- Top of page
- Materials and Methods
Ectomycorrhizal (ECM) fungi play a fundamental role in forest ecosystems by facilitating nutrient and water uptake by their host plants (Smith & Read, 1997). Cenococcum geophilum Fr. [Syn. Cenococcum graniforme (Sow.) Ferd & Winge] is an ECM fungus that is distributed worldwide, from arctic or alpine areas (Gardes & Dahlberg, 1996) to tropical rain forests (Lee et al., 1997), and colonizes a wide variety of host species. Moreover, this fungus is the most common and most readily recognized ectomycorrhizal fungus in the world (Trappe, 1964; LoBuglio et al., 1991; Massicotte et al., 1992; Shinohara et al., 1999), dominating the underground ECM communities of many forest ecosystems (Vogt et al., 1981, 1982). This fungus is more resilient to drought stress than are other ECM fungi (Mexal & Reid, 1973; Coleman et al., 1989) and also protects host plants from drought (Pigott, 1982a,b; Wu et al., 1999).
C. geophilum does not form detectable sexual or asexual spores, which are important taxonomic criteria for fungi. While it forms abundant sclerotia that serve as dormant propagules during times of environmental stress (Massicotte et al., 1992), its sexual stage has yet to be observed. Worldwide molecular surveys of C. geophilum isolates conducted to understand the genetic diversity of this common and functional ECM fungus have revealed its significant and unexpectedly high genetic diversity (LoBuglio et al., 1991, 1996; Shinohara et al., 1999; Panaccione et al., 2001; Jany et al., 2002; LoBuglio & Taylor, 2002). Using RAPD, PCR/RFLP of the rDNA internal transcribed spacer (ITS), and other molecular analyses, Jany et al. (2002) observed a high degree of genetic diversity between and within C. geophilum populations in beech stands and suggested a high rate of mitotic or meiotic recombination. LoBuglio & Taylor (2002) also analyzed polymorphisms at nine loci in two C. geophilum populations by PCR-single strand conformation polymorphism (SSCP) techniques and found that genotypic variation occurred on a fine scale, in some cases even within one soil sample (equivalent to a volume of c. 500 ml). However, the reason for such high genotypic variation in this fungus is not currently understood, given that its sexual stage has never been observed. Analyses of the early developmental process of a C. geophilum population may help to explain its high genetic variation.
On the south-eastern slope of Mount Fuji, succession is still in the early primary stages in a volcanic desert following the Hoei eruption of 1707; vegetation is recovering patchily, forming islands on the scoria desert. The vegetation islands are usually initiated by Polygonum cuspidatum L. establishment and thereafter are invaded by other herbaceous species and the pioneer woody shrub Salix reinii Franch. et Savat (Lian et al., 2003; Zhou et al., 2003). At this site, Nara et al. (2003b) used terminal restriction fragment length polymorphism (terminal-RFLP) and sequence analyses of the ITS region in nuclear r-DNA to identify 21 ECM fungal species in the underground ECM community associated with S. reinii. The underground ECM community in undeveloped patches (coverage < 0.5 m2) was dominated by mushroom-fungi like Laccaria and Inocybe, and no C. geophilum was found there. In developed patches (coverage > 45 m2), however, C. geophilum colonized S. reinii even as a minor constituent. The C. geophilum population at this site seems to be at an early stage of establishment, and a genetic investigation of this population would be informative toward understanding the genotypic differentiation among C. geophilum populations.
In this study, we analyzed the genetic structure of C. geophilum populations in early successional volcanic deserts on Mount Fuji. First, we developed new polymorphic simple sequence repeat (SSR) markers for C. geophilum, because SSR markers often show higher polymorphism than do other markers and have advantages in population genetics as a result of their codominance (Byrne et al., 1996; Zhou et al., 2001a,b; Lian et al., 2003). We also studied C. geophilum populations in different areas on the mountain to compare the population structure between early and later developmental stages.
- Top of page
- Materials and Methods
A phylogenetic analysis of C. geophilum by nuclear small subunit ribosomal RNA analysis revealed that this fungus is a lineage of ascomycetes (LoBuglio et al., 1996). As proliferating hyphae of ascomycetes are usually haploid, the C. geophilum mycelium may also consist of haploid cells. In this study, a single band was always amplified from every examined C. geophilum root tip by each of the five markers. This is in accordance with the haploidy in C. geophilum.
This study provides information on the establishment and development of a C. geophilum population during primary succession. C. geophilum colonization increased as succession proceeded. C. geophilum root tips were quite rare in the early successional site and became more common at the other three sites where the vegetation was more developed. While C. geophilum was detected in only 22 soil samples out of the 311 soil samples at Gotenba (7.1%), it was detected in nine, five, and eight soil samples out of the 20 soil samples at Fujinomiya, Subashiri A, and Subashiri B (45%, 25%, and 40%), respectively. Such low occurrence of this fungus at the early successional site might result in part from the smaller supply of inocula from outside sources.
