Forest Ecology and Management 216 (2005) 175–186

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Nitrogen form preference of six dipterocarp species

Mariko Norisada *, Katsumi Kojima
Asian Natural Environmental Science Center, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan
Received 20 November 2004; received in revised form 8 April 2005; accepted 10 May 2005

Abstract

We investigated the nitrogen form preference of six dipterocarp species: Anisoptera costata Korth., Dipterocarpus
obtusifolius Teijsm. ex Miq., Hopea odorata Roxb., Neobalanocarpus heimii (King) P. Ashton, Shorea faguetiana Heim,
and Shorea roxburghii G. Don. Seedlings were supplied with nitrogen as nitrate, ammonium, or both in sand culture in a
controlled environment. Except for N. heimii, all species showed greater shoot growth when supplied with ammonium than with
nitrate. Higher root mass ratios were observed in all species with nitrate, which would be an adaptive response to limited nitrogen
uptake. The five species, which preferred ammonium, showed a higher light-saturated photosynthetic rate with ammonium
supply. The lower light-saturated photosynthetic rate with nitrate supply was a result of lower photosynthetic capacity, as
indicated by a lower CO2-saturated photosynthetic rate. The lower leaf nitrogen content in seedlings supplied with nitrate would
be the cause of the lower photosynthetic performance. Nitrate reductase activity in leaf and root of D. obtusifolius, N. heimii, and
S. roxburghii showed generally low inducibility with nitrate.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Nitrate; Ammonium; Growth; Photosynthesis; Nitrate reductase; Dipterocarpaceae

1. Introduction (Richter and Gottwald, 1996). Examination of

The members of the Dipterocarpaceae are pre-
dominant tree species of the upper canopy of tropical
rain forests in Southeast Asia (Symington, 1974;
Whitmore, 1984). They are the most important timber
species in the region, and depletion of the stock is now
of concern as a result of overexploitation since their
entrance on the international market in the 1950s
* Corresponding author. Tel.: +81 3 5841 2785;
fax: +81 3 5841 2785.
E-mail address: norisada@fr.a.u-tokyo.ac.jp (M. Norisada).
sustainable use and enrichment of existing resources
has increased the need for knowledge of the
environmental responses of the species.
Light and water are the two main factors covered in
studies of the environmental responses of dipterocarp
and other tropical tree species (Chazdon et al., 1996;
Mulkey and Wright, 1996; Whitmore, 1996). These
two factors have crucial roles in species distribution
and thus the species richness of tropical forests.
Studies of temperate tree species have shown a
correspondence of the site preference of a species with
its nutritional characteristics, so the nutrient regime

