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Serpentine Plants in Hokkaido and their Chemical Characteristics
Takafumi Mizuno, Kenji Horie, Shiro Nosaka, Hitoshi Obata, and Naoharu Mizuno

Northeastern Naturalist, Volume 16, Special Issue 5 (2009): 65–80

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Soil and Biota of Serpentine: A World View 2009 Northeastern Naturalist 16(Special Issue 5):65–80 Serpentine Plants in Hokkaido and their Chemical Characteristics Takafumi Mizuno1,*, Kenji Horie2, Shiro Nosaka3, Hitoshi Obata1, and Naoharu Mizuno4 Abstract - In the serpentine area in Hokkaido, 46 taxa of serpentine plant species were recognized, and 44 of them were endemic to Hokkaido. The P concentration in the serpentine plants was lower, while the concentrations of K, Ca, and N were higher, than those in nonserpentine plants and trees. The Ni concentration of the serpentine plants increased proportionally to that of the exchangeable Ni concentration in the soil up to 10 mg kg-1 soil, but did not increase further. Among the plants investigated, a nonserpentine plant, Thlaspi japonicum, was recognized for its extraordinary Ni accumulation (1300 mg kg-1 on average), indicating that this plant is the first Ni-hyperaccumulator identified in Japan. Introduction Serpentine regions are widespread in Japan (Fig. 1A). Especially in Hokkaido, the northern part of Japan, serpentine outcrops and soils are found along the central mountain chain extending from north to south. It is well known that serpentine soils contain excessively high Mg and Ni concentrations and can cause Ni toxicity in plants, and that plants which grow there are forced to tolerate these severe conditions (Hunter and Vergnano 1952). Similar to the serpentine areas in other countries, unique plant floras in Hokkaido serpentine regions have attracted many researchers for a long time (Hara, 1934–1939; Inagaki et al. 1966–1969; Nosaka 1960–1962, 1969, 1974; Nosaka and Horie, 1993, 1994; Toyokuni 1955–1960). However, only a very small number of investigations have included analyses of chemical characteristics of serpentine plants (Horie et al. 2000, Yamagata et al. 1960). In this paper, we summarize Dr. Horie Kenji’s series of studies on the serpentine floras in the Hokkaido area. His 39-year-long extensive research includes statistical data of serpentine floras and analyses of their chemical components. Field-site Description There are two metamorphic belts along the N–S axis in Hokkaido. One is a serpentine-rich Kamuikotan metamorphic belt in the north, and the second is a periodite-rich Hidaka metamorphic belt in the south. As shown in Figure 1B, the serpentine rocks in these belts are located 100 to 1900 m above sea 1Graduate School of Bioresources, Mie University, Tsu, Mie, 514-8507 Japan. 2Northern Wild Plants Garden, Asahikawa, Hokkaido, 071-1248 Japan. 3Aichi University of Education, Kariya, Aichi, 448-8542 Japan. 4Rakuno Gakuen University, Ebetsu, Hokkaido, 069-8501 Japan. *Corresponding author - tmizuno@bio.mie-u.ac.jp. 66 Northeastern Naturalist Vol. 16, Special Issue 5 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 67 level from 42 to 45ºN latitude, 142 to 143ºE longitude. For the purpose of clarifying the distribution of serpentine plants, we divided the serpentine areas on Kamuikotan belt into five districts: northern areas (Toikanbetsu, Mt. Shirikoma, Nakatonbetsu, Otoineppu), central areas (Horokanai, Mt. Shiratori, Shibetsu, Wassamu, Asahikawa-Kamuikotan, Furano), southern areas (Hobetsu, Mt. Bouzu, Shimekappu, Hidaka), Mt. Yupari areas (Mt. Yupari, Mt. Furanonishi), and Mt. Tottabetsu areas (Mt. Tottabetsu, Mt. Chiroro); and the Hidaka belt into two: Mt. Apoi areas (Mt. Apoi), and Shizunai areas (Shizunai and Mitsuishi). We examined these regions for their serpentine vegetation (Horie et al. 2000). From October to April, these areas are covered with snow, and snow remains until June in high mountain areas. Methods For the floristic survey and taxonomy, works by Kitamura (1993), Nosaka (1960–1962, 1969, 1974), and Nosaka and Horie (1993, 1994) were cited. Plant samples were collected at their blooming time, and only their leaves were used for chemical analysis. Dried samples were digested with a mixture of sulfuric acid and hydrogen peroxide, then five methods were applied to determine the concentration of sixteen atoms or ions in the sample solution: atomic absorption spectrometry (Hitachi 180-50 spectrometer, Hitach, Japan) (Na, K, Mg, Ca, Fe, Mn, Zn, Cu, Ni, Cr, Al, and Ti); Kjeldahl method (N); molybdenum blue method (P); AAS with methylisobutylketone-abstraction of dimethyldithiocarbamic acid-complex (Co); and isopropyl ether extraction- 475nm absorption measurement of thiocyanic acid-treated sample (Mo). Samples of serpentine rocks from 30 locations and soils from 75 locations of serpentine areas in Hokkaido were collected at a point within 10 cm from the sampled plants (Horie 2002). Soil samples were collected from the land surface (upper 15 cm) or around the plant roots on the rocks, then passed through 2-mm mesh and pounded in a mortar, and passed again through 1.2- mm mesh. Rock samples were fractured and passed through 0.05-mm mesh. These samples were decomposed using either the hydrogen fluoride-perchloric acid or alkali-fusion method (Purvis and Peterson 1956). Exchangeable Figure 1 (opposite page). Distribution of serpentine areas in Japan (A) and Map of sampling locations of serpentine plants in Hokkaido (B). (A) Localities—1: Toikanbetsu, 2: Mt. Bouzu, 3: Asahikawa-Kamuikotan, 4: Mt. Yupari, 5: Tomamu, 6: Mt. Apoi, 7: Hayachine, 8: Sawame, 9:Miyamori, 10: Mt. Shibutsu, 11: Mt. Tanikawa, 12: Mt. Hakuba, 13: Ooshika, 14: Shibukawa,15: Yana, 16: Is. Suga, 17: Mt. Asama, 18: Mt. Ooe,19: Sekinomiya, 20: Mt. Ryuumon, 21: Mt. Kurosawa, 22: Jinryou, 23: Hukuhara, 24: Miyahama, 25: Engyouji, 26: Mt. Higashiakashi, 27: Mt. Nishiki, 28: Mt. Rakan, and 29: Tanoura. (B) The 7 sampling areas, containing 19 sampling locations, are as follows. Northern areas (1: Toikanbetsu, 2: Mt. Shirikoma, 3: Nakatonbetsu, 4: Otoineppu), central areas (5: Horokanai, 6: Mt. Shiratori, 7: Shibetsu, 8: Wassamu, 9: Asahikawa-Kamuikotan, 10: Furano), southern areas (11: Hobetsu, 12: Mt. Bouzu, 13: Shimekappu, 14: Hidaka), Mt. Yupari areas (15: Mt. Yupari, 16: Mt. Furanonishi), Mt. Tottabetsu areas (17: Mt. Tottabetsu, 18: Mt. Chiroro), Mt. Apoi areas (19: Mt. Apoi), and Shizunai areas (20: Shizunai, Mitsuishi). 68 Northeastern Naturalist Vol. 16, Special Issue 5 Na, K, Mg, Ca, and Ni were extracted following the Shollenberger method. Chemical components were analyzed with an atomic absorption spectrometer. Results and Discussion Distribution of serpentine plants The serpentine vascular plants in Hokkaido comprised 46 taxa, including 17 families, 32 genera, 40 species, 5 varieties, and 1 form (Appendix 1). The Hokkaido serpentine flora contained 70.8% of the serpentine species found in Japan (65 taxa of 22 families). The majority of the 46 taxa in Hokkaido belong to Compositae (13 taxa), Primulaceae (4 taxa), Ranunculaceae (3 taxa), Rosaceae (3 taxa), Violaceae (3 taxa), or Scrophulariaceae (3 