American Journal of Botany 88(10): 18131817. 2001.

PERSISTENCE

OF SEED BANK UNDER THICK VOLCANIC

DEPOSITS TWENTY YEARS AFTER ERUPTIONS OF

MOUNT USU, HOKKAIDO ISLAND, JAPAN1

SHIRO TSUYUZAKI2
AND

MASAKI GOTO

Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810 Japan The topsoil that contained the seed bank became buried under thick tephra after the eruptions of Mount Usu during 1977 and 1978. To determine the seed bank potential of the topsoil 20 yr after the eruptions, i.e., in 1998, 408 100-cm3 samples were excavated under 115185 cm of volcanic deposits. The topsoil was collected at 10-cm intervals along the horizontal scale and was divided into a 0 5 cm deep upper layer and a 510 cm deep lower layer. The seed bank was estimated by both the germination (GM) and otation (FM) methods. In total, 23 species with an average seed density of 1317 seeds/m2 were identied by GM, and 30 species with a density of 2986 seeds/m2 were extracted by FM. The dominant species was Rumex obtusifolius, and perennial herbs, such as Carex oxyandra, Viola grypoceras, and Poa pratensis, were common. For nine species this study provided the rst records for eld seed longevity 20 yr. The seed density in the upper layer was double that in the lower layer, and the horizontal distribution was heterogeneous even at 10-cm intervals. We concluded that the seed bank has retained the original structure of the seed bank under the tephra and will persist longer with soil water content between 20 and 40%, no light, and low temperature uctuations ( 0.17 C of standard deviation in a day). Key words: buried seed populations; former topsoil; long seed longevity; Mount Usu; seed bank.

Seed longevity in nature is an important determinant of plant community dynamics because the wide range of seed dormancy strategies has evolved to adapt to various environmental changes and to maintain populations (Thompson and Grime, 1979). Furthermore, species with longer lived seeds have lower local extinction rates (Stocklin and Fischer, 1999). There have been numerous studies conducted to detect seed longevity using experimental seed burial tests (Leck, Parker, and Simpson, 1989; Baskin and Baskin, 1998). Many seed longevity experiments have been conducted under somewhat unnatural conditions. For example, seeds collected from elds and placed into milk bottles for 100 yr would experience altered soil moisture and related factors when buried and periodically tested (Kivilaan and Bandruski, 1981). Evaluation of seed banks buried under natural conditions, e.g., volcanic ash, would provide more realistic data (Whittaker, Partomihardjo, and Riswan, 1995). The seed bank buried under thick volcanic deposits persisted for 10 yr after the 19771978 eruptions on Mount Usu, northern Japan (Tsuyuzaki, 1991). This seed bank had been conserved because predators were few, the movements of seeds by erosion and animal carriers rare, and contamination from the vegetation did not occur due to thick volcanic deposits. Therefore, this seed bank provides a chance for the long-term monitoring of buried seed populations under natural conditions, and this paper reports the seed bank status 20 yr after the burial. STUDY AREA AND METHODS
Mount Usu is located on Hokkaido Island (42 32 N, 140 50 E, 727 m in altitude in 1978) and is one of the most active volcanoes in Japan. The volManuscript received 14 September 2000; revision accepted 8 March 2001. The authors thank K. Narita and Y. Yamada for eld assistance; H. Okada and all staff members of Usu Volcano Observatory for providing facilities; Muroran Forest Management Ofce for permission to conduct research in a restricted area; and M. A. Leck and E. O. Guerrant Jr. for their critical reading of the manuscript. Funds for this study were partly provided by the Ministry of Education, Science, and Culture of Japan. 2 Author for reprint requests (e-mail: tsuyu@ees.hokudai.ac.jp).
1

