Ectomycorrhizal sporocarp succession and production. during early primary succession on Mount Fuji

Transcription

1 Research Ectomycorrhizal sporocarp succession and production Blackwell Publishing Ltd. during early primary succession on Mount Fuji Kazuhide Nara, Hironobu Nakaya and Taizo Hogetsu Asian Natural Environmental Science Center, The University of Tokyo, Midori-cho, NishiTokyo, Tokyo , Japan Summary Author for correspondence: Kazuhide Nara Tel: Fax: Received: 29 August 2002 Accepted: 22 November 2002 doi: /j x The species composition, succession and biomass production of ectomycorrhizal (ECM) sporocarps were studied during early primary succession on Mount Fuji, Japan, with special reference to developmental stages and the growth of associated hosts. Weekly sporocarp surveys were conducted over 2 yr on a volcanic desert, where the total vegetation coverage was about 5%. We also quantified the growth of associated hosts in terms of size, photosynthesis, and leaf N and P concentration. A total of sporocarps of 23 species were recorded. They were associated almost exclusively with an alpine dwarf willow, Salix reinii. Two Laccaria and one Inocybe species were the first colonizers; subsequent fungal species were added as the host grew. There was no evidence of any fungus disappearing and being replaced in the sere of ECM fungal succession. The biomass production of ECM sporocarps was exceptionally large, in general, and amounted to 19% of leaf biomass in the most productive associations. Annual ECM sporocarp production in individual ECM associations was strongly correlated with the growth of the associated host, especially with the photosynthetic rate, which appeared to be determined by leaf N and P concentration. Key words: ectomycorrhizal fungi (ECM), early primary succession, volcanic desert, fungal succession, alpine dwarf willow (Salix reinii), photosynthesis, sporocarp production, leaf N concentration. New Phytologist (2003) 158: Introduction The succession of vegetation is an important concept in terrestrial ecology. Many studies, using a variety of approaches, have been conducted worldwide but, until a few decades ago, most studies had neglected the existence of mycorrhizal fungi. With increasing awareness of the importance of mycorrhizal functions in nutrient and water uptake, the effects of mycorrhizal fungi on vegetation succession began to be studied (Miller, 1979; Reeves et al., 1979). The majority of these studies targeted herbaceous plant species in combination with vesicular arbuscular mycorrhiza (VA) mycorrhizal fungi. A basic model of early primary succession has been developed to demonstrate that nonmycotrophic plants are followed by mycotrophic plants, and that succession is driven, in the main, by the invasion of VA mycorrhizal fungi, facilitating nutrient and water uptake (e.g. Allen & Allen, 1984). Ectomycorrhizal (ECM) host plants are typically observed not only in climax forests but also during early primary succession (Allen et al., 1992; Helm & Allen, 1995). Because available nutrients such as nitrogen (N) and phosphorus (P) are quite limited at this stage, symbiosis with ECM fungi is supposed to be advantageous for the survival and growth of host plants, as in the case of VA mycorrhizal fungi. Species of ECM fungi are quite diverse and vary in host range. Furthermore, different ECM fungi have different effects on a host species. To evaluate the effects of ECM fungi on host growth and competition during early primary succession, it is important to determine the existing ECM species or communities at this stage. As many ECM fungi produce conspicuous sporocarps, sporocarp production has been studied in a variety of forests to obtain information about existing ECM fungal species. Several researchers have studied ECM sporocarp communities during early primary succession. Allen et al. (1992) noted New Phytologist (2003) 158:

2 194 Research that no ECM sporocarps were observed until 10 yr after the eruption of Mount St Helens, although some ECM tree species had already invaded. Glacier fronts are another area where primary succession occurs. Helm et al. (1996) noted that species of Inocybe, Laccaria and Hebeloma were the most common sporocarps associated with host species of Salicaceae in early successional stages at Exit Glacier (Alaska, USA). Jumpponen et al. (1999, 2002) reported the presence of 13 ECM sporocarps at the front of Lyman Glacier ( Washington, USA). Because none of these studies have quantitative descriptions, more detailed investigations are needed to understand the ECM sporocarp communities in early primary successional sites. The ECM sporocarp community associated with a pioneer host species can be assumed to change along with the host as it ages and develops, even in the early stages of primary succession. Models of ECM sporocarp succession corresponding to tree growth were developed from investigations in temperate forests and plantations, particularly for fungi in association with Betula, Picea and Pinus (Mason et al., 1982, 1983; Deacon et al., 1983; Fleming, 1983; Fleming et al., 1984; Last et al., 1984; Dighton et al., 1986). The models included the initial appearance of early stage fungi, the subsequent appearance of late-stage fungi and aspects of soil development, such as the increase of organic matter and N content, which is supposed to greatly affect the sporocarp succession. However, most of ECM sporocarp succession studies have been conducted in secondary successional sites where soils are more or less already developed. Succession of ECM sporocarps in primary succession has been reported in only a few studies. Jumpponen et al. (1999, 2002) noted that ECM sporocarp succession at the retreating glacier followed the early to late-stage model, and that fungal species increased with host vegetation development. In these studies, sporocarp production was quite rare and insufficient as a basis for discussion of the sere of ECM sporocarp communities. The succession of ECM sporocarps after volcanic eruptions has not yet been studied during early primary succession. To construct a successional model for ECM sporocarps during early primary succession, further intensive study in more appropriate sites is needed. The relationship between host growth and ECM sporocarp production gives some indication of the role of ECM associations in nature, and has been illustrated in several studies. Defoliation or tree girdling, which reduces the supply of assimilates underground, decreases sporocarp production of ECM fungi (Last et al., 1979; Högberg et al., 2001). Thus, host growth activities, such as photosynthesis, are assumed to have a great impact on sporocarp production. Hendrix et al. (1985) noted that transplanted loblolly pines that produce sporocarps