0378-1127/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.foreco.2005.05.020
176 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186
likely will also be important for seedling establish-
ment (Kronzucker et al., 1997). The nutritional
characteristics of trees are of great importance in
silviculture as well. Nevertheless, our knowledge of
the influence of nutrient conditions on tropical tree
species is less than that of light and water (Whitmore,
1996). Nutritional studies in dipterocarp species are
mostly limited to some fertilizer experiments (e.g.,
Fetcher et al., 1996; Gunatilleke et al., 1997).
However, a study by Bungard et al. (2000) showed
the effects of nitrogen availability on the photosyn-
thetic characteristics of four dipterocarp species under
different light regimes and on responses of these
characteristics to sudden changes of light regime. The
results suggested the importance of variations in
nitrogen availability in regeneration dynamics and in
the distribution of canopy-dominating dipterocarp
species.
Both the amount and the form of nitrogen affect
tree growth by affecting nitrogen uptake. Nitrate and
ammonium are the major inorganic forms of nitrogen
taken up by plant roots (Marschner, 1995). Ammo-
nium can be readily assimilated into amino acids, but
nitrate has to be first reduced to ammonium via
nitrate reductase followed by nitrite reductase. In
studies of closed- and open-forest communities of
Australian rainforests, Stewart et al. (1988, 1990)
reported low levels of nitrate reductase in the roots
and shoots of most of the closed-forest species
examined but high levels in the leaves of pioneer
species.
In the field, the soil nitrogen regime depends on
climate, soil type, vegetation, and microenvironment
(Vitousek and Matson, 1988; Maggs, 1991; Smith
et al., 1998; Silver et al., 2000). The composition of
nitrogen can vary over time (Maithani et al., 1998) and
change in response to disturbances (Vitousek et al.,
1989). Investigations of nitrogen composition changes
with succession have found ammonium to dominate as
a result of low nitrification at late successional stages
(Attiwill and Adams, 1993). Studies of changes of
nitrogen composition after burning or clear-cutting
have reported an increase in nitrogen mineralization
and nitrification, after such disturbances (Matson
et al., 1987; Attiwill and Adams, 1993). Most of the
limited studies of tropical tree species have examined
the effects of quantity (e.g., Lawrence, 2001) but not
quality of nitrogen. Consequently, responses of
tropical tree species to qualitative changes in soil
nitrogen remain to be shown.
Understanding of nitrogen characteristics of dip-
terocarps is essential for clarifying mineral cycling
and species establishment in tropical forests in
Southeast Asia and for development of silvicultural
techniques. The effects of nitrogen on dipterocarps,
however, have been reported only quantitatively (e.g.,
Mirmanto et al., 1999; Bungard et al., 2000, 2002), not
qualitatively. Given that dipterocarps are climax-
forest species, it would seem that they should prefer
ammonium to nitrate, as in the case of Australian
rainforest species. However, considering that the
Dipterocarpaceae consist of nearly 500 species with a
broad range of light demand (Symington, 1974),
variation in nitrogen characteristics cannot be ruled
out. Here, we report responses of growth and
photosynthesis to ammonium, nitrate, or a mixture
in six dipterocarp species: Anisoptera costata Korth.,
Dipterocarpus obtusifolius Teijsm. ex Miq., Hopea
odorata Roxb., Neobalanocarpus heimii (King) P.
Ashton, Shorea faguetiana Heim, and Shorea roxbur-
ghii G. Don. All the six species are distributed in
Thailand and chosen because of the availability of
seeds. The objective of the study was to clarify
whether each species prefers ammonium to nitrate for
shoot growth. Owing to the irregular fruiting habit of
dipterocarps, we carried out two independent experi-
ments with three species each. In Experiment II, we
assessed in vivo nitrate reductase activities (NRA),
growth, and photosynthesis.
2. Materials and methods
2.1. Plant materials and treatments
2.1.1. Experiment I
Seeds of three dipterocarp species—A. costata, H.
odorata, and S. roxburghii—were collected in south-
ern Thailand and sown in a greenhouse (28/25 8C,
natural light) in Tokyo, Japan. One-year-old seedlings
were transplanted into a 1/10000-a Wagner pot
(100 cm2 surface area and 18.5 cm depth) in sand.
Two to three weeks after transplanting, 10 pots of each
species were set in each of six watering systems.
Seedlings were watered with a nutrient solution at the
level of sand surface of pots, differing in nitrogen
 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186 177

Table 1 measured at ambient CO2 concentration and at

Nutrient solution composition of the three nitrogen form treatments
saturated CO2 concentration at 64 and 65 DAT and
Treatment at 71 and 72 DAT, respectively.
NH4 NH4 + NO3 NO3
NaNO3 0 0 0.3 2.1.2. Experiment II
KNO3 0 1.5 3 Seeds of three dipterocarp species—D. obtusifo-
Ca(NO3)2
4H2O 0 0.25 0.35 lius, N. heimii, and S. faguetiana—were collected in
(NH4)2SO4 2 1 0
southern Thailand and sown in a greenhouse as above.
Na2SO4
10H2O 0.15 0.15 0
K2SO4 1.5 0.75 0 Each 2.5-month-old seedling of D. obtusifolius and N.
CaCl2
2H2O 0.35 0.1 0 heimii and each 9-month-old seedling of S. faguetiana
MgSO4
7H2O 0.25 0.25 0.25 was transplanted into a 1/10000-a Wagner pot in sand.
NaH2PO4
2H2O 0.6 0.6 0.6 The same three nitrogen treatments as in Experiment I
FeSO4
7H2O 0.01 0.01 0.01
were applied for 127 days. Height and diameter of
H3BO3 0.02 0.02 0.02
MnCl2
4H2O 0.002 0.002 0.002 stem were measured once a week. The number of
ZnSO4
7H2O 0.002 0.002 0.002 newly developed leaves was counted weekly as well.
CuSO4
5H2O 0.002 0.002 0.002 At the end of the experiment, photosynthesis was
Na2MoO4
2H2O 0.0005 0.0005 0.0005 measured at ambient and saturated CO2 concentration
CoCl2
6H2O 0.0005 0.0005 0.0005
in a fully developed leaf in each of 10 seedlings per
Concentrations are in mM. treatment (123–126 DAT), and NRA was measured in
leaves and roots (127 DAT). Plants were then
form, three times a day for 15 min. Three treatments of harvested and separated into leaf, stem, and root as
different forms of 4 mM nitrogen were provided: above for dry mass determination.
ammonium, nitrate, and both. The nutrient solution
composition is shown in Table 1. All nutrient contents 2.2. Photosynthesis
other than nitrogen, sulfur, and chloride were the same
among the treatments. The pH of nutrient solutions Photosynthesis of fully developed leaves was
was adjusted to 5.8 with HCl or NaOH. Nutrient measured with a portable photosynthesis system
solutions were replaced once a week. Two watering (LI6400, Li-Cor). Light-saturated photosynthetic rates
systems were provided for each treatment, and were measured at 350 mmol mol 1 CO2 concentration
watering systems were exchanged within each of and 1000 mmol m 2 s 1 PAR. Photosynthetic capa-
the treatments 3 days after solution refreshment and city was determined at saturated CO2 concentration
exchanged across the treatments every week to (1000 mmol m 2 s 1) under the same PAR. The leaf
eliminate effects of watering system. Pots were chamber temperature was controlled to 28 8C. The
rotated within each watering system every 3–4 days relative humidity ranged from 70 to 90%.
to eliminate the effects of location effects. Treatment
was continued for 72 days. Height and diameter of 2.3. Chlorophyll
stem were measured once a week. At 41 and 42 days
after treatment (DAT), photosynthesis at ambient CO2 The chlorophyll content of the leaves in which
concentration under saturated light was measured on photosynthesis was measured was determined with a
fully developed leaves of 10–15 seedlings per chlorophyll meter (SPAD-502, Minolta) in Experi-
treatment, then all seedlings in one watering system ment I or spectrophotometrically in Experiment II. In
in each treatment were harvested for dry weight spectrometric measurement, a 1-cm leaf disc was
measurement. The harvested seedlings were separated homogenized in 80% acetone, and the homogenate
into leaf, stem and root, then dried to a constant weight was centrifuged for 10 min at 1870  g. The pellet
at 80 8C for biomass determination. The remaining was extracted with 80% acetone again, and the
seedlings were divided among the two watering supernatants were bulked. Acetone (80%) was added
systems per treatment for further growth. Photosynth- to the supernatant to give a final volume of 10 mL.
esis of fully developed leaves under saturated light was A645 and A663 of the supernatant was measured, and
178 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186