cano is composed of two peaks, O-Usu (727 m) and Ko-Usu (609 m), enclosed by a caldera rim and crater basin. Before the 19771978 eruptions, the vegetation was dominated by broad-leaved forests consisting mostly of Populus maximowiczii and Betula platyphylla var. japonica and articial meadows represented by Dactylis glomerata and Trifolium repens (Hara, 1978). Due to the thick volcanic ash deposits in 19771978, vegetation was completely destroyed on the summit area. Soon after the eruptions, vegetation recovery began as the result of vegetative reproduction, seeds in the former topsoil, seed immigration, and articial seeding (Tsuyuzaki, 1987). Observations showed that seedlings of specic species were conspicuous in the gully where the former topsoil was exposed but did not appear in areas without the former topsoil (Tsuyuzaki, 1989). These seedlings apparently originated from the seed bank present in the former topsoil. Ten yeas after the eruptions, soil blocks were collected from the former topsoil that was buried 65140 cm under the volcanic deposits. The seed bank consisted of at least 25 species of which seed density was 2000 seeds/m2, and the dominant species was the nonnative Rumex obtusifolius (Tsuyuzaki, 1991). In June 1998, we excavated four sites and collected 408 100-cm3 topsoil samples from the crater basin. To avoid the contamination of fresh seeds from the ground surface owing to the movements of volcanic deposits, gullies and adjacent areas were not selected for the excavations. Sites were resultingly 1030 m from each other. When the ground surface of the former topsoil was reached, the thickness of volcanic deposits was measured. Dedpending on the conditions of volcanic deposits, the sizes of quadrats were determined as 40 40 cm (two quadrats) or 50 50 cm (two quadrats). The quadrat was divided into 10 10 cm subquadrats. Two soil samples were collected from the upper layer (05 cm deep) in each subquadrat, and then two soil samples were taken from the lower layer (510 cm). In each layer, the two samples were collected from upper-left and lower-right corners of each subquadrat to link upper and lower layers. One sample was used to test germination and another for the otation test. Each soil sample was 20 cm2 in surface area and 5 cm in depth by a soil tin. Due to the collapse of one pit during the collection of soils in a 50 50 cm quadrat, we did not collect samples from the lower layer in one quadrat. Owing to this, we used the samples collected from both layers for the sum total of upper and lower layers. In each site, three 100-cm3 topsoil samples were also collected to measure water content. To estimate the species composition and seed density, two methods were used: the germination method (GM) and the otation method (FM). Nomenclature follows Ohwi and Kitagawa (1983). The germination method was

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1 SD. A few unidentied species

TABLE 1. Seeds detected by germination and otation methods. Values indicate mean seed density (no./m2) might have been germinated using both methods.
Germination Species Life form Upper Lower Upper

Flotation Lower

Total number of species Rumex obtusifolius (N) Carex oxyandra Viola grypoceras Poa pratensis (N) Betula spp. Juncus effusus var. decipiens Carex sp. Aralia cordata Geum macrophyllum var. sachalinense Rumex acetosella (N) Hypericum erectum Trifolium repens (N) Hydrocotyle ramiora Cerastium fontanum Taraxacum ofcinale (N) Epilobium cephalostigma Sagina japonica Youngia japonica Eragrostis multicaulis Erigeron annuus (N) Ranunculus repens Luzula capitata Unidentied Total seeds No. species/sample Total species

P P P P W P P P P P P P P P P P A A A A P P

17 659 49 24 55 85 18 24

14* 6NS 2NS 2NS 4* 2NS 1ND

15 180 16 8 8 16 25 41 8 8 82 8 8

6 2 1 1 2 2 2 1 1 8 1 1

* 24 2NS 6 1ND * 6 1ND 6 1ND 6 1ND ND 6 1ND 6 1ND 6 1ND ND 12 1NS(2) 994 16* 0.98 0.99* 17

8 1 16 2(2) 434 13 0.66 0.96 15

25 1055 18* 177 6* 61 2NS 43 2NS 91 3* ND 18 2ND 18 2ND 12 1ND 6 1ND * 49 2* 61 3* 37 2* 970 29*(14) 2598 47* 2.09 2.25* 26

18 467 22 57 3 49 3 16 2 16 2 6 1 8 1 66 3 402 21(10) 1082 39 0.93 1.28 18

Note: Under life form, A annuals (including biennials), P perennials, and W trees. Number of unidentied species is shown in parentheses. A dash indicates that no seeds were detected. N 82 for the upper layer, and N 61 for the lower layer. An asterisk indicate number of seeds were signicantly different between upper and lower layers at P 0.05 (binomial test after Bonferroni corrections). NS: not signicant, ND: not determined, N: nonnative species.

