Increased CO2 Concentration and Temperature on Growth and Yield of Winter Wheat at Two Levels of Nitrogen Application. *Plant, Cell and Environment*, Vol. 16,

southern U.S. rice cultivars differing in their tolerance to enhanced ultraviolet-B radiation. *Environmental and Experimental Botany*, Vol. 70, pp. 174-184, ISSN 0098-

Physiological Responses to α-Tocopherol, Glycine Betaine or Salicylic Acid

Productivity through Altered Pollen Germination and Spikelet Fertility.

Respiration, Membrane Stability, Antioxidant Capacity, and Yield of Rice Plants.

turnover, and Metabolism of Reactive Oxygen Species. *Annual Review of Plant*

Plants, *Ananas comosus* (L.) Merr. II. Effects of Variation of the Day/Night Temperature Regime. *Australian Journal of Plant Physiology*, Vol. 7, pp. 375-385, ISSN

168 the Economy, Human Health, and on Urban and Natural Environments

John, E. (2002). Candidate Effector and Regulator Genes Activated by Glycine Betaine in

Jones, P.D.; New, M.; Parker, D.E.; Martin, S. & Rigor, I.G. (1999). Surface Air Temperature

Kang, G.Z., Z.X. Wang, K.F. Xia, and G.C. Sun, 2007: Protection of Ultrastructure in Chilling-

Keeling, P.L.; Banisadr, R.; Barone, L.; Wasserman, B.P. & Singletary, G.W. (1994). Effect of

Khan, N.A.; Syeed, S.; Masood, A.; Nazar, R. & Iqbal, N. (2010). Application of Salicylic Acid

Khan, W.; Prithiviraj, B. & Smith, A. (2003). Photosynthetic Responses of Corn and Soybean

Kondo, S. & Takahashi, Y. (1989). Relation between Early Drop of Apple Fruit and Ethylene

Kreiner, M.; Harvey, L.M. & McNeil, B. (2002). Oxidative Stress Response of a Recombinant

Lambers, H. (1985). Respiration in Tack Plants and Tissues: Its Regulation and Dependence

Larkindale, J. & Huang, B. (2004). Thermotolerance and Antioxidant Systems in *Agrostis*

Larkindale, J. & Knight, M.R. (2002). Protection Against Heat Stress-induced Oxidative

Larque-Saavedra, A. (1978). The Antitranspirant Effect of Acetylsalicylic Acid on *Phaseolus*

Lee, C.B.; Hayashi, H. & Moon, B.Y. (1997). Stabilization by Glycinebetaine of

Loka, D.A., Oosterhuis, D.M., 2010. Effect of High Night Temperatures on Cotton

Acid. *Plant Physiology*, Vol. 128, pp. 682-695, ISSN 0032-0889.

*vulgaris. Plant Physiology*, Vol. 43, pp. 126-128, ISSN 0032-0889.

PCC7002. *Molecular Cell*, Vol. 30, pp. 296-299, ISSN 1097-2765.

*Experimental Botany*, Vol. 68, pp. 258-263, ISSN 0098-8472.

*Horticultural Science*, Vol. 58, pp. 1-8, ISSN 1882-3351.

*Technology*, Vol. 30, pp. 346-353, ISSN 0141-0229.

Vol. 18, Springer-Verlag, Berlin, Gremany.

html.

8, pp. 277-282, ISSN 1009-3095.

Vol. 1, pp. e1, ISSN 2037-0156.

ISSN 0310-7841.

ISSN 0176-1617.

1617.

Arabidopsis. Available from http://abstracts.aspb.org/pb2002/public/P63/0153.

and its Changes over the Past 150 Years. *Reviews of Geophysics*, Vol. 37, pp. 173–199.

stressed Banana Leaves by Salicylic Acid. *Journal of Zhejiang University Science*, Vol.

Temperature on Enzymes in the Pathway of Starch Biosynthesis in Developing Wheat and Maize Grain. *Australian Journal of Plant Physiology*, Vol. 21, pp. 807-827,

Increases Contents of Nutrients and Antioxidative Metabolism in Mungbean and Alleviates Adverse Effects of Salinity Stress. *International Journal of Plant Biology*,

to Foliar Application of Salicylates. *Journal of Plant Physiology*, Vol. 160, pp. 485-492,

Evolution under High Night-temperature Conditions. *Journal of Japanese Society for*

*Aspergillus niger* to Exogenous Menadione and H2O2 Addition. *Enzyme and Microbial*

on Environmental Factors, Metabolism and Invaded Organism. In: *Higher Plant Cell Respiration. Encyclopedia of Plant Physiology*, R. Douce & D.A. Day (Eds.), New series,

*stolonifera*: Involvement of Salicylic Acid, Abscisic Acid, Calcium, Hydrogen Peroxide and Ethylene. *Journal of Plant Physiology*, Vol. 161 pp. 405-413, ISSN 0176-

Damage in Arabidopsis Involves Calcium, Abscisic Acid, Ethylene, and Salicylic

Photosynthetic Oxygen Evolution by Thylakoid Membranes from Synechococcus

Respiration. ATP Levels, and Carbohydrate Content. *Environmental and*

Effects of High Night Temperature on

1011-1019, ISSN 0167-4412.

633-638, ISSN 0022-0957.

*Journal*, Vol. 76, pp. 824-828, ISSN 0002-1962.

*Physiology*, Vol. 21, pp. 829-841, ISSN 0310-7841.

243, Bios Scientific Publishers, Oxford, UK.

263-281, John Wiley & Sons, New York, USA.

*Research*, Vol. 8, pp. 17-33, ISSN 0378-4290.

ISSN 0960-7412.

191-202, ISSN 0168-1923.

*Environment*, Vol. 25, pp. 1729-1737, ISSN 0140-7791.

Tobacco. *Planta*, Vol. 193, pp. 372-376, ISSN 0032-0935.

*Growth Regulation*, Vol. 30, pp. 157-161, ISSN 0167-6903.

Crop Physiology and Productivity: Plant Growth Regulators Provide a Management Option 171

Sakamoto, H. & Murata, N. (1998). Metabolic Engineering of Rice Leading to Biosynthesis of

Sayed, O.H.; Earnshaw, M.J. & Emes, M.J. (1989). Photosynthetic Response of Different

Seddigh, M. & Jolliff, G.D. (1984). Night Temperature Effects on Morphology, Phenology,

Senaratna, T.; Touchell, D.; Bunn, T. & Dixon, K. (2000). Acetyl salicylic acid (aspirin) and

Shirasu K.; Nakajima H. & Rajashekar K. (1997). Salicylic Acid Potentiates an Agonist-

Smirnoff, N. (1995). Antioxidant Systems and Plant Response to the Environment. In:

Sullivan, C.Y. (1972). Mechanisms of Heat and Drought Resistance in Grain Sorghum and

(Eds.), 247-264, Oxford and IPH Publishing Company, New Delhi, India. Sullivan, C.Y. & Ross, W.M. (1979). Selecting for Drought and Heat Resistance in Grain

Turnbull, M.H.; Murthy, R. & Griffin, K.L. (2002). The Relative Impacts of Daytime and

Vanacker, H.; Lu, H.; Rate, D.N. & Greenberg, J.T. (2001). A Role for Salicylic Acid and

Warrag, M.O.A. & Hall, A.E. (1984). Reproductive Responses of Cowpea (*Vigna unguiculata*

Willits, D.H. & Peet, M. (1998). The Effect of Night Temperature on Greenhouse Grown

Yalpani, N.; Enyedi, A.J.; Leon, J. & Raskin, I. (1994). Ultraviolet Light and Ozone Stimulate

Defense Mechanisms. *Plant Cell*, Vol. 9, pp. 261-270, ISSN 1040-4651. Singletary, G.W.; Banisadr, R. & Keeling, P.L. (1994). Heat Stress during Grain Filling in

Glycinebetaine and Tolerance to Salt and Cold. *Plant Molecular Biology*, Vol. 38, pp.

Varieties of Wheat to High Temperature. *Journal of Experimental Botany*, Vol. 40, pp.

Yield and Yield Components of Indeterminate Field-grown Soybean. *Agronomy*

salicylic acid induce multiple stress tolerance in bean and tomato plants. *Plant*

dependent Gain Control that Amplifies Pathogen Signal in the Activation of

Maize: Effects on Carbohydrate Storage and Metabolism. *Australian Journal of Plant*

*Environment and Plant Metabolism: Flexibility and Acclimation*, N. Smirnoff (Ed.), 217-

Methods of Measurement. In: *Sorghum in the Seventies*, N.G.P. Rao & L.R. House

Sorghum. In: *Stress Physiology in Crop Plants*, H. Mussell, H. & R.C. Staples (Eds.),

Night-time Warming on Photosynthetic Capacity in *Populus detoides*. *Plant Cell and*

NPR1 in Regulating Cell Growth in Arabidopsis. *Plant Journal*, Vol. 28, pp. 209-216,

(L.) Walp.) to Heat Stress. II. Responses to Night Air Temperature. *Field Crops*

Tomato Yields in Warm Climates. *Agricultural and Forest Meteorology*, Vol. 92, pp.

Accumulation of Salicylic Acid, Pathogen-related Proteins and Virus Resistance in

170 the Economy, Human Health, and on Urban and Natural Environments

Paembonan, S.A.; Hagihara, A. & Hozumi, K. (1992). Long-term Respiration in Relation to

Paleg, L.G.; Douglas, T.J.; van Daal, A. & Keech, D.B. (1981). Proline, Betaine and Other

Pan, Q.; Zhan, J.; Liu, H.; Zhang, J.; Chen, J.; Wen, P. & Huang, W. (2006) Salicylic Acid

Peng, S.; Huang, J.; Sheehy, J.E.; Laza, R.C.; Visperas, R.M.; Zhong, X.; Centeno, G.S.; Khush,

Penning de Vries, F.W.T. (1975). The Cost of Maintenance Process in the Cell. *Annals of*

Peterson, R.B. & Zelitch, I. (1982). Relationship between Net CO2 Assimilation and Dry

Pospisil, P. & Dau, H. (2000). Chlorophyll Fluorescence Transients of Photosystem II

Prasad, P.V.V.; Boote, K.J.; Allen Jr, L.H.; Sheehy, J.E. & Thomas, J.M.G. (2006). Species,

Prasad, P.V.V; Craufurd, P.Q. & Summerfield, R.J. (1999). Fruit Number in Relation to

Rajasekaran, L.R.; Kriedemann, P.E.; Aspinall, D. & Paleg, L.G. (1997). Physiological

NaCl Stress in Wheat. *Photosynthetica*, Vol. 34, pp. 357-366, ISSN 0300-3604. Rao, M.V.; Paliyath, G.; Ormrod, P.; Murr, D.P. & Watkins, C.B. (1997). Influence of Salicylic

Raskin, I. (1992). Role of Salicylic Acid in Plants. *Annual Review of Plant Physiology and Plant*

Reynolds, M.P.; Balota, M.; Delgado, M.I.B.; Amani, I. & Fisher, R.A. (1994). Physiological

Ryals, J.; Uknes, S. & Ward, E. (1994). Systematic Acquired Resistance. *Plant Physiology*, Vol.

Ryan, M.G. (1991). Effects of Climate Change on Plant Respiration. *Ecological Applications*,

Tress. *Tree Physiology*, Vol. 10, pp. 101-110, ISSN 0829-318X.

*Plant Physiology*, Vol. 8, pp. 107-114, ISSN 0310-7841.

*States of America* Vol. 101, pp. 9971-9975, ISSN 0027-8424.

*Photosynthesis Research*, Vol. 65, pp. 41-52, ISSN 0166-8595.

Stress. *Annals of Botany*, Vol. 84, pp. 381-386, ISSN 0305-7364.

*Physiology*, Vol. 115, pp. 137-149, ISSN 0032-0889.

104, pp. 1109-1112, ISSN 0032-0889.

Vol. 1, pp. 157-167, ISSN 1051-0761.

*Molecular Biology*, Vol. 43, pp. 439-463, ISSN 1040-2519.

*Botany*, Vol. 39, pp. 77-92, ISSN 0305-7364.

685, ISSN 0032-0889.

ISSN 0378-4290.

7841.

Growth and Maintenance Processes of the Aboveground Parts of a Hinoki Forest

Organic Solutes Protect Enzymes Against Heat Inactivation. *Australian Journal of*

Synthesized by Benzoic Acid 2 Hydroxylase Participates in the Development of Thermotolerance in Pea Plants. *Plant Science*, Vol. 171, pp. 226-233, ISSN 0168-9452.

G.S. & Cassman, K.G. (2004). Rice Yields Decline with Higher Night Temperature from Global Warming. *Proceedings of the National Academy of Sciences of the United*

Weight Accumulation in Field-grown Tobacco. *Plant Physiology*, Vol. 70, pp. 677-

Membrane Particles as a Tool for Studying Photosynthetic Oxygen Evolution.

Ecotype and Cultivar Differences in Spikelet Fertility and Harvest Index of Rice in Response to High Temperature Stress. *Field Crops Research*, Vol. 95, pp. 398-411,

Pollen Production and Viability in Groundnut Exposed to Short Episodes of Heat

Significance of Proline and Glycinebetaine: Maintaining Photosynthesis during

Acid on H2O2 Production, Oxidative Stress, and H2O2-metabolizing Enzymes. *Plant*

and morphological traits associated with spring wheat yield under hot irrigated conditions. *Australian Journal of Plant Physiology*, Vol. 21, pp. 717–730, ISSN 0310-

**Part 5**

**Impacts on Forests,**

**Global Warming and Ecological Changes:**

**Mangroves and Sea Ecosystems**

### **Part 5**

**Global Warming and Ecological Changes: Impacts on Forests, Mangroves and Sea Ecosystems**

Global Warming Impacts – Case Studies on

172 the Economy, Human Health, and on Urban and Natural Environments

Yamane, Y.; Kashino, Y. & Koike, H. (1997). Increase in Fluorescence Fo Level and Reversible

Yilmaz, J.L. (2004). Abiotic Stress Tolerance: Metabolic and Physiological Effects of

Zhang, J. & Kirkham, M.B. (1996). Antioxidant Responses to Drought in Sunflower and Sorghum Seedlings. *New Phytologist*, Vol. 132, pp. 361-373, ISSN 0028-646X. Zheng, S.H.; Nakamoto, H.; Yoshikawa, K.; Furuya, T. & Fukuyama, M. (2002). Influence of

Zhou, X.M., Mackenzie, A.F., Madramootoo, C.A., & Smith, D.L.J. (1999). Effects of Some

*Agronomy and Crop Science*, Vol. 183, pp. 103-110, ISSN 0931-2250.

*Biochemistry*, J.L. Yilmaz (Ed), 1-40, Lund University, Lund, Sweden. Yoshida, S. (1981). Physiological Analysis of Rice Yield. In: *Fundamentals of Rice Crop Science*,

Plants. *Photosynthetic Research*, Vol. 52, pp. 57-64.

*Production Science*, Vol. 5, pp. 215-218, ISSN 1343-943X.

Philippines.

