Characterization of the reproductive mode and life cycle of the whitish truffle <Emphasis Type="Italic">T. borchii</Emphasis>

Truffles are the fruiting structures of ascomycetes in the genus Tuber. Because of their economic importance, truffles have been cultivated for many years using artificially inoculated host plants. Nevertheless, the life cycle and reproductive mode of Tuber spp. are still poorly understood. In filamentous ascomycetes, sexual reproduction is genetically controlled by the mating-type (MAT) locus. Among Tuber spp., the MAT locus has been recently characterized in the black truffles Tuber melanosporum and Tuber indicum. Here, by using sequence information derived from these species and from a Tuber borchii expressed sequence tag (EST) showing similarity to the mat1 gene of Alternaria brassicicola, we embarked on a chromosome-walking procedure to sequence the complete MAT region of T. borchii. This fungus produces highly commercialized whitish truffles and represents a model species for addressing basic questions concerning the life cycle of Tuber spp. We show that T. borchii is heterothallic, as its MAT locus is organized into two idiomorphs, each harbored by different mycelial strains. The alignment of the MAT locus from black truffles and T. borchii reveals that extensive sequence rearrangements and inversions occurred between these species. Moreover, by coupling mating-type analyses to karyological observation, we show that mycelia isolated from ascocarps and mycorrhizae are formed by homokaryotic hyphae.     

A review of research on Chinese <Emphasis Type="Italic">Tuber</Emphasis> species

Truffles are abundant in some regions of China. Nevertheless, it was not until the 1980s that Tuber species were discovered by Chinese mycologists. In recent years, international truffle markets have shown an increasing interest in the import of Chinese Tuber. These truffles serve as a complement to European truffles due to their lower prices and their greater availability in a deficit market. However, Chinese truffles have been the subject of fraudulent commercial practices, and these could have a negative effect on truffle culture. These concerns have been behind numerous recent studies designed to obtain detailed information about Chinese Tuber species. Unfortunately, many of these studies are not published in English, and are dispersed throughout the specific local or national bibliography and proceedings of specialized truffle conferences. In view of the need to expand current knowledge of Chinese Tuber species, we present a comprehensive summary of the taxonomy, ecology, mycorrhizae, genetics, biochemistry, and cultivation of Chinese Tuber species. We also provide a synthetic taxonomy and morphological characterization of 16 Chinese Tuber species in order to assist in their verification and monitoring.     

Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia,Canada

Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.     

Host species effects on bacterial communities associated with the fruiting bodies of <Emphasis Type="Italic">Tuber</Emphasis> species from the Sichuan Province in Southwest China

Tuber species produce highly sought-after truffles and host a wide diversity and high abundance of bacteria. It has been suggested that some of these bacteria contribute to the growth, maturity, and aromatic properties of truffles. Here, we characterized and compared the microbiomes of several species of truffles from the Sichuan Province in Southwest China using high-throughput sequencing of bacterial community 16S rRNA genes. Two T. pseudoexcavatum ascocarp samples had relatively similar bacterial communities, as indicated by PCoA analysis. In contrast, three T. indicum samples collected at different maturity stages did not contain similar communities, suggesting that the maturity stage of ascocarps affects community composition in addition to host phylogenetic background. Despite the variation seen among species and maturity stages, the Proteobacteria phylum dominated all communities, which is consistent with previous studies of Tuber-associated bacteria. Moreover, Bradyrhizobium was the dominant genus in most Tuber ascocarps, which is also consistent with previous studies, although the functional role of this genus within truffles is unclear. Notably, Serratia, which are essential producers of thiophene volatiles within T. borchii were dominant in all of our samples. This finding supports the hypothesis that the ability to produce thiophene volatiles is widespread among these bacteria.     

Reevaluation of the Life Cycle of Tuber magnatum

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.     