conducted in a greenhouse in the Faculty of Science, Hokkaido University, Sapporo, Japan, immediately after the soil collections. The soils were sprayed over vermiculite in a 1 cm thick layer (except for large volcanic particles contained therein) in a pot (25 20 cm in surface area, 10 cm in depth). The observations were continued for 5 mo until germination was no longer observed. After the species could be identied, the seedlings were clipped out. Seedlings that could not be identied were transplanted to another pot and grown until identication could be made. For FM samples, we used a centrifuged otation method following (Tsuyuzaki, 1994). The soil samples were agitated with 50% K2CO3 otation solution (1.54 g/cm3). The mixture was centrifuged ( 4000 g) for 45 min, and then nearly all organic debris oated. All organic debris was decanted and ltered with two layers of miracloth (Calbiochem, California). The seeds were rinsed with distilled water and kept in a refrigerator at 5 C until used. The seeds were identied by morphological traits using voucher seed collections. After the identication, the length, width, and thickness of seeds were measured using a binocular stereomicroscope. The seed volume was evaluated as if the shape were oval sphere. Any seeds that could not be identied by morphological traits were germinated in an incubator at 15 /25 C (12 h/12 h). The viability of seeds extracted by the otation test was not estimated by tetrazolium tests because of overestimation due probably to the staining of bacteria and/or fungi (Tsuyuzaki, 1991). Instead, the viability was estimated from their rmness and intact appearance using a seed-crushing technique, i.e., the albumen of seeds crushed by a needle was not juicy and/or became brown; those seeds were considered to have died (Naka and Yoda, 1984; Tsuyuzaki, 1991). To estimate the spatial heterogeneity of the seed bank, the vertical distribution of the number of seeds was estimated by Wilcoxons binomial test after Bonferroni corrections (Zar, 1996). Coefcients of variation (CV) were calculated to investigate the spatial heterogeneity of seed density (Thompson,

1986). When CV is high, the distribution is contagious and heterogeneous. To determine the difference of detection sensitivity between GM and FM, Spearmans rank correlation coefcients were obtained on the total number of seeds and species.

RESULTS The thickness of volcanic deposits over seed bank sample sites ranged from 115 to 185 cm. The surface of the former topsoil was clearly dened, indicating that the surface of the former topsoil had not been disturbed. A few ant nests were established on the ground surface but were 50 cm deep. Visible animals were not observed in 50 cm deep volcanic deposits. Therefore, seeds produced by the present vegetation could not percolate into the former topsoil. Water content in the former topsoil ranged from 22.3% to 36.4%. In total, 23 and 30 species were detected by GM and FM, respectively (Table 1). Twenty-two species were identied, of which six were nonnative. There were 1.31 0.17 species/m2 by GM and 2.87 0.36 species/m2 by FM in the 10 cm deep layer. Mean number of seeds or seedlings per sample was 2.13 0.30 according to the GM and 8.25 1.49 according to the FM. Densities of seedlings and seeds were estimated to be 1317 469 individuals/m2 and 2986 1996 individuals/m2 as determined by GM and FM, respectively. Spearmans rank correlation coefcients were 0.423 on number of seeds between GM and FM (signicant at P 0.01, N 61) and 0.462 on number of species (P 0.01). Therefore, the two methods were roughly comparable, although total number of

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Fig. 1. Spatial distribution of the total number of seeds in a 50 50 10 cm quadrat of which density was the highest in the four quadrats surveyed. The sizes of circles indicate that number of seeds (otation method) or seedlings (germination method) in each 10 10 cm subquadrat. The maximum circle indicates 52 seeds. The numeral within each box represents the number of species.