Inhibition of Photosystem 2 Reaction by High-temperature Treatments in Higher

Compatible Solutes and Calmodulin on *E. coli* and Tobacco. In: *Pure and Applied*

S. Yoshida (Ed.), 231-251. International Rice Research Institute, Los Banos,

High Night Temperature on Flowering and Pod Setting in Soybean. *Plant*

Injected Plant Growth Regulators, With or Without Sucrose, on Grain Production, Biomass and Photosynthetic Activity of Field–grown Corn Plants. *Journal of*

**11**

*Japan*

**Effects of Temperature and Light Conditions on**

**Growth of Current-Year Seedlings of Warm-**

**Temperate Deciduous Tree Species**

*2Yatsugatake Forest, Agricultural and Forestry Research Center,*

*Graduate School of Life and Environmental Sciences, University of Tsukuba, Minamimaki, Nagano,*

*1Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano,*

Koichi Takahashi1, Hiroyuki Kobori1 and Tatsuyuki Seino2

**Temperate Evergreen Tree Species and Cool-**

It is suggested that global warming affects plant distribution along latitudinal and altitudinal gradients because vegetation changes with thermal conditions. Simulation studies predicted that global warming largely affects plant distribution (e.g., Morin et al., 2008). Actually, vegetation change during several decades has been observed (Penuelas et al., 2007; Lenoir et al., 2008). By contrast, some other studies did not observe vegetation changes (Holtmeier & Broll, 2007; Harsch et al., 2009). Interpretation of results of simulation models also needs caution because simulation results are different according to modeling methods even for same species (Thuiller, 2003). Therefore, there is still uncertainty of effects

Plant distribution is determined by integrated demographic processes such as seed dispersal, seed germination, growth and survival of individual plants. Since early demographic phase such as seedling establishment is more susceptible to environmental conditions than the adult phase (Kullman, 2002), it is important to clarify effects of temperature on seedling growth to predict effects of global warming on plant distribution. There are many experimental studies that examined effects of temperature on growth of tree seedlings (Danby & Hik, 2007; Hoch & Körner, 2009; Munier et al., 2010). For example, Yin et al. (2008) reported that seedling growth of *Betula albo-sinensis* increased in the warm condition with 0.51ºC higher than the ambient air condition. Many experimental studies that examined effects of temperature were conducted at bright conditions (e.g. Danby & Hik, 2007; Way & Sage, 2008). However, most seedlings distribute in dark closed-canopy conditions in forests. Therefore, it is necessary to examine effects of temperature on seedling

Plants plastically change morphology according to light conditions. For example, relative biomass allocation to leaves is greater in dark conditions than in bright conditions,

**1. Introduction**

of global warming on plant distribution.

growth not only in bright conditions but also in dark conditions.

### **Effects of Temperature and Light Conditions on Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species and Cool-Temperate Deciduous Tree Species**

Koichi Takahashi1, Hiroyuki Kobori1 and Tatsuyuki Seino2

*1Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano, 2Yatsugatake Forest, Agricultural and Forestry Research Center, Graduate School of Life and Environmental Sciences, University of Tsukuba, Minamimaki, Nagano, Japan*

#### **1. Introduction**

It is suggested that global warming affects plant distribution along latitudinal and altitudinal gradients because vegetation changes with thermal conditions. Simulation studies predicted that global warming largely affects plant distribution (e.g., Morin et al., 2008). Actually, vegetation change during several decades has been observed (Penuelas et al., 2007; Lenoir et al., 2008). By contrast, some other studies did not observe vegetation changes (Holtmeier & Broll, 2007; Harsch et al., 2009). Interpretation of results of simulation models also needs caution because simulation results are different according to modeling methods even for same species (Thuiller, 2003). Therefore, there is still uncertainty of effects of global warming on plant distribution.

Plant distribution is determined by integrated demographic processes such as seed dispersal, seed germination, growth and survival of individual plants. Since early demographic phase such as seedling establishment is more susceptible to environmental conditions than the adult phase (Kullman, 2002), it is important to clarify effects of temperature on seedling growth to predict effects of global warming on plant distribution. There are many experimental studies that examined effects of temperature on growth of tree seedlings (Danby & Hik, 2007; Hoch & Körner, 2009; Munier et al., 2010). For example, Yin et al. (2008) reported that seedling growth of *Betula albo-sinensis* increased in the warm condition with 0.51ºC higher than the ambient air condition. Many experimental studies that examined effects of temperature were conducted at bright conditions (e.g. Danby & Hik, 2007; Way & Sage, 2008). However, most seedlings distribute in dark closed-canopy conditions in forests. Therefore, it is necessary to examine effects of temperature on seedling growth not only in bright conditions but also in dark conditions.

Plants plastically change morphology according to light conditions. For example, relative biomass allocation to leaves is greater in dark conditions than in bright conditions,

Effects of Temperature and Light Conditions on

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 177

Fig. 1. Distributions (areas surrounded by red lines) of *Quercus myrsinaefolia*, *Q. crispula* and *Betula platyphylla* var. *japonica* in Japan. Distributions of the two *Quercus* species and *B. platyphylla* var. *japonica* were redrawn from Horikawa (1972) and Okuyama (1982), respectively. Blue and green dots indicate locations of two study sites, Matsumoto and

Nobeyama, respectively.

accompanied with reduction of leaf mass per area (LMA) (Ellsworth & Reich, 1992; Gould, 1993; Niinemets et al., 1999; Takahashi et al., 2005). These morphological changes increase light capture per plant, which is an adaptive strategy in dark conditions. Thus, plants adapt to changing environments through morphological plasticity. This study compared growth responses to temperature and light conditions among three species with different climatically distribution ranges. This comparison is important to clarify effects of global warming on distribution shift of vegetation.

Central Japan is a latitudinal vegetation ecotone between warm-temperate evergreen broadleaved forests and cool-temperate deciduous broad-leaved forests. Ishigami et al. (2003) simulated net primary production (NPP) of forests by the modified model of BIOME3 (Haxeltine & Prentice, 1996). They predicted that NPP of warm-temperate evergreen broadleaved forests is greater than that of cool-temperate deciduous broad-leaved forests above the current northern distribution limit of warm-temperate evergreen broad-leaved forests if air temperature is increased by global warming. According to the simulation result of NPP, Ishigami et al. (2003) suggested that the northern distribution limit of warm-temperate evergreen broad-leaved forests will move to the north. However, the northern distribution limit would not move to the north easily because of competition with existing northern vegetation (Kohyama & Shigesada, 1995). Therefore, it is important to compare growth responses of seedlings to temperature and light conditions between warm-temperate evergreen broad-leaved species and cool-temperate deciduous broad-leaved species to clarify vegetation changes due to global warming.

It is considered that warm-temperate evergreen broad-leaved species cannot distribute in the cool-temperate zone due to low winter temperature, not due to low summer temperature for the growth (Kira, 1949). Cold winter temperature decreases photochemical efficiency of leaves in warm-temperate evergreen broad-leaved species (Aranda et al., 2005; Taneda & Tateno, 2005). Reduction of photochemical efficiency due to cold temperature is more conspicuous in bright conditions than dark conditions (Matsuki et al., 2003). Therefore, it is necessary to examine growth responses to temperature and light conditions not only during growth period but also during dormant period (winter) to clarify how temperature affects growth of warm-temperate evergreen broad-leaved species.

This study examined effects of temperature and light conditions on seedling growth of a warm-temperate evergreen broad-leaved species and two cool-temperate deciduous broadleaved species to answer the following two questions.

(1) Do the warm-temperate evergreen and cool-temperate deciduous broad-leaved species grow more in higher temperature and light conditions?

(2) Do high temperature conditions mitigate reduction of photochemical efficiency in winter for the warm-temperate evergreen broad-leaved species?

#### **2. Materials and methods**

#### **2.1 Study site**

This study was conducted at the two sites in Nagano Prefecture, central Japan: the campus of Shinshu University (N3615'04'', E13758'41'', 630 m above sea level) in Matsumoto, and the Yatsugatake experimental forest (N3556'40'', E13828'11'', 1350 m a.s.l.) of Tsukuba University in Nobeyama (Fig. 1). The mean annual precipitation of Matsumoto was 1035.7 mm during 2000–2009. The mean annual temperature was 12.2C. The mean monthly

176 the Economy, Human Health, and on Urban and Natural Environments

accompanied with reduction of leaf mass per area (LMA) (Ellsworth & Reich, 1992; Gould, 1993; Niinemets et al., 1999; Takahashi et al., 2005). These morphological changes increase light capture per plant, which is an adaptive strategy in dark conditions. Thus, plants adapt to changing environments through morphological plasticity. This study compared growth responses to temperature and light conditions among three species with different climatically distribution ranges. This comparison is important to clarify effects of global

Central Japan is a latitudinal vegetation ecotone between warm-temperate evergreen broadleaved forests and cool-temperate deciduous broad-leaved forests. Ishigami et al. (2003) simulated net primary production (NPP) of forests by the modified model of BIOME3 (Haxeltine & Prentice, 1996). They predicted that NPP of warm-temperate evergreen broadleaved forests is greater than that of cool-temperate deciduous broad-leaved forests above the current northern distribution limit of warm-temperate evergreen broad-leaved forests if air temperature is increased by global warming. According to the simulation result of NPP, Ishigami et al. (2003) suggested that the northern distribution limit of warm-temperate evergreen broad-leaved forests will move to the north. However, the northern distribution limit would not move to the north easily because of competition with existing northern vegetation (Kohyama & Shigesada, 1995). Therefore, it is important to compare growth responses of seedlings to temperature and light conditions between warm-temperate evergreen broad-leaved species and cool-temperate deciduous broad-leaved species to

It is considered that warm-temperate evergreen broad-leaved species cannot distribute in the cool-temperate zone due to low winter temperature, not due to low summer temperature for the growth (Kira, 1949). Cold winter temperature decreases photochemical efficiency of leaves in warm-temperate evergreen broad-leaved species (Aranda et al., 2005; Taneda & Tateno, 2005). Reduction of photochemical efficiency due to cold temperature is more conspicuous in bright conditions than dark conditions (Matsuki et al., 2003). Therefore, it is necessary to examine growth responses to temperature and light conditions not only during growth period but also during dormant period (winter) to clarify how temperature

This study examined effects of temperature and light conditions on seedling growth of a warm-temperate evergreen broad-leaved species and two cool-temperate deciduous broad-

(1) Do the warm-temperate evergreen and cool-temperate deciduous broad-leaved species

(2) Do high temperature conditions mitigate reduction of photochemical efficiency in winter

This study was conducted at the two sites in Nagano Prefecture, central Japan: the campus of Shinshu University (N3615'04'', E13758'41'', 630 m above sea level) in Matsumoto, and the Yatsugatake experimental forest (N3556'40'', E13828'11'', 1350 m a.s.l.) of Tsukuba University in Nobeyama (Fig. 1). The mean annual precipitation of Matsumoto was 1035.7 mm during 2000–2009. The mean annual temperature was 12.2C. The mean monthly

warming on distribution shift of vegetation.

clarify vegetation changes due to global warming.

affects growth of warm-temperate evergreen broad-leaved species.

leaved species to answer the following two questions.

grow more in higher temperature and light conditions?

for the warm-temperate evergreen broad-leaved species?

**2. Materials and methods**

**2.1 Study site**

Fig. 1. Distributions (areas surrounded by red lines) of *Quercus myrsinaefolia*, *Q. crispula* and *Betula platyphylla* var. *japonica* in Japan. Distributions of the two *Quercus* species and *B. platyphylla* var. *japonica* were redrawn from Horikawa (1972) and Okuyama (1982), respectively. Blue and green dots indicate locations of two study sites, Matsumoto and Nobeyama, respectively.

Effects of Temperature and Light Conditions on

\*: *p* < 0.05, \*\*: *p* < 0.01, \*\*\*: *p* < 0.001.

growth.

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 179

**Species Variables T L T × L** *n* **Qm** Stem height 56.1\*\*\* < 0.1 0.3 99 Stem diameter 16.1\*\*\* 12.5\*\*\* 0.2 46 Dry mass 24.2\*\*\* 15.1\*\*\* 1.5 46 LMA 3.5\* 7.0\* 1.2 47 **Qc** Stem height 46.2\*\*\* 3.1 1.3 94 Stem diameter 18.0\*\*\* 36.2\*\*\* 1.6 94 Dry mass 11.6\*\*\* 14.2\*\*\* 0.6 94 LMA 32.4\*\*\* 67.4\*\*\* 0.4 93 **Bp** Stem height 517.5\*\*\* 0.4 1.0 120 Stem diameter 768.0\*\*\* 11.5\*\*\* 1.3 120 Dry mass 595.9\*\*\* 49.5\*\*\* 8.3\*\*\* 117 LMA 2.3 52.9\*\*\* 2.3 109

Table 1. ANOVA results of stem height, stem diameter, seedling dry mass and leaf mass per area (LMA) for *Quercus myrsinaefolia* (Qm), *Quercus crispula* (Qc) and *Betula platyphylla* var. *japonica* (Bp) to test three temperature conditions (T), two light conditions (L) and the interaction (T × L) on the four variables. *F*-values and the number of samples (*n*) are shown. Seeds of *Q. myrsinaefolia*, *B. platyphylla* var. *japonica* and *Q. crispula* were collected in 2008. Seeds were sown in plastic pots (diameter 15 cm × height 13 cm) filled with horticulture soil and leaf mold. The number of pots was 20 for each light condition (L1 and L2) at each temperature condition (T1, T2 and T3) for each species. Seed sowing was conducted between late March and early April in 2009. However, seeds of *B. platyphylla* var. *japonica* were sown again at T1 in early June because seeds did not germinate. Pots were watered once a week or according to the need during the experiment period. All pots were randomly moved within each treatment once a week to minimize effects of pot position on seedling

Seedlings of the three species were excavated and washed carefully in early October in 2009 after the cease of the current-year growth. In terms of *Q. myrsinaefolia*, only the half number of seedlings was excavated at each light and temperature condition, and the other seedlings were remained for the measurement of photochemical efficiency (see next paragraph). Stem diameter at the base and stem height were measured at the harvest for the three species. Seedlings were divided into stem, root and leaf, and then all leaves were scanned by using the free graphic software ImageJ (http://rsbweb.nih.gov/ij/) to measure total leaf area per

The ratio of light-induced variable to maximum fluorescence of chlorophyll (Fv/Fm) of *Q. myrsinaefolia* was measured as a surrogate for photochemical efficiency of PSII (Demmig-Adams et al., 1989). Photochemical efficiency was measured around the noon once a month from summer of 2009 to the next summer by using a chlorophyll fluorometer OS-30p (Opti-Science, NH, USA). The measurement was conducted after 30 min of dark acclimation. Two-way ANOVA was done to examine effects of temperature and light conditions on stem height, stem diameter, LMA and seedling dry mass (including root) for the three species (*Q.*

seedling. Each organ was oven-dried at 80C for two days, and was weighed.

*myrsinaefolia*, *B. platyphylla* var. *japonica* and *Q. crispula*).

temperatures in January and in August were –0.3 and 24.8C, respectively. The mean annual precipitation of Nobeyama was 1435.3 mm during 2000–2009. The mean annual temperature was 7.2C. The mean monthly temperatures in January and in August were –5.4 and 19.2C, respectively.