Effect of culture conditions on mycelial growth, antibacterial activity, and metabolite profiles of the marine-derived fungus Arthrinium c.f. saccharicola

The effects of culture conditions and competitive cultivation with bacteria on mycelial growth, metabolite profile, and antibacterial activity of the marine-derived fungus Arthrinium c.f. saccharicola were investigated. The fungus grew faster at 30°C, at pH 6.5 and in freshwater medium, while exhibited higher antibacterial activity at 25°C, at pH 4.5, 5.5, and 7.5, and in 34 ppt seawater medium. The fungus grew faster in a high-nitrogen medium that contained 0.5% peptone and/or 0.5% yeast extract, while exhibiting higher bioactivity in a high-carbon medium that contained 2% glucose. The fungal growth was inhibited when it was co-cultured with six bacterial species, particularly the bacterium Pseudoalteromonas piscida. The addition of a cell free culture broth of this bacterium significantly increased the bioactivity of the fungus. Metabolite profiles of the fungus revealed by gas chromatography (GC)-mass spectrometry showed clear difference among different treatments, and the change of relative area of three peaks in GC profile followed a similar trend with the bioactivity variation of fungal extracts. Our results showed clear differences in the optimal conditions for achieving maximal mycelial growth and bioactivity of the fungus, which is important for the further study on the mass cultivation and bioactive compounds isolation from this fungus.     

The phylogenetic position of Tuber glabrum sp. nov. and T. sinomonosporum nom. nov., two Paradoxa-like truffle species from China

Based on morphological and molecular phylogenetic analysis, the new species Tuber glabrum is described from China, and the new name T. sinomonosporum is proposed for Paradoxa sinensis. Tuber glabrum is similar to T. gigantosporum but differs by its glabrous ascomata. The ITS sequences of the two Paradoxa-like truffle species, which invariably have one-spored asci, fall into the lineage of Tuber and cluster in a distinct clade together with the European T. macrosporum and the North American T. canaliculatum.     

Mating Type Locus of Chinese Black Truffles Reveals Heterothallism and the Presence of Cryptic Species within the T. indicum Species Complex

Tuber spp. are filamentous ascomycetes which establish symbiosis with the roots of trees and shrub species. By virtue of this symbiosis they produce hypogeous ascocarps, known as truffles. Filamentous ascomycetes can reproduce by homothallism or heterothallism depending on the structure and organization of their mating type locus. The first mating type locus in a truffle species has been recently characterized in Tuber melanosporum and it has been shown that this fungus, endemic in Europe, is heterothallic. The availability of sequence information for T. melanosporum mating type genes is seminal to cloning their orthologs from other Tuber species and assessing their reproductive mode. Here we report on the organization of the mating type region in T. indicum, the black truffle species present in Asia, which is the closest relative to T. melanosporum and is characterized by an high level of morphological and genetic variability. The present study shows that T. indicum is also heterothallic. Examination of Asiatic black truffles belonging to different genetic classes, sorted according to the sequence polymorphism of the internal transcribed spacer rDNA region, has revealed sequence variations and rearrangements in both coding and non-coding regions of the mating type locus, to suggest the existence of cryptic species within the T. indicum complex. The presence of transposable elements within or linked to the mating type region suggests a role of these elements in generating the genotypic diversity present among T. indicum strains. Overall, comparative analyses of the mating type locus have thus allowed us to tackle taxonomical and phylogenetic issues within black truffles and make inferences about the evolution of T. melanosporum-T. indicum lineage. Our results are not only of fundamental but also of applied relevance as T. indicum produces edible fruit bodies that are imported also into Europe and thus may represent a biological threat for T. melanosporum.     