taxa). Several photos of typical serpentine plants are shown in Figure 2. The nonserpentine plant families that we observed frequently on the serpentine soils were Rosaceae, Violaceae, Umbelliferae, Ericaceae, and Compositae. Alpine plants such as Pinus pumila Regel (Dwarf Pine), Geum pentapetalum (L.) Makino (Aleutian Avens), and Vaccinium vitis-idaea L.var. minus Loddiges (Bilberry) were growing on serpentine soils in the low altitudes of mountains in the northern and central parts of Hokkaido, but not in the southern parts. The distribution and genetic variations of the serpentine floras in Hokkaido are unique. In these floras are several rare endemic plants and alpine plants. It is speculated that the cold climate in Hokkkaido preserved these plants since the last glacial epoch. Nakata and Kojima (1987) also suggested that the cool and humid climate, as well as undulating relief of northern Hokkaido, are important factors for the development of the unique phytogeocoenosis. Kitamura (1993) pointed out that many kinds of serpentine plants inhabiting Mt. Yupari and Mt. Apoi were also found in Mt. Hayachine, Iwate Prefecture, Japan (Fig. 1A, point 7), but only three taxa (Leontopodium hayachinense Hara & Kitam., Polygonum hayachinensis Makino, Primula macrocarpa Maxim.) were endemic to this mountain. Chemical characteristics of serpentine soil Table 1 shows the average concentrations of the chemical components and heavy metals , Table 2 gives the total element average concentrations of the total elements, and Table 3 gives the average concentrations of the exchangeable elements in serpentine rocks in each sampled area in Hokkaido. The serpentine soils were distinctively very low in K, Ca, and Al; however, they were very high in Mg, Ni, and Cr. A highly significant positive correlation (r = 0.903, P < 0.001) was observed between the Ca and Al concentrations in serpentine rocks, and there was a significant negative correlation (r = -0.527, P < 0.01) between Ni and Mg concentrations. The pH of the serpentine soil samples ranged from 5.7 to 9.4, and the mean value was 6.9. Less than 10% of the soil samples showed a pH higher than 8.0. The concentration of exchangeable K in the serpentine soil samples ranged from an undetectable amount to 4.7 mmol+ kg-1. Even compared with 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 69 the lowest level of K in agricultural fields, the average exchangeable K was very low (1.2 mmol+ kg-1), especially in the southern areas (0.05–0.54 mmol+ Figure 2. Typical serpentine plants endemic to the serpentine areas in Hokkaido. Plant names and their localities are as follows (clockwise from top left): Japonolirion osense var. saitoi (northern-area endemic), Aconitum ito-seiyanum (northern-area endemic), Adenophora pereskiifolia var. uryuensis (central-area endemic), Taraxacum yuparense var. grandisquamatum (southern-area endemic), Viola yubariana (Mt. Yupari-area endemic) Primula yuparensis (Mt. Yupari area endemic), Callianthemum miyabeanum (Mt. Apoi-area endemic), and Hypochoeris crepidioides (Mt. Apoi-area endemic). 