of Pisolithus are twice the height and diameter of those not associated with sporocarps. Host growth might be correlated with production of the associated sporocarps. The relationship between ECM sporocarp production and the growth of an individual host has never been studied during early primary succession. Scattered vegetation patches, which are usually observed in early successional sites, enable us to identify easily an individual host associated with each ECM sporocarp. At such sites, we can investigate the relationship between sporocarp production and host growth in individual associations. These investigations may improve our knowledge of ECM associations in nature. In the present study, we investigated the sporocarp production of ECM fungi in a volcanic desert on Mt Fuji, Japan, where the vegetation is still in the early stage of primary succession. We quantitatively described the sporocarp composition at this site and proposed a model of ECM sporocarp succession in an early primary successional site. Host tree growth parameters such as leaf biomass, photosynthesis and leaf nutrient concentrations were also quantified to determine the relationship between host growth and the production of the associated sporocarps. The significance of ECM symbioses in the early stage of primary succession is discussed. Materials and Methods Research site Mount Fuji, the highest and most famous mountain in Japan, erupted in 1707, and its south-eastern side was completely covered with scoria (i.e. tephra, typically 2 30 mm in diameter), up to 10 m deep. The existing vegetation was completely destroyed, and is now recovering. While the tree line is generally located at 2500 m above sea level on the other sides of the mountain, it is located at around 1300 m on the south-eastern side and continues to rise after 300 yr of vegetation recovery (Fig. 1). Our research site is located between 1500 m and 1600 m above sea level (35 20 N, E), and is in the upper montane zone or the lower subalpine zone. Vegetation in this area is patchily distributed, and the total vegetation coverage is approximately 5%, indicating the early stages of primary succession. Several perennial herbs belonging to Polygonaceae, Asteraceae and Brassicaceae were the first colonizers because of their ability to adapt to the unstable scoria surface (Masuzawa, 1997). Polygonum cuspidatum plays an important role in subsequent vegetation succession. This species forms large patches, up to 10 m in diameter, by vegetative and sexual reproduction, thus providing stable habitats for subsequent plant species on the unstable scoria desert (Adachi et al., 1996). Many pioneer herbaceous and woody species grow in these patches. We established a research quadrat ( m) for our sporocarp surveys. At 1400 m above sea level on the south-eastern side of Mt Fuji, the annual mean air temperature is 8.6 C, and the monthly mean temperature ranges from 1.9 C in January to 19.1 C in August (Tateno & Hirose, 1987). Mean annual precipitation at Tarobo (altitude 1300 m on the same slope) New Phytologist (2003) 158:

3 Research 195 Fig. 1 Location of the research site established in an area of early primary succession on Mount Fuji, Japan. is 4854 mm, which is considerably more than the 1650 mm that falls at Kawaguchiko (northern foothills, altitude 800 m). The high precipitation at Tarobo is mainly due to its location between the wet, warm air masses from the sea to the south and the cool air masses flowing down across the northern slope (Ohsawa, 1984). The soil at a depth of 5 cm remains moist throughout the fruiting seasons because of the high precipitation and scoria substrates. More soil nutrients are available within the vegetation patches than on the bare ground. With the development of the vegetation patches, the amount and forms of soil N have been changed dramatically due to bacterial activity and the N preferences of existing plants (Hirose & Tateno, 1984). Vegetation Within the research quadrat, 159 vegetation patches were larger than 50 cm in diameter. These patches were mapped using a survey laser instrument (Criterion 400; Laser Technology, CO, USA) (Fig. 2). Many smaller patches of Polygonum weyrichii var. alpinum were distributed among the 159 vegetation patches, but they were excluded from this study because their total coverage was small and no ECM associations were observed during our preliminary study. Each vegetation patch that we investigated was numbered and the plant species, and their coverage in each patch, were recorded in August In order to identify the host species that produced ECM sporocarps, plant species composition was compared between sporocarp-producing and nonproducing patches. Five different root systems of each of the 10 plant species that were predominant in coverage within Fig. 2 The spatial distribution of vegetation patches in the quadrat with reference to ectomycorrhizal symbionts. Each circle represents a vegetation patch. Closed circles indicate vegetation patches containing Salix reinii. The patches in which ectomycorrhizal sporocarps were recorded in 2000 or 2001 are flagged. The occurrence of ectomycorrhizal sporocarps completely coincides with the presence of S. reinii in vegetation patches. sporocarp-producing patches were collected and their mycorrhizal formation was examined under a dissecting microscope. Sporocarp survey A total of 34 sporocarp surveys were conducted from May to November in 2000 and We surveyed weekly from late June to middle October in both years, because almost all ECM sporocarps are produced in these seasons. Each survey covered the entire quadrat, and required 1 3 d to complete. Each sporocarp was marked with a small flag to avoid double counting, and to record spatial distribution for future New Phytologist (2003) 158:

4 196 Research research. Because most of the sporocarps were produced within or close to the vegetation patches, the number of sporocarps of each species was summed to give a total for each patch during each survey. These figures were used to estimate the annual sporocarp production of each patch. The macroscopic characteristics of ECM sporocarps at various developmental stages were recorded, and specimens of all species were freeze-dried (FDU-540; EYELA, Tokyo, Japan) for microscopic observation, future DNA analysis and deposition in a public museum. Microscopic characteristics were observed using the procedure of Breitenbach and Kränzlin (1991). The nomenclature generally followed Hansen and Knudsen (1992, 1997). Because there were no regional monographs of ECM fungal taxa in association with alpine dwarf willows in Japan, we used specific literature of other regions and world monographs for the identification of the following genera: Cortinarius (Brandrud et al ), Hebeloma (Breitenbach & Kränzlin, 2000), Inocybe (Stangl, 1989), Laccaria (Mueller, 1992), Russula (Sarnari, 1998), Scleroderma (Guzman, 1970) and Leccinum (Lannoy & Estades, 1995). Species belonging to Entolomataceae were excluded from the present study because their ECM status at the research site was uncertain and some species appeared to be saprophytic. The mean dry weight of sporocarps of each species was determined from 20 fully developed sporocarps, or from all sporocarps for those species with less than 20 sporocarps. The mean dry weight was multiplied by the number of sporocarps to calculate the biomass production of each species. Quantification of Salix reinii status The area covered by S. reinii was a parameter of its size and considered to be a good index of its developmental stage in our research site (Lian et al., 2003). Thus, the cover of S. reinii in each vegetation patch was measured with a digital planimeter on photographs that were taken from 7 m above the patch, and used as an index of host size. To investigate the relationship between sporocarp production and host growth, nine patches with similar amounts of S. reinii cover were selected. The whole-leaf biomass of S. reinii in each of the nine patches was estimated from the dry weight of leaves harvested from a square (20 20 cm) in the center of the S. reinii coverage, in October We also randomly sampled five current-year shoots from each of the nine patches in November 2000, and recorded the length and number of winter buds for each shoot. To measure photosynthesis in S. reinii, we randomly chose five fully developed canopy leaves in each of the nine patches. The photosynthetic rate of each leaf was measured on a fine day in August 2000 with a portable photosynthesis system (LI-6400; LiCor, Lincoln, NE, USA). Fixed chamber conditions were used with saturated light (25 C, 400 p.p.m. CO 2, 1500 µmol m 2 s 1 light intensity with a red and blue LED combination). The area and dry weight of each leaf were recorded after photosynthesis was measured. The N and P concentrations of each leaf were then colorimetrically determined after digestion with H 2 SO 4 and H 2 O 2 using the indophenol blue method and the ascorbic acid deoxidizing molybdenum blue method, respectively. In November 2000, six soil cubes ( cm) were sampled from each patch, three from the periphery and three from the inside, and 200 root tips within each sample were randomly collected. The rate of ECM formation (number of mycorrhizal root tips/200 root tips 100) was determined under a dissecting microscope. Statistics The statistical analyses were performed with the SPSS 11.0 software package (SPSS, Chicago, IL, USA) for Windows. All regression relationships were fitted using a least-squares regression, and their statistical significance was tested. The data on host parameters, such as current-year shoot length, winter bud number, photosynthesis, leaf N and P concentrations, and mycorrhizal formation are presented as means ± SE. These data were tested for statistically significant differences among the nine selected patches by one-way analysis of variance, and Tukey s multiple comparison test was applied when appropriate. Spearman s rank correlation coefficients were used to evaluate the relationship between the ECM sporocarp production and the host parameters. Results Host species A total of 19 herbaceous plant species and eight tree species were confirmed in 159 vegetation patches in the research quadrat (Table 1). Polygonum cuspidatum and the alpine P. weyrichii var. alpinum were observed in 155 and 104 patches, respectively. Several species belonging to Rosaceae and Cyperaceae were also found in many patches. The families, such as Polygonaceae, Rosaceae and Cyperaceae, have been described to have some ECM herbaceous species in other geographical areas (Massicotte et al., 1998). Any of above herbaceous species did not form ectomycorrhizae in our research site (Table 1). Woody species were a relatively minor constituent. Salix reinii and Spiraea japonica var. alpina were observed in 37 and 30 patches, respectively. As a result of weekly sporocarp surveys over 2 yr, ECM sporocarps were found in 38 of the 159 patches (Fig. 2). The most apparent difference in plant species composition between the sporocarp-producing and nonproducing patches was in S. reinii (Table 1). Of 38 patches in which mycorrhizal sporocarps were recorded, 37 contained S. reinii. The remaining patch with mycorrhizal sporocarps had Salix bakko. Of the 10 major plant species that dominated the sporocarpproducing patches (Table 1), nine species did not have ectomycorrhizae; S. reinii was the exception. New Phytologist (2003) 158:

5 Research 197 Table 1 Plant communities in sporocarp-producing and nonproducing patches on the volcanic desert of Mount Fuji Species Sporocarp-producing patches (n = 38) Sporocarp-nonproducing patches (n = 121) Total (n = 159) Woody species Salix reinii a Spiraea japonica a Rosa fujisanensis Betula ermanii Larix kaempferi Ligustrum obtusifolium Salix bakko Weigela decora Herbaceous species Polygonum cuspidatum a Cirsium purpuratum a Arabis serrata a Polygonum weyrichii var. alpinum a Campanula punctata var. hondoensis Calamagrostis hakonensis a Picris hieracioides ssp. japonica Artemisia princeps Miscanthus olygostachyus a Aster ageratoides ssp. ovatus a Clematis stans a Anaphalis margaritacea Senecio nemorensis Angelica hakonensis Hedysarum vicioides Carex doenitzii Fragaria nipponica Artemisia pedunculosa Cirsium effusum Figures indicate the number of patches in which each species appeared. a Indicates the 10 predominant species in sporocarp-producing patches; five root systems of each were investigated for mycorrhizal formation. The area covered by S. reinii in each patch varied greatly, ranging from a several-year-old seedling of m 2 to large bushes over 50 m 2. Although S. reinii was the exclusively dominant host plant for ECM fungi in our early successional site, the total area covered by this species was only 502 m 2, less than 1% of the whole quadrat. The subsequent successional tree species, Betula ermanii and Larix kaempferi, were observed on only four and three patches, respectively. These species were minor constituents on the patches, and were always accompanied by S. reinii. The total area covered by B. ermanii and L. kaempferi was 0.77 m 2 and 0.85 m 2, respectively. Species composition of ECM sporocarps The ECM sporocarps appeared between July and November in both years. In total, sporocarps of 23 species were recorded over 2 yr (Table 2). The total sporocarp production in 2001 was significantly lower than in 2000, probably because of the severe above-ground disturbance from avalanches and windstorms that occurred in the winter between the two fruiting seasons. Cortinariaceae