Trichoderma eijii and T. pseudolacteum, two new species from Japan

Trichoderma eijii and T. pseudolacteum, are described here as new species from Japan. These species were isolated from decaying wood in the Tomakomai Experimental Forest in Hokkaido Prefecture and from bedlogs on shiitake mushroom (Lentinula edodes) farms, respectively. The species were characterized using a combined approach that included cultural studies, holomorph morphology, and phylogenetic analyses of internal transcribed spacer and protein coding gene sequences (RNA polymerase subunit II, translation elongation factor 1-α, endochitinase, and actin). The results of phylogenetic analyses of these gene sequences indicate that T. eijii belongs to the Hamatum clade and is closely related to Hypocrea pezizoides, H. flaviconidia, and H. atroviridis/T. atroviride, from which it differs mainly in part-ascospore size and anamorphic characteristics. Trichoderma pseudolacteum, which was previously recognized as H. lactea sensu Doi, is morphologically distinct from H. lactea (= Hypocrea citrina) and is strongly supported as a separate lineage based on our phylogenetic analyses.     

Truffle diversity (<Emphasis Type="Italic">Tuber</Emphasis>, Tuberaceae) in British Columbia

To improve baseline data for the developing truffle industry in British Columbia, we compiled existing Tuber species sequences from published and unpublished studies and generated new ITS sequences for truffles belonging to Tuber collected in the province. In doing so, we obtained evidence that 13 species of Tuber occur in the province, including six introduced and seven native species, two of which are putative undescribed species. Of the native species, the Tuber anniae species complex is widely distributed in the province while Tuber beyerlei appears to be much more restricted in distribution. Four of the introduced species have commercial value (Tuber melanosporum, Tuber aestivum, Tuber brumale, and Tuber borchii) as do two of the native species (Tuber gibbosum and Tuber oregonense). Focused sampling on likely tree hosts, both hardwood and Pinaceae species, as well as in currently unexplored parts of the province seems likely to expand our knowledge of the diversity and distribution of Tuber species in British Columbia.     

Morphological,mycorrhizal and molecular characterization of Finnish truffles belonging to the Tuber anniae species-complex

The truffle species Tuber anniae was originally described from the U.S. Pacific Northwest and is purported to be uncommon. Here, we report for the first time on the fruiting of closely related taxa in Baltic Rim countries. These truffles were found in a forest dominated by Scots pine in eastern Finland. Mycorrhizal analyses confirmed its symbiosis with Pinus sylvestris. Morphological observations of ascomata and mycorrhizae, and phylogenetic analyses confirmed that these white truffles belong within the group of Tuber puberulum (i.e., Puberulum clade). Further, they group in Clades II and III of the T. anniae species-complex. With the inclusion of sequences from GenBank we are able to demonstrate that the previously unnamed environmental clade (Clade II) has been found as ectomycorrhiza in symbiosis with pine, birch, oak, aspen and even orchids in Europe. Thus, the T. anniae species-complex as a whole (and two of the three clades within) exhibit considerable geographic disjuncts: Northwestern North America and the Baltic Rim of Europe. Clade II, which was collected in agricultural soils in Finland and along roadsides in Alaska, may also be adapted for colonization into new habitats. This may help to explain its presence in New Zealand (where Tuber is not native), which most likely resulted from human-mediated dispersal of these fungi through forestry or the nursery trade. Based on our results, we hypothesize that management practices such as organic and lime amendments, along with aeration, are beneficial to the fruiting of T. anniae. Further research is needed to determine the edibility of these species and whether commercial markets can be developed.     

Azospirillum himalayense sp. nov., a nifH bacterium isolated from Himalayan valley soil,India