70 Northeastern Naturalist Vol. 16, Special Issue 5 Table 1. The average concentrations of the chemical components and heavy metals in serpentine rocks in each sampled area in Hokkaido, Japan. N.D. = not detected; tr. = trace. Average of chemical components (%) No. of Locality samples H2O SiO2 MgO FeO Na2O K2O Al2O3 CaO NiO Cr2O3 TiO2 Total Northern areas 7 12.49 41.53 38.46 5.91 0.35 tr. 0.16 0.07 0.34 0.22 0.02 99.58 Central areas 8 13.96 41.71 35.03 7.55 0.30 tr. 0.48 0.21 0.45 0.15 0.02 99.86 Southern areas 4 14.81 39.25 37.93 6.03 0.24 0.01 0.46 0.18 0.29 0.52 0.02 99.71 Mt. Yupari areas 3 11.23 42.90 37.67 6.49 0.37 0.01 0.5 0.08 0.39 0.21 0.01 99.86 Mt. Tottabetsu areas 1 6.21 41.40 43.10 6.81 0.32 N.D. 0.65 0.87 0.34 0.11 tr. 99.81 Mt. Apoi areas 4 3.94 43.08 39.55 7.38 0.40 0.02 2.35 1.88 0.29 0.26 0.05 99.20 Shizunai areas 2 13.12 40.85 38.60 5.12 0.39 0.05 0.83 0.26 0.36 0.25 0.01 99.76 Total average 11.81 41.60 37.60 6.65 0.33 0.01 0.68 0.41 0.37 0.24 0.02 99.69 Standard deviation 4.00 1.70 3.00 1.02 0.07 0.01 0.76 0.65 0.09 0.18 0.01 0.51 Average of heavy metal concentrations (mg kg-1) No. of Locality samples Ni Cr Ti Mn Zn Cu Co Northern areas 7 2720 1795 94.0 723 33.6 8.5 13.1 Central areas 8 3551 1051 115.8 1006 46.8 9.5 43.9 Southern areas 4 2279 3555 74.0 782 51.3 11.1 55.4 Mt. Yupari areas 3 3048 1485 54.3 860 45.3 8.9 101.4 Mt. Tottabetsu areas 1 2689 672 2.0 893 38.0 10.1 116.1 Mt. Apoi areas 4 2323 1802 246.0 1116 63.3 25.9 100.0 Shizunai areas 2 2954 1758 47.5 906 51.0 12.4 95.5 Total average 2905 1737 109.0 900 46.0 11.8 57.3 Standard deviation 672 1455 96.0 280 14.0 6.0 42.3 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 71 kg-1). Similarly, exchangeable Ca was also very low (0.7–126.5 mmol+ kg-1; average 18.0 mmol+ kg-1), especially in the southern areas and Mt. Tottabetu areas (or under 5.0 mmol+ kg-1, average). In contrast, the average exchangeable Mg in the serpentine soils was high (73.7 mmol+ kg-1), about twice that in agricultural fields. The concentration of exchangeable Ni was also high (0.02–0.65 mmol+ kg-1; 0.24 mmol+ kg-1 average), although little of the Ni in the soil was exchangeable. One-fourth of the soil samples contained more than 0.3 mmol+ kg-1 exchangeable Ni, the level which causes Ni-excess symptoms in plants (Mizuno 1968). None Table 2. Average concentration of the total elements in serpentine soil in each sampled area in Hokkaido, Japan. Ni Mg Ca Fe Al Na K Locality (mg kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) Northern areas 1965.0 186.2 1.7 38.2 4.8 3.1 0.5 Central areas 1944.3 137.8 4.0 40.8 14.7 3.9 2.4 Southern areas 2051.0 190.2 3.7 23.8 9.4 14.0 0.3 Mt. Yupari areas 1534.0 160.5 6.7 44.5 13.9 6.0 1.1 Mt. Tottabetsu areas 2589.0 260.0 6.9 64.9 8.1 1.4 0.2 Mt. Apoi areas 1889.7 174.5 9.8 46.0 17.6 4.0 1.2 Shizunai areas 1333.7 119.3 21.2 35.9 21.0 20.9 1.9 Total average 1844.0 161.0 6.1 40.6 13.1 6.7 1.3 Standard deviation 598.0 52.0 6.1 16.1 10.2 8.8 1.8 P2O5 Mn Zn Ti SiO2 Mo Locality (g kg-1) (mg kg-1) (mg kg-1) (g kg-1) (g kg-1) (g kg-1) Northern areas 0.2 478.1 39.0 0.3 391.6 1.6 Central areas 0.6 847.1 58.9 9.7 469.6 1.0 Southern areas 0.1 487.4 39.9 1.4 426.2 0.9 Mt.Yupari areas 0.3 850.6 61.0 2.7 488.3 0.7 Mt.Tottabetsu areas 0.2 886.0 55.0 0.5 429.5 0.6 Mt.Apoi areas 0.2 845.5 63.5 2.5 560 0.7 Shizunai areas 0.0 874.7 75.0 10.5 475.8 0.6 Total average 0.3 757.0 54.0 3.0 464.5 1.0 Standard deviation 0.5 351.0 21.0 7.8 77.4 0.5 Table 3. Average concentration of exchangeable elements in serpentine soil in each sampled area in Hokkaido, Japan. No. of pH Na K Mg Ca Ni Locality samples (H2O) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) Northern areas 14 7.4 25.1 40.9 815 260.2 11.5 Central areas 22 6.7 19.4 79.2 1051.7 354.7 7.3 Southern areas 9 7.3 5.6 10.6 412.3 104 3.8 Mt. Yupari areas 15 6.6 6.5 34.8 697.2 231 7.7 Mt. Tottabetsu areas 1 6.1 4.1 13.3 343.4 121.4 3.4 Mt. Apoi areas 11 6.6 18.5 36.7 776.5 298.7 3.4 Shizunai areas 3 7.6 12.2 15 385.5 1347.6 2.8 Total average 75 6.9 16.0 47.0 895.8 360.7 7.0 Standard deviation 0.7 21.8 50.1 690.4 