was the most species-rich family, with 12 species. Inocybe was the most species-rich genus, with seven species, although some of them were quite rare. Hebeloma, Laccaria and Russula were the next species-rich genera; each contained three species. Boletus and Cantharellus were also found, but were rare. Leccinum was observed under the coverage of B. ermanii surrounded by S. reinii. Laccaria laccata was the most abundant species, producing 3955 (43% of the total) and 762 (34% of the total) sporocarps in 2000 and 2001, respectively. Although small numbers of its sporocarps were found throughout the fruiting seasons, there was a conspicuous production peak in summer of each year. In a single survey at the peak of 2000, the sporocarp number of L. laccata exceeded 90% of the annual production. Sporocarps of Laccaria amethystina and Laccaria murina were also abundant, and their production peaks were quite similar to that of L. laccata. Cortinarius decipens and Hebeloma mesophaeum sporocarps were also major species, and their production peaks were in late autumn. Inocybe lacera and Scleroderma bovista were major species that did not have prominent production peaks. A substantial number of their sporocarps were found continuously New Phytologist (2003) 158:

6 198 Research Table 2 Ectomycorrhizal sporocarps recorded during early primary succession on Mount Fuji species Number Patch Dry weight (g) Number Patch Dry weight (g) Boletus pulverulentus Opat Boletus cf. rubellus Krombh Cantharellus cibarius Fr Cortinarius alboviolaceus (Pers. Fr.) Fr Cortinarius decipens (Pers. Fr.) Fr Hebeloma leucosarx Orton Hebeloma mesophaeum (Pers.) QuÈl Hebeloma pusillum Lange Inocybe acuta Boud Inocybe calospora Quèl. a a a Inocybe dulcamara (Pers.) Kumm Inocybe fastigiata (Schaeff.) Quèl Inocybe lacera (Fr.) Kumm Inocybe sp. 1 a a a Inocybe sp Laccaria amethystina Cooke Laccaria laccata (Scop. Fr.) Berk. & Br Laccaria murina Imai Leccinum scabrum (Bull. Fr.) S.F. Gray Russula norvegica Reid Russula pectinatoides Peck Russula sororia (Fr.) Romell Scleroderma bovista Fr Total Data in the Number and Dry weight columns indicate the total number and the total dry weight, respectively, of sporocarps of each species in the quadrat. Data in the Patch columns indicate the total number of patches in which each species was recorded. a Inocybe calospora and Inocybe sp.1 were not distinguished from Inocybe lacera in throughout the fruiting seasons, from July to November, of both years. The total biomass production of all ECM sporocarps in the quadrat amounted to 3089 g and 1563 g in 2000 and 2001, respectively. Among all ECM species, S. bovista dominated the dry weight. This species amounted to 2505 g (81% of the total) and 1430 g (92% of the total) dry weight for 2000 and 2001, respectively (Table 2). In comparison, the other species were minor constituents in terms of dry weight. Succession of ECM sporocarps with host development The species composition of ECM sporocarps changed according to host size enlargement (Fig. 3). Inocybe lacera appeared in association with hosts whose size ranged from m 2 to 154 m 2. Sporocarps of L. laccata and L. amethystina were observed under hosts larger than m 2 and 0.15 m 2, respectively. There were six patches in which the area covered by hosts was smaller than 0.5 m 2. These small and young hosts had only one or two of the three ECM species, I. lacera, L. laccata and L. amethystina, and were not associated with other ECM fungal species. Thus, these three species were first-stage fungi that appear first on the sere of the ECM fungal succession during early primary succession on Mt Fuji. The number of sporocarps on these first-stage fungi increased monotonically with the increase of host size (Fig. 4a c). Sporocarps of L. murina and S. bovista appeared first in association with larger hosts (> 0.6 m 2 ) (Fig. 3), and usually in the area surrounding each vegetation patch. These fungi were usually accompanied by members of the first-stage fungi, indicating that L. murina and S. bovista were second-stage fungi in the sere of the ECM fungal succession. The number of sporocarps on these second-stage fungi also increased monotonically with host size development (Fig. 4d,e). With further increases in host size, Hebeloma spp. (> 1.2 m 2 ), Cortinarius spp. (> 2.1 m 2 ), Russula spp. (> 2.4 m 2 ) and Inocybe sp. 4 (> 2.4 m 2 ) appeared (Fig. 3). Sporocarps of these species were always observed inside each vegetation patch, where organic materials accumulated. These fungi were usually accompanied by the first- and second-stage fungi. Thus, species of Hebeloma, Cortinarius, Russula and Inocybe sp. 4 were relatively late-stage fungi in the sere of the ECM fugal succession in our early successional site. The number of sporocarps on these late-stage fungi also increased with host size development (Fig. 4f h). As new fungal species joined the existing fungal communities, the number of ECM sporocarp species increased along with host development (Fig. 5). Thirteen species were associated with the largest S. reinii. New Phytologist (2003) 158:

7 Research 199 Fig. 3 The recruitment of ectomycorrhizal fungal species with host size enlargement. Each mark represents sporocarp occurrence in relation to the associated host size. Only major fungal species are shown. The x-axis, shown logarithmically, indicates the area covered by Salix reinii in each vegetation patch. ECM sporocarp production related to host growth The biomass production of ECM sporocarps in each patch ranged from 0.27 g to 1208 g dry weight over 2 yr, and increased with host size (Fig. 6). In association with mid-sized hosts (2 10 m 2 ), however, sporocarp productivity varied greatly; several unproductive patches produced less than 1 g of sporocarp dry weight, and many productive patches produced more than 50 g. To determine the possible causes of this large variation, we chose nine patches that had mid-sized hosts for further investigation. The total dry weight of the ECM sporocarps in the nine selected patches ranged from 0.41 g to 264 g in 2000 (Table 3). No significant correlation between host size and sporocarp biomass was observed in these nine patches (Table 3). Even in the unproductive patches, a large part of the host root tips were ectomycorrhizae. Their mycorrhizal formation rates were not significantly different from those in productive patches (Table 3). In the most productive of the nine patches, sporocarp biomass recorded in 2000 amounted to 264 g, or 19% of the host leaf biomass. This was followed by patches 120 and 61, which had sporocarp biomass of 9% and 8% of the leaf biomass, respectively. The sporocarp production in the least productive patch was only 0.41 g, or 0.1% of the host leaf biomass, a ratio that was 140 times