Gram-negative, free-living bacterial strain ptl-3^T was isolated from Himalayan valley soil, India. Polyphasic taxonomy was performed including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analyses. 16S rRNA gene sequence analysis showed that the strain ptl-3^T belonged to the genus Azospirillum and was closely related to A. brasilense (98.7 % similarity) and A. rugosum (97 % similarity). 16S rRNA gene sequence similarity (96–95 %) was shown with other members of the genus Azospirillum. Major fatty acid 18:1ω7c was also similar to the genus Azospirillum. DNA–DNA relatedness value between strain ptl-3^T and A. brasilense was found to be 47 %. Various biochemical tests showed that the strain ptl-3^T differed from its closely related species A. brasilense. On the basis of phenotypic, chemotaxonomic and molecular genetics evidence, a bacterium with the type strain ptl-3^T is proposed as a novel species of the genus Azospirillum. The name of bacterial strain ptl-3^T has been proposed as Azospirillum himalayense sp. nov. The type strain of ptl-3^T (CCUG 58760^T, KCTC 23189^T) has been submitted to two culture collection centres. The accession numbers for 16S rRNA and nifH gene are GQ 284588 and GQ 249665. respectively.     

Paludibacter jiangxiensis sp. nov., a strictly anaerobic,propionate-producing bacterium isolated from rice paddy field

A mesophilic, obligately anaerobic, propionate-producing fermentative bacterium, designated strain NM7^T, was isolated from rural rice paddy field. Cells of strain NM7^T are Gram-negative, non-motile, non-spore-forming, short rods, and negative for catalase. The strain grew optimally at 37 °C (the range for growth 15–40 °C) and pH 7.0 (pH 5.0–7.5). The strain could grow fermentatively on various sugars, including arabinose, xylose, fructose, galactose, glucose, mannose, cellobiose, lactose, maltose, sucrose, pectin and starch. The main end products of glucose fermentation were acetate and propionate. Yeast extract was not required but stimulated the growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, and Fe(III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of genomic DNA was 42.8 mol%. The major cellular fatty acids were C_15:0, anteiso-C_15:0, C_16:0, and C_17:0. The most abundant polar lipid of strain NM7^T was phosphatidylethanolamine. 16S rRNA gene sequence analysis revealed that it belongs to the family Porphyromonadaceae of the phylum Bacteroidetes. The closest recognized species was Paludibacter propionicigenes (91.4 % similarity in 16S rRNA gene sequence). A novel species, Paludibacter jiangxiensis sp. nov., is proposed to accommodate strain NM7^T (=JCM 17480^T = CGMCC 1.5150^T = KCTC 5844^T).     

Truffle biogeography—A case study revealing ecological niche separation of different Tuber species

Ecology of hypogeic mycorrhizal fungi, such as truffles, remains largely unknown, both in terms of their geographical distribution and their environmental niches. Occurrence of true truffles (Tuber spp.) was therefore screened using specific polymerase chain reaction (PCR) assays and subsequent PCR amplicon sequencing in tree roots collected at 322 field sites across the Czech Republic. These sites spanned a wide range of climatic and soil conditions. The sampling was a priori restricted to areas thought to be suitable for Tuber spp. inasmuch as they were characterized by weakly acidic to alkaline soils, warmer climate, and with tree species previously known to host true truffles. Eight operational taxonomic units (OTUs) corresponding to Tuber aestivum, T. borchii, T. foetidum, T. rufum, T. indicum, T. huidongense, T. dryophilum, and T. oligospermum were detected. Among these, T. borchii was the OTU encountered most frequently. It was detected at nearly 19% of the sites. Soil pH was the most important predictor of Tuber spp. distribution. Tuber borchii preferred weakly acidic soils, T. foetidum and T. rufum were most abundant in neutral soils, and T. huidongense was restricted to alkaline soils. Distribution of T. aestivum was mainly dictated by climate, with its range restricted to the warmest sites. Host preferences of the individual Tuber spp. were weak compared to soil and climatic predictors, with the notable exception that T. foetidum appeared to avoid oak trees. Our results open the way to better understanding truffle ecology and, through this new knowledge, also to better‐informed trufficulture.     