seeds detected by FM was signicantly higher than by GM. Rumex obtusifolius was dominant and accounted for 40% of the total number of individuals. Perennial herbs such as Carex oxyandra, Viola grypoceras, and Poa pratensis were common. Four annuals were germinated, but their density was very low, and only one woody species was detected. The habitat preferences of the species present showed a wide array of habitats: open forest (e.g., Betula spp. and Aralia cordata), grassland (Rumex obtusifolius and Poa pratensis), and wet sites (Ranunculus repens and Juncus effusus var. decipiens). Using the FM, the vertical distribution showed that the upper layer contained more species and seeds (Table 1). Two species, Hypericum erectum and an unidentied species, were distributed only in the lower layer. The seed sizes ranged from 0.03 to 2.75 mm3 for all 30 species detected by FM. Hypericum erectum had the smallest seeds, and the unidentied species had the second smallest seeds. Species of which the seed volume was 0.15 mm3 represented 5.4% of the total seeds in the upper layer, while they accounted for 22.7% in the lower layer, indicating that smaller seeds tended to be distributed more in the lower layer. In contrast, species of which the seed volume was 1.05 mm3, i.e., three unidentied species, were detected in the upper layer only. Horizontal distribution of total seeds showed high spatial heterogeneity (Fig. 1). The CV was 2.56 on GM and 16.5 on FM for total number of seeds, and CV of R. obtusifolius, the dominant species, showed 2.15 on GM and 7.05 on FM (Table 2), indicating that the spatial heterogeneity was very high and
TABLE 2. Coefcients of variation (CV) on number of seeds buried in the former topsoil. N: number of samples.
Layer Germination method Flotation method

clump sizes were irregular. The CV in the upper layer was higher than in the lower layers. DISCUSSION Because the site locations of the present survey were 50 m distant from the survey sites used for the study 10 yr after the eruptions, an exact comparison is not possible. However, the dominant species was the same, i.e., Rumex obtusifolius, and the seed number and density did not differ greatly between 10 yr (Tsuyuzaki, 1991) and 20 yr after the eruptions (Table 1). Most seed banks have been shown to be heterogeneously distributed due to microtopography difference (Thompson, 1986; Bigwood and Inouye, 1988). More seeds were distributed in the upper layers of soil as shown in references in other studies (e.g., Kramer and Johnson, 1987), and thus CV is lower in the lower layers (Kellman, 1978). However, species that produced the smallest seeds were detected only in the lower layers, perhaps because smaller seeds are more easily moved to the lower layer (Fenner, 1985). Requirements for seed germination include temperature, temperature uctuation, light, etc. While seed germination for many species is enhanced by daily temperature uctuations (Baskin and Baskin, 1998), the standard deviation of temperature uctuation for a single day was 0.23 C under 50 cm of ash on Mount Usu (Tsuyuzaki, 1991). Also, the mean daily temperature was 15 C in August, and light could not penetrate to the former topsoil. Wet soil occasionally maintains seed germination ability longer than dry soil (Toole and Toole, 1953; Lewis, 1973). The soil was wet and the temperature low on Mount Usu (Tsuyuzaki, 1991), and thus the activity of bacteria was considered to be low. Therefore, the conditions of wet soil, no light, and low temperature uctuation of the former topsoil may be adequate for seed survival for many species. The seed density was similar to the active seed bank observed in the meadow in Tohoku District, Japan (Hayashi and

Upper (N 82) Lower (N 61) Upper and lower (N

61)

3.43 2.97 2.56

11.5 10.3 16.5

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TABLE 3. Previous reports on species that were found in the former topsoil buried underneath volcanic deposits from Mount Usu, northern Japan, 20 yr after the 19771978 eruptions.
Species Longevity

Betula spp. Cerastium fontanum Erigeron annuus Geum macrophyllum var. sachalinense Juncus effusus var. decipiens Poa pratensis Ranunculus repens Rumex obtusifolius Rumex acetosella Taraxacum ofcinale Trifolium repens Viola grypoceras

5 ( B. maximowiciana, Osumi and Sakurai, 1997) 10 (Tsuyuzaki; 1991), 20 (Poschlod and Jordan, 1992)* 90 (Roberts and Vankat, 1991) 10 (Tsuyuzaki, 1991) 3073 (Granstrom, 1988) 10 (Tsuyuzaki, 1991), 39 (Toole and Brown, 1946) 10 (Tsuyuzaki, 1991), 20 (Lewis, 1973) 10 (Tsuyuzaki, 1991), 39 (Toole and Brown, 1946) 10 (Tsuyuzaki, 1991) 90 (Roberts and Vankat, 1991) 10 (Tsuyuzaki, 1991), 30 (Toole and Brown, 1946), 90 (Roberts and Vankat, 1991) 10 (Tsuyuzaki, 1991)

* Paper not directly referred (cited in Thompson, Bakker, and Bekker, 1997).