#### **2.2 Study species**

This study examined a warm-temperate evergreen broad-leaved species (*Quercus myrsinaefolia* Blume) and two cool-temperate deciduous broad-leaved species (*Betula platyphylla* var. *japonica* Hara and *Quercus crispula* Blume) (Fig. 1). *Q. myrsinaefolia* is a shadetolerant tall tree species that distributes in the northern part of warm-temperate zone (Horikawa, 1972). *B. platyphylla* var. *japonica* and *Q. crispula* are shade-intolerant and midshade-tolerant tall tree species, respectively (Samejima, 1979; Koike, 1988; Masaki et al., 1992). *B. platyphylla* var. *japonica* and *Q. crispula* often form pure stands after disturbances such as strong wind, forest fire and clear cutting (Samejima, 1979; Kamitani, 1993; Namikawa et al., 1997).

*Q. myrsinaefolia* distributes below 700 m a.s.l. in the warm-temperate zone, and the northern distribution limit is about N38 (Horikawa, 1972). Distribution of *Q. myrsinaefolia* is limited to below 500 m a.s.l. in the north of N36. Although the southern part of Nagano Prefecture was the northern distribution limit of *Q. myrsinaefolia* (Horikawa, 1972), the distribution of *Q. myrsinaefolia* is recently observed in central part (Matsumoto area) of Nagano Prefecture probably because of escapes of seeds from planted *Q. myrsinaefolia* trees (Otsuka & Ozeki, 2008). *B. platyphylla* var. *japonica* distributes in central and northern Japan (Okuyama, 1981). *B. platyphylla* var. *japonica* distributes mainly at about 1000 m and 1300 m a.s.l. in Matsumoto and Nobeyama, respectively (Editorial Board of Flora of Nagano Prefecture, 1997). *Q. crispula* is one of the dominant species in cool-temperate deciduous broad-leaved forests, and widely distributes in the cool-temperate zone of Japan (Horikawa, 1972). *Q. crispula* distributes mainly at about 1000 m and 1300 m a.s.l. in Matsumoto and Nobeyama, respectively, like *B. platyphylla* var. *japonica*. Therefore, Matsumoto is the northern distribution limit for the warm-temperate evergreen species *Q. myrsinaefolia*, and Nobeyama is optimal thermal conditions for the two cool-temperate deciduous species *B. platyphylla* var. *japonica* and *Q. crispula*.

#### **2.3 Seedling growth experiments**

Growth experiments were conducted at Matsumoto and Nobeyama to examine effects of temperature and light conditions on the current-year seedling growth of *Q. myrsinaefolia*, *B. platyphylla* var. *japonica* and *Q. crispula*. A greenhouse experiment was also conducted at Matsumoto to make optimal temperature conditions for the warm-temperate evergreen *Q. myrsinaefolia* because Matsumoto is the northern distribution limit of this species. Windows of the greenhouse were opened in summer to avoid extreme rising of air temperature inside the greenhouse. Increase of air temperature in the greenhouse was 3C on the average. Therefore, air temperature increased in the order of Nobeyama, Matsumoto and the greenhouse at Matsumoto. Nobeyama, Matsumoto and the greenhouse at Matsumoto are referred to T1, T2 and T3, respectively, in this study. Two light conditions (20% and 54% light) were set at each temperature condition by using shade cloth. 20% and 54% light conditions are referred to L1 and L2, respectively.

\*: *p* < 0.05, \*\*: *p* < 0.01, \*\*\*: *p* < 0.001.

Global Warming Impacts – Case Studies on

178 the Economy, Human Health, and on Urban and Natural Environments

temperatures in January and in August were –0.3 and 24.8C, respectively. The mean annual precipitation of Nobeyama was 1435.3 mm during 2000–2009. The mean annual temperature was 7.2C. The mean monthly temperatures in January and in August were –5.4 and 19.2C,

This study examined a warm-temperate evergreen broad-leaved species (*Quercus myrsinaefolia* Blume) and two cool-temperate deciduous broad-leaved species (*Betula platyphylla* var. *japonica* Hara and *Quercus crispula* Blume) (Fig. 1). *Q. myrsinaefolia* is a shadetolerant tall tree species that distributes in the northern part of warm-temperate zone (Horikawa, 1972). *B. platyphylla* var. *japonica* and *Q. crispula* are shade-intolerant and midshade-tolerant tall tree species, respectively (Samejima, 1979; Koike, 1988; Masaki et al., 1992). *B. platyphylla* var. *japonica* and *Q. crispula* often form pure stands after disturbances such as strong wind, forest fire and clear cutting (Samejima, 1979; Kamitani, 1993;

*Q. myrsinaefolia* distributes below 700 m a.s.l. in the warm-temperate zone, and the northern distribution limit is about N38 (Horikawa, 1972). Distribution of *Q. myrsinaefolia* is limited to below 500 m a.s.l. in the north of N36. Although the southern part of Nagano Prefecture was the northern distribution limit of *Q. myrsinaefolia* (Horikawa, 1972), the distribution of *Q. myrsinaefolia* is recently observed in central part (Matsumoto area) of Nagano Prefecture probably because of escapes of seeds from planted *Q. myrsinaefolia* trees (Otsuka & Ozeki, 2008). *B. platyphylla* var. *japonica* distributes in central and northern Japan (Okuyama, 1981). *B. platyphylla* var. *japonica* distributes mainly at about 1000 m and 1300 m a.s.l. in Matsumoto and Nobeyama, respectively (Editorial Board of Flora of Nagano Prefecture, 1997). *Q. crispula* is one of the dominant species in cool-temperate deciduous broad-leaved forests, and widely distributes in the cool-temperate zone of Japan (Horikawa, 1972). *Q. crispula* distributes mainly at about 1000 m and 1300 m a.s.l. in Matsumoto and Nobeyama, respectively, like *B. platyphylla* var. *japonica*. Therefore, Matsumoto is the northern distribution limit for the warm-temperate evergreen species *Q. myrsinaefolia*, and Nobeyama is optimal thermal conditions for the two cool-temperate deciduous species *B. platyphylla*

Growth experiments were conducted at Matsumoto and Nobeyama to examine effects of temperature and light conditions on the current-year seedling growth of *Q. myrsinaefolia*, *B. platyphylla* var. *japonica* and *Q. crispula*. A greenhouse experiment was also conducted at Matsumoto to make optimal temperature conditions for the warm-temperate evergreen *Q. myrsinaefolia* because Matsumoto is the northern distribution limit of this species. Windows of the greenhouse were opened in summer to avoid extreme rising of air temperature inside the greenhouse. Increase of air temperature in the greenhouse was 3C on the average. Therefore, air temperature increased in the order of Nobeyama, Matsumoto and the greenhouse at Matsumoto. Nobeyama, Matsumoto and the greenhouse at Matsumoto are referred to T1, T2 and T3, respectively, in this study. Two light conditions (20% and 54% light) were set at each temperature condition by using shade cloth. 20% and 54% light

respectively.

**2.2 Study species**

Namikawa et al., 1997).

var. *japonica* and *Q. crispula*.

**2.3 Seedling growth experiments**

conditions are referred to L1 and L2, respectively.

Table 1. ANOVA results of stem height, stem diameter, seedling dry mass and leaf mass per area (LMA) for *Quercus myrsinaefolia* (Qm), *Quercus crispula* (Qc) and *Betula platyphylla* var. *japonica* (Bp) to test three temperature conditions (T), two light conditions (L) and the interaction (T × L) on the four variables. *F*-values and the number of samples (*n*) are shown.

Seeds of *Q. myrsinaefolia*, *B. platyphylla* var. *japonica* and *Q. crispula* were collected in 2008. Seeds were sown in plastic pots (diameter 15 cm × height 13 cm) filled with horticulture soil and leaf mold. The number of pots was 20 for each light condition (L1 and L2) at each temperature condition (T1, T2 and T3) for each species. Seed sowing was conducted between late March and early April in 2009. However, seeds of *B. platyphylla* var. *japonica* were sown again at T1 in early June because seeds did not germinate. Pots were watered once a week or according to the need during the experiment period. All pots were randomly moved within each treatment once a week to minimize effects of pot position on seedling growth.

Seedlings of the three species were excavated and washed carefully in early October in 2009 after the cease of the current-year growth. In terms of *Q. myrsinaefolia*, only the half number of seedlings was excavated at each light and temperature condition, and the other seedlings were remained for the measurement of photochemical efficiency (see next paragraph). Stem diameter at the base and stem height were measured at the harvest for the three species. Seedlings were divided into stem, root and leaf, and then all leaves were scanned by using the free graphic software ImageJ (http://rsbweb.nih.gov/ij/) to measure total leaf area per seedling. Each organ was oven-dried at 80C for two days, and was weighed.

The ratio of light-induced variable to maximum fluorescence of chlorophyll (Fv/Fm) of *Q. myrsinaefolia* was measured as a surrogate for photochemical efficiency of PSII (Demmig-Adams et al., 1989). Photochemical efficiency was measured around the noon once a month from summer of 2009 to the next summer by using a chlorophyll fluorometer OS-30p (Opti-Science, NH, USA). The measurement was conducted after 30 min of dark acclimation.

Two-way ANOVA was done to examine effects of temperature and light conditions on stem height, stem diameter, LMA and seedling dry mass (including root) for the three species (*Q. myrsinaefolia*, *B. platyphylla* var. *japonica* and *Q. crispula*).

Effects of Temperature and Light Conditions on

positive standard deviations are shown.

**3.1 Growth of the three species**

**3. Results**

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 181

Fig. 3. Stem diameter for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values with

Effect of temperature on stem height was significant for the three species (Table 1), i.e., stem height was greater in higher temperature conditions, irrespective of light conditions (Fig. 2). However, stem height of *Q. crispula* was not different between T1 and T2 because of the shoot re-growth from the base after the shoot die-back in late May at T2. The difference in stem height of *Q. myrsinaefolia* between T2 and T3 was also not large (Fig. 2). Light conditions did not affect stem height for the three species (Table 1). By contrast, stem diameter of the three species was different not only among the three temperature conditions

Fig. 2. Stem height for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values with positive standard deviations are shown.

Fig. 3. Stem diameter for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values with positive standard deviations are shown.

#### **3. Results**

Global Warming Impacts – Case Studies on

180 the Economy, Human Health, and on Urban and Natural Environments

Fig. 2. Stem height for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values with

positive standard deviations are shown.

#### **3.1 Growth of the three species**

Effect of temperature on stem height was significant for the three species (Table 1), i.e., stem height was greater in higher temperature conditions, irrespective of light conditions (Fig. 2). However, stem height of *Q. crispula* was not different between T1 and T2 because of the shoot re-growth from the base after the shoot die-back in late May at T2. The difference in stem height of *Q. myrsinaefolia* between T2 and T3 was also not large (Fig. 2). Light conditions did not affect stem height for the three species (Table 1). By contrast, stem diameter of the three species was different not only among the three temperature conditions

Effects of Temperature and Light Conditions on

large (Fig. 5).

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 183

LMA was greater at L2 than L1 for the three species (Fig. 5, Table 1). LMA of *Q. myrsinaefolia* and *Q. crispula* was significantly different among the three temperature conditions (Table 1). Although LMA of the two species was greater at T2 than T1 and T3, the difference was not

Fig. 5. Leaf mass per area (LMA) for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values

Values of photochemical efficiency (Fv/Fm) differed, according to temperature and light conditions and season (Fig. 6). In August of 2009, Fv/Fm values were similar between the two light conditions for the three temperature conditions. Although Fv/Fm value was about 0.8 at T3 in August, Fv/Fm values slightly decreased as temperature decreased from T3 to T1. Fv/Fm value was about 0.7 at T1 in August of 2009. Fv/Fm value decreased from autumn to winter in each temperature condition. This tendency was more conspicuous at L2

with positive standard deviations are shown.

**3.2 Photochemical efficiency of** *Q. myrsinaefolia*

but also between the two light conditions (Table 1). Stem diameter of the three species was greater at L2 than L1 for each temperature condition (Fig. 3). Stem diameter was greater at T2 and T3 than T1 for *Q. crispula*. Stem diameter of *B. platyphylla* var. *japonica* increased in the order of T1, T2 and T3. However, stem diameter of *Q. myrsinaefolia* was slightly smaller at T3 than T2 for the two light conditions.

Fig. 4. Seedling dry mass for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values with positive standard deviations are shown.

Effects of temperature and light conditions on seedling dry mass were significant for the three species (Table 1), i.e., seedling dry mass was greater at higher temperature and brighter light conditions (Fig. 4). However, seedling dry masses of *Q. myrsinaefolia* at T3 were similar to and smaller than those at T2 for L1 and L2, respectively.

182 the Economy, Human Health, and on Urban and Natural Environments

but also between the two light conditions (Table 1). Stem diameter of the three species was greater at L2 than L1 for each temperature condition (Fig. 3). Stem diameter was greater at T2 and T3 than T1 for *Q. crispula*. Stem diameter of *B. platyphylla* var. *japonica* increased in the order of T1, T2 and T3. However, stem diameter of *Q. myrsinaefolia* was slightly smaller

Fig. 4. Seedling dry mass for the three species at the three temperature and two light

were similar to and smaller than those at T2 for L1 and L2, respectively.

with positive standard deviations are shown.

conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values

Effects of temperature and light conditions on seedling dry mass were significant for the three species (Table 1), i.e., seedling dry mass was greater at higher temperature and brighter light conditions (Fig. 4). However, seedling dry masses of *Q. myrsinaefolia* at T3

at T3 than T2 for the two light conditions.

LMA was greater at L2 than L1 for the three species (Fig. 5, Table 1). LMA of *Q. myrsinaefolia* and *Q. crispula* was significantly different among the three temperature conditions (Table 1). Although LMA of the two species was greater at T2 than T1 and T3, the difference was not large (Fig. 5).

Fig. 5. Leaf mass per area (LMA) for the three species at the three temperature and two light conditions. Blue and red bars indicate L1 and L2 light conditions, respectively. Mean values with positive standard deviations are shown.

#### **3.2 Photochemical efficiency of** *Q. myrsinaefolia*

Values of photochemical efficiency (Fv/Fm) differed, according to temperature and light conditions and season (Fig. 6). In August of 2009, Fv/Fm values were similar between the two light conditions for the three temperature conditions. Although Fv/Fm value was about 0.8 at T3 in August, Fv/Fm values slightly decreased as temperature decreased from T3 to T1. Fv/Fm value was about 0.7 at T1 in August of 2009. Fv/Fm value decreased from autumn to winter in each temperature condition. This tendency was more conspicuous at L2

Effects of Temperature and Light Conditions on

observed on March 11.

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 185

species, except for the effect of light on stem height, and (2) that high temperature mitigated reduction of photochemical efficiency (Fv/Fm) of *Q. myrsinaefolia* in winter. Furthermore, winter temperature was so cold at T1 for *Q. myrsinaefolia* that all seedlings of this species died in winter. Therefore, this study showed that high temperature and light conditions increased the seedling growth of the three species and survival of *Q. myrsinaefolia* in winter.