Table 2
Nitrogen form effects on biomass (root, stem, leaf, shoot, and total) and allocation (root mass ratio, stem mass ratio, and leaf mass ratio) of three
dipterocarp species, A. costata, H. odorata, and S. roxburghii, at DAT 42
Species Parameter Treatment F P
NH4 NH4 + NO3 NO3
A. costata Biomass (g DW)
Root 2.94 (1.72) b 4.55 (2.84) a 4.55 (2.61) a 4.91 0.015
Stem 2.45 (1.79) a 2.69 (1.65) a 2.66 (1.80) a 0.30 0.741
Leaf 4.99 (3.50) a 5.17 (2.89) a 2.75 (1.89) b 9.65 <0.001
Shoot 7.44 (5.24) ab 7.86 (4.49) a 5.41 (3.66) b 5.92 0.008
Total 10.38 (6.93) a 12.41 (7.25) a 9.96 (6.24) a 2.33 0.117
Allocation (% of total biomass)
Root 0.30 (0.05) c 0.37 (0.05) b 0.48 (0.05) a 29.82 <0.001
Stem 0.23 (0.04) ab 0.21 (0.02) b 0.26 (0.04) a 4.95 0.015
Leaf 0.46 (0.07) a 0.42 (0.06) a 0.26 (0.04) b 29.68 <0.001
H. odorata Biomass (g DW)
Root 0.71 (0.34) a 0.96 (0.42) a 0.79 (0.46) a 0.90 0.421
Stem 0.73 (0.38) a 0.66 (0.32) ab 0.51 (0.39) b 10.97 <0.001
Leaf 1.67 (0.74) a 1.42 (0.50) a 0.92 (0.49) b 13.42 <0.001
Shoot 2.40 (1.12) a 2.07 (0.82) a 1.43 (0.87) b 13.06 <0.001
Total 3.12 (1.43) a 3.03 (1.04) a 2.22 (1.31) b 6.24 0.006
Allocation (% of total biomass)
Root 0.23 (0.04) b 0.32 (0.09) a 0.35 (0.04) a 9.31 <0.001
Stem 0.23 (0.03) a 0.21 (0.04) a 0.22 (0.03) a 0.73 0.493
Leaf 0.54 (0.03) a 0.47 (0.06) b 0.43 (0.05) b 14.23 <0.001
S. roxburghii Biomass (g DW)
Root 0.58 (0.17) a 0.60 (0.17) a 0.56 (0.18) a 0.18 0.836
Stem 0.63 (0.24) a 0.66 (0.17) a 0.44 (0.11) b 5.16 0.013
Leaf 1.81 (0.63) a 1.83 (0.50) a 0.93 (0.28) b 11.87 <0.001
Shoot 2.45 (0.86) a 2.49 (0.66) a 1.37 (0.37) b 10.20 <0.001
Total 3.02 (0.97) a 3.09 (0.79) a 1.93 (0.53) b 7.31 0.003
Allocation (% of total biomass)
Root 0.20 (0.05) b 0.20 (0.03) b 0.29 (0.04) a 17.99 <0.001
Stem 0.21 (0.03) a 0.22 (0.02) a 0.23 (0.03) a 1.62 0.216
Leaf 0.60 (0.03) a 0.59 (0.03) a 0.48 (0.03) b 52.07 <0.001
One-year-old seedlings were grown for 72 days with supply of nitrogen as ammonium, nitrate, or both. Means are presented, with standard
deviation in parentheses. F and P values of ANCOVA are presented as well. Means with significant treatment effect (P < 0.05) are shown in bold.
Different letters indicate significant difference between N forms (Tukey HSD, P < 0.05).