The genotype richness of C. geophilum was also lower at Gotenba than at the other three sites. Seven, 10, eight, and 10 genotypes of C. geophilum were detected at Gotenba, Fujinomiya, Subashiri A, and Subashiri B, respectively; the numbers of C. geophilum genotypes per C. geophilum-colonized root tip were 0.13, 0.37, 0.50, and 0.42, respectively. The frequency distribution of genotypes shown in Table 2 indicates that dominance by the most frequent genotype was much more prominent at Gotenba. These results suggest that the genotype richness of C. geophilum increased as succession proceeded.
Increased genotype richness of C. geophilum accompanying advanced host development was also observed on a smaller scale. At Gotenba, small patches were generally colonized by a single genotype of C. geophilum, but big patches were colonized by several genotypes. As patch size roughly represents the time since patch establishment (Zhou et al., 2003), this indicates that C. geophilum genotype richness increased with patch development. At Gotenba, Nara et al. (2003a,b) demonstrated above-ground and underground ECM fungal succession; a few fungal species first colonize small S. reinii, and fungal species richness increases with host size development. The reason for this is species enrichment by the accumulation of species introduced from the surrounding communities. The genotype richness of C. geophilum might follow a similar pattern to fungal species richness by a similar mechanism.
Allelic analysis of the SSR markers provides some insight into genetic interrelations between populations. High similarities among some genotypes were found over different populations (Fig. 4), including between Gotenba and Fujinomiya (in Groups 1 and 3), between Fujinomiya and both Subashiri populations (in Group 8), and between the two Subashiri populations (in Groups 5, 7, and 8). The south-eastern slope of Mount Fuji, including the Gotenba site, was newly covered by volcanic scoria produced by the Hoei eruption in 1707. Therefore, the C. geophilum population at Gotenba was initially established by propagules introduced from populations surrounding the scoria desert. The similarity analysis indicates that the genotypes at Gotenba might have been transferred after the eruption from the direction of Fujinomiya, but not from Subashiri. While none of the seven C. geophilum genotypes at Gotenba was detected at Subashiri A and B, four genotypes (B, D, E, and F) out of the seven were common to Fujinomiya. This fact also supports the above inference.
This fungus is thought to form no meiospores or mitospores, and known propagules for C. geophilum are only sclerotia, that is aggregations of mycelium, that may be dispersed by wind, water, and animals, including insects and birds (Trappe, 1969; Massicotte et al., 1992). Therefore, genetic flow may be mostly caused by the dispersal of sclerotia. A sclerotium of C. geophilum is relatively large, so its long-distance dispersal could be quite limited compared with that of far smaller and lighter propagules, like the basidiospores of many ECM fungi. Both the lower frequency and the reduced genotype richness of C. geophilum at Gotenba might largely be owing to this limited propagule supply from the outside, although the long-distance dispersal of C. geophilum propagules is prerequisite for the initial colonization of the fungus in this volcanic desert that might have occurred only occasionally.
A spreading mechanism of a C. geophilum genotype on a smaller scale can be seen in the Gotenba population that might be at the early colonization stage. Genotype B was associated with many patches and predominantly colonized the host willows, and the maximum distance between two patches that contained genotype B was 78 m. The distribution patterns of genotypes E and F widely overlapped genotype B along the slope, although their occurrence was much less than that of the latter. At this site, many C. geophilum sclerotia were found in soil samples containing C. geophilum root tips. Frequent avalanches scoop up scoria at a depth of several tens of centimeters and carry them to lower positions; these avalanches may contribute to the dispersal of sclerotia.
A short and inconspicuous sexual process or parasexuality is suggested to occur in the C. geophilum life cycle based on its significant genetic diversity (Panaccione et al., 2001; Jany et al., 2002; LoBuglio & Taylor, 2002). However, this does not seem to be the case in this study. Although the genotypes were diverse even at the early stage of primary succession at Gotenba, all of them were highly similar to those at Fujinomiya. This suggests that the genotypic diversity at Gotenba only reflects the diversity of the surrounding populations supplying the propagules and that sexual events such as recombination did not contribute to genotypic diversity. Two discrete genotypic groups (Groups 1 and 3 in Fig. 4) have been maintained at Gotenba, and we were unable to find intermediate genotypes generated by the recombination of these two genotypes. This result also supports the absence of frequent recombination in the Gotenba population. High similarity within each genotype group at the Gotenba population might be an indication of mutation events. Higher genotypic variety was found at three other developed sites surrounding the Gotenba site. In these sites, periods after establishment of the C. geophilum populations should be longer than the Gotenba site. Thus, the higher variety might be caused by recombination or mutation events accumulated during the longer period as suggested in a study of mature forests (Jany et al., 2002). Some other features such as geographic factors might also contribute to the genotypic diversity among the sites. For example, the low similarity of genotypes between the closely situated sites Subashiri A and B might be caused by the separation of a valley.
In conclusion, this study demonstrates that a C. geophilum population under primary succession on Mount Fuji shows a significant genetic diversity, but that the diversity may not be directly caused by genetic recombination.