Fig. 7. A current-year seedling of *Quercus myrsinaefolia* at T1 with L1 light condition on February 16 and March 11, 2010. These photos were taken for a same seedling. Die-back was

than stem diameter in dark conditions as compared with bright conditions.

Light conditions did not affect stem height of the three species. Seiwa & Kikuzawa (1989) also reported that stem height of current-year seedlings was not different between bright and dark conditions for deciduous broad-leaved species with small seeds, while stem height was smaller in bright conditions than dark conditions for those of large seeds. Thus, stem height is not always taller in brighter conditions for current-year seedlings. This may reflect a survival strategy in forests. Litter accumulation on forest floor is a factor reducing seed germination and seedling survival (Goldberg & Werner, 1983). Therefore, the current-year seedlings germinated below litter layer have to grow over the litter layer. Furthermore, high growth of stem height is also advantageous for survival because of competition with other plants. Therefore, seedlings of the three species would preferentially grow stem height more

LMA was smaller at darker conditions for the three species at the three temperature conditions, which is advantageous for light capture efficiency per unit leaf mass. LMA of *Q. myrsinaefolia* and *Q. crispula* was also affected by temperature. Although LMA of the two

than L1 in each temperature condition. In addition, Fv/Fm value decreased to almost zero in February at T1, and all seedlings died by March (Fig. 7). By contrast, although Fv/Fm values decreased in winter at T2 and T3, these values increased again from spring to summer (Fig. 6) and the seedlings grew well by autumn (Fig. 8).

Fig. 6. Seasonal change of Fv/Fm of *Quercus myrsinaefolia* at three temperature and two light conditions. Blue and red circles indicate L1 and L2 light conditions, respectively. Mean values with standard deviations are shown.

#### **4. Discussion**

This study showed (1) that high temperature and light conditions increased the current-year seedling growth of the warm-temperate evergreen and the two cool-temperate deciduous

184 the Economy, Human Health, and on Urban and Natural Environments

than L1 in each temperature condition. In addition, Fv/Fm value decreased to almost zero in February at T1, and all seedlings died by March (Fig. 7). By contrast, although Fv/Fm values decreased in winter at T2 and T3, these values increased again from spring to

Fig. 6. Seasonal change of Fv/Fm of *Quercus myrsinaefolia* at three temperature and two light conditions. Blue and red circles indicate L1 and L2 light conditions, respectively. Mean

This study showed (1) that high temperature and light conditions increased the current-year seedling growth of the warm-temperate evergreen and the two cool-temperate deciduous

values with standard deviations are shown.

**4. Discussion**

summer (Fig. 6) and the seedlings grew well by autumn (Fig. 8).

species, except for the effect of light on stem height, and (2) that high temperature mitigated reduction of photochemical efficiency (Fv/Fm) of *Q. myrsinaefolia* in winter. Furthermore, winter temperature was so cold at T1 for *Q. myrsinaefolia* that all seedlings of this species died in winter. Therefore, this study showed that high temperature and light conditions increased the seedling growth of the three species and survival of *Q. myrsinaefolia* in winter.

Fig. 7. A current-year seedling of *Quercus myrsinaefolia* at T1 with L1 light condition on February 16 and March 11, 2010. These photos were taken for a same seedling. Die-back was observed on March 11.

Light conditions did not affect stem height of the three species. Seiwa & Kikuzawa (1989) also reported that stem height of current-year seedlings was not different between bright and dark conditions for deciduous broad-leaved species with small seeds, while stem height was smaller in bright conditions than dark conditions for those of large seeds. Thus, stem height is not always taller in brighter conditions for current-year seedlings. This may reflect a survival strategy in forests. Litter accumulation on forest floor is a factor reducing seed germination and seedling survival (Goldberg & Werner, 1983). Therefore, the current-year seedlings germinated below litter layer have to grow over the litter layer. Furthermore, high growth of stem height is also advantageous for survival because of competition with other plants. Therefore, seedlings of the three species would preferentially grow stem height more than stem diameter in dark conditions as compared with bright conditions.

LMA was smaller at darker conditions for the three species at the three temperature conditions, which is advantageous for light capture efficiency per unit leaf mass. LMA of *Q. myrsinaefolia* and *Q. crispula* was also affected by temperature. Although LMA of the two

Effects of Temperature and Light Conditions on

*Q. myrsinaefolia* in the northern distribution limit.

period.

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 187

temperate evergreen broad-leaved forests tend to be determined by coldness in winter, not by warmth during the growth period (Kira, 1991). Thus, winter temperature may be

Increase of temperature clearly mitigated reduction of photochemical efficiency (Fv/Fm) of *Q. myrsinaefolia* during winter. This tendency was more conspicuous at brighter conditions. In forests, seedlings hardly grow in understory dark conditions, and grow vigorously in canopy gaps (Takahashi et al., 2001; Takahashi & Rustandi, 2006). Therefore, it is suggested that increase of photochemical efficiency of *Q. myrsinaefolia* by increase of temperature would enhance the growth in bright conditions. This effect may be large in the northern distribution limit of *Q. myrsinaefolia* because *Q. myrsinaefolia* co-dominates with many deciduous broad-leaved species there (Otsuka et al., 2004). Much light penetrates into forest floor in winter because of leaf fall of deciduous broad-leaved species. Understory seedlings and saplings of *Q. myrsinaefolia* can assimilate during winter after leaf fall of deciduous broad-leaved species, and the photosynthetic production during winter increases the growth and survival of *Q. myrsinaefolia* in the understory (Takenaka, 1986). Therefore, increase of winter temperature would enhance the growth and photochemical efficiency of

The growth of the two cool-temperate deciduous broad-leaved species (*Q. crispula* and *B. platyphylla* var. *japonica*) was greater at T2 and T3 conditions than T1 (the optimal temperature conditions). Many studies showed that tree growth is greater nearer the southern and lower distribution limits along latitudinal and altitudinal gradients, respectively (Persson, 1998; Mäkinen et al., 2000; Li et al., 2003; Takahashi & Yoshida, 2009). Although the results of the previous studies were conducted in natural distribution ranges, the result of this study showed that tree growth increases in the temperature conditions warmer than the natural distribution range. Drought stress increases near southern and lower distribution limits (Buckley et al., 1997; Takahashi et al., 2003a; Adams & Kolb, 2005; Hart et al., 2010; Lebourgeois et al., 2010). On the contrary, increase of temperature prolongs the growth period (Sparks et al., 2000; Chmielewski & Rotzer, 2001; Fujimoto, 2008). Although increase of temperature induces drought stress to some extent, it would increase annual growth of plants by increasing annual carbon gain through increase of growth

The high growth of *Q. crispula* and *B. platyphylla* var. *japonica* at the two temperature conditions (T2 and T3) warmer than the current distribution range suggests that the natural distribution ranges of the two species are not determined by optimal temperature conditions alone (i.e., the concept of ecological niche). Although *Q. crispula* is a common species in oldgrowth deciduous broad-leaved forests (Masaki et al., 1992; Takahashi et al., 2003b), *Q. crispula* is recognized as a gap-dependent species (Yamamoto, 1989). It is reported that saplings of deciduous broad-leaved species distributed mainly in canopy gaps in an evergreen broad-leaved forest (Miura et al., 2001). In deciduous broad-leaved forests, understory saplings increases carbon gain by leafing before leaf expansion of canopy trees (Seiwa & Kikuzawa, 1996). However, understory of evergreen broad-leaved forests is dark conditions throughout a year, which have restricted saplings of deciduous broad-leaved species into canopy gaps. Therefore, actual distribution ranges are largely affected not only by temperature conditions but also by competition with other species (Takahashi, 2003).

important for the distribution of *Q. myrsinaefolia* along latitudinal gradients.

species tended to be greater at T2 than T1 and T3, these differences were not large. By contrast, Woodward (1979) observed that low temperature increased LMA of *Phleum bertolonii* and *P. alpinum* because of increase of cell diameter. Unfortunately, this study did not examine cell diameter of leaves at the three temperature conditions. However, it is possible that light dominates the control of LMA of the current-year seedlings of the three species, compared with temperature.

Fig. 8. A 1-year old seedling of *Quercus myrsiaefolia* at T3 with L1 light condition on October 14, 2010.

Many previous studies about effects of temperatures on plant growth have been conducted so far in cold environments such as alpine region and tundra (Chapin & Shaver, 1985; Molau, 1997; Arft et al., 1999; Hollister & Webber, 2000). It is often reported that high temperature increases plant growth in such cold environments (Chapin & Shaver, 1996; Takahashi, 2005), meaning that cold temperature limits plant growth. Increase of temperature enhances photosynthetic rates in cold environments because of temperature dependency of photosynthetic rates (DeLucia & Smith, 1987). Furthermore, high temperature throughout a year expands the growth period of plants (Chmielewski & Rotzer, 2001). Nevertheless, seedling dry mass, stem height and diameter of *Q. myrsinaefolia* at T3 were similar to or smaller than those at T2 (the temperature conditions of the northern distribution limit). This suggests that temperature during the growth period is not a major factor limiting the growth of *Q. myrsinaefolia*. Northern distribution limits of warm-

186 the Economy, Human Health, and on Urban and Natural Environments

species tended to be greater at T2 than T1 and T3, these differences were not large. By contrast, Woodward (1979) observed that low temperature increased LMA of *Phleum bertolonii* and *P. alpinum* because of increase of cell diameter. Unfortunately, this study did not examine cell diameter of leaves at the three temperature conditions. However, it is possible that light dominates the control of LMA of the current-year seedlings of the three

Fig. 8. A 1-year old seedling of *Quercus myrsiaefolia* at T3 with L1 light condition on October

Many previous studies about effects of temperatures on plant growth have been conducted so far in cold environments such as alpine region and tundra (Chapin & Shaver, 1985; Molau, 1997; Arft et al., 1999; Hollister & Webber, 2000). It is often reported that high temperature increases plant growth in such cold environments (Chapin & Shaver, 1996; Takahashi, 2005), meaning that cold temperature limits plant growth. Increase of temperature enhances photosynthetic rates in cold environments because of temperature dependency of photosynthetic rates (DeLucia & Smith, 1987). Furthermore, high temperature throughout a year expands the growth period of plants (Chmielewski & Rotzer, 2001). Nevertheless, seedling dry mass, stem height and diameter of *Q. myrsinaefolia* at T3 were similar to or smaller than those at T2 (the temperature conditions of the northern distribution limit). This suggests that temperature during the growth period is not a major factor limiting the growth of *Q. myrsinaefolia*. Northern distribution limits of warm-

species, compared with temperature.

14, 2010.

temperate evergreen broad-leaved forests tend to be determined by coldness in winter, not by warmth during the growth period (Kira, 1991). Thus, winter temperature may be important for the distribution of *Q. myrsinaefolia* along latitudinal gradients.

Increase of temperature clearly mitigated reduction of photochemical efficiency (Fv/Fm) of *Q. myrsinaefolia* during winter. This tendency was more conspicuous at brighter conditions. In forests, seedlings hardly grow in understory dark conditions, and grow vigorously in canopy gaps (Takahashi et al., 2001; Takahashi & Rustandi, 2006). Therefore, it is suggested that increase of photochemical efficiency of *Q. myrsinaefolia* by increase of temperature would enhance the growth in bright conditions. This effect may be large in the northern distribution limit of *Q. myrsinaefolia* because *Q. myrsinaefolia* co-dominates with many deciduous broad-leaved species there (Otsuka et al., 2004). Much light penetrates into forest floor in winter because of leaf fall of deciduous broad-leaved species. Understory seedlings and saplings of *Q. myrsinaefolia* can assimilate during winter after leaf fall of deciduous broad-leaved species, and the photosynthetic production during winter increases the growth and survival of *Q. myrsinaefolia* in the understory (Takenaka, 1986). Therefore, increase of winter temperature would enhance the growth and photochemical efficiency of *Q. myrsinaefolia* in the northern distribution limit.

The growth of the two cool-temperate deciduous broad-leaved species (*Q. crispula* and *B. platyphylla* var. *japonica*) was greater at T2 and T3 conditions than T1 (the optimal temperature conditions). Many studies showed that tree growth is greater nearer the southern and lower distribution limits along latitudinal and altitudinal gradients, respectively (Persson, 1998; Mäkinen et al., 2000; Li et al., 2003; Takahashi & Yoshida, 2009). Although the results of the previous studies were conducted in natural distribution ranges, the result of this study showed that tree growth increases in the temperature conditions warmer than the natural distribution range. Drought stress increases near southern and lower distribution limits (Buckley et al., 1997; Takahashi et al., 2003a; Adams & Kolb, 2005; Hart et al., 2010; Lebourgeois et al., 2010). On the contrary, increase of temperature prolongs the growth period (Sparks et al., 2000; Chmielewski & Rotzer, 2001; Fujimoto, 2008). Although increase of temperature induces drought stress to some extent, it would increase annual growth of plants by increasing annual carbon gain through increase of growth period.

The high growth of *Q. crispula* and *B. platyphylla* var. *japonica* at the two temperature conditions (T2 and T3) warmer than the current distribution range suggests that the natural distribution ranges of the two species are not determined by optimal temperature conditions alone (i.e., the concept of ecological niche). Although *Q. crispula* is a common species in oldgrowth deciduous broad-leaved forests (Masaki et al., 1992; Takahashi et al., 2003b), *Q. crispula* is recognized as a gap-dependent species (Yamamoto, 1989). It is reported that saplings of deciduous broad-leaved species distributed mainly in canopy gaps in an evergreen broad-leaved forest (Miura et al., 2001). In deciduous broad-leaved forests, understory saplings increases carbon gain by leafing before leaf expansion of canopy trees (Seiwa & Kikuzawa, 1996). However, understory of evergreen broad-leaved forests is dark conditions throughout a year, which have restricted saplings of deciduous broad-leaved species into canopy gaps. Therefore, actual distribution ranges are largely affected not only by temperature conditions but also by competition with other species (Takahashi, 2003).

Effects of Temperature and Light Conditions on

Portugal. *Planta*, 177, 377-387.

75-87 (in Japanese).

Mainichi Shinbunsha, Nagano, Japan (in Japanese).

environments. *Functional Ecology*, 6, 423-435.

533.

155.

1040-1049.

Japanese).

*Research*, 6, 185-200.

America. *Trees*, 24, 621-633.

*Landscape Online*, 1, 1-33.

*Meteorology*, 59, 269-276 (in Japanese).

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 189

DeLucia, E. H. & Smith, W. K. (1987) Air and soil temperature limitations on photosynthesis

Demmig-Adams, B., Adams, W.I., Winter, K., Meyer, A., Schreiber, U., Pereira, J. S., Krüger,

Editorial Board of Flora of Nagano Prefecture (1997) Flora of Nagano Prefecture. Shinano

Ellsworth, D. S. & Reich, P. B. (1992) Leaf mass per area, nitrogen content and

Fujimoto, S. (2008) Estimating the impact of thermal change on broad-leaved tree leaf

Goldberg, D. E. & Werner, P. A. (1983) The effects of size of opening in vegetation and litter

Gould, K. S. (1993) Leaf heteroblasty in *Pseudopanax crassifolius*: functional significance of

Harsch, M. A., Hulme, P. E., McGlone, M. & Duncan, R. P. (2009) Are treelines advancing? A

Hart, J. L., Gevel, S. L., Sakulich, J. & Grissino-Mayer, H. D. (2010) Influence of climate and

Haxeltine, A. & Prentice, I. C. (1996) BIOME3: An equilibrium terrestrial biosphere model

Hollister, R. D. & Webber, P. J. (2000) Biotic validation of small open-top chambers in a

Holtmeier, F.-K. & Broll, G. (2007) Treeline advance -driving processes and adverse factors.