chlorophyll-a and -b contents were determined 2.5. Nitrate reductase activity

according to Arnon (1949).
2.4. Leaf nitrogen
In Experiment II, the nitrogen content of the leaves
in which photosynthesis was measured was deter-
mined with an NC analyzer (NA1500, Carlo Erba).
Leaf discs (diameter, 4 mm) were taken from the
leaves and dried at 80 8C for further analysis.
Measurement was duplicated for each sample.
The in vivo NRA of leaf and root samples was
determined according to Gebauer et al. (1998) with
some modifications. Ten 7-mm leaf discs were taken
from each of the leaves in which photosynthesis was
measured and immersed immediately in 5 mL of
incubation buffer in a 15-mL tube. Leaf discs were
incubated for 2 h at 28 8C in the dark in a N2
atmosphere. Incubation was terminated by boiling at
100 8C for 1 min. Then, 0.6 mL of 5% (w/v)
 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186 179

sulfanilamide in 3N HCl, 0.6 mL of 0.1% (w/v) N-

(1-naphthyl) ethylene-diamine-dihydrochloride, and
0.8 mL of Milli-Q water were added to 2 mL of
incubated solution, and the mixture was kept at
room temperature for 30 min. A540 was measured,
and the generated nitrite was determined against a
series of standards. About 200–300 mg of fresh fine
roots were chopped and similarly treated for NRA
measurement.

2.6. Statistical analysis

Generally, differences among treatments were

evaluated with ANOVA or Kruskal–Wallis test and
Scheffé’s test for each species. Analysis of covar-
iance was used for data obtained repeatedly from the
same seedlings over time. Differences in height
and diameter increment and leaf production among
N forms in Experiment II were tested with the
Tukey–Welsch method. Differences in biomass
were tested by ANCOVA with initial plant size
(d2h for leaf, stem, and total biomass and d2 for root
biomass) as the covariate. Data were log- or arcsine-
transformed as necessary to ensure homogeneity of
variance.