lower than that of the most productive patch. The host leaf biomass, the length of a current-year shoot, and the number of winter buds on a current-year shoot, were also significantly correlated with ECM sporocarp production (Table 3). Leaf N concentration of each host ranged from 1.8 ± 0.1% to 2.9 ± 0.1%, and was significantly higher in the productive patches than in the unproductive patches (Table 3). Leaf P concentration ranged from 0.10 ± 0.01% to 0.20 ± 0.02%, and was also significantly higher in the productive patches than in the unproductive patches (Table 3). Host photosynthetic rate ranged from ± to ± µmol CO 2 s 1 g 1 leaf dry weight, and was significantly higher in productive than in unproductive patches (Table 3). Furthermore, a significant correlation was confirmed between sporocarp production and the photosynthetic rate of their associated hosts in the nine patches (Table 3). The relationship between photosynthetic rate and leaf N concentration is shown in Fig. 7a. The photosynthetic rate of each leaf increased linearly with leaf N concentration, and the correlation was statistically significant. The leaf N concentration ranged from 1.62% to 3.19%, and was low in the unproductive hosts and high in the productive hosts. The photosynthetic rate also increased linearly with leaf P concentration, and the correlation was statistically significant (Fig. 7b). The P concentration of the productive hosts exceeded 0.15%, and photosynthetic rates were higher than 0.15 µmol CO 2 s 1 g 1 leaf dry weight. These values were clearly higher than for the leaves of the hosts with low and middle productivity. The species composition of ECM sporocarps was apparently different between the productive and unproductive patches (Table 3). Scleroderma bovista was recorded primarily New Phytologist (2003) 158:

8 200 Research New Phytologist (2003) 158:

9 Research 201 Fig. 5 The number of ectomycorrhizal species in relation to the associated host size. The x-axis, shown logarithmically, indicates the area covered by Salix reinii in each vegetation patch. The y-axis represents the number of confirmed ectomycorrhizal species in each vegetation patch in 2000 and The correlation is statistically significant (P < 0.01). in the productive patches and H. mesophaeum was relatively frequent in the unproductive patches, although the number of sporocarps in the latter was quite limited. Discussion Ectomycorrhizal fungal communities during early primary succession An alpine dwarf willow, S. reinii, was the exclusively dominant host species of ECM fungi in our early successional site on Mt Fuji, despite occupying less than 1% of the ground area. This alpine dwarf willow is usually distributed up to an elevation of 3000 m on Mt Fuji. Our research quadrat is situated on the south-east slope, where the effects of the last eruption are evident, and it is located at the upper limit of the species. B. ermanii and L. kaempferi also formed ectomycorrhizae and produced some sporocarps. However, the total ground area covered by both of these species was 0.003%, and their contribution to overall sporocarp production was negligible compared with that of S. reinii. In the present study, 23 ECM fungal species, representing sporocarps, were confirmed predominantly in Fig. 6 The biomass production of ectomycorrhizal sporocarps in relation to associated host size. The x-axis, shown logarithmically, indicates the area covered by Salix reinii in each patch. The y-axis, shown logarithmically, represents the total ectomycorrhizal sporocarp biomass produced in each vegetation patch in 2000 and The correlation is statistically significant (P < 0.01). association with S. reinii. Each species had a relatively short fruiting period and a different peak season. More than half of the annual sporocarp production by each fungal species was often counted during a single survey. Thus, weekly surveys throughout the entire fruiting season are essential to obtain an accurate account of sporocarp composition and productivity, as suggested by Vogt et al. (1992). Because our surveys covered the whole fruiting season, with measurements taken at weekly intervals, the fungal list obtained should reflect the genuine features of sporocarp production at our study site (Table 2). Long-year research is also recommended to assess fungal species (Vogt et al., 1992); however, this would greatly depend on site conditions. In our research site, the recorded species were almost same between each of the two years, and 2247 out of 2248 sporocarps belonged to the species that had been recorded in These results indicate that the two years would be enough for the evaluation of ECM sporocarp composition in our research site, probably because of the favorable environmental conditions for sporocarp formation in our research site. No other studies to date have described comparable fungal communities during early primary succession after a volcanic eruption. In another example of primary succession, Jumpponen et al. (1999, 2002) reported 13 ECM fungal species at the Fig. 4 The number of sporocarps of each ectomycorrhizal fungal species or group in relation to host size. The x-axis, shown logarithmically, indicates the area covered by Salix reinii in each vegetation patch. The y-axis represents the total number of sporocarps on each species recorded in each vegetation patch in 2000 and The R-values followed by * and ** indicate significant correlation at P < 0.05 and P < 0.01, respectively. (a) Inocybe lacera; (b) Laccaria laccata; (c) Laccaria amethystea; (d) Laccaria murina; (e) Scleroderma bovista; (f) Hebeloma spp.; (g) Cortinarius spp., (h) Russula spp. New Phytologist (2003) 158:

10 202 Research Table 3 Ectomycorrhizal sporocarp production in relation to various host growth parameters Mycorrhizal sporocarps in 2000 Species (number) (%) Mycorrhizal rate (% root tips) Leaf P concentration (%) Leaf N concentration (%) Winter bud Number (/CYS) Current year shoot (CYS) (cm) Photosynthesis ( 10 3 µmol CO 2 s 1 g 1 leaf dry wt) Leaf biomass (kg per patch) Host size (m 2 ) Sporocarp biomass (g) Patch No ± 9 a 24.5 ± 2.6 a 20.4 ± 2.3 a 2.9 ± 0.1 a 0.19 ± 0.01 ab 67.8 ± 4.8 a Sb(109) L(192) I(15) H(4) C(7) ± 10 ab 27.7 ± 1.2 a 18.2 ± 0.7 a 2.5 ± 0.2 abc 0.20 ± 0.02 a 73.2 ± 4.9 a Sb(60) L(10) H(7) ± 13 ab 11.4 ± 0.6 bc 10.4 ± 0.8 bc 2.7 ± 0.2 ab 0.16 ± 0.00 abc 67.2 ± 6.0 a Sb(36) L(16) I(1) C(1) ± 5 bc 14.2 ± 0.6 b 14.6 ± 1.0 ab 2.5 ± 0.1 abc 0.15 ± 0.01 bcd 54.1 ± 8.0 ab Sb(16) L(120) I(19) R(1) ± 2 d 6.1 ± 0.7 cd 6.8 ± 1.0 cd 2.0 ± 0.1 d 0.11 ± 0.00 cd 42.0 ± 10.9 ab Sb(3) L(4) B(1) ± 11 d 6.5 ± 1.1 cd 5.8 ± 0.6 cd 1.8 ± 0.1 d 0.11 ± 0.00 cd 59.3 ± 8.7 ab L(1) H(4) B(7) ± 13 cd 5.0 ± 0.9 cd 7.6 ± 1.1 cd 2.1 ± 0.1 bc 0.12 ± 0.01 cd 59.4 ± 2.6 ab L(1) I(1) H(4) C(1) ± 7 d 2.7 ± 0.4 d 4.2 ± 0.4 d 2.0 ± 0.1 d 0.12 ± 0.00 cd 54.1 ± 6.8 ab L(1) R(1) ± 6 d 3.0 ± 0.5 d 3.2 ± 0.8 d 2.0 ± 0.1 d 0.10 ± 0.01 d 28.7 ± 6.4 b H(3) r s * 0.912* 0.921* 0.912* * *Significant correlation at P < Each figure in columns 5 10 indicates a mean ± SEM. Figures followed by different letters within a column are statistically different by Tukey s HSD test (P < 0.01). r s is the Spearman s rank correlation coefficient paired with sporocarp biomass. Abbreviations for the mycorrhizal fungi are as follows: B, Boletus cf. rubellus; C, Cortinarius spp.; H, Hebeloma spp.; I, Inocybe spp., L, Laccaria spp.; R, Russula spp.; Sb, Scleroderma bovista. Fig. 7 The relationships between photosynthetic rate and the concentration of leaf nitrogen (a) and phosphorus (b) of Salix reinii with reference to the production of associated ectomycorrhizal sporocarps. Closed circles indicate leaves collected from patches in which ectomycorrhizal sporocarp production was relatively high, open triangles indicate those from patches with middle productivity, and open squares indicate those from unproductive patches. The R-values in (a) and (b) indicate statistically significant correlation (P < 0.01). front of the retreating Lyman Glacier. There, many ECM host species, including members of Abies, Alnus, Larix, Picea, Pinus, Salix and Tsuga, readily became established from adjacent ridges in a short period after the deglaciation. In our early successional site, S. reinii was exclusively dominant as the host plant, and had associations with at least 22 species of ECM fungi. Ectomycorrhizal fungi might be more diverse than previously thought during early primary succession. New Phytologist (2003) 158:

11 Research 203 Species of Cortinarius, Hebeloma, Inocybe, Laccaria and Russula dominate alpine and arctic dwarf willow stands (Graf, 1994; Gardes & Dahlberg, 1996) or S. repens stands (van der Heijden et al., 1999). Because a significant number of sporocarps of these genera were also observed at our site, different Salix species might have some similarities in ECM fungal communities. Although S. bovista, (as well as other Scleroderma species) has rarely been reported in association with Salix species, it was one of the dominant fungal species at our site, comprising 14.6% of the total number and 84.6% of the total dry weight of all ECM sporocarps. Species of Scleroderma are usually observed in disturbed areas, or on immature soil (Ingleby et al., 1985). The dominance of S. bovista would partly be due to the early successional conditions, as well as geographical factors and different host species. In the present study, we investigated the sporocarp community of ECM fungi in the early stage of primary succession. The presence of sporocarps indicated the underground presence of their ectomycorrhizae; however, the reverse is not always true. In many cases, an abundance of above-ground ECM fungi does not reflect an abundance of ectomycorrhizae underground (Horton & Bruns, 2001; Zhou & Hogetsu, 2002). The sporocarps obtained would be important keys to underground ECM communities, providing their DNA to be matched with ECM root tips. Succession of ECM sporocarps during early primary succession Ectomycorrhizal sporocarps were observed within each vegetation patch and its surrounding area. The vegetation patches were sparsely distributed on the volcanic desert, and therefore, individual associations between hosts and ECM sporocarps were clearly identified. Salix reinii grows to form a dwarf patch within a vegetation patch, and its coverage area increases by vegetative growth of each genet and recruitment of new genets (Lian et al., 2003). Thus, the coverage area is not only an index of its developmental stages but also an acceptable index of the periods after the first colonization. The periodic aerial photographs that show vegetation patch enlargement in the last 40 yr strongly support this. These features in our research site enabled us to investigate the succession of ECM sporocarps along with the developmental stages of a single host species, S. reinii, during early primary succession on Mt Fuji. In the sere of ECM sporocarps, the first-stage fungi (L. laccata, L. amethystina and I. lacera) were joined with the secondstage fungi (L. murina and S. bovista), and later the relatively late-stage fungi (Hebeloma spp., Cortinarius spp. and Russula spp.) appeared together with the first- and second-stage fungi. Small hosts (< 0.5 m 2 ) were always observed in a small area on the periphery of vegetation patches. The fungal communities associated with these small hosts were simple, being dominated by one or two first-stage fungi. Along with host size increment, S. reinii extends its root both inside and outside vegetation patches over several meters. The second-stage fungi were usually observed on the outsides of each patch, where no litter had accumulated. The relatively late-stage species were observed only inside the vegetation patches, where some litter had accumulated. The soil N content is known to increase with development of vegetation patches, accompanied by a shift from inorganic to organic N (Tateno & Hirose, 1987). The succession of ECM sporocarps may be driven by the diversification of soil conditions created by the host growth and the different preference among ECM fungi to the soil environment. The above successional model of ECM sporocarps was able to explain the absence of second-stage and relatively late-stage fungi in association with small hosts. However, this might also be explained by the lower probability of finding sporocarps in the small sampling area of small-size hosts, which was incomparably smaller than that of large-size hosts. To evaluate this possibility, we tested the null hypothesis that a fungal species (or group) is distributed evenly over the whole S. reinii coverage and the absence of a species results from the lower collecting chance there. In this hypothesis, the probability that a single sporocarp of a species is not observed in any of the small host is (1 A S /A T ), where A S is the total host area of the smallsize hosts, which are smaller than the smallest host producing sporocarps of this species, and A T is the total area of all hosts within the quadrat. Thus, if N is the total number of recorded sporocarps of this species during 2000 and 2001, the probability that no sporocarp appears in any of the small hosts is P species = (1 A S /A T ) N. Although P L. murina (0.22) and P Russula spp. (0.39) were greater than 0.05, P Cortinarius spp., P Hebeloma spp. and P S. bovista were , and 0.014, respectively, and less than Thus, at least, the sporocarp absence of the latter three fungal taxa in smaller hosts could not be explained simply by the lower probability of finding sporocarps in the small sampling area