441.2 5.0 72 Northeastern Naturalist Vol. 16, Special Issue 5 of the samples contained more than 0.7 mmol+ kg-1 exchangeable Ni, which would cause strong Ni toxicity (Mizuno 1968). This upper limit is the result of Ni in serpentine soil being hardly soluble due to the high Mg concentration and high pH (Mizuno 1979). We also analyzed the chemical components of soils in several serpentine areas in Yana (Fig. 1A, point 14), Suga island (16), and Mt. Asama (17) in the central Japan areas. Their pH ranged from 6.86–7.23, the total Ni content in the soil samples ranged from 1500–3670 mg kg-1, and other chemical components were not extremely different from those of the Hokkaido area (data not shown). So the general pattern of the metal concentrations—low K and Ca, and high Mg and Ni—is one of the characteristics of the serpentine soil in Hokkaido, but also in other serpentine areas in Japan. Chemical characteristics of serpentine plants Horie (2002) analyzed chemical components in the serpentine plants (37 taxa), nonserpentine plants (33 taxa), and trees (5 taxa) growing on the serpentine soil in Hokkaido. As a result, the average of five macroelements and five microelements of serpentine plants, nonserpentine plants, and trees growing on serpentine soils were obtained (Table 4). The P concentration in the serpentine plants (1.4 g kg-1) was lower than those of the nonserpentine plants (1.7 g kg-1) and trees (3.1 g kg-1), while the concentrations of K, Ca, and N were higher. The serpentine plants were found to contain N, K, Ca, and Mg concentrations that were two times as high as non-serpentine plants, and four-times as high as trees. The concentration of P in the serpentine plants was at a minimum level within the range that does not cause P deficiency. Such physiological characteristics enabled them to grow on the nutrient-poor serpentine soil. The Fe concentration of the serpentine plants ranged from 62–1210 mg kg-1, (mean = 290 mg kg-1), which is 1.3 times higher than the average concentration of Fe in nonserpentine plants (219 mg kg-1) and 4.9 times higher than that of trees growing on serpentine soils (59 mg kg-1). Likewise, Mn, Zn, and Cu concentrations in the serpentine plants were all higher than those of nonserpentine plants and trees. The Mn, Zn, and Cu concentration ratios of serpentine plants were 1.7, 1.3, and 1.1 times those of nonserpentine plants, and 1.5, 2.8, and 2.0 times those of trees. On the contrary, the Ni concentration in serpentine plants (53 mg kg-1 on average) was lower than that of nonserpentine plants (67 mg kg-1 on average). The Ni concentrations of the serpentine plants increased proportionally to that of the exchangeable Ni concentrations in the soils up to a level of 15 mg kg-1 in the soil, but did not increase further (Fig. 3a–d). On the other hand, Ni concentration in the nonserpentine plants increased linearly in proportion to the amount of the exchangeable Ni in the soil (Fig. 3e,f). Hence, the serpentine plants in Hokkaido are estimated to have a mechanism to control the uptake and accumulation of Ni in their aerial tissue, in order to avoid the damage associated with excessive Ni. The mechanisms of xylem loading of Ni and/or Ni-transport mechanisms to upper parts of plants have not been clearly identified, but some 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 73 Table 4. Concentration of macro- and microelements of native plants found growing on serpentine soil in Hokkaido, Japan. Macroelements Microelements (g kg-1 D.W.) (mg kg-1 D.W.) Plant name N P K Mg Ca Fe Mn Zn Cu Ni Deschampsia caespitosa 