3. Results

3.1. Experiment I

3.1.1. Growth
All the three dipterocarp species showed less
height growth when supplied with nitrate as a sole
nitrogen source (Fig. 1). Similar nitrogen form effects
were also observed in diameter growth (data not
shown). Aboveground biomass, especially leaf
biomass, was less when supplied with nitrate as a
sole nitrogen source (Table 2). Root biomass, on the
other hand, was less affected in H. odorata and S.
roxburghii seedlings or increased in A. costata
seedlings, resulting in higher root mass ratio for all
the three species when supplied with nitrate (Table 2).
Fig. 1. Nitrogen form effects on height increment of three dipterocarp
Less growth of H. odorata and S. roxburghii seedlings species: A. costata (a), H. odorata (b), and S. roxburghii (c). One-year-
supplied with nitrate was observed on total plant old seedlings were grown with supply of nitrogen as ammonium,
biomass as well, while A. costata seedlings showed nitrate, or both. Heights are expressed relative to initial size. Error bars
similar total plant biomass between the two nitrogen denote standard deviations. Different letters indicate significant dif-
forms (Table 2). ference between nitrogen forms at the end of the experiment (Scheffé,
P < 0.05).
180 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186
Fig. 2. Nitogen form effects on light-saturated photosynthetic rate
(Pn) (a), stomatal conductance (gs) (b), internal CO2 concentration (Ci)
(c), and CO2-saturated light-saturated photosynthetic rate (Pnsat) (d) of
fully developed leaves in three dipterocarp species: Ac, A. costata; Ho,
H. odorata; Sr: S. roxburghii. One-year-old seedlings were grown with
supply of nitrogen as ammonium, nitrate, or both and gas exchange
was measured at 64 and 65 DAT under ambient CO2 concentration and
at 71 and 72 DAT under saturated CO2 condition. Error bars denote
standard deviations. Different letters indicate significant difference
between nitrogen forms for each species (Scheffé, P < 0.05). NS: not
significant (ANOVA, P < 0.05).
3.1.2. Photosynthesis
All the three dipteorcarp species showed lower Pn
when supplied with nitrate as a sole nitrogen source,
which was accompanied with higher internal CO2
concentration (Ci) and lower CO2-saturated photo-
synthetic rate under saturated light (Pnsat; Fig. 2).
Stomatal conductance (gs) was not affected by the
nitrogen forms (Fig. 2b).
Fig. 3. Nitrogen form effects on chlorophyll content of fully
developed leaves in three dipterocarp species: Ac, A. costata; Ho,
H. odorata; Sr, S. roxburghii. One-year-old seedlings were grown
with supply of nitrogen as ammonium, nitrate, or both for 72 days
and chlorophyll content was measured at the end of the experiment.
Chlorophyll content is shown as value from SPAD meter. Error bars
denote standard deviations. Different letters indicate significant
difference between nitrogen forms for each species (Scheffé,
P < 0.05).
3.1.3. Chlorophyll
The chlorophyll content of fully developed leaves
was lower in the seedlings supplied with nitrate for all
the three dipterocarp species (Fig. 3).
3.2. Experiment II
3.2.1. Growth
D. obtusifolius and S. faguetiana seedlings showed
less height growth when supplied with nitrate as a sole
nitrogen source (Fig. 4). Diameter growth in the two
species was also less when supplied with nitrate (data
not shown). Aboveground biomass was less in the two
species when nitrate was supplied as a sole nitrogen
source (Table 3). In contrast, root biomass was
increased in D. obtusifolius or less affected in S.
faguetiana when supplied with nitrate as a sole
nitrogen source, resulting in higher root mass ratio
(Table 3). In contrast to the two species, N. heimii
seedlings showed similar growth between the two
nitrogen forms and more growth when supplied with
ammonium plus nitrate (Fig. 4; Table 3). Root mass
ratio of N. heimii seedlings was higher when nitrate
was supplied as a sole nitrogen source (Table 3).
3.2.2. Photosynthesis
D. obtusifolius and S. faguetiana seedlings showed
lower Pn when supplied with nitrate as a sole nitrogen
source (Fig. 5a). Both species showed lower Pnsat
when supplied with nitrate as a sole nitrogen source
(Fig. 5d). D. obtusifolius seedlings showed lower gs
 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186
Fig. 4. Nitrogen form effects on height increment of three dipterocarp
species: D. obtusifolius (a), S. faguetiana (b), and N. heimii (c). 2.5-
month-old seedlings of D. obtusifolius and N. heimii, and 9-month-old
S. faguetiana seedlings were grown with supply of nitrogen as
ammonium, nitrate, or both. Heights are expressed as relative to
initial size. Error bars denote standard deviations. Different letters
indicate significant difference between nitrogen forms at the end of the
experiment (Tukey–Welsch, P < 0.05). NS: not significant.
181
Fig. 5. Nitrogen form effects on light-saturated photosynthetic rate
(Pn) (a), stomatal conductance (gs) (b), internal CO2 concentration
(Ci) (c), and CO2-saturated photosynthetic rate (Pnsat) (d) of fully
developed leaves in three dipterocarp species: Do, D. obtusifolius;
Sf, S. faguetiana; Nh, N. heimii. 2.5-month-old seedlings of D.
obtusifolius and N. heimii and 9-month-old S. faguetiana seedlings
were grown with supply of nitrogen as ammonium, nitrate, or both
and gas exchange was measured at 123–126 DAT. Error bars denote
standard deviations. Different letters indicate significant difference
between nitrogen forms for each species (Scheffé, P < 0.05). NS:
not significant (P < 0.05).
(Fig. 5b) and higher Ci (Fig. 5c) when supplied with
nitrate, but S. faguetiana seedlings showed no
difference in the two parameters by nitrogen form
treatments (Fig. 5b and c). N. heimii seedlings showed
similar Pn and Pnsat between the two nitrogen forms
and highest Pn when supplied with ammonium plus
nitrate (Fig. 5a and d).
182 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186

