the first 5 m, confirming the existence of isolation by distance at a small scale for T. melanosporum, as has already been highlighted by Bertault et al. (2001). This result indicates that spore propagation is predominant within the few first metres surrounding the below-ground fruit bodies. The observed SGS may reflect the dispersal strategy of the truffles. Indeed, a stronger SGS has been found in species utilizing animal rather than wind dispersal (Vekemans & Hardy, 2004). The SGS was higher for the ECMs when compared with the ascocarp sampling, reflecting the local vegetative growth of the mycelium needed to colonize the root system. In fact, with one exception, the ECM at the same sampling point always exhibited the same MLG (Table S1). Our results raise doubts about analysing several ECMs from the same sampling point in future investigations, as the same genet could be sampled repetitively.
Subpopulations can occur with geographical barriers on a large scale but also with genetic factors at a local scale, as is the case for L. amethystina (Hortal et al., 2012). In Rollainville, Geneland did not identify any subpopulations when analysing SSR data alone. Thus, there was no major bottleneck for gene flow in this orchard. Consistently, positive kinship coefficient values were observed between genets separated by 20 and 90 m in Rollainville and Montemartano, respectively, suggesting that gene flow occurred in both truffle orchards. However, when Geneland was run using SSR and mating type data, two subpopulations in Rollainville were found. Interestingly, these two subpopulations differed based on their mating type. This result points to a nonrandom distributional pattern of genets with respect to their mating type (see later).
In Rollainville, the genetic diversity of the ascocarps, ECMs and soil samples was compared under trees F10, F11 and E10 in the 2010–11-harvesting season. A fairly good correspondence was observed between the genetic structure of ascocarps and ECMs, as six out of the 10 genets were shared between the two structures. Such a correspondence between the ECM and fruiting bodies has been observed previously in a natural T. melanosporum site (Rubini et al., 2011a) and in other ectomycorrhizal species, such as Hebeloma cylindrosporum (Guidot et al., 2001), Suillus grevillei (Zhou et al., 2001), Suillus pictus (Hirose et al., 2004) and Tricholoma matsutake (Lian et al., 2006).
Strains of different mating type are not randomly distributed in the truffle orchards
Very few studies have investigated the in situ distribution of filamentous ascomycetes according to their mating type. Zaffarano et al. (2011) investigated heterothallic endophytes with opposite mating types belonging to the Phialocephala fortinii sensu lato–Acephala applanata species complex. They did not observe a patchy distribution, as both mating types of these endophytes were found in the same location. Conversely, analyses carried out on a natural truffle stand showed that each individual plant hosted a single T. melanosporum strain (Rubini et al., 2011a). The MAT-based screening of ECMs from the rootlets of nursery-inoculated seedlings growing in pots demonstrated that the coexistence of both mating types on the same root apparatus is a possible but transient condition, because one mating type tends to outcompete the other over time (Rubini et al., 2011a).
In both the Montemartano and Rollainville orchards, large soil patches (up to 15 m2) were detected where all of the T. melanosporum mycorrhizas displayed the same mating type. Nevertheless, plants from these sites harboured different genets, suggesting a nonrandom distribution of T. melanosporum strains of opposite mating type. Geneland analysis carried out in Rollainville also supports the same conclusion (see earlier). Furthermore, Linde & Selmes (2012) recently reported that 50% of plants screened from three T. melanosporum orchards in Australia harboured ECMs of the same mating type, as well as a skewed frequency of the two mating types in these sites. The presence of a single mating type has primarily been observed on older plants from either natural or cultivated sites, rather than on nursery seedlings (Rubini et al., 2011a; Linde & Selmes, 2012; present study). This nonrandom distribution of genotypes could reflect competition between genets of different mating types. Future investigations are needed to corroborate this hypothesis and assess whether mating-type-linked genes mediate this phenomenon.
The problem of finding a sexual partner in the field
Our results raise the question of how sexual reproduction can occur and how strains of opposite mating type can encounter each other in truffle orchards where large patches of ECMs of the same mating type are present. These large patches are destined to be infertile unless a sexual partner arrives. In Daldinia loculata, a nonectomycorrhizal heterothallic ascomycete, the two sexual partners can often be quite distant, but this fungus differentiates microconidia that are dispersed by wind over long distances (Guidot et al., 2003). Truffles live and fruit underground, and the production of mitotic conidia has been reported for Tuber borchii, Tuber dryophilum and three undescribed Tuber sp. (Urban et al., 2004; Healy et al., 2012). If T. melanosporum produces mitotic conidia, environmental factors, such as wind and water, might contribute to conidia dissemination, as in other fungal species (e.g. for Coniothyrium minitans conidia; Yang et al., 2009). A sexual partner can also be brought into the proximity of root-resident strains by the soil microfauna or by animals (Kendrick, 1985; Lehmkuhl et al., 2004; Hohmann & Huckschlag, 2005; Kataržyt≐ & Kutorga, 2011) that are attracted by truffle volatiles (Pacioni et al., 1991; Hochberg et al., 2003; Splivallo et al., 2011). Finally, humans can spread truffle spores or any detached cells via agricultural machinery and soil practices in truffle grounds (Agrios, 2004). In case gametic limitation occurs, any cultivation practice aimed at supplying the sexual partners to root-resident strains is predicted to promote truffle mating and production. We note that, long before this truffle mating strategy was discovered, many truffle growers were inoculating soil with mature ascocarps, although this is not the case for the two truffle orchards analysed here. Whether inoculating soil each year with spores will be sufficient to promote production remains to be elucidated.
In conclusion, this study shows that gene flow in truffle orchards occurs between both close (a few metres apart) and distant plants (separated by > 90 m) and that several T. melanosporum genets can be found on the same host plant. This distributional pattern, however, appears to be related to the allelic configuration of the MAT locus.
The work is part of the doctoral work of H. De la Varga, financed by the Instituto Nacional de Investigaciones Agroalimentarias (INIA, Spain). CNR IGV was funded by Regione Umbria and MIUR, Progetto di ricerca di interesse nazionale (PRIN 2008) ‘Il ciclo biologico del tartufo: interazioni genotipo-ambiente’. This study benefited from funding from the Lorraine Region, ANR SYSTERRA SYSTRUF (ANR-09-STRA-10) and by the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-12- LABXARBRE-01). The authors are grateful to three anonymous referees for their comments and to Mr D. Manna, the owner of the Montemartano truffle orchard.