Ishigami, Y., Shimizu, Y. & Omasa, K. (2003) Projection of climatic change effects on

Kamitani, T. (1993) Ecological studies on regeneration of beech coppice forests in heavy

Kira, T. (1949) Forest zones of Japan. Ringyo Gizyutu Kyokai, Tokyo, Japan (in Japanese). Kira, T. (1991) Forest ecosystems of east and southeast Asia in a global perspective. *Ecological*

potential natural vegetation distribution in Japan. *Journal of Agricultural*

snowfall region. *Memoirs of the Faculty of Agriculture, Niigata University*, 30, 1-108 (in

among plant functional types. *Global Biogeochemical Cycles*, 10, 693-709. Hoch, G. & Körner, C. (2009) Growth and carbon relations of tree line forming conifers at

constant vs. variable low temperatures. *Journal of Ecology*, 97, 57-66.

tundra ecosystem. *Global Change Biology*, 6, 835-842.

Horikawa, Y. (1972) Atlas of the Japanese flora. Gakken, Tokyo, Japan.

leaf morphology and anatomy. *Annals of Botany*, 71, 61-70.

in Engelmann spruce during summer. *Canadian Journal of Forest Research*, 17, 527-

A., Czgan, F. C. & Lange, O. L. (1989) Photochemical efficiency of photosystem II, photon yield of O2 evolution, photosynthetic capacity, and carotenoid composition during the midday depression of net CO2 uptake in *Arbutus unedo* growing in

photosynthetic carbon gain in *Acer saccharum* seedlings in contrasting forest light

phenology in the warm temperate zone. *Japanese Journal of Conservation Ecology*, 13,

cover on seedling establishment of goldenrods (*Solidatgo* spp.). *Oecologia*, 60, 149-

global meta-analysis of treeline response to climate warming. *Ecology Letters*, 12,

disturbance on the growth of *Tsuga canadensis* at its southern limit in eastern North

based on ecophysiological constraints, resource availability, and competition

### **5. Conclusion**

This study predicted that high winter temperature mitigates the reduction of photochemical efficiency and reduces winter mortality of *Q. myrsinaefolia*. Furthermore, this study showed that increase of temperature enhances the growth of not only *Q. myrsinaefolia* but also *Q. crispula* and *B. platyphylla* var. *japonica*. It is possible that global warming intensifies competition between warm-temperate and cool-temperate broad-leaved species at their latitudinal ecotones. Therefore, it is unclear whether *Q. myrsinaefolia* will gain parts of the habitat now covered by *B. platyphylla* var. *japonica* and *Q. crispula* under global warming. This study examined only one warm-temperate broad-leaved species and two cooltemperate broad-leaved species, and the results of this study are not enough to show general responses of the two life forms to temperature and light conditions because of large variations in responses to temperature and light conditions within a life form (Parsons et al., 1994; Takahashi et al., 2003a; van der Werf et al., 2007). Therefore, it is necessary to analyze many species for each life form to clarify general growth responses to temperature and light conditions.

#### **6. Acknowledgment**

This study was partially supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Nos. 19580168, 22580160). We thank Dr. H. Kobayashi for constructive comments.

#### **7. References**

188 the Economy, Human Health, and on Urban and Natural Environments

This study predicted that high winter temperature mitigates the reduction of photochemical efficiency and reduces winter mortality of *Q. myrsinaefolia*. Furthermore, this study showed that increase of temperature enhances the growth of not only *Q. myrsinaefolia* but also *Q. crispula* and *B. platyphylla* var. *japonica*. It is possible that global warming intensifies competition between warm-temperate and cool-temperate broad-leaved species at their latitudinal ecotones. Therefore, it is unclear whether *Q. myrsinaefolia* will gain parts of the habitat now covered by *B. platyphylla* var. *japonica* and *Q. crispula* under global warming. This study examined only one warm-temperate broad-leaved species and two cooltemperate broad-leaved species, and the results of this study are not enough to show general responses of the two life forms to temperature and light conditions because of large variations in responses to temperature and light conditions within a life form (Parsons et al., 1994; Takahashi et al., 2003a; van der Werf et al., 2007). Therefore, it is necessary to analyze many species for each life form to clarify general growth responses to temperature and light

This study was partially supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Nos. 19580168, 22580160). We thank Dr. H. Kobayashi for

Adams, H. D. & Kolb, T. E. (2005) Tree growth response to drought and temperature in a

Aranda, I., Castro, L., Alia, R., Pardos, J. A. & Gil, L. (2005) Low temperature during winter

Arft, A. M., Walker, M. D., Gurevitch, J., Alatalo, J. M., Bret-Harte, M. S., Dale, M., Diemer,

Buckley, B. M., Cook, E. R., Peterson, M. J. & Barbetti, M. (1997) A changing temperature

Chapin, F. S., III & Shaver, G. R. (1985) Individualistic growth response of tundra plant species to environmental manipulations in the field. *Ecology*, 66, 564-576. Chmielewski, F. & Rotzer, T. (2001) Response of tree phenology to climate change across

Danby, R. K. & Hik, D. S. (2007) Responses of white spruce (*Picea glauca*) to experimental warming at a subarctic alpine treeline. *Global Change Biology*, 13, 437-451.

cork oak (*Quercus suber*). *Tree Physiology*, 25, 1085-1090.

Europe. *Agricultural and Forest Meteorology*, 108, 101-112.

experiment. *Ecological Monographs*, 69, 491-511.

mountain landscape in northern Arizona, USA. *Journal of Biogeography*, 32, 1629-

elicits differential responses among populations of the Mediterranean evergreen

M., Gugerli, F., Henry, G. H. R., Jones, M. H., Hollister, R. D., Jónsdóttir, I. S., Laine, K., Lévesque, E., Marion, G. M., Molau, U., Mølgaard, P., Nordenhäll, U., Raszhivin, V., Robinson, C. H., Starr, G., Stenström, A., Totland, Ø., Turner, P. L., Walker, L. J., Webber, P. J., Welker, J. M. & Wookey, P. A. (1999) Responses of tundra plants to experimental warming: meta-analysis of the international tundra

response with elevation for *Lagarostrobos franklinii* in Tasmania, Australia. *Climatic*

**5. Conclusion**

conditions.

**6. Acknowledgment**

constructive comments.

1640.

*Change*, 36, 477-498.

**7. References**

Effects of Temperature and Light Conditions on

Growth of Current-Year Seedlings of Warm-Temperate Evergreen Tree Species… 191

Otsuka, K., Ozeki, M. & Maekawa, M. (2004) Distribution of *Quercus myrsinaefolia* Blume

Parsons, A. N., Welker, J. M., Wookey, P. A., Press, M. C., Callaghan, T. V. & Lee, J. A. (1994)

Penuelas, J., Ogaya, R., Boada, M. & Jump, A. S. (2007) Migration, invasion and decline:

Persson, B. (1998) Will climate change affect the optimal choice of *Pinus sylvestris*

Samejima, J. (1979) Useful hard-wood in Hokkaido and its characters for improvement (II).

Seiwa, K. & Kikuzawa, K. (1989) Seasonal growth patterns of seedling height in relation to

Seiwa, K. & Kikuzawa, K. (1996) Importance of seed size for the establishment of seedlings

Sparks, T. H., Jeffree, E. P. & Jeffree, C. E. (2000) An examination of the relationship between

Takahashi, K. (2005) Effects of artificial warming on shoot elongation of alpine dwarf pine

Takahashi, K., Azuma, H. & Yasue, K. (2003a) Effects of climate on the radial growth of tree

Takahashi, K., Mitsuishi, D., Uemura, S., Suzuki, J. & Hara, T. (2003b) Stand structure and

Takahashi, K. & Rustandi, A. (2006) Responses of crown development to canopy openings

Takahashi, K., Seino, T. & Kohyama, T. (2001) Responses to canopy openings in architectural

Takahashi, K., Seino, T. & Kohyama, T. (2005) Plastic changes of leaf mass per area and leaf

comparison with cool temperate trees. *Annals of Botany*, 97, 559-569.

of five deciduous broad-leaved tree species. *Vegetatio*, 123, 51-64.

records from the UK. *International Journal of Biometeorology*, 44, 82-87. Takahashi, K. (2003) Effects of climatic conditions on shoot elongation of alpine dwarf pine

Conservation Research Institute, Nagano, Japan (in Japanese).

beech forest in Catalonia (NE Spain). *Ecography*, 30, 829-837.

*Forest Tree Breeding of Hokkaido*, 22, 6-14 (in Japanese).

Norikura, central Japan. *Ecological Research*, 18, 549-558.

northern Japan. *Forest Ecology and Management*, 174, 39-50.

broad-leaved tree species. *Ecological Research*, 20, 17–23.

*Conservation Research Institute*, 7, 17-22 (in Japanese).

change. *Journal of Ecology*, 82, 307-318.

provenances? *Silva Fennica*, 32, 121-128.

*Antarctic, and Alpine Research*, 35, 1-7.

*Journal of Forest Research*, 31, 1336-1347.

15 (in Japanese).

*Research*, 37, 620-625.

*actual conditions of global warming at Nagano Prefecture*, 35-41, Nagano Environmental

(Fagaceae), escaped evergreen broad-leaved tree, in mid and downstream region of the Chikuma River, northern Nagano Prefecture. *Bulletin of Nagano Environmental*

Growth responses of four sub-Arctic dwarf shrubs to simulated environmental

changes in recruitment and forest structure in a warming-linked shift of European

seed mass in deciduous broad-leaved tree species. *Japanese Journal of Ecology*, 39, 5-

flowering times and temperature at the national scale using long-term phenological

(*Pinus pumila*) at its upper and lower altitudinal limits in central Japan. *Arctic,*

(*Pinus pumila*) on Mt. Shogigashira, central Japan. *Arctic, Antarctic, and Alpine*

species in the upper and lower distribution limits of an altitudinal ecotone on Mt.

dynamics during a 16-year period in a sub-boreal conifer-hardwood mixed forest,

by saplings of eight tropical submontane forest tree species in Indonesia: a

development of saplings in eight deciduous broad-leaved tree species. *Canadian*

nitrogen content in response to canopy openings in saplings of eight deciduous

190 the Economy, Human Health, and on Urban and Natural Environments

Kohyama, T. & Shigesada, N. (1995) A size-distribution-based model of forest dynamics

Koike, T. (1988) Leaf structure and photosynthetic performance as related to the forest succession of deciduous broad-leaved trees. *Plant Species Biology*, 3, 77-87. Kullman, L. (2002) Rapid recent range-margin rise of tree and shrub species in the Swedish

Lebourgeois, F., Rathgeber, C. B. K. & Ulrich, E. (2010) Sensitivity of French temperate

Lenoir, J., Gegout, J. C., Marquet, P. A., de Ruffray, P. & Brisse, H. (2008) A significant

Li, M. H., Yang, J. & Krauchi, N. (2003) Growth responses of *Picea abies* and *Larix decidua* to

Mäkinen, H., Nöjd, P. & Mielikäinen, K. (2000) Climatic signal in annual growth variation of

Masaki, T., Suzuki, W., Niiyama, K., Iida, S., Tanaka, H. & Nakashizuka, T. (1992)

Matsuki, S., Ogawa, K., Tanaka, A. & Hara, T. (2003) Morphological and photosynthetic

Miura, M., Manabe, T., Nishimura, N. & Yamamoto, S. (2001) Forest canopy and community

Molau, U. (1997) Responses to natural climatic variation and experimental warming in two

Morin, X., Viner, D. & Chuine, I. (2008) Tree species range shifts at a continental scale: new predictive insights from a process-based model. *Journal of Ecology*, 96, 784-794. Munier, A., Hermanutz, L., Jacobs, D. M. & Lewis, K. (2010) The interacting effects of

Niinemets, Ü., Kull, O., Tenhunen, J. D. (1999) Variability in leaf morphology and chemical

Okuyama, S. (1982) *Wild plants in Japan (spring to early summer)*, Seibundo-Shinkosha

Otsuka, K. & Ozeki, M. (2008) Monitoring of warm-temperate evergreen broad-leaved

Japan: a 7-year study of a 4-ha plot. *Journal of Ecology*, 89, 841-849.

*nivalis*. *Global Change Biology*, 3 (Suppl. 1), 97-107.

trees. *International Journal of Plant Science*, 160, 837-848.

Publishling Co., Tokyo, Japan (in Japanese).

coniferous forests to climate variability and extreme events (A*bies alba*, *Picea abies*

upward shift in plant species optimum elevation during the 20th century. *Science*,

elevation in subalpine areas of Tyrol, Austria. *Canadian Journal of Forest Research*, 33,

Norway spruce (*Picea abies*) along a transect from central Finland to the Arctic

Community structure of a species-rich temperate forest, Ogawa Forest Reserve,

responses of *Quercus crispula* seedlings to high-light conditions. *Tree Physiology*, 23,

dynamics in a temperate old-growth evergreen broad-leaved forest, south-western

tundra plant species with contrasting life forms: *Cassiope tetragona* and *Ranunculus*

temperature, ground disturbance, and herbivory on seedling establishment: implications for treeline advance with climate warming. *Plant Ecology*, 210, 19-30. Namikawa, K., Ishikawa, Y. & Sano, J. (1997) Stand dynamics during a 12-year period in a

second-growth stand in a cool temperate forest in northern Japan. *Ecological*

composition as a function of canopy light environment in coexisting deciduous

species *Quercus myrsinaefolia* in Nagano Prefecture, In: *Investigation report for the*

along a latitudinal environmental gradient. *Vegetatio*, 121, 117-126.

and *Pinus sylvestris*). *Journal of Vegetation Science*, 21, 364-376.

timberline. *Canadian Journal of Forest Research*, 30, 769-777.

Scandes. *Journal of Ecology*, 90, 68-77.

central Japan. *Vegetatio*, 98, 97-111.

320, 1768-1771.

653-662.

769-775.

*Research*, 12, 277-287.

*actual conditions of global warming at Nagano Prefecture*, 35-41, Nagano Environmental Conservation Research Institute, Nagano, Japan (in Japanese).