Table 3
Nitrogen form effects on biomass (root, stem, leaf, shoot, and total) and allocation (root mass ratio, stem mass ratio, and leaf mass ratio) of three
dipterocarp species, D. obtusifolius, S. faguetiana, and N. heimii, at the end of the experiment
Species Parameter Treatment F P
NH4 NH4 + NO3 NO3
D. obtusifolius Biomass (g DW)
Root 1.53 (0.55) b 1.35 (0.54) b 2.06 (0.75) a 4.71 0.013
Stem 0.95 (0.37) a 0.75 (0.29) b 0.58 (0.23) b 15.08 <0.001
Leaf 3.46 (1.34) a 2.72 (1.09) a 1.31 (0.43) b 25.77 <0.001
Shoot 4.41 (1.69) a 3.48 (1.37) a 1.89 (0.64) b 23.27 <0.001
Total 5.94 (2.21) a 4.82 (1.86) ab 3.95 (1.32) b 9.69 <0.001
Allocation (% of total biomass)
Root 0.26 (0.03) b 0.29 (0.05) b 0.52 (0.06) a 161.44 <0.001
Stem 0.16 (0.03) a 0.16 (0.02) a 0.15 (0.03) a 1.70 0.192
Leaf 0.58 (0.04) a 0.56 (0.05) a 0.33 (0.04) b 178.76 <0.001
S. faguetiana Biomass (g DW)
Root 1.01 (0.62) a 1.42 (1.44) a 0.84 (0.47) a 0.94 0.396
Stem 1.88 (1.33) a 2.53 (2.32) a 1.33 (0.76) a 2.65 0.080
Leaf 2.39 (1.88) a 3.40 (2.82) a 1.51 (1.18) a 2.45 0.096
Shoot 4.28 (3.15) ab 5.94 (5.08) a 2.84 (1.92) b 2.88 0.065
Total 5.29 (3.75) a 7.36 (6.48) a 3.68 (2.38) a 2.53 0.089
Allocation (% of total biomass)
Root 0.21 (0.05) ab 0.20 (0.04) b 0.24 (0.04) a 5.50 <0.001
Stem 0.38 (0.08) a 0.35 (0.05) a 0.38 (0.06) a 0.87 0.424
Leaf 0.41 (0.12) a 0.45 (0.09) a 0.38 (0.08) a 2.68 0.078
N. heimii Biomass (g DW)
Root 0.47 (0.25) b 0.61 (0.27) ab 0.66 (0.14) a 3.46 0.039
Stem 0.75 (0.30) b 0.97 (0.44) a 0.98 (0.29) a 4.47 0.016
Leaf 1.11 (0.61) b 1.95 (0.97) a 1.22 (0.44) ab 6.96 0.002
Shoot 1.86 (0.87) b 2.92 (1.38) a 2.20 (0.68) ab 7.11 0.002
Total 2.33 (1.10) b 3.53 (1.62) a 2.85 (0.77) ab 4.36 0.018
Allocation (% of total biomass)
Root 0.20 (0.03) b 0.18 (0.03) b 0.24 (0.05) a 12.67 <0.001
Stem 0.34 (0.07) ab 0.29 (0.07) b 0.34 (0.05) a 4.84 0.012
Leaf 0.46 (0.08) b 0.54 (0.07) a 0.42 (0.07) b 13.50 <0.001
2.5-month-old seedlings of D. obtusifolius and N. heimii and 9-month-old S. faguetiana seedlings were grown for 127 days with supply of
nitrogen as ammonium, nitrate, or both. Means are presented, with standard deviation in parentheses. F and P values of ANCOVA are presented
as well. Means with significant treatment effect (P < 0.05) are shown in bold. Different letters indicate significant difference between N forms
(Tukey HSD, P < 0.05).