23.0 1.4 7.3 1.2 1.1 104 86 21 6.7 7 Hierochloe pluriflora 21.9 1.2 11.1 0.9 0.7 62 14 17 5.2 23 Japonolirion osense 21.2 1.6 23.3 2.5 8.3 93 21 35 7.0 9 Betula apoiensis 18.8 1.1 7.3 2.1 3.1 85 68 114 6.9 7 Quercus mongolica var. grosseserrata 20.9 1.4 6.0 3.6 4.1 130 190 13 6.0 14 Arenaria katoana var. katoana 26.6 1.4 12.1 4.5 3.6 221 65 31 5.0 21 Silene repens var. apoiensis 30.2 1.5 31.3 4.4 4.7 294 86 21 5.2 19 Aconitum ito-seiyanum 22.0 1.2 14.6 12.1 9.6 155 86 20 6.4 62 Callianthemum miyabeanum 22.7 1.6 20.7 2.8 10.8 121 52 85 6.8 24 Ranunculus acris var. nipponicus 21.2 1.2 18.8 5.0 3.2 214 86 128 12.4 126 Draba japonica 29.1 1.8 13.8 12.0 4.1 910 54 32 6.4 131 Aruncus dioicus var. subrotundatus 24.9 1.3 12.2 4.0 4.8 205 57 31 8.9 18 Potentilla apoiensis 22.4 1.4 12.8 2.3 9.2 1080 76 72 20.0 24 Potentilla matsumurae var. yuparensis 21.4 1.2 11.4 6.2 4.4 130 89 38 5.4 90 Geranium erianthum f. glabriusculum 25.9 1.8 10.2 1.2 5.9 103 31 17 6.8 4 Euonymus alatus f. kakurensis 12.6 1.5 9.5 4.2 6.5 116 108 15 4.4 6 Hypericum tatewakii 22.1 1.1 6.2 3.0 2.7 82 29 22 5.7 58 var. nigro-punctatum Viola sacchalinensis var. alpina 24.9 1.5 11.5 6.5 3.3 766 64 33 6.0 123 Viola yubariana 32.2 1.5 21.9 6.6 2.9 256 44 30 4.6 248 Angelica acutiloba var. lanceolata 28.6 1.5 22.6 3.2 9.4 205 97 30 6.2 19 Peucedanum multivittatum 39.5 1.8 31.0 6.2 8.7 194 93 45 10.1 43 var. linearilobum Primula hidakana 15.8 1.0 11.3 3.4 3.5 170 18 22 11.4 8 Primula modesta var. samanimontana 19.3 1.4 25.7 4.9 3.7 1210 57 40 15.6 23 Primula takedana 39.0 1.7 24.6 3.3 1.7 336 38 35 9.7 34 Primula yuparensis 14.8 1.0 15.1 9.3 1.5 126 35 36 6.7 108 Lagotis takedana 19.1 1.3 18.7 5.1 2.4 299 36 21 8.2 172 Veronica schmidtiana var. yezoalpina 18.2 1.5 12.1 4.0 2.9 310 31 18 5.8 24 Adenophora pereskiifoliar 16.8 1.3 21.7 5.7 9.9 141 56 28 5.6 32 var. uryuensis Achillea ptarmica var. yezoensis 15.6 1.6 19.1 3.9 7.9 223 117 54 7.4 12 Cirsium apoense 33.7 1.4 25.0 8.6 9.3 412 40 16 10.0 75 Crepis gymnopus 19.8 1.7 30.3 6.3 5.0 108 25 31 6.3 11 Erigeron thunbergii var. angustifolius 22.2 1.8 24.4 2.8 6.2 212 38 24 10.8 22 Hypochoeris crepidioides 23.7 1.6 31.3 5.3 5.6 298 34 48 8.3 54 Picris hieracioides var. jessoensis 22.1 1.3 26.2 4.8 3.1 314 29 30 6.8 27 Saussurea chionophylla 24.6 1.5 21.1 6.6 5.6 506 38 19 9.0 118 Saussurea kudoana var. uryuensis 15.7 1.1 23.2 5.8 5.3 175 22 16 7.9 53 Taraxacum yuparense var. yuparense 39.8 1.9 29.0 6.3 3.4 361 30 28 7.9 74 Total average 23.6 1.4 18.3 4.9 5.1 290 58 36 7.8 53 Standard deviation 6.7 0.2 7.8 2.6 2.7 273 35 26 3.1 54 recent reports showed the roles of nicotianamine for Ni tolerance and accumulation (Callahan et al. 2007, Kim et al. 2005). It is possible that the level of production of metal chelators in roots may be related to the difference of Ni accumulation level between serpentine and nonserpentine plants. 74 Northeastern Naturalist Vol. 16, Special Issue 5 Figure 3. Relationships between exchangeable nickel in soils and nickel concentrations in dried leaves of serpentine and nonserpentine plants. According to the criteria established by Brooks et al. (1977), a plant containing 100 to 1000 mg kg-1 Ni in dry matter is a “strong Ni accumulator” and one containing more than 1000 mg kg-1 Ni is a “Ni 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 75 Figure 4. Ranges of Ni concentrations in native plants growing on serpentine areas in Hokkaido. 