**12**

*Taiwan*

**Decreasing of Population Size of**

*1Department of Life Science, National Taiwan University, Taipei, 2Institute of Plant Biology, National Taiwan University, Taipei, 3Graduate Institute of Ecology and Evolutionary Biology,*

A very recent on-going trend worldwide is that sea level is found rising up due to global warming (Cazenave and Llovel, 2010). With one to two degrees temperature elevated, the polar iceberg will melt which eventually will lead to significant sea-level rise. There is increasing interest in knowing how mangrove population changes under sea-level rising. Mangrove forest (mangrove) is usually termed "kidney of the earth". The morphology and anatomy of these plants have unique structure, such as breathing roots, knee roots. Viviparous seedlings of the Rhizophoraceae plants further drift with the sea which results in implantation and reproduction. The origin of the name mangrove is derived from plants of a kind of mangrove tree *Rhizophora mucronata*. The plant's timber, tree trunks, branches, flowers are red. These trees grow in tropical and subtropical regions of rivers and coastal

According to the literature, mangrove forests are mainly distributed in the western coastal estuary in Taiwan (Hsueh and Lee, 2000). Taiwan's largest and most well-known mangrove protected areas are in Danshui district, including Chuwei mangrove conservation area. These areas are along the Danshui River. The main object of the protected areas is *Kandelia* mangrove forests. *Kandelia candel* (L.) Druce is grouped into Rhizophoraceae *Kandelia* genera. Kandelia is the dominant species and turned out to be a precious national treasure along the Danshui River in Taipei, Taiwan. In addition to *Kandelia*, there are a wide range of plants associated with its surrounding area. These plants have also evolved special morphological and physiological characteristics to adapt to flooding and high salinity environment. These halophytes include *Phragmites austrakis* (reed), *Sporobolus virginicus* (salt sage), and *Imperata cylindrica* (cogon grass). In 1982 Lue carried out an ecological survey of Chuwei mangrove

*Imperata cylindrica*, a perennial herb, is a C4-type plant. It has 10 pairs of chromosomes. A single *Imperata* individual can generate many seeds. Seed dispersal can be assisted by wind. *Imperata* can also use their underground rhizome for asexual reproduction. It is adapted to a

**1. Introduction**

salt marshes, evergreen shrub or tree forest.

forest, and found the population of *Imperata cylindrica* (Lue, 1982).

Ping Kao1, Ting-Ying Wu1, Chia-Lun Chang2, Chang-Hung Chou3 and Ing-Feng Chang1,2

**and Sea-Level Rising**

*China Medical University, Taichung,*

*Imperata cylindrica* **Mangrove Ecotype**

### **Decreasing of Population Size of**  *Imperata cylindrica* **Mangrove Ecotype and Sea-Level Rising**

Ping Kao1, Ting-Ying Wu1, Chia-Lun Chang2, Chang-Hung Chou3 and Ing-Feng Chang1,2 *1Department of Life Science, National Taiwan University, Taipei, 2Institute of Plant Biology, National Taiwan University, Taipei, 3Graduate Institute of Ecology and Evolutionary Biology, China Medical University, Taichung, Taiwan*

#### **1. Introduction**

Global Warming Impacts – Case Studies on

192 the Economy, Human Health, and on Urban and Natural Environments

Takahashi, K. & Yoshida, S. (2009) How the scrub height of *Pinus pumila* decreases at the

Takenaka, A. (1986) Comparative ecophysiology of two representative *Quercus* species appearing in different stages of succession. *Ecological Research*, 1, 129-140. Taneda, H. & Tateno, M. (2005) Hydraulic conductivity, photosynthesis and leaf water

Thuiller, W. (2003) BIOMOD – optimizing predictions of species distributions and projecting potential future shifts under global change. *Global Change Biology*, 9, 1353-1362. van der Werf, G. W., Sass-Klaassen, U. G. W. & Mohren, G. M. J. (2007) The impact of the

Way, D. A. & Sage, R. F. (2008) Elevated growth temperatures reduce the carbon gain of black spruce [*Picea mariana* (Mill.) B.S.P.]. *Global Change Biology*, 14, 624-636. Woodward, F. I. (1979) The differential temperature responses of the growth of certain plant

Yamamoto, S. (1989) Gap dynamics in climax *Fagus crenata* forests. *Botanical Magazine, Tokyo*,

Yin, H., Lai, T., Cheng, X., Jiang, X. & Liu, Q. (2008) Warning effect on growth and

balance in six evergreen woody species from fall to winter. *Tree Physiology*, 25, 299-

2003 summer drought on the intra-annual growth pattern of beech (*Fagus sylvatica* L.) and oak (*Quercus robur* L.) on a dry site in the Netherlands. *Dendrochronologia*,

species from different altitudes. II. Analyses of the control and morphology of leaf extension and specific leaf area of *Phleum bertolonii* D.C. and *P. alpinum* L. *New*

physiology of seedling of *Betula albo-sinensis* and *Abies faxoniana* under two contrasting light conditions in subalpine coniferous forests of western Sichuan,

treeline. *Ecological Research*, 24, 847-854.

306.

25, 103-112.

102, 93-114.

*Phytologist*, 82, 397-405.

China. *Journal of Plant Ecology*, 32, 1072-1083.

A very recent on-going trend worldwide is that sea level is found rising up due to global warming (Cazenave and Llovel, 2010). With one to two degrees temperature elevated, the polar iceberg will melt which eventually will lead to significant sea-level rise. There is increasing interest in knowing how mangrove population changes under sea-level rising. Mangrove forest (mangrove) is usually termed "kidney of the earth". The morphology and anatomy of these plants have unique structure, such as breathing roots, knee roots. Viviparous seedlings of the Rhizophoraceae plants further drift with the sea which results in implantation and reproduction. The origin of the name mangrove is derived from plants of a kind of mangrove tree *Rhizophora mucronata*. The plant's timber, tree trunks, branches, flowers are red. These trees grow in tropical and subtropical regions of rivers and coastal salt marshes, evergreen shrub or tree forest.

According to the literature, mangrove forests are mainly distributed in the western coastal estuary in Taiwan (Hsueh and Lee, 2000). Taiwan's largest and most well-known mangrove protected areas are in Danshui district, including Chuwei mangrove conservation area. These areas are along the Danshui River. The main object of the protected areas is *Kandelia* mangrove forests. *Kandelia candel* (L.) Druce is grouped into Rhizophoraceae *Kandelia* genera. Kandelia is the dominant species and turned out to be a precious national treasure along the Danshui River in Taipei, Taiwan. In addition to *Kandelia*, there are a wide range of plants associated with its surrounding area. These plants have also evolved special morphological and physiological characteristics to adapt to flooding and high salinity environment. These halophytes include *Phragmites austrakis* (reed), *Sporobolus virginicus* (salt sage), and *Imperata cylindrica* (cogon grass). In 1982 Lue carried out an ecological survey of Chuwei mangrove forest, and found the population of *Imperata cylindrica* (Lue, 1982).

*Imperata cylindrica*, a perennial herb, is a C4-type plant. It has 10 pairs of chromosomes. A single *Imperata* individual can generate many seeds. Seed dispersal can be assisted by wind. *Imperata* can also use their underground rhizome for asexual reproduction. It is adapted to a

Decreasing of Population Size of

**2.2 Hypotheses to be tested**

tolerant (Chang and Chou, 2006).

**2.3 Proline content measurement**

statistical package.

**2.4 RNA extraction**

*Imperata cylindrica* Mangrove Ecotype and Sea-Level Rising 195

flooding pressure threatens plant survival was chosen as a sampling site (Figure 1, Figure 2). Sarlun sandy beach was chosen as a control site (Figure 1, Figure 3) (Figure 1, 2, and 3 were adapted and modified as previously described (Chang, 1996)). These two sampling sites are close to each other in Danshui area, Taipei as shown in Figure 1. Plant leaves from Chuwei were harvested between July and October in 1995 and 2011 for proline content assay. Plant samples were collected on spring-tide days (high tide and flooding; Jul 14, Aug 13, Sep 11, Sep 28 and Oct 12 in 1995; Feb 20 in 2011). During harvesting, each leaf sample was excised by sterilized scissors and stored in zip-block in ice bucket with dry ice to keep it fresh and brought back to lab immediately for study the same day. Plant leaves harvested from each site were assay for water proline. Plants rhizomes collected from the field were washed by sterilized water and cultured in pots (60× 20 ×20 cm3) in greenhouse for two weeks, then transplanted to Kimura's culture solution (Chang & Chou, 2006) to grow plantlet, the vegetative shoot. The culture solution was aerated with air pump without break. The culture solution was changed every week. These plantlets were used for differential display assay.

We have two hypotheses for the interpretation of possible reasons for the population size decrease of Chuwei ecotype. The first hypothesis is a synecology hypothesis. This hypothesis address that the overpopulated *Kandelia* may threaten the survival of Chuwei Imperata. The second hypothesis is an autoecology hypothesis. This hypothesis address the local flooding may affect the survival of Chuwei Imperata. We hypothesized that Chuwei ecotype may respond to local flooding in a manner different from *Kandelia*. We suspected that local flooding may impact the survival of Chuwei ecotype even though it is flooding

L-proline content was measured by the method as previously described (Bates *et al*., 1973). Half a gram of plant fresh leaf tissues were chopped into pieces and quick-frozen in liquid nitrogen in 5 g sea sand and 5mL extraction buffer containing 3 % (w/v) 5-sulfosalicylic acid for grinding and homogenization. The homogenates were centrifugated at 538×g (Sigma, Model 2K15, Taiwan) for 10 minutes. The supernatant was obtained as proline crude extract. The reaction mixture contained 2 ml acid-ninhydrin solution, 2ml proline crude extract. Acid-ninhydrin solution contained a mixture of 0.14 M ninhydrin, 60 % (v/v) acetic acid, 2.4 M phosphatic acid and 2 ml acetic acid. The reaction took place at 100℃ for 1 hour. Samples were then put into -20℃ refrigerator to stop the reaction immediately. Four ml methane was mixed with samples and vortex viguously. The solution was divided into two layers, the upper methane layer and the lower water layer. Three ml upper ethane layer solution was placed in a cuvette. Proline content was analyzed spectrophotometrically at wavelength 520nm using spectrophotometer (Beckman, Model DU-50, Taiwan). Ten replicas were

RNA extraction was conducted by use of pine tree method as previously described (Chang *et al.,* 1993). Leaves of Sarlun and Chuwei population of *Imperata cylindrica* were ground in liquid nitrogen using mortar and pestle. About 2 grams of leaf powder were added into 15 mL extraction buffer containing 100 mM Tris-HCl pH 8.0, 25 mM EDTA, 2 M NaCl, 0.5 g/L

s pairwise t-test in SPSS

included for each data set. Data were analyzed by Student�

wide range of environmental factors and confers a high degree of stress tolerance. The underground rhizome is a commonly used medicinal herb. There are about 10 species of *Imperata* worldwide. In Taiwan there is only one genus one species, namely *Imperata cylindrica* (L.) Beauv. var. *major* (Nees) Hubb. (Hsu, 1975). Cheng & Chou (1997a) examined the leaves of *I. cylindrica* from Chuwei using scanning electron microscope (SEM) and found that Chuwei population differs from others in anatomy and morphology. The stele was empty instead of solid. The lower stem was surfaced with white wax instead of trichomes. In addition, polymorphism among populations was also analyzed by rapid amplify polymorphic DNA (RAPD) (Cheng & Chou, 1997b) and restriction fragment length polymorphism (RFLP) (Chou & Tsai, 1999) on ribosomal DNA (rDNA) (Chiang *et al*., 1998; Tsai & Chou, 1999). In 2006 Chang found that Chuwei population is salt and flood tolerant (Chang & Chou, 2006). In 2008 Chang utilized proteomics approach and identified differentially expressed protein among Chuwei, Sarlun and Neihu Imperata populations (Chang, 2008). All these results revealed that Chuwei population was found to be a distinct ecotype.

Under salt pressure, plants accumulated various organic compounds *i.e.* proline, glycinebetaine, choline, glycerol, and sorbitol as compatible solutes in their bodies. Barley (Stewart & Michelle, 1983), tobacco (Binzel *et al*., 1987), eggplant (Jain *et al*., 1987), and spinach (Coughlan & Wyn Jones, 1980) accumulated large amounts of proline in response to salt treatment. The amount of proline increased with increased salinity. In addition, the expression of proline was regulated by abscisic acid (ABA) (Stewart, 1980; Stewart & Voeberg, 1985). Proline played diverse roles in stress physiology. Stewart & Lee (1974) pointed out that the accumulation of proline in plants correlated its tolerance to salt stress. They also indicated that high level proline would protect many N metabolism-related enzymes from harm. Moreover, large amount of proline would inhibit ACC from converting to ethylene so that plant would not hurt by ethylene (Chrominski *et al*., 1988; Chrominski *et al*., 1989). Proline could also be osmo-protectant which functions to maintain osmotic potential balance (Jain, 1987). It is suggested that proline in *I. cylindrica* from Chuwei may function as an osmo-regulator against salt stress.

Mangrove swamp environment is very unique. It is not only a salty land but is also subjected to periodic flooding which is very detrimental to plant growth. It has been discussed that sealevel rise may impact the survival of mangrove forests (Feller *et al*., 2010). The rises of sea water may impact the survival of mangrove trees in the world. We suspect that the rise of sea water may be one of the causes of the population size decrease of Chuwei mangrove population of *Imperata*. A field work survey discovered that the population size of Chuwei ecotype is decreasing. The proline content of Chuwei population in the field before and after flooding was measured. In present study, we tried to investigate the relationship between the decrease of population size of *Imperata* Chuwei ecotype and proline content changes. We hypothesized that the dynamics of proline content may be related to the population size decrease. The study of how flooding affects proline accumulation may elucidate the possible impact of sea-level rise using Imperata mangrove population as an example.

#### **2. Materials and methods**

#### **2.1 Sampling sites and plant materials**

*Imperata cylindrica* (L.) Beauv. var. *major* (Nees) Hubb, Cogon grass, was used as plant materials. Chuwei mangrove salt marsh wetland (Hwang & Chen, 1995) where periodical flooding pressure threatens plant survival was chosen as a sampling site (Figure 1, Figure 2). Sarlun sandy beach was chosen as a control site (Figure 1, Figure 3) (Figure 1, 2, and 3 were adapted and modified as previously described (Chang, 1996)). These two sampling sites are close to each other in Danshui area, Taipei as shown in Figure 1. Plant leaves from Chuwei were harvested between July and October in 1995 and 2011 for proline content assay. Plant samples were collected on spring-tide days (high tide and flooding; Jul 14, Aug 13, Sep 11, Sep 28 and Oct 12 in 1995; Feb 20 in 2011). During harvesting, each leaf sample was excised by sterilized scissors and stored in zip-block in ice bucket with dry ice to keep it fresh and

brought back to lab immediately for study the same day. Plant leaves harvested from each site were assay for water proline. Plants rhizomes collected from the field were washed by sterilized water and cultured in pots (60× 20 ×20 cm3) in greenhouse for two weeks, then transplanted to Kimura's culture solution (Chang & Chou, 2006) to grow plantlet, the vegetative shoot. The culture solution was aerated with air pump without break. The culture solution was changed every week. These plantlets were used for differential display assay.