3.2.3. Chlorophyll and nitrogen
D. obtusifolius and S. faguetiana seedlings showed
lower chlorophyll content in leaves when nitrate was
supplied as a sole nitrogen source (Fig. 6a). N. heimii
seedlings showed similar leaf chlorophyll contents
between the two nitrogen forms (Fig. 6a). Chlorophyll-
a/b ratio was lower in D. obtusifolius and N. heimii
seedlings when supplied with nitrate, but not affected in
S. faguetiana seedlings (Fig. 6b). For all the three
species, leaf nitrogen content was lower when supplied
with nitrate as a sole nitrogen source (Fig. 6c).
3.2.4. In vivo nitrate reductase activity in leaves
and roots
In vivo NRA was detected in leaves of all three
species (Fig. 7a). S. faguetiana seedlings showed
higher leaf NRA when supplied with nitrate as a sole
nitrogen source, while N. heimii seedlings showed
lower leaf NRA under the condition (Fig. 7a). D.
obtusifolius seedlings showed no response in leaf
NRA to nitrogen form treatments (Fig. 7a). Similar or
lower levels of NRA were detected in roots than in
leaves of all three species (Fig. 7b). D. obtusifolius
 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186
Fig. 6. Nitrogen form effects on chlorophyll content (a), chloro-
phyll-a/b ratio (b), and nitrogen content (c) of fully developed leaves
in three dipterocarp species: Do, D. obtusifolius; Sf, S. faguetiana;
Nh, N. heimii. 2.5-month-old seedlings of D. obtusifolius and N.
heimii and 9-month-old S. faguetiana seedlings were grown for 127
days with supply of nitrogen as ammonium, nitrate, or both. Error
bars denote standard deviations. Different letters indicate significant
difference between nitrogen forms for each species (Scheffé,
P < 0.05). NS: not significant (ANOVA, P < 0.05).
seedlings showed higher root NRA when nitrate was
supplied as a sole nitrogen source, while the other two
species showed no response in root NRA to nitrogen
forms (Fig. 7b).
4. Discussion
4.1. Growth of the six dipterocarp species with
different nitrogen forms
Except for N. heimii, all of the dipterocarp species
we examined showed growth reduction in shoots,
183
Fig. 7. Nitrogen form effects on in vivo nitrate reductase activity
(NRA) in leaves (a) and roots (b) of three dipterocarp species: Do, D.
obtusifolius; Sf, S. faguetiana; Nh, N. heimii. 2.5-month-old seed-
lings of D. obtusifolius and N. heimii and 9-month-old S. faguetiana
seedlings were grown for 127 days with supply of nitrogen as
ammonium, nitrate, or both. Error bars denote standard deviations.
Different letters indicate significant difference between nitrogen
forms for each species (Scheffé, P < 0.05). NS: not significant
(ANOVA for leaf, Kruskal–Wallis for root, P < 0.05).
especially in leaves, when N was supplied as nitrate,
which indicates a preference of these species for
ammonium in shoot growth (Tables 2 and 3; Figs. 1 and
4). N. heimii seedlings did not show a clear preference
between the two nitrogen forms (Tables 2 and 3;
Figs. 1 and 4). N. heimii grows comparatively slowly
(Soerianegara and Lemmens, 1994). Its relatively low
demand for nitrogen might have hidden its preference
of nitrogen form. Bungard et al. (2000) reported the
involvement of nitrogen availability in the growth
response of dipterocarp species to gap formation, and
these investigators pointed out that nutrient conditions
affect regeneration dynamics and the distribution of
canopy-dominating dipterocarp species. Considering
that nutrient regime varies across and within site,
nutritional traits would also play a role in ecological
patterns of distribution, which have been mostly related
to physiological traits in terms of light and water
acquisition. Since nutrient availability is determined by
both amount and form, the nitrogen form regime seems
also to play an important role in these ecological
aspects. Information about natural habitat relating to
soil nitrogen regime is lacking for the examined species
so far, but the outcome of this study will shed a new light
184 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186
on their distribution and regeneration in future research.
An increase in the ratio of ammonium to nitrate with
succession progress has been reported in tropical
(Vitousek and Matson, 1988) and temperate forests
(e.g., Robertson and Vitousek, 1981). Stewart et al.
(1988, 1990) reported a low level of nitrate assimilation
in closed forest species in Australia. A preference for
ammonium would be an advantage at sites where
ammonium dominates. On the other hand, given that
soil nitrate content increases after disturbance (e.g.,
Vitousek et al., 1982; Denslow et al., 1998), a low
ability to use nitrate would be a disadvantage in
competition for nitrogen.
Nitrate caused a higher root mass ratio in seedlings
of all the examined species (Tables 2 and 3). A higher
root mass ratio under nitrogen-limiting conditions has
been well documented (e.g., Andrews et al., 1999; Cruz
et al., 2003a; de Groot et al., 2003; Nguyen et al., 2003;
for review, see Andrews et al., 2001). More photo-
assimilate distribution to roots could have compensated
for the lower nitrogen uptake when ammonium-
preferring species were supplied with nitrate, while it
reduced carbon gain at the same time. Interestingly, N.
heimii, which showed no apparent preference between
ammonium and nitrate, also showed higher root mass
ratio when supplied with nitrate as a sole nitrogen
source (Table 3), while the reason is unclear.