Numata, 1971) and abandoned pastures in Hokkaido Island, northern Japan (Tsuyuzaki and Kanda, 1996). Those seed banks are persistent (Grime, 1979). Species composition in the abandoned pasture resembles the seed bank in the former topsoil of Mount Usu, i.e., there are Poa pratensis, Trifolium repens, Cerastium fontanum, Viola grypoceras, Rumex obtusifolius, Rumex acetosella, and Geum macrophyllum var. sachalinense. Most seeds are small, i.e., 12 mm long, in the study of Tohoku District. Nonnative species seem to be common in the various seed banks of seminatural vegetation, such as abandoned meadows and pastures, in Japan. These patterns, i.e., species composition, seed size, density, and spatial patterns of seed bank, appear to have been retained in the seed bank under the volcanic deposits 20 yr after the burial and indicate that conditions are optimal for seed storage and the seed bank will persist longer. Of species detected in the present study, seeds that have been reported as long lived, i.e., over 20 yr, under (semi-)natural conditions are (Table 3): R. obtusifolius, Trifolium repens, Poa pratensis, Taraxacum ofcinale, Ranunculus repens, Juncus effusus var. decipiens, and Erigeron annuus. Except for E. annuus and J. effusus var. decipiens, all of these species were also extracted from the former topsoil on Mount Usu 10 yr after the eruptions. Additional species were detected both 10 and 20 yr after the eruptions on Mount Usu; these were Rumex acetosella, Cerastium fontanum, Viola grypoceras, and Geum macrophyllum var. sachalinense (Table 3). The other nine species have not been recorded for seed longevity, or the longevity was reported as 20 yr: Carex oxyandra, Aralia cordata, Hypericum erectum, Hydrocotyle ramiora, Epilobium cephalostigma, Sagina japonica, Youngia japonica, Eragrostis multicaulis, and Luzula capitata. Of those, the longevity of closely related species are reported as: Hypericum histum and H. perforatum 5 yr (Granstrom 1987; Osumi and Sakurai, 1997), and Viola arvensis 20 yr (Chapman and Anderson, 1987). The seeds of Sagina decumbens, Luzula parviora, and L. campestris survive for more than a few decades in various regions (Thompson, Bakker, and Bekker, 1997). This suggests that phylogenetic constraints may exist on seed longevity. Seeds and resulting seedlings might be used to determine how the genetic heterogeneity of the seed bank changes under natural storage conditions. LITERATURE CITED
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THOMPSON, K., AND J. P. GRIME. 1979. Seasonal variation on the seed banks of herbaceous species in ten contrasting habitats. Journal of Ecology 67: 893921. TOOLE, E. H., AND E. BROWN. 1946. Final results of the Duvel buried seed experiment. Journal of Agricultural Research 72: 201210. TOOLE, V. K., AND E. H. TOOLE. 1953. Seed dormancy in relation to seed longevity. Proceedings of International Seed Testing Associations 18: 325328. TSUYUZAKI, S. 1987. Origin of plants recovering on the volcano Usu, northern Japan, since the eruptions of 1977 and 1978. Vegetatio 73: 5358. TSUYUZAKI, S. 1989. Analysis of revegetation dynamics on the volcano Usu, northern Japan, deforested by 19771978 eruptions. American Journal of Botany 76: 14681477.

TSUYUZAKI, S. 1991. Survival characteristics of buried seeds 10 years after the eruption of the Usu volcano in northern Japan. Canadian Journal of Botany 69: 22512256. TSUYUZAKI, S. 1994. Rapid seed extraction from soils by a otation method. Weed Research 34: 433436. TSUYUZAKI, S., AND F. KANDA. 1996. Revegetation patterns and seedbank structure on abandoned pastures in northern Japan. American Journal of Botany 83: 14221428. WHITTAKER, R. J., T. PARTOMIHARDJO, AND S. RISWAN. 1995. Surface and buried seed banks from Krakatau, Indonesia: implications for the sterilization hypothesis. Biotropica 27: 346354. ZAR, J. H. 1996. Biostatistical analysis, 3rd ed. Prentice Hall, Englewood Cliffs, New Jersey, USA.