#### **2.2 Hypotheses to be tested**

Global Warming Impacts – Case Studies on

194 the Economy, Human Health, and on Urban and Natural Environments

wide range of environmental factors and confers a high degree of stress tolerance. The underground rhizome is a commonly used medicinal herb. There are about 10 species of *Imperata* worldwide. In Taiwan there is only one genus one species, namely *Imperata cylindrica* (L.) Beauv. var. *major* (Nees) Hubb. (Hsu, 1975). Cheng & Chou (1997a) examined the leaves of *I. cylindrica* from Chuwei using scanning electron microscope (SEM) and found that Chuwei population differs from others in anatomy and morphology. The stele was empty instead of solid. The lower stem was surfaced with white wax instead of trichomes. In addition, polymorphism among populations was also analyzed by rapid amplify polymorphic DNA (RAPD) (Cheng & Chou, 1997b) and restriction fragment length polymorphism (RFLP) (Chou & Tsai, 1999) on ribosomal DNA (rDNA) (Chiang *et al*., 1998; Tsai & Chou, 1999). In 2006 Chang found that Chuwei population is salt and flood tolerant (Chang & Chou, 2006). In 2008 Chang utilized proteomics approach and identified differentially expressed protein among Chuwei, Sarlun and Neihu Imperata populations (Chang, 2008). All these results revealed that Chuwei population was found to be a distinct

Under salt pressure, plants accumulated various organic compounds *i.e.* proline, glycinebetaine, choline, glycerol, and sorbitol as compatible solutes in their bodies. Barley (Stewart & Michelle, 1983), tobacco (Binzel *et al*., 1987), eggplant (Jain *et al*., 1987), and spinach (Coughlan & Wyn Jones, 1980) accumulated large amounts of proline in response to salt treatment. The amount of proline increased with increased salinity. In addition, the expression of proline was regulated by abscisic acid (ABA) (Stewart, 1980; Stewart & Voeberg, 1985). Proline played diverse roles in stress physiology. Stewart & Lee (1974) pointed out that the accumulation of proline in plants correlated its tolerance to salt stress. They also indicated that high level proline would protect many N metabolism-related enzymes from harm. Moreover, large amount of proline would inhibit ACC from converting to ethylene so that plant would not hurt by ethylene (Chrominski *et al*., 1988; Chrominski *et al*., 1989). Proline could also be osmo-protectant which functions to maintain osmotic potential balance (Jain, 1987). It is suggested that proline in *I. cylindrica* from Chuwei may

Mangrove swamp environment is very unique. It is not only a salty land but is also subjected to periodic flooding which is very detrimental to plant growth. It has been discussed that sealevel rise may impact the survival of mangrove forests (Feller *et al*., 2010). The rises of sea water may impact the survival of mangrove trees in the world. We suspect that the rise of sea water may be one of the causes of the population size decrease of Chuwei mangrove population of *Imperata*. A field work survey discovered that the population size of Chuwei ecotype is decreasing. The proline content of Chuwei population in the field before and after flooding was measured. In present study, we tried to investigate the relationship between the decrease of population size of *Imperata* Chuwei ecotype and proline content changes. We hypothesized that the dynamics of proline content may be related to the population size decrease. The study of how flooding affects proline accumulation may elucidate the possible

*Imperata cylindrica* (L.) Beauv. var. *major* (Nees) Hubb, Cogon grass, was used as plant materials. Chuwei mangrove salt marsh wetland (Hwang & Chen, 1995) where periodical

impact of sea-level rise using Imperata mangrove population as an example.

ecotype.

function as an osmo-regulator against salt stress.

**2. Materials and methods**

**2.1 Sampling sites and plant materials**

We have two hypotheses for the interpretation of possible reasons for the population size decrease of Chuwei ecotype. The first hypothesis is a synecology hypothesis. This hypothesis address that the overpopulated *Kandelia* may threaten the survival of Chuwei Imperata. The second hypothesis is an autoecology hypothesis. This hypothesis address the local flooding may affect the survival of Chuwei Imperata. We hypothesized that Chuwei ecotype may respond to local flooding in a manner different from *Kandelia*. We suspected that local flooding may impact the survival of Chuwei ecotype even though it is flooding tolerant (Chang and Chou, 2006).

#### **2.3 Proline content measurement**

L-proline content was measured by the method as previously described (Bates *et al*., 1973). Half a gram of plant fresh leaf tissues were chopped into pieces and quick-frozen in liquid nitrogen in 5 g sea sand and 5mL extraction buffer containing 3 % (w/v) 5-sulfosalicylic acid for grinding and homogenization. The homogenates were centrifugated at 538×g (Sigma, Model 2K15, Taiwan) for 10 minutes. The supernatant was obtained as proline crude extract. The reaction mixture contained 2 ml acid-ninhydrin solution, 2ml proline crude extract. Acid-ninhydrin solution contained a mixture of 0.14 M ninhydrin, 60 % (v/v) acetic acid, 2.4 M phosphatic acid and 2 ml acetic acid. The reaction took place at 100℃ for 1 hour. Samples were then put into -20℃ refrigerator to stop the reaction immediately. Four ml methane was mixed with samples and vortex viguously. The solution was divided into two layers, the upper methane layer and the lower water layer. Three ml upper ethane layer solution was placed in a cuvette. Proline content was analyzed spectrophotometrically at wavelength 520nm using spectrophotometer (Beckman, Model DU-50, Taiwan). Ten replicas were included for each data set. Data were analyzed by Student� s pairwise t-test in SPSS statistical package.

#### **2.4 RNA extraction**

RNA extraction was conducted by use of pine tree method as previously described (Chang *et al.,* 1993). Leaves of Sarlun and Chuwei population of *Imperata cylindrica* were ground in liquid nitrogen using mortar and pestle. About 2 grams of leaf powder were added into 15 mL extraction buffer containing 100 mM Tris-HCl pH 8.0, 25 mM EDTA, 2 M NaCl, 0.5 g/L

Decreasing of Population Size of

*Imperata cylindrica* Mangrove Ecotype and Sea-Level Rising 197

Fig. 2. Sampling sites of *Imperata cylindrica* in Chuwei, Taipei, Taiwan. *Imperata cylindrica* is populated in area D. In area D, sampling site 1, 2, and 3 were labeled as arrows indicated.

Site 2 and 3 were lost just very recently for unknown reasons.

Fig. 1. Sampling sites of *Imperata cylindrica* in Danshui area, Taipei, Taiwan. *Imperata cylindrica* sampling sites were labeled as arrows indicated. A: Neihu; B: Sarlun; C: Chuwei.

196 the Economy, Human Health, and on Urban and Natural Environments

Fig. 1. Sampling sites of *Imperata cylindrica* in Danshui area, Taipei, Taiwan. *Imperata cylindrica* sampling sites were labeled as arrows indicated. A: Neihu; B: Sarlun; C: Chuwei.

Fig. 2. Sampling sites of *Imperata cylindrica* in Chuwei, Taipei, Taiwan. *Imperata cylindrica* is populated in area D. In area D, sampling site 1, 2, and 3 were labeled as arrows indicated. Site 2 and 3 were lost just very recently for unknown reasons.

Decreasing of Population Size of

**2.5 Differential display assay**

**2.7 Database searching**

transcription immediately followed by storage under -80℃.

bands specific to either Chuwei or Sarlun population.

**2.6 Isolation of electrophoretic bands for DNA cloning and sequencing**

were selected from each plate to perform colony PCR and subsequent sequencing.

*Imperata cylindrica* Mangrove Ecotype and Sea-Level Rising 199

spermidine, 2% beta-mercaptoethanol, 2% CTAB, and 2% PVP. The homogenate was mixed well and subjected to water bath at 65℃ for 10 minutes. The mixture was then centrifuged at 13000 rpm for 15 minutes at room temperature. The upper layer was recovered to a new centrifuge tube. Equal volume of chloroform:isoamyl alcohol (24:1) was added. After thorough mixing, centrifugation was carried out at 13000 rpm for 15 minutes at room temperature. This step was repeated twice in order to remove proteins and metabolites. The upper water layer was transferred to a new centrifuge tube. One third volumes of 10 M LiCl was added, mix well and subjected to incubation under 4℃ overnight. The solution was centrifuged at 13000 rpm for 30 minutes at 4℃. The pellet was washed by 1mL 70% (v/v) ethanol. The solution was transferred to a sterilized microcentrifuge tube for centrifugation at 13000 rpm for 15 minutes at 4℃. The wash step was repeated twice to remove salt. The pellet was air dried. One hundred μL sterilized ddH2O was added to resuspend RNA. RNA concentration was measured by NanoDrop spectrometer. The RNA integrity was checked by 2% agarose gel electrophoresis, and the solution was used as templates for reverse-

Differential display and the subsequent silver stain were carried out by following the manufacturer instruction (RNAimage Kit 5, GenHunter Corporation, Cat. No. G505; SILVER SEQUENCE™ Staining Reagents, Promega, Cat. No. Q4132). After reverse-transcription, cDNAs were used as templates to perform another PCR with AP primers provided by the manufacturer. PCR products were loaded into denature acryl-amide gel for electrophoresis at 1700V for 4 hours. The gel was stained by silver stain immediately after the electrophoresis was completed. The Air-dried gel was scanned by scanner (EPSON PERFECTION V750 PRO, EPSON). The electrophoretic bands were compared to identify gel

The electrophoretic bands were excised from the acrylamide gel by sterilized dissection knife, and collected in 1.5 mL eppendorfs. The recovery step was conducted by use of QIAGEN II Gel Extraction Kit (QIAGEN, Cat. No. 28704). The DNA concentrations were detected by NanoDrop spectrometer. The recovered DNA was used for TA cloning. Cloning was conducted using TA Cloning Vector (RBC, # 200-09205) as cloning vector. T4 DNA ligase was used for ligation between TA vector and DNA. *E. coli* (DH5α) was used as hosts. The ligated plasmids were transformed into DH5α competent cells by heat-shock, and incubated under 37℃ on LB medium (with ampicillin) for 14 hours. Five to eight colonies

Sequences acquired were searched against NCBI database using BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul *et al*., 1990). Plant Genome Database (PlantGDB, http://www.plantgdb.org/), the Gene Ontology(http://www. geneontology.org/), the Arabidopsis Information Resource(TAIR, http:// www.arabidopsis.org/), and MIPS *Oryza sativa* database(MOsDB, http:// mips.helmholtz-muenchen.de/plant/rice/)were also used. In nucleotide sequence searching, the cloned sequences were compared against databases by BLASTn program and

Fig. 3. Sampling sites of *Imperata cylindrica* in Sarlun, Taipei, Taiwan. Sampling sites were labeled as arrows indicated.

spermidine, 2% beta-mercaptoethanol, 2% CTAB, and 2% PVP. The homogenate was mixed well and subjected to water bath at 65℃ for 10 minutes. The mixture was then centrifuged at 13000 rpm for 15 minutes at room temperature. The upper layer was recovered to a new centrifuge tube. Equal volume of chloroform:isoamyl alcohol (24:1) was added. After thorough mixing, centrifugation was carried out at 13000 rpm for 15 minutes at room temperature. This step was repeated twice in order to remove proteins and metabolites. The upper water layer was transferred to a new centrifuge tube. One third volumes of 10 M LiCl was added, mix well and subjected to incubation under 4℃ overnight. The solution was centrifuged at 13000 rpm for 30 minutes at 4℃. The pellet was washed by 1mL 70% (v/v) ethanol. The solution was transferred to a sterilized microcentrifuge tube for centrifugation at 13000 rpm for 15 minutes at 4℃. The wash step was repeated twice to remove salt. The pellet was air dried. One hundred μL sterilized ddH2O was added to resuspend RNA. RNA concentration was measured by NanoDrop spectrometer. The RNA integrity was checked by 2% agarose gel electrophoresis, and the solution was used as templates for reversetranscription immediately followed by storage under -80℃.

#### **2.5 Differential display assay**

Global Warming Impacts – Case Studies on

198 the Economy, Human Health, and on Urban and Natural Environments

Fig. 3. Sampling sites of *Imperata cylindrica* in Sarlun, Taipei, Taiwan. Sampling sites were

labeled as arrows indicated.

Differential display and the subsequent silver stain were carried out by following the manufacturer instruction (RNAimage Kit 5, GenHunter Corporation, Cat. No. G505; SILVER SEQUENCE™ Staining Reagents, Promega, Cat. No. Q4132). After reverse-transcription, cDNAs were used as templates to perform another PCR with AP primers provided by the manufacturer. PCR products were loaded into denature acryl-amide gel for electrophoresis at 1700V for 4 hours. The gel was stained by silver stain immediately after the electrophoresis was completed. The Air-dried gel was scanned by scanner (EPSON PERFECTION V750 PRO, EPSON). The electrophoretic bands were compared to identify gel bands specific to either Chuwei or Sarlun population.

#### **2.6 Isolation of electrophoretic bands for DNA cloning and sequencing**

The electrophoretic bands were excised from the acrylamide gel by sterilized dissection knife, and collected in 1.5 mL eppendorfs. The recovery step was conducted by use of QIAGEN II Gel Extraction Kit (QIAGEN, Cat. No. 28704). The DNA concentrations were detected by NanoDrop spectrometer. The recovered DNA was used for TA cloning. Cloning was conducted using TA Cloning Vector (RBC, # 200-09205) as cloning vector. T4 DNA ligase was used for ligation between TA vector and DNA. *E. coli* (DH5α) was used as hosts. The ligated plasmids were transformed into DH5α competent cells by heat-shock, and incubated under 37℃ on LB medium (with ampicillin) for 14 hours. Five to eight colonies were selected from each plate to perform colony PCR and subsequent sequencing.

#### **2.7 Database searching**

Sequences acquired were searched against NCBI database using BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul *et al*., 1990). Plant Genome Database (PlantGDB, http://www.plantgdb.org/), the Gene Ontology(http://www. geneontology.org/), the Arabidopsis Information Resource(TAIR, http:// www.arabidopsis.org/), and MIPS *Oryza sativa* database(MOsDB, http:// mips.helmholtz-muenchen.de/plant/rice/)were also used. In nucleotide sequence searching, the cloned sequences were compared against databases by BLASTn program and

Decreasing of Population Size of

unit: micro g gfw-1

stress response.

2011.

1 Values showed significant difference in Student'

**- A cross-talk between flooding and salt stress**

accumulated in response to salt treatment.