4.2. Photosynthesis of six dipterocarp species
with different nitrogen forms
The five dipterocarp species which showed pre-
ference for ammonium had a higher light-saturated
photosynthetic rate with ammonium supply (Figs. 2a
and 5a). This is likely the cause of the better growth
of these species when nitrogen was supplied as
ammonium. Lower photosynthetic rate in leaves of the
five species supplied with nitrate was not accompanied
by lower stomatal conductance (Figs. 2b and 5b),
indicating reduction in photosynthetic capacity as the
cause of the lower photosynthetic rate. An impairment
in photosynthetic capacity was suggested by the
higher internal CO2 concentration (Figs. 2c and 5c)
and apparently revealed by the lower CO2-saturated
photosynthetic rate (Figs. 2d and 5d) with nitrate
supply. A higher internal CO2 concentration suggests a
lower carboxylation efficiency resulting from lower
rubisco activity. A lower CO2-saturated photosyn-
thetic rate indicates lower RuBP regeneration rate and/
or lower rubisco activity. Lower rubisco activity in
seedlings supplied with nitrate likely resulted from
lower rubisco content, since the rubisco activation
state increases as leaf nitrogen decreases (Cheng and
Fuchigami, 2000).
The effects of nitrogen form on leaf chlorophyll
content correspond well with those on photosynthetic
rate (Figs. 2(a and d), 3, 5(a and d), 6a). It is not clear
whether lower chlorophyll content is a cause or a
result of the lower photosynthetic activity. In any case,
although it was examined in only three of the six
species, the lower leaf nitrogen content with nitrate
supply (Fig. 6c) suggests that insufficient nitrogen
uptake caused by inability to assimilate nitrate would
be a cause of the lower chlorophyll content with nitrate
supply. The lower chlorophyll-a/b ratio in leaves of D.
obtusifolius and N. heimii seedlings supplied with
nitrate resulted from a greater decrease in chlorophyll-
a than in chlorophyll-b. The response of the
chlorophyll-a/b ratio to nitrogen limitation is not
well documented, and contradictory results have been
obtained so far. A decrease in the chlorophyll-a/b ratio
under nitrogen deficiency was reported in cassava
(Manihot esculenta Crantz) plants (Cruz et al., 2003b),
but a common response of increased chlorophyll-a/b
ratio under nitrogen deficiency was reported in four
tropical tree species (Kitajima and Hogan, 2003).
4.3. Nitrate assimilation ability of three
dipterocarp species
NRA was detected in leaves and roots, with similar
or somewhat higher levels in leaves than in roots of all
three species examined (Fig. 7). The levels of NRAs in
roots and leaves are comparable to those of closed-
forest species in Australia (Stewart et al., 1988, 1990).
The ratio of leaf NRA to root NRA differs among
species and may change depending on nitrogen
availability (Downs et al., 1993). Ratios of leaf to
root nitrate reductase decreased in D. obtusifolius and
increased in S. faguetiana seedlings with nitrate
supply (data not shown). Leaf nitrate reduction uses a
photosynthetic derivative reductant, whereas root
nitrate reduction uses a reductant produced through
carbohydrate breakdown. The increase in the leaf to
root nitrate reductase ratio in S. faguetiana may have
some advantage in this context.
 M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186
Although nitrate reductase is a substrate-inducible
enzyme (Sivasankar and Oaks, 1996), such induction
was not found except in leaves of S. faguetiana and
roots of D. obtusifolius. Stewart et al. (1988) also
reported low inducibility of nitrate reductase in
closed-forest species, whereas the pioneer species
they tested showed substrate induction of the enzyme.
Soil nitrate content increases after disturbance
(Vitousek et al., 1982; Brouwer and Riezebos,
1998). Even though S. faguetiana was revealed to
prefer ammonium, the increase in leaf nitrate
reductase in response to nitrate may have some
advantage by consuming excess light energy when
leaves are exposed to excess light by gap formation.
Lavoie et al. (1992) concluded that nitrate uptake and
not nitrate assimilation via nitrate reductase limited
the growth of jack pine (Pinus banksiana Lamb.) with
nitrate as the sole N source. We cannot tell from our
results whether assimilation via nitrate reductase
limited the growth of the five dipterocarp species when
nitrate was supplied as the sole N source. Compre-
hensive investigation of the use of nitrogen in different
forms is needed in order to address this question.
In conclusion, we showed that all of the dipterocarp
species examined except for N. heimii prefer ammo-
nium to nitrate as a nitrogen source. The greater growth
with supply of ammonium was due to greater
photosynthesis, which was the result of greater nitrogen
absorption. Future work should examine the response of
the species to different forms of nitrogen at lower
concentrations. Physiological aspects of nitrogen
uptake and assimilation have recently been well
documented in temperate tree species, especially in
coniferous species (e.g., Kronzucker et al., 1996).
Further research on nitrogen characteristics of dipter-
ocarp species with the help of knowledge and tools
derived from studies of temperate tree species would be
important for a better understanding of tropical forests
and for reforestation in Southeast Asia.
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