Coexistence of three canopy tree species in a riparian forest in the Chichibu Mountains, central Japan

Three canopy tree species (Fraxinus platypoda, Pterocarya rhoifolia, andCercidiphyllum japonicum) coexist in riparian forests in the Chichibu Mountains of central Japan. We compared the forest structure and the reproductive characteristics of these species.F. platypoda was the dominant canopy species. It produced many saplings and grew in abandoned channels and floodplains, and was able to invade both large and small disturbance sites.P. rhoifolia was a subdominant species that occurred on the deposits of large-scale landslides and grew in patches containing even-aged trees.C. japonicum was the other subdominant species that produced few saplings and invaded large disturbance sites together withP. rhoifolia. Establishment sites ofC. japonicum were restricted to fine mineral soils and fallen logs. We found tradeoffs in reproductive characteristics (seed size, seed number, irregular seed production, and sprouting) among the three canopy species.F. platypoda andP. rhoifolia had large seeds and produced fruits irregularly.C. japonicum produced many small seeds every year and sprouted prolifically around the main stem. The causes of the coexistence mechanism of the three riparian canopy tree species may be both niche- and chance-determined to varying degrees. In riparian areas, the three canopy species were well-adapted to disturbances throughout their life-history.     

Isolation and characterization of polysaccharides with the antitumor activity from Tuber fruiting bodies and fermentation system

Fifty-two polysaccharides were isolated from the fermentation systems of Tuber melanosporum, Tuber indicum, Tuber sinense, Tuber aestivum and the fruiting bodies of Tuber indicum, Tuber himalayense, Tuber sinense by elution with an activated carbon column. Polysaccharides from Tuber fermentation system exhibited relatively higher in vitro antitumor activity against HepG2, A549, HCT-116, SK-BR-3, and HL-60 cells than those from Tuber fruiting bodies. All polysaccharides were mainly composed of d-mannose, d-glucose, and d-galactose, which suggested that the polysaccharides from Tuber fruiting bodies and fermentation system have identical chemical compositions. The results of antitumor activity and structural identification indicated that the polysaccharide fractions could promote antitumor activity. Tuber polysaccharides from Tuber fermentation system exhibited relatively higher than that from Tuber fruiting bodies. These results confirm the potential of Tuber fermentation mycelia for use as an alternative resource for its fruiting bodies.     

Effects of temperature,salinity and their interaction on growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a seafood poisoning that is widespread in tropical, subtropical and temperate regions of the world. In the main island of Japan, distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3, have been reported. To discuss the bloom dynamics of these Japanese species/phylotypes of Gambierdiscus, first we tested six culture media to optimize growth conditions and then clarified the effects of temperature and salinity and temperature–salinity interactions on growth. All strains of the species/phylotypes tested showed the highest cell yields when they were cultivated in IMK/2 medium. G. australes, G. scabrosus and Gambierdiscus sp. type 2 grew in the range 17.5–30 °C, whereas Gambierdiscus sp. type 3 grew in 15–25 °C. The semi-optimal temperature ranges (≥80% of the maximal growth rate) of the former three species/phylotypes were 19–28 °C, 24–31 °C and 21–28 °C, respectively, whereas that of the latter phylotype was 22–25 °C. Hence, Gambierdiscus sp. type 3 may be adapted to relatively lower water temperatures of ≤25 °C. In contrast, G. australes, G. scabrosus and Gambierdiscus sp. type 2 presumably possess adaptability to relatively high water temperatures. The optimal temperature for G. scabrosus was 30 °C, whereas the optimal temperature for the others was 25 °C. G. australes and Gambierdiscus sp. type 3 grew in a salinity range of 25–40 whereas G. scabrosus and Gambierdiscus sp. type 2 grew in salinity 20–40. Furthermore, the semi-optimal salinity range of G. australes, G. scabrosus, Gambierdiscus spp. type 2 and type 3 were salinity 27–38, 24–36, 22–36 and 29–37, respectively. Among the species/phylotypes, G. scabrosus and Gambierdiscus sp. type 2 grew even at salinity 20 where the others did not grow, thus possessing adaptability to low salinity waters. Our results clearly demonstrate that the optimal and tolerable temperature–salinity conditions differ among Japanese Gambierdiscus species/phylotypes. Considering these results, temperature–salinity interactions may play an important role in bloom dynamics and the distribution of the Gambierdiscus species/phylotypes in Japanese coastal waters.     