76 Northeastern Naturalist Vol. 16, Special Issue 5 hyperaccumulator.” Among the 75 taxa inhabiting the serpentine soils in Hokkaido, 8 qualify as strong Ni accumulators and only Thlaspi japonicum H. Boiss qualifies as a Ni hyperaccumulator (Fig. 4). Both strong Ni accumulators and the Ni hyperaccumulator were found growing on Mt. Yupari. Among the 8 strong Ni accumulators, 7 taxa (Ranunculus acris var. nipponicus Hara f. yuparensis (Miyabe) Toyokuni, Viola yubariana Nakai, Lagotis takedana Miyabe & Tatewaki, Draba japonica Maximowicz, Viola sacchalinensis var alpine Hara, Saussurea chionophylla Takeda, and Primula yubariensis Takeda) were the serpentine plants, and one taxon (Allium shoenoprasum var. shibutuense Kitamura) was a nonserpentine plant inhabiting serpentine soil. These plants were reported for the first time as strong Ni accumulators in Japan. Thlaspi japonicum was reported for its Ni hyperaccumulation ability (Reeves 1988) and for its Ni accumulation in the epidermal cells in crystal form and Ni elimination in guttation fluid (Mizuno et al. 2003). Horie (2002) showed the Ni concentration in T. japonicum ranged from 820 to 1955 mg kg-1 (mean = 1299 mg kg-1). This result confirms T. japonicum as a hyperaccumulator. T. japonicum is the sole Ni-hyperaccumulator identified in Japan at present, and our further research on T. japonicum is under way. In addition to physiological research on Ni tolerance and accumulation in T. japonicum (Mizuno et al. 2003, 2005a), we have isolated several candidate transporters (TjZNT1 and 2) that are responsible for Ni tolerance (Mizuno et al. 2005b). Several genes related to Ni tolerance in plants have been reported (Kim et al. 2005, Schaaf et al. 2006). However, no other study investigating the participation of ZIP family metal transporters for Ni tolerance has been reported, and their Ni tolerance mechanism has not been identified. Detailed investigations of these genes are now in progress (Mizuno et al. 2007, Nishida et al. 2008). Summary Conclusion Soils of Hokkaido serpentine areas showed high Ni, Cr, and Mg and low Ca, and their chemical components are almost in the same range of those of other serpentine areas in Japan. Serpentine plants showed low Ni content in their shoots compared to non-serpentine plants, and no Ni hyperaccumulator was found among them. Only a non-serpentine plant, T. japonicum, was considered to be a hyperaccumulator. Acknowledgments We wish to thank Dr. Horie Kenji for generously providing us with the extensive data which he collected over the thirty-nine years since 1969, including his doctoral thesis. We hope this paper introduces readers to the serpentine flora in Hokkaido. The full work of Dr. Horie is available (currently only in Japanese ) in the Journal of Rakuno Gakuen University (see citation listing below). 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 77 Literature Cited Brooks R.R., J. Lee, and R.D. Reeves. 1977. Detection of nickliferous rocks by analysis of herbarium species of indicator plants, Journal of Geochemical Exploration 7:49–77. Callahan D.L., S.D. Kolev, R.A.J. O’Hair, D.E. Salt, and A.J.M. Baker. 2007. 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Journal Of Geobotany 4:97–101. 1955; ibidem 5:12–15, 81–84, 115–116. 1956; ibidem 6:17–20, 63–67. 1957; ibidem 7:37–38. 1958; ibidem 9:10–13, 38–41. 1960. Yamagata, N., Y. Murakami, and T. Torii. 1960. Biogeochemical investigations in serpentine-chromite ore district. Geochimica et Cosmochimica Acta 18:23–35. 