*Imperata cylindrica* Mangrove Ecotype and Sea-Level Rising 201

**Sampling date Before flooding After flooding** 07.14.1995 138.99+ 7.53 64.62+ 1.581 08.13.1995 130.94 + 8.29 70.49+ 4.841 10.12.1995 156.82+ 14.84 52.16+ 2.571 02.20.2011 150.45 + 2.37 89.97+ 3.681

Table 1. Average proline content in leaves of *I. cylindrica* from Chuwei population before and after flooding on spring-tide (high tide) days. Sampling was performed in 1995 and

**3.3 Proline serves more than one function in** *Imperata cylindrica* **Chuwei ecotype?**

In a previous study, *I. cylindrica* from Chuwei was tolerant to 1% salt treatment. Therefore, it was grouped as a salt-tolerant ecotype (Chang & Chou, 2006). The tolerance ability was even stronger than *Kandelia candel* (tolerant to 0.8 % salt), a dominant mangrove forest species in Chuwei salt marsh wetland (Hwang & Chen, 1995). Since Chuwei sampling site is a mangrove salt marsh land, how plants survived in such a high salt land is poorly known. According to our study, proline content of *I. cylindrica* from Chuwei was much higher than those from others in the field (Chang & Chou, 2006). They were also higher under salt treatment. In the greenhouse experiments, proline of *I. cylindrica* from Chuwei was highly

However, does proline serve only single role in plants? It has been reported that the regulation of ion uptake and production of organic solutes (*i.e*. methyl proline) is related to combined salt and flood stress tolerance in some wetland plants (Carter *et al.*, 2006). In addition, mechanism of how some halophytes tolerate flooding is reviewed (Colmer and Flowers, 2008). In present study, proline content dropped dramatically (almost 50 %) after local flooding (only in one hour) in Chuwei ecotype, suggesting that proline may serve as a signal molecule in response to flooding. The dynamic of proline in leaves of Chuwei *Imperata* was suggested which might not only be involved in salt stress, but also in flooding

The decrease of proline content in response to flooding can be explained by two mechanisms. The first mechanism is that proline acts as a direct response to flooding. It has been reported the accumulation of proline was regulated by abscisic acid (ABA) (Stewart, 1980; Stewart & Voeberg, 1985). Alcohol dehydrogenase *ADH* gene expression was also known regulated by ABA (de Bruxelles *et al*., 1996) through G-box, a promoter region (Walker *et al*., 1987; Lu *et al.*, 1996). It is possible that *ADH* gene expression and proline accumulation cross-talks, and is regulated through ABA. Since proline accumulated in response to salt stress in Chuwei ecotype, decrease in proline content may be an easier way for cell to recognize to activate fermentation pathway, including activation of *ADH* gene. An alternative mechanism is an indirect response of proline to flooding. The accumulated proline transported downward toward root (shoot to root) to protect root from salt damage, and anti-transpirated with a flooding signal molecule from root to shoot (Else *et al.,* 1996) for stomata closes in response to flooding. This pathway might overlap with water-deficit pathway. Besides, a long distance transport of proline as a signal molecule was required. However, we did not confirm any of the possibility because it was very complicated to simulate a physiology experiment with two stresses acting at the same time. Timing makes the experiment even difficult to resolve. Maybe

s t-test at 5% level. S.E. was at 5% level. Proline count

BLASTtx program for matches. In protein sequence searching, the acquired sequences were first translated by ExPASy Translate Tool (http://au.expasy.org/tools/dna.html), and then screen for possible protein sequences manually. The translated protein sequences were compared against databases by BLASTp program.

#### **3. Results and discussion**

#### **3.1 Population size of Chuwei ecotype is much smaller than before**

*Imperata cylindrica* (L.) Beauv. var. *major* (Nees) Hubb., a top-ten weed (Holm *et al.,* 1977), was found in Taiwan all around the island (Hsu, 1975). In America, *Imperata cylindrica* was found as a non-endemic species dominating Florida and neighboring states in both dry and wetlands (King & Grace, 2000). Interestingly, it was found growing in Chuwei mangrove salt marsh wet land as well. To track the dynamics of population size of Chuwei ecotype, we carried out field survey since 2008. Surprising, we observed decrease of population size of Chuwei ecotype. Two of the sampling sites (Chuwei and Sarlun) we selected for the field work (site 2 and site 3 as shown in Figure 2) before year 2000 were found missing since the beginning of 2009. Only sampling site 1 (around 30 m2) still existed so far (Figure 2). In addition, the size of sampling site 1 is only less than half of the size as before. Therefore, we concluded that Chuwei ecotype is now decreasing its population and is endangered. However, the reason why the population size decreased is unknown.

#### **3.2 Proline content in leaves of Chuwei ecotype decreased dramatically after flooding in the field**

We have two hypotheses for the interpretation of possible reasons for the population size decrease of Chuwei ecotype. The first hypothesis is supported by the evidence that more and more *Kandelia* trees are invading the habitat of Chuwei Imperata. These *Kandelia* trees may threaten the survival of Imperata. It is expected that in ten years, this area will be dominated by *Kandelia* trees and Imperata Chuwei ecotype will eventually be extinct. In order to test the second hypothesis, we compare proline content in leaves of *I. cylindricain* in the field before and after local flooding on the spring tide (high tide) date. Samples were collected at monthly interval from August to December in 1995. For proline content analysis, leaves sampling was performed with 20 individuals within a day. To test if proline content changed in response to flooding, same leaf samples were collected before and after flooding (1 hr difference) on spring-tide days. Proline content analysis showed that proline content dropped dramatically after flooding. On July 14 in 1995, proline content decreased 53 % after flooding. On August 13, proline content decreased 46 % after flooding. On October 12, proline content decreased 67 % after flooding. Moreover, on February 20 in 2011, proline content decreased 40% after flooding (Table 1).

The decrease of proline content in response to local flooding in Chuwei supported our second hypothesis. In the salt marsh wetland, high level of proline content is helpful in maintaining the osmotic equilibrium in the cells. When local flooding occurred, proline dropped rapidly, which is harmful to plants since osmotic equilibrium was not maintained in the cells. This might eventually lead to cell death. In order to determine if proline content in leaves of plantlet increases in responses to flooding, flooding treatment was performed three months in the greenhouse to see long-term effect on proline. Leaf samples were collected and analyzed. Results showed that the accumulation of proline was not found in leaves of flooding-treated plantlet in the greenhouse (data not shown).

200 the Economy, Human Health, and on Urban and Natural Environments

BLASTtx program for matches. In protein sequence searching, the acquired sequences were first translated by ExPASy Translate Tool (http://au.expasy.org/tools/dna.html), and then screen for possible protein sequences manually. The translated protein sequences were

*Imperata cylindrica* (L.) Beauv. var. *major* (Nees) Hubb., a top-ten weed (Holm *et al.,* 1977), was found in Taiwan all around the island (Hsu, 1975). In America, *Imperata cylindrica* was found as a non-endemic species dominating Florida and neighboring states in both dry and wetlands (King & Grace, 2000). Interestingly, it was found growing in Chuwei mangrove salt marsh wet land as well. To track the dynamics of population size of Chuwei ecotype, we carried out field survey since 2008. Surprising, we observed decrease of population size of Chuwei ecotype. Two of the sampling sites (Chuwei and Sarlun) we selected for the field work (site 2 and site 3 as shown in Figure 2) before year 2000 were found missing since the beginning of 2009. Only sampling site 1 (around 30 m2) still existed so far (Figure 2). In addition, the size of sampling site 1 is only less than half of the size as before. Therefore, we concluded that Chuwei ecotype is now decreasing its population and is endangered.

**3.2 Proline content in leaves of Chuwei ecotype decreased dramatically after flooding**

We have two hypotheses for the interpretation of possible reasons for the population size decrease of Chuwei ecotype. The first hypothesis is supported by the evidence that more and more *Kandelia* trees are invading the habitat of Chuwei Imperata. These *Kandelia* trees may threaten the survival of Imperata. It is expected that in ten years, this area will be dominated by *Kandelia* trees and Imperata Chuwei ecotype will eventually be extinct. In order to test the second hypothesis, we compare proline content in leaves of *I. cylindricain* in the field before and after local flooding on the spring tide (high tide) date. Samples were collected at monthly interval from August to December in 1995. For proline content analysis, leaves sampling was performed with 20 individuals within a day. To test if proline content changed in response to flooding, same leaf samples were collected before and after flooding (1 hr difference) on spring-tide days. Proline content analysis showed that proline content dropped dramatically after flooding. On July 14 in 1995, proline content decreased 53 % after flooding. On August 13, proline content decreased 46 % after flooding. On October 12, proline content decreased 67 % after flooding. Moreover, on February 20 in 2011, proline

The decrease of proline content in response to local flooding in Chuwei supported our second hypothesis. In the salt marsh wetland, high level of proline content is helpful in maintaining the osmotic equilibrium in the cells. When local flooding occurred, proline dropped rapidly, which is harmful to plants since osmotic equilibrium was not maintained in the cells. This might eventually lead to cell death. In order to determine if proline content in leaves of plantlet increases in responses to flooding, flooding treatment was performed three months in the greenhouse to see long-term effect on proline. Leaf samples were collected and analyzed. Results showed that the accumulation of proline was not found in

leaves of flooding-treated plantlet in the greenhouse (data not shown).

compared against databases by BLASTp program.

content decreased 40% after flooding (Table 1).

**3.1 Population size of Chuwei ecotype is much smaller than before**

However, the reason why the population size decreased is unknown.

**3. Results and discussion**

**in the field**

1 Values showed significant difference in Student' s t-test at 5% level. S.E. was at 5% level. Proline count unit: micro g gfw-1

Table 1. Average proline content in leaves of *I. cylindrica* from Chuwei population before and after flooding on spring-tide (high tide) days. Sampling was performed in 1995 and 2011.

#### **3.3 Proline serves more than one function in** *Imperata cylindrica* **Chuwei ecotype? - A cross-talk between flooding and salt stress**

In a previous study, *I. cylindrica* from Chuwei was tolerant to 1% salt treatment. Therefore, it was grouped as a salt-tolerant ecotype (Chang & Chou, 2006). The tolerance ability was even stronger than *Kandelia candel* (tolerant to 0.8 % salt), a dominant mangrove forest species in Chuwei salt marsh wetland (Hwang & Chen, 1995). Since Chuwei sampling site is a mangrove salt marsh land, how plants survived in such a high salt land is poorly known. According to our study, proline content of *I. cylindrica* from Chuwei was much higher than those from others in the field (Chang & Chou, 2006). They were also higher under salt treatment. In the greenhouse experiments, proline of *I. cylindrica* from Chuwei was highly accumulated in response to salt treatment.

However, does proline serve only single role in plants? It has been reported that the regulation of ion uptake and production of organic solutes (*i.e*. methyl proline) is related to combined salt and flood stress tolerance in some wetland plants (Carter *et al.*, 2006). In addition, mechanism of how some halophytes tolerate flooding is reviewed (Colmer and Flowers, 2008). In present study, proline content dropped dramatically (almost 50 %) after local flooding (only in one hour) in Chuwei ecotype, suggesting that proline may serve as a signal molecule in response to flooding. The dynamic of proline in leaves of Chuwei *Imperata* was suggested which might not only be involved in salt stress, but also in flooding stress response.

The decrease of proline content in response to flooding can be explained by two mechanisms. The first mechanism is that proline acts as a direct response to flooding. It has been reported the accumulation of proline was regulated by abscisic acid (ABA) (Stewart, 1980; Stewart & Voeberg, 1985). Alcohol dehydrogenase *ADH* gene expression was also known regulated by ABA (de Bruxelles *et al*., 1996) through G-box, a promoter region (Walker *et al*., 1987; Lu *et al.*, 1996). It is possible that *ADH* gene expression and proline accumulation cross-talks, and is regulated through ABA. Since proline accumulated in response to salt stress in Chuwei ecotype, decrease in proline content may be an easier way for cell to recognize to activate fermentation pathway, including activation of *ADH* gene. An alternative mechanism is an indirect response of proline to flooding. The accumulated proline transported downward toward root (shoot to root) to protect root from salt damage, and anti-transpirated with a flooding signal molecule from root to shoot (Else *et al.,* 1996) for stomata closes in response to flooding. This pathway might overlap with water-deficit pathway. Besides, a long distance transport of proline as a signal molecule was required. However, we did not confirm any of the possibility because it was very complicated to simulate a physiology experiment with two stresses acting at the same time. Timing makes the experiment even difficult to resolve. Maybe

Decreasing of Population Size of

*Imperata cylindrica* Mangrove Ecotype and Sea-Level Rising 203

(Strittmauer and Kossel, 1984, Alexandrov *et al.*, 2009, Wu *et al*., 2009) was identified. Moreover, BLAST search result of band S4 protein sequence showed that ATP synthase CF0 subunit III of many eudicotyledons such as *Geranium carolinianum* and *Erodium texanum* (Guisinger *et al.*, 2008) was identified. For BLAST search result of band S8 nucleotide sequence, the most likely candidate was a hypothetical protein of *Sorghum bicolor* (Paterson *et al.*, 2009). Similar sequences were also found in *Zea mays* and *Oryza sativa*, still hypothetical proteins (Yu *et al.,* 2005, Lai *et al.*, 2004). Moreover, for BLAST search result of band S8 protein sequence, eight possible queries out of six reading frames were blast against NCBI database. Five of these queries had no significant similarities, the others matched to

For band S9 nucleotide BLAST search, a putative casein kinase of *Oryza sativa* was found. This sequence was also similar to another putative serine/threonine protein kinase of *Oryza sativa* (Sasaki *et al*., 2002). For band S9 protein BLAST search, eight possible queries out of six reading frames were submitted to NCBI database. Two of them had matches indicating serine/threonine protein kinase of *Oryza sativa*; four were found to share similarities with hypothetical proteins among various vascular plants; the rest had no significant similarities found. For band S6 nucleotide BLAST search, no significant similarities were found among all databases mentioned above. For band S6 protein BLAST search, four possible queries

**Band # Size (bp) Accession # Identity Annotation of**

**Band # Size (bp) Accession # Identity Annotation of**

S8 468 AK333595 96% *Sorghum bicolor*,

S9 383 AAT44311 79% *Oryza sativa*,

*Imperata* Chuwei ecotype encountered both flooding and salt pressure. In order to survive, plants must possess adaptation mechanism against flooding and salt stress. The increase of

**matched gene**

*Zea mays* chloroplast, 16S rRNA

*Triticum aestivum*, hypothetical

**matched gene**

*Zea mays*, ATP synthase C chain

hypothetical

casein kinase

hypothetical proteins of *Sorghum bicolor, Zea mays* and *Oryza sativa*.

C3 776 AY928077 99%

C12 553 AK333595 82%

S4 404 AY928077 98%

Table 2. Differential display bands specific to Chuwei ecotype.

Table 3. Differential display bands specific to Sarlun population.

**3.5 Adaptational divergence of** *Imperata cylindrica* **Chuwei ecotype**

were submitted. No significant similarities were found.

an easier way to keep tract on proline dynamic was to label proline using isotope as a tracer. Although the role of proline as signal molecule in response to salt stress has been proposed (Maggio *et al*., 2002; Hare & Cress, 1997), the involvement of proline in flooding response has not been discovered before. Anyway, due to the fact that proline functioned in response to both flood and salt stress in present study, a crosstalk between two distinct pathways, salt and flooding response, was suggested.

#### **3.4 Differential display reverse transcription assay revealed five genes which were differentially expressed in Chuwei ecotype as compared to Sarlun population**

Differential display was first introduced In 1992, by Liang and Pardee as an easy application for comparison, identification and isolation of genes expressed as mRNA in various cells under designated conditions (Liang and Pardee, 1995). The basic steps of differential display include isolation of intact RNA from sample, reverse transcription of the RNA using an arbitrary primer to produce ssDNA, several rounds of polymerase chain reactions using the same primer to amplify the corresponding cDNAs, and display of the cDNAs as bands on a matrix gel, such as a DNA sequencing gel. In order to identify differentially expressed genes in Chuwei ecotype, differential display (DD) was introduced. The differences of gene expression between Chuwei ecotype and Sarlun population were compared. By comparing and collecting the bands uniquely appeared to either Chuwei or Sarlun populations. Population-specific ESTs was isolated and identified.