Optimal conditions of in vitro mycelial growth of <Emphasis Type="Italic">Lentinus strigosus</Emphasis>, an edible mushroom isolated in the Brazilian Amazon

The protocols of in vitro cultivation described in the literature for mushrooms are usually correlated with temperate climate habitat, but it is necessary to study protocols for species of tropical climates. In this article, we collected, isolated, and evaluated the conditions of in vitro mycelial growth of Lentinus strigosus and correlated these with the characteristics of its habitat. These results indicate, as optimal conditions of in vitro mycelial growth for L. strigosus, the use of 35°C for incubation, initial pH from 5 to 7, without illumination, Sabouraud dextrose agar medium, and agitation for culture in liquid medium.     

Desulfovibrio oceani subsp. oceani sp. nov., subsp. nov. and Desulfovibrio oceani subsp. galateae subsp. nov., novel sulfate-reducing bacteria isolated from the oxygen minimum zone off the coast of Peru

Two deltaproteobacterial sulfate reducers, designated strain I.8.1^T and I.9.1^T, were isolated from the oxygen minimum zone water column off the coast of Peru at 400 and 500 m water depth. The strains were Gram-negative, vibrio-shaped and motile. Both strains were psychrotolerant, grew optimally at 20°C at pH 7.0–8.0 and at 2.5–3.5% NaCl (w/v). The strains grew by utilizing hydrogen/acetate, C_3–4 fatty acids, amino acids and glycerol as electron acceptors for sulfate reduction. Fumarate, lactate and pyruvate supported fermentative growth. Sulfate, sulfite, thiosulfate and taurin supported growth as electron acceptors. Both strains were catalase-positive and highly oxygen-tolerant, surviving 24 days of exposure to atmospheric concentrations. MK6 was the only respiratory quinone. The most prominent cellular fatty acid was iso-17:1-ω9c (18%) for strain I.8.1^T and iso-17:0-ω9c (14%) for strain I.9.1^T. The G+C contents of their genomic DNA were 45–46 mol%. Phylogenetic analysis of 16S rRNA and dsrAB gene sequences showed that both strains belong to the genus Desulfovibrio. Desulfovibrio acrylicus DSM 10141^T and Desulfovibrio marinisediminis JCM 14577^T represented their closest validly described relatives with pairwise 16S rRNA gene sequence identities of 98–99%. The level of DNA-DNA hybridization between strains I.8.1^T and I.9.1^T was 30–38%. The two strains shared 10–26% DNA-DNA relatedness with D. acrylicus. Based on a polyphasic investigation it is proposed that strains I.8.1^T and I.9.1^T represent a novel species for which the name Desulfovibrio oceani sp. nov. is proposed with the two subspecies D. oceani subsp. oceani (type strain, I.8.1^T = DSM 21390^T = JCM 15970^T) and D. oceani subsp. galateae (type strain, I.9.1^T = DSM 21391^T = JCM 15971^T).     

Phylogenetic and populational study of the Tuber indicum complex

When examined using SEM, Chinese samples of Tuber indicum and T. sinense displayed the same ascospore ornamentation as that of T. pseudohimalayense, T. indicum, collected in India by Duthie in 1899, and samples renamed T. himalayense in 1988. The different authors who named the four taxa (T. indicum, T. himalayense, T. sinense, T. pseudohimalyense) described differences in the surface of the peridium which could be considered as usual variations within a single species. We consider T. indicum, T. himalayense, T. sinense and T. pseudohimalayense as one species, T. indicum. Within this T. indicum complex, according to ITS and β-tubulin sequences, there are two groups in China, which could be considered as geographical ecotypes. This study is the first to identify a genetic and phylogeographical structure within the Chinese Tuber species.