2009 T. Mizuno, K. Horie, S. Nosaka, H. Obata, and N. Mizuno 79 Appendix 1. Distribution of serpentine plants by region in Hokkaido, Japan. Type = distribution type (A = northern areas endemic, B = Central areas endemic, C = northern and central areas endemic, D = southern areas endemic. E = Mt. Yupari endemic, F = Mt. Apoi endemic, G1 = widely distributed in low mountain areas, G2 = widely distributed in low to high mountain areas, G3 = widely distributed in high and partial low mountain areas, G4 = widely distributed in partial high mountain areas, and H = plants found in Hokkaido and Honshu areas). N = northern area, C = central area, S = southern area, MY = Mt. Yupari area, MT = Mt. Tottabetsu area, MA = Mt. Apoi area, and S = Shizunai area. + = present; – = not found. Locality Type Species N C S MY MT MA S A Japonolirion osense var. saitoi (Makino & Tatewaki) Ohwi + – – – – – – A Aconitum ito-seiyanum Miyabe & Tatewaki + – – – – – – A Primula takedana Tatewaki + – – – – – – B Adenophora pereskiifoliar var. uryuensis (Miyabe &Tatewaki) Toyokuni & Nosaka – + – – – – – C Quercus mongolica var. grosseserrata Rehder et Wilson f. integerrima Inagaki + + – – – – – C Geranium erianthum f. glabriusculum Inagaki + + – – – – – C Euonymus alatus f. kakurensis Inagaki + + – – – – – C Achillea ptarmica var. yezoensis Kitamura + + – – – – – C Picris hieracioides var. jessoensis (Tatewaki) Ohwi + + – – – – – D Taraxacum yuparense var. grandisquamatum H. Koidzumi – – + – – – – E Deschampsia caespitosa var. levis (Takeda) Ohwi – – – + – – – E Hierochloe pluriflora Koidzumi – – – + – – – E Potentilla matsumurae var. yuparensis Kudo ex Miyabe & Tatewaki – – – + – – – E Viola yubariana Nakai – – – + – – – E Primula yuparensis Takeda – – – + – – – E Lagotis takedana Miyabe & Tatewaki – – – + – – – E Erigeron thunbergii var. glabratus (A. Gray) A. Gray f. haruoi – – – + – – – E Saussurea riederi var. yuparensis Kitamura – – – + – – – E Taraxacum yuparense var. yuparense H. Koidzumi – – – + – – – F Betula apoiensis Nakai – – – – – + – F Arenaria katoana var. lanceolata Tatewaki – – – – – + – 80 Northeastern Naturalist Vol. 16, Special Issue 5 Locality Type Species N C S MY MT MA S F Silene repens var. apoiensis (Hara) Hara – – – – – + – F Callianthemum miyabeanum Tatewaki – – – – – – + – F Aruncus dioicus var. subrotundatus (Tatewaki) Hara – – – – – + – F Peucedanum multivittatum var. linearilobum Tatewaki – – – – – + – F Tilingia ajanensis var. angustissima (Nakai) Kitagawa – – – – – + – F Primula modesta var. samanimontana Tatewaki – – – – – + – F Veronica schmidtiana var. yezoalpina (Koidzumi) Yamazaki f. exigua – – – – – + – F Cirsium apoense Nakai – – – – – + – F Hypochoeris crepidioides (Miyabe & Kudo) Tatewaki & Kitamura – – – – – + – F Saussurea kudoana var. kudoana Tatewaki & Kitamura – – – – – + – G1 Hypericum tatewakiivar. tatewakii S.Watanabe + + – – – – – G1 Hypericum tatewakii var. nigro-punctatum S. Watanabe + + + – – – – G1 Saussurea kudoana var. uryuensisTatewaki & Kitamura + + + – – – – G2 Viola sacchalinensis var. alpina Hara + + + + + + – G2 Primula hidakana Miyabe & Kudo ex Tatewaki – – – – + + + G2 Veronica schmidtiana var. yezoalpina (Koidzumi) Yamazaki f. yezoalpina + + + + + – – G2 Crepis gymnopus Koidzumi + + + + + + – G3 Ranunculus acris var. nipponicus Hara f. yuparensis (Miyabe) Toyokuni – + – + – – – G3 Viola brevistipulata var. hidakana (Nakai) S. Watanabe – + – – – + – G3 Angelica acutiloba var. lanceolata (Tatewaki) Ohwi f. lineariloba (Koidzumi) – – + + + + – S. Watanabe & Kawano ex Ohwi G3 Erigeron thunbergii var. angustifolius (Tatewaki) Hara – – – – – + – G4 Potentilla apoiensis Nakai – – – – + + – G4 Saussurea chionophylla Takeda – – – + + – – H Arenaria katoana var. katoana Makino – – + + + – – H Draba japonica Maximowicz – – – + + – – Total 14 14 8 17 9 19 1