of these hosts at the P < 0.05 level of significance. The number of sporocarps of each major ECM fungal species or group monotonically increased with host size development, and every regression in Fig. 4 is statistically significant by the test of the regression (P < 0.05). In this figure, the x-value at which each regression line crosses with y = 1 is regarded as the expected smallest size in which sporocarps of each species are recorded (A E ). The A E of I. lacera, L. laccata, L. amethystina, L. murina, S. bovista, Hebeloma spp., Cortinarius spp. and Russula spp. are 0.000, 0.040, 0.158, 0.307, 0.001, 1.135, and m 2, respectively. Although the A E value of S. bovista is smaller than the values of two firststage fungi, the A E value of L. murina is larger than the values of all first-stage fungi and smaller than the values of the relatively late-stage fungi. The A E values of the relatively late-stage fungi are larger than the values of both the first-stage fungi and the second-stage fungi. Thus, the order of A E values of above fungal taxa except S. bovista is consistent with the New Phytologist (2003) 158:

12 204 Research observed sequential appearance of ECM sporocarps along with the host size development (Fig. 3). These A E results, combined with the P species results, would further support the ECM sporocarp succession model presented above. There was no indication of fungal replacement in the ECM sporocarp succession in our early successional site. Sporocarps of all major fungal taxa did not decrease and become more numerous with host size increment (Fig. 4). The ECM fungal succession in secondary successional sites usually consists of the decrease or replacement of some fungal species. Sporocarp observations around transplanted birch trees have demonstrated a clear serial change in sporocarp species; initially an early stage species, H. crustuliniforme, appears and is gradually decreased in number and replaced with the late-stage species, Lactarius, Cortinarius and Russula (Last et al., 1984). Similar replacements or decrease of ECM fungi in association with many other tree species have also been described (Dighton et al., 1986). Despite the limited information about the ECM fungal succession during early primary succession, the lack of fungal replacement is also noted on the forefront of the retreating Lyman Glacier (Jumpponen et al., 2002). Although the mechanisms of species replacement in fungal succession remain unclear, competition between ECM fungi could be a factor (Wu et al., 1999). The lack of species replacement of ECM fungi at our early successional site might indicate that there has been little or no competition in combination with limited inoculum supply of new competitors in a vast barren desert. Sporocarp production of ECM fungi during early primary succession The annual sporocarp biomass production by ECM fungi per hectare of S. reinii coverage was estimated to be 61.5 kg dry weight in 2000 calculated from all hosts in our quadrat. This index was raised to 633 kg calculated from the sporocarp production in the most productive host. No comparable data are available for other areas in early primary succession. The annual biomass production of epigeous sporocarps per alpine dwarf shrub coverage is kg ha 1 dry weight at alpine mire communities (Senn-Irlet, 1993). Sporocarp production of ECM fungi has been studied more in temperate forests, in which host trees have incomparably larger biomass than dwarf shrubs. Annual dry weight production of epigeous mycorrhizal sporocarps is kg ha 1 in Scots pine forests, kg ha 1 in spruce forests, and kg ha 1 in Pacific silver fir forests (Vogt et al., 1992). Thus, the figure estimated for ECM sporocarps associated with S. reinii is exceptionally large. This large sporocarp production suggests huge production of spores by species compatible with S. reinii and tolerant of the severe environmental conditions there. In early primary succession after volcanic eruption, colonizable ECM spores from distant forests are quite limited (Allen et al., 1992). Therefore, the large sporocarp production in our research site would be indispensable to improve ectomycorrhiza formation on incoming and existing S. reinii by supplying a huge number of colonizable spores on-site. In the most productive of the nine patches investigated intensively, annual sporocarp production amounted to 264 g dry weight (i.e. 19% of the host leaf biomass) (Table 3). This figure is surprisingly large in comparison with sporocarp production in other forest stands; for example, epigeous sporocarp production was 0.2% and 0.1% of leaf biomass in a 23-yr-old and a 180-yr-old Abies amabilis forest, respectively (Vogt et al., 1982). The biomass of hyphae and mycorrhizal sheaths is hundreds of times larger than the sporocarp biomass, and the turnover of fungal components is relatively rapid (Forgel & Hunt, 1979; Vogt et al., 1982). Thus, the fungal contribution to carbon and nutrient cycling even in those forest ecosystems is large despite such small proportions of sporocarps relative to the total forest biomass (Vogt et al., 1982). Our results suggest that the contribution of mycorrhizal fungi to carbon and nutrient cycling during early primary succession could be much greater than previously thought. There is a large variation in photosynthetic rates of S. reinii, ranging from ± to ± µmol CO 2 s 1 g 1 leaf dry weight among nine similar-size hosts (Table 3). The ECM sporocarp biomass production of the highest photosynthesizing host was 264 g, which was 644 times greater than that of the lowest one, and the biomass production of ECM sporocarps was significantly correlated with the photosynthetic rates among similar-size hosts (Table 3). Ectomycorrhizal sporocarp production appears to be determined by host photosynthetic rate even during early primary succession, as demonstrated in vitro (Lamhamedi et al., 1994) and in forest stands (Last et al., 1979; Högberg et al., 2001). In addition to the ECM biomass production, the species composition of ECM fungi was apparently different between actively photosynthesizing hosts and low-photosynthesizing hosts (Table 3). This may indicate that photosynthate supply to ECM fungi influences ECM sporocarp communities. Scleroderma bovista was dominant in actively photosynthesizing hosts, but not in low-photosynthesizing hosts. Because the habitats of S. bovista have no litter accumulation and an extremely low organic matter in soil, its carbon appears to be mostly derived from host photosynthates. A sporocarp of S. bovista was exceptionally large (e.g. 75 times heavier than that of L. laccata in dry weight) and it would need a lot of photosynthates for fruiting. The low-photosynthesizing hosts could not support the sporocarp formation of this species. Although the number of sporocarps was small in low-photosynthesizing hosts, Hebeloma spp. and B. cf. rubellus, which mainly appeared on relatively humus-rich soil inside vegetation patches, were relatively dominant. These species might have the ability to use other carbon sources in addition to the photosynthates of S. reinii. The photosynthetic rate of each S. reinii leaf increased linearly with leaf N and P concentration (Fig. 7). This result New Phytologist (2003) 158:

13 Research 205 indicates that leaf nutrient status would be the main limiting factor of the photosynthetic activity of S. reinii, taking account of our research site conditions, such as the sufficient rainfall, mild temperature and enough sunlight in growth seasons. Furthermore, the leaves having higher photosynthetic rates with higher concentrations of N and P distinctly belonged to the hosts that produced larger amounts of sporocarps (Fig. 7). It is supposed that nutrient supply from ECM fungi is essential to active photosynthesis of hosts, and conversely enough photosynthate supply from hosts is indispensable for the activity of ECM fungi. Our results may indicate that the magnitude of such bidirectional interaction could determine the activity of both symbionts in each ECM association and, as a result, be expressed in its ECM sporocarp production. Acknowledgements This work was supported in part by a grant from PROBRAIN and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Adachi N, Terashima I, Takahashi M Central die-back of monoclonal stands of Reynoutria japonica in an early stage of primary succession on Mount Fuji. Annals of Botany 77: Allen EB, Allen MF Competition between plants of different successional stages: mycorrhizae as regulators. Canadian Journal of Botany 62: Allen MF, Crisafulli C, Friese CF, Jeakins SL Re-formation of mycorrhizal symbioses on Mount St Helens, : Interactions of rodents and mycorrhizal fungi. Mycological Research 96: Brandrud TE, Lindström H, Marklund H, Melot J, Muskos S Cortinarius flora photographica. Matfors, Sweden: Cortinarius HB. Breitenbach J, Kränzlin F Fungi of Switzerland, 3. Lucerne, Switzerland: Mykologia Lucerne. Breitenbach J, Kränzlin F Fungi of Switzerland, 5. Lucerne, Switzerland: Mykologia Lucerne. Deacon JW, Donaldson SL, Last FT Sequences and interactions of mycorrhizal fungi of birch. Plant and Soil 71: Dighton J, Poskitt JM, Howard DM Changes in occurrence of basidiomycete fruit-bodies during forest stand development: with specific reference to mycorrhizal species. Transactions of the British Mycological Society 87: Fleming LV Succession of mycorrhizal fungi on birch: Infection of seedlings planted around mature trees. Plant and Soil 71: Fleming LV, Deacon JW, Last FT, Donaldson SJ Influence of propagating soil on the mycorrhizal succession of birch seedlings transplanted to a field site. Transactions of the British Mycological Society 82: Forgel R, Hunt G Fungal and arboreal biomass in a western Oregon Douglas-fir ecosystem: distribution patterns and turnover. Canadian Journal of Forest Research. 9: Gardes M, Dahlberg A Mycorrhizal diversity in arctic and alpine tundra: an open question. New Phytologist 133: Graf F Ecology and sociology of macromycetes in snow-beds with Salix herbacea L. in the alpine Valley of Radönt (Grisons, Switzerland). Dissertationes Botanicæ 235: Guzman G Monografía del género Scleroderma Pers. emend. Fr. Darwiniana 16: Hansen L, Knudsen H Nordic Macromycetes, 2. Copenhagen, Denmark: Nordsvamp. Hansen L, Knudsen H Nordic Macromycetes, 3. Copenhagen, Denmark: Nordsvamp. van der Heijden EW, de Vries FW, Kuyper ThW Mycorrhizal associations of Salix repens L. communities in succession of dune ecosystems. I. Above-ground and below-ground views of ectomycorrhizal fungi in relation to soil chemistry. Canadian Journal of Botany 77: Helm DJ, Allen EB Vegetation chronosequence near Exit Glacier, Kenai Fjords National Park, Alaska, USA. Arctic and Alpine Research 27: Helm DJ, Allen EB, Trappe JM Mycorrhizal chronosequence near Exit Glacier, Alaska. Canadian Journal of Botany 74: Hendrix JW, Hunt CS, Maronek DM Relationship between the ectomycorrhizal fungus Pisolithus tinctorius associated with loblolly pine and acid-generating Thiobacillus spp. on an acidic strip mine site. Canadian Journal of Microbiology 31: Hirose T, Tateno M Soil nitrogen patterns induced by colonization of Polygonum cuspidatum on Mt Fuji. Oecologia 61: Högberg P, Nordgren A, Buchmann N, Taylor Andrew FS, Ekblad A, Högberg Mona N, Nyberg G, Ottosson Löfvenius M, Read David J Large-scale forest girdling shows that current photosynthesis drives soil respiration. Nature 411: Horton TR, Bruns TD The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology 10: Ingleby K, Last FT, Mason PA Vertical distribution and temperature relations of sheathing mycorrhizas of Betula spp. growing on coal spoil. Forest Ecology and Management 12: Jumpponen A, Trappe JMC, Cázares E Ectomycorrhizal fungi in Lyman Lake Basin: a comparison between primary and secondary successional sites. Mycologia 91: Jumpponen A, Trappe JMC, Cázares E Occurrence of ectomycorrhizal fungi on the forefront of retreating Lyman Glacier (Washington, USA) in relation to time since deglaciation. Mycorrhiza 12: Lamhamedi MS, Godbout C, Fortin JA Dependence of Laccaria bicolor basidiome development on current photosynthesis of Pinus strobus seedlings. Canadian Journal of Forest Research 24: Lannoy G, Estades A Monographie des Leccinum d Europe. Paris, France: Chevallier Imprimeurs. Last FT, Pelham J, Mason PA, Ingleby K Influence of leaves on sporophore production by fungi forming sheathing mycorrhizas with Betula spp. Nature 280: Last FT, Mason PA, Ingleby K, Fleming LV Succession of fruitbodies of sheathing mycorrhizal fungi associated with Betula pendula. Forest Ecology and Management 9: Lian C, Oishi R, Miyashita N, Nara K, Nakaya H, Zhou Z, Wu B, Hogetsu T Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mt. Fuji, as revealed by nuclear and chloroplast microsatellite analysis. Molecular Ecology (in press.) Mason PA, Last FT, Pelham J, Ingleby K Ecology of some fungi associated with an ageing stand of birches (Betula pendula and B. pubescens). Forest Ecology and Management 4: Mason PA, Wilson J, Last FT, Walker C The concept of succession in relation to the spread of sheathing mycorrhizal fungi on inoculated tree seedlings growing in unsterile soils. Plant and Soil 71: Massicotte HB, Melville LH, Peterson RL, Luoma DL Anatomical aspects of field ectomycorrhizas on Polygonum viviparum (Polygonaceae) and Kobresia bellardii (Cyperaceae). Mycorrhiza 7: Masuzawa T Ecology of the alpine plants. Tokyo, Japan: University of Tokyo Press. Miller FM Some occurrences of vesicular-arbuscular mycorrhiza in New Phytologist (2003) 158: