Leaf nitrogen to phosphorus ratios of tropical trees:
experimental assessment of physiological and
environmental controls
Lucas A. Cernusak1,2, Klaus Winter1 and Benjamin L. Turner1
1Smithsonian Tropical Research Institute, PO Box 0843-03092, Balboa, Ancon, Republic of Panama; 2Present address: School of Environmental and Life
Sciences, Charles Darwin University, Darwin, NT 0909, Australia
Author for correspondence:
Lucas A. Cernusak
Tel: +61 8 8946 7630
Email: lucas.cernusak@cdu.edu.au
Received: 28 August 2009
Accepted: 8 October 2009
New Phytologist (2010) 185: 770?779
doi: 10.1111/j.1469-8137.2009.03106.x
Key words: leaf nitrogen concentration, leaf
nitrogen to phosphorus ratio, leaf
phosphorus concentration, relative growth
rate, water-use efficiency.
Summary
? We investigated the variation in leaf nitrogen to phosphorus ratios of tropical
tree and liana seedlings as a function of the relative growth rate, whole-plant
water-use efficiency, soil water content and fertilizer addition.
? First, seedlings of 13 tree and liana species were grown individually in 38-l pots
prepared with a homogeneous soil mixture. Second, seedlings of three tree species
were grown in 19-l pots at high or low soil water content, and with or without
added fertilizer containing nitrogen, phosphorus and potassium.
? For plants grown under common soil conditions, leaf nitrogen to phosphorus
ratios showed a unimodal, or hump-shaped, relationship with the relative growth
rate. The leaf nitrogen to phosphorus ratio increased in response to low soil water
content in three species, and increased in response to fertilizer addition in two of
the three species. Across all species and treatments, the leaf nitrogen to phospho-
rus ratio was positively correlated with the water-use efficiency.
? The results suggest that the within-site variation among tropical tree species in
the leaf nitrogen to phosphorus ratio may be caused by associations between this
ratio and the relative growth rate. Modification of the soil environment changed
the leaf nitrogen to phosphorus ratio, but underlying associations between this
ratio and the relative growth rate were generally maintained. The observed corre-
lation between the leaf nitrogen to phosphorus ratio and water-use efficiency has
implications for linking nutrient stoichiometry with plant transpiration.
Introduction
The nitrogen to phosphorus (N : P) ratio in terrestrial plant
leaves can provide important information about nutrient
limitations to primary productivity (Sterner & Elser, 2002;
A?gren, 2008). For example, it has been suggested that leaf
N : P ratios above a given threshold (c. 16 on a mass basis)
indicate phosphorus limitation to biomass production, and
those below a given threshold (c. 14 on a mass basis) indi-
cate nitrogen limitation (Koerselman & Meuleman, 1996;
Aerts & Chapin, 2000; Tessier & Raynal, 2003; Gu?sewell,
2004). This offers a powerful tool for ecological and physio-
logical investigations by providing a straightforward means
of characterizing the relative availability of nitrogen vs phos-
phorus at a given site. However, some terrestrial ecosystems
appear not to conform to this expectation (Craine et al.,
2008), suggesting that the interpretation of terrestrial plant
N : P ratios may be more complex.
At a basic level, it is clear that terrestrial plants exer-
cise some level of homeostatic control over their N : P
ratios. For example, the N : P ratio of plants grown
experimentally reflects the N : P supply ratio of the
nutrient solution fed to the plants, but the range of val-
ues in the former is several fold less than that of the lat-
ter (Gu?sewell & Koerselman, 2002). Thus, the challenge
to the interpretation of plant N : P ratios in nature is to
understand the relative partitioning of control between
intrinsic physiology and external environment (A?gren,
2008). A recent study has illustrated this point with
respect to tropical rainforests, finding a large variation in
leaf N : P ratio among tropical tree species at a given
site (Townsend et al., 2007); the authors concluded that
New
PhytologistResearch
770 New Phytologist (2010) 185: 770?779
www.newphytologist.org
 The Authors (2009)
Journal compilation  New Phytologist (2009)
the dominant control over leaf N : P ratios in the tro-
pics is probably the identity of the species or mixture of
species under examination. What causes the variation in
leaf N : P ratios of different tropical tree species growing
in the same environment? The answer to this question is
important for the interpretation of leaf N : P ratios in
tropical forests, and would provide general insight into
the rapidly emerging field of ecological stoichiometry
(Sterner & Elser, 2002). It may also contribute towards
a better understanding of the effect of species diversity
on relationships between nitrogen and phosphorus avail-
ability and biomass production in tropical forests (Kitay-
ama, 2005).
A?gren (2004) proposed a conceptual model for the
understanding of the C : N : P stoichiometry of photoau-
totrophic organisms. The model is predicated on the basis
that photosynthetic organisms require proteins to carry out
photosynthesis and growth. On the other hand, they require
ribosomes to synthesize proteins. Proteins are nitrogen rich
and phosphorus poor, whereas ribosomes contain approxi-
mately equal amounts of protein and rRNA, which is phos-
phorus rich; this gives them a very low N : P mass ratio of
c. 3.3 (Sterner & Elser, 2002). Thus, photosynthetic organ-
isms require nitrogen-rich proteins to capture carbon and
grow, but also require phosphorus-rich ribosomes to syn-
thesize proteins. According to these constraints, the growth
rate of a photosynthetic organism can be described as
(A?gren, 2004):
dC
dt
? /CNNp; Eqn 1
dNp
dt
? /NPPri; Eqn 2
(C, amount of carbon in the plant; t, time; NP, amount of
nitrogen in proteins used for growth; Pri, amount of phos-
phorus in ribosomes used for protein synthesis; /CN and
/NP, rate factors). Plants contain nitrogen and phosphorus
in compounds other than those used for growth and protein
synthesis, respectively, such that the total amounts of nitro-
gen (N) and phosphorus (P) in the plant can be expressed
as:
N ? Np ? bNC ; Eqn 3
P ? Pri ? bpC ; Eqn 4
(bN and bP, proportionality constants). Eqns 3 and 4 state
that the amounts of nitrogen and phosphorus in the plant
not associated with growth and protein synthesis, respec-
tively, but still necessary for normal functioning, are pro-
portional to the amount of plant carbon. Under stable and
balanced growth, such that the plant has a constant relative
chemical composition and a constant relative growth rate
(l), the following definitions exist:
l ? 1
C
dC
dt
? 1
N
dN
dt
? 1
P
dP
dt
? 1
M
dM
dt
; Eqn 5
(M, total plant mass). Equations 1?5 can be used to predict
plant C : N : P stoichiometry as a function of l (A?gren,
2004):
N
C
? l
/CN
? bN; Eqn 6
P
C
? l
2
/CN/NP
? bP; Eqn 7
N
P
? l/NP ? bN/CN/NP
l2 ? bp/CN/NP
: Eqn 8
Equation 6 predicts that, for nitrogen-limited growth,
the N : C ratio should increase linearly as a function of l.
On the other hand, Eqn 7 predicts that, for phosphorus-
limited growth, the P : C ratio should increase curvilinearly
as a quadratic function of l. Eqn 8 predicts that the whole-
plant critical N : P ratio, where both nitrogen and phos-
phorus are simultaneously limiting growth, is expected to
show a unimodal relationship with l, increasing at low val-
ues of l to a maximum, and then decreasing as l increases
further. Example predictions based on Eqns 6, 7 and 8 are
shown in Fig. 1. Although the model described above
(A?gren, 2004) predicts the N : P ratio on a whole-plant
basis, leaf N : P ratios have been shown to correlate with
those in other plant organs (Kerkhoff et al., 2006), and the
model may therefore provide useful insight into the varia-
tion in the N : P ratios of leaves. If excess uptake of nitro-
gen or phosphorus takes place when the supply of one of
these nutrients limits growth but the supply of the other
does not, plant N : P ratios would be expected to differ
from the prediction of Eqn 8. Variation in the leaf N : P
ratio, however, might be partly buffered against such an
effect if the storage of excess nitrogen or phosphorus were
to take place mostly in the roots and stems in woody peren-
nial plants (e.g. Dyckmans & Flessa, 2001).
Cernusak et al. (2007) observed that the leaf N : P ratios
in seedlings of a tropical pioneer tree species, Ficus insipida,
correlated positively with the whole-plant water-use effi-
ciency (WUE). The WUE can be expressed in mass units of
g C kg)1 H2O, and is a measure of the amount of carbon
accumulated in the plant biomass for a given amount of
water transpired to the atmosphere. The plant N : P ratio
might be expected to correlate with WUE according to the
New
Phytologist Research 771
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
following argument: plant carbon gain relates positively to
the amount of nitrogen in proteins associated with growth,
as shown above in Eqn 1; on the other hand, the transport
of phosphorus to the surfaces of roots in the soil, where it
can be subsequently absorbed by the plant, partly depends
on the mass flow of the soil solution resulting from transpi-
ration by the plant (Barber, 1995; Tinker & Nye, 2000;
Cernusak et al., 2007; Scholz et al., 2007; Cramer et al.,
2008, 2009). Thus, carbon uptake should correlate with
NP, and phosphorus uptake should correlate with transpira-
tion. Consequently, it could be hypothesized that the N : P
ratio would correlate with the C : T ratio, where T is the
cumulative transpiration, and C : T is equal to WUE.
There are some limitations to this line of reasoning that
should be recognized. These include the observation that
WUE appears to increase with increasing phosphorus avail-
ability, not just with increasing nitrogen availability (Raven
et al., 2004), and the relative immobility of phosphorus in
soils, which might minimize its transport to root surfaces by
mass flow (Barber, 1995; Tinker & Nye, 2000). Fortu-
nately, the hypothesis is amenable to experimental testing.
In this article, we report the relationship between the leaf
N : P ratio and l for seedlings of 13 tropical tree and liana
species grown in a homogeneous soil environment. In addi-
tion, we examined the effects of a variation in soil water
content and fertilizer addition on the leaf N : P ratios in
seedlings of three tropical tree species with contrasting l
values. Finally, we tested the hypothesis that the leaf N : P
ratio correlates with WUE across a diverse range of species
and soil conditions.
Materials and Methods
Plants were grown at the Santa Cruz Experimental Field
Facility of the Smithsonian Tropical Research Institute in
Gamboa, Panama (907?N, 7942?W). The altitude at the
site is c. 28 m above sea level. Leaf N : P ratios considered
in this article are from plants grown for two experiments. In
the first experiment, several individuals each of 13 tropical
tree and liana species were grown in 38-l pots, with one
plant in each pot, for several months. The pots were filled
with a homogenized soil mixture, comprising 60% by vol-
ume of dark, air-dried topsoil and 40% by volume of air-
dried rice husks. The rice husks were added to the soil mix-
ture to improve soil structure and drainage. Soil water con-
tent throughout the experiment was maintained between
field capacity and 60% of field capacity, such that the plants
were well watered. This experiment included three conifer-
ous tree species [Cupressus lusitanica Mill. (Cupressaceae),
Pinus caribaea Morelet (Pinaceae) and Thuja occidentalis L.
(Cupressaceae)], seven angiosperm tree species [Calophyl-
lum longifolium Willd. (Clusiaceae), Clusia pratensis Seem.
(Clusiaceae), Hyeronima alchorneoides Allema?o (Euphorbia-
ceae), Luehea seemannii Triana & Planch. (Tiliaceae), Swie-
tenia macrophylla King (Meliaceae), Tabebuia rosea (Bertol.)
A. DC. (Bignoniaceae), Tectona grandis Linn. (Verbena-
ceae)] and three angiosperm liana species [Gouania lupulo-
ides (L.) Urb. (Rhamnaceae), Mikania leiostachya Benth.
(Asteraceae), Stigmaphyllon hypargyreum Triana & Planch.
(Malphigiaceae)]. Additional information about the experi-
ment can be found in Cernusak et al. (2008).
The second experiment employed three angiosperm tree
species [Platymiscium pinnatum (Jacq.) Dugand (Fabaceae),
S. macrophylla and T. grandis]. Twenty seedlings of each
(a)
(b)
(c)
Fig. 1 Representative predictions of relationships between the plant
N : C (a), P : C (b) and N : P (c) ratios and the relative growth rate.
Predicted relationships are for the case in which the nutrient or nutri-
ents involved are limiting growth. Predictions are according to the
C : N : P model of A?gren (2004), using the following parameter
values: /CN = 1 g C g
)1 N d)1; /NP = 0.5 g P g
)1 N d)1;
bN = 8 mg N g
)1 C; bP = 2 mg P g
)1 C.
772 Research
New
Phytologist
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
species were planted individually in 19-l pots. The pots
were filled with a homogenized soil mixture. The soil mix-
ture for this experiment comprised 80% by volume of dark,
air-dried topsoil and 20% by volume of air-dried rice husks.
At the beginning of the experiment, 10 of the 20 pots for
each species were randomly chosen to receive c. 12 g Osmo-
cote-Plus controlled-release fertilizer (Scotts-Sierra, Mary-
ville, OH, USA). The fertilizer contained by weight 15%
nitrogen, 9% phosphorus and 12% potassium, and had an
estimated release time of 5?6 months at a temperature of
32C according to the manufacturer. Five fertilized and five
unfertilized pots from each species were then randomly allo-
cated to receive a reduced water supply. All pots started the
experiment watered to field capacity. Those receiving the
full water allocation were weighed each week and re-watered
to near field capacity. Later in the experiment, pots were
weighed and re-watered at shorter intervals, depending on
the water loss rates. Those receiving the reduced water allo-
cation were allowed to dry down to pot water contents of
< 2.5 kg, or c. 40% of field capacity, over several weeks.
Thereafter, they were weighed and re-watered to this pot
water content each week, or at shorter intervals, as neces-
sary. Pots were weighed to the nearest 5 g with a 64-kg
capacity balance (Sartorius QS64B; Thomas, Swedesboro,
NJ, USA). Additional information about the experiment
can be found in Cernusak et al. (2009).
The initial plant dry mass at the beginning of the experi-
ments was estimated by harvesting several representative
individuals of each species. At the conclusion of the experi-
ments, leaves, stems and roots were harvested separately and
dried to constant weight at 70C. The mean l value of each
plant was calculated as l = [loge(M2) ) loge(M1)] ? t, where
loge(M2) and loge(M1) are the natural logarithms of plant
dry mass at the end and beginning of the experiment,
respectively, and t is the duration of the experiment (Black-
man, 1919). Cumulative plant water use over the course of
the experiments was determined by weighing the pots at
weekly, or subweekly, intervals, as necessary. Pots were
closed such that they did not drain during the experiments.
At each weighing, the pots were replenished with water to a
previously determined target weight. Pots without plants
were deployed to estimate soil evaporation. The WUE was
calculated as (C2 ) C1 + CL) ?T, where C2 and C1 are the
plant carbon masses at the end and beginning of the experi-
ment, respectively, CL is the leaf litter carbon mass abscised
during the experiment and T is the cumulative transpira-
tion.
The leaf dry matter of each plant was ground to a fine,
homogeneous powder in a Cyclotec 1093 sample mill with
a 0.5-mm screen (FOSS; Eden Prairie, MN, USA). Samples
of c. 3 mg were analyzed for carbon and nitrogen concen-
tration with an elemental analyzer (ECS 4010; Costech
Analytical Technologies, Valencia, CA, USA) coupled to an
isotope ratio mass spectrometer (Delta XP; Finigan MAT,
Bremen, Germany). In the first experiment, leaf dry matter
was analyzed for phosphorus concentration by acid diges-
tion and detection on an inductively coupled plasma optical
emission spectrometer (Perkin Elmer Inc., Wellesley, MA,
USA). Leaf samples were prepared by digesting c. 200 mg
of sample material under pressure in polytetrafluoroethyl-
ene vessels with 2 ml of concentrated nitric acid. In the sec-
ond experiment, leaf dry matter was analyzed for
phosphorus concentration by ashing at 550C, followed by
dissolution in 1 M H2SO4, with phosphate detection by
automated molybdate colorimetry using a Lachat Quick-
chem 8500 (Hach Ltd, Loveland, CO, USA). Species? mean
leaf nitrogen and phosphorus concentrations for the first
experiment have been reported previously (Cernusak et al.,
2008), but the variation in the leaf N : P ratio was not
assessed as a function of l or WUE in that paper. Leaf
phosphorus concentrations and leaf N : P ratios for the sec-
ond experiment are presented for the first time here.
The dependence of the leaf N : C, P : C and N : P ratios
on l for the first experiment was assessed using the linear
and nonlinear regression routines in Systat 12.0 (SPSS, Chi-
cago, IL, USA). Variation among species and treatments in
leaf N : C, P : C and N : P ratios for the second experi-
ment was assessed by analysis of variance. The number of
observations was 60, the degree of freedom for species was
two, the degrees of freedom for nutrient and water treat-
ments were one each and the degree of freedom error was
48. Pair-wise comparisons among species following analyses
of variance were made according to Tukey?s method.
Results were considered to be statistically significant at
P < 0.05.
Results
The leaf N : C ratio varied linearly as a function of l across
the dataset for the first experiment (Fig. 2a), consistent with
the prediction of Eqn 6. The l value explained 44% of the
variation in the leaf N : C ratio. The regression equation
relating the two was leaf N : C = 0.671l + 17.1
(R2 = 0.44, P < 0.001, n = 88), where the leaf N : C ratio
is in mg g)1 and l is in mg g)1 d)1. On the other hand,
the leaf P : C ratio varied as a nonlinear function of l
(Fig. 2b), qualitatively consistent with the prediction of
Eqn 7. The nonlinear regression equation relating the leaf
P : C ratio to l2 was leaf P : C = 0.0053l2 + 3.55 (R2 =
0.31, n = 88), where the leaf P : C ratio is in mg g)1 and l
is in mg g)1 d)1. For comparison, a linear regression of the
leaf P : C ratio with l as independent variable had R2 of
0.27. The leaf N : P ratio also varied as a nonlinear func-
tion of l (Fig. 2c). The leaf N : P ratio increased with
increasing l at low values of l, reached a maximum, and
then decreased as l increased further. This unimodal pat-
tern of variation was qualitatively consistent with that pre-
dicted by Eqn 8. A nonlinear regression, taking the form of
New
Phytologist Research 773
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
Eqn 8, resulted in the following equation: leaf
N : P = [(258l ) 1510) ? (l2 + 62)] (R2 = 0.23, n = 88).
Mean values for the second experiment are shown in
Table 1 for the leaf N : C, P : C and N : P ratios for each
species by treatment combination. Analysis of variance indi-
cated significant variation among species in the leaf N : C,
P : C and N : P ratios (P < 0.001). The leaf N : C ratio
was higher in P. pinnatum than in T. grandis or S. macro-
phylla, and the leaf P : C ratio was highest in P. pinnatum,
intermediate in T. grandis and lowest in S. macrophylla. In
general, treatment effects on the leaf N : C ratio were more
pronounced than treatment effects on the leaf P : C ratio.
The leaf N : C ratio increased significantly in response to
low soil water content and fertilizer addition (P < 0.001).
By contrast, the leaf P : C ratio decreased slightly in
response to low soil water content and increased slightly in
response to fertilizer addition, but these effects were only
moderately significant (P = 0.04 and P = 0.06, respec-
tively). The leaf N : P ratio varied significantly by nutrient
treatment (P < 0.001) and by water treatment (P < 0.001).
The mean leaf N : P ratio was lowest in T. grandis, whereas
S. macrophylla and P. pinnatum showed similar mean val-
ues. There was a marked increase in the leaf N : P ratio
with decreasing soil water content, and this pattern was con-
sistent across all three species (Table 1). The leaf N : P ratio
also increased in response to fertilizer addition. There was a
significant interaction between nutrient treatment and spe-
cies (P < 0.001), such that S. macrophylla showed a strong
response of the leaf N : P ratio to fertilizer addition,
whereas T. grandis showed a weaker response and P. pinna-
tum showed no response (Table 1).
The leaf N : P ratio from the second experiment is
shown as a function of l in Figs 3 and 4. Platymiscium
pinnatum and S. macrophylla were slower growing species
compared with T. grandis. Variation in the nitrogen and
phosphorus availability of the soil environment, associated
with either a variation in soil water content or fertilizer
addition, caused the leaf N : P ratios to shift up or down,
with the shift caused by the treatment superimposed on the
background variation in the leaf N : P ratio associated with
the variation in l. The effect of soil water content on the
leaf N : P ratio can be seen in Fig. 3, for both unfertilized
(Fig. 3a) and fertilized (Fig. 3b) soil. Similarly, the effect of
fertilizer addition on the leaf N : P ratio can be seen in
Fig. 4, at both high (Fig. 4a) and low (Fig. 4b) soil water
contents. In general, for a given soil treatment in the second
experiment, the leaf N : P ratio showed a relationship with
l qualitatively consistent with that predicted by Eqn 8.
When data from both experiments were combined, the
leaf N : P ratio showed a positive correlation with WUE
(Fig. 5). Across the combined dataset, WUE explained 42%
of the variation in the leaf N : P ratio. The regression equa-
tion relating the two was leaf N : P = 8.28WUE + 0.26
(R2 = 0.42, P < 0.001, n = 148). The positive correlation
between the leaf N : P ratio and WUE resulted from the
contributions of both a positive correlation between leaf
nitrogen concentration and WUE (R2 = 0.15, P < 0.001,
n = 148) and a negative correlation between leaf phospho-
rus concentration and WUE (R2 = 0.24, P < 0.001,
n = 148).
(b)
(c)
(a)
Fig. 2 Leaf N : C (a), P : C (b) and N : P (c) ratios plotted as func-
tions of the relative growth rate for 13 tropical tree and liana species.
Open symbols, coniferous species; filled symbols, angiosperm tree
species; semi-filled symbols, angiosperm liana species.
774 Research
New
Phytologist
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
Discussion
The results presented in Figs 2?4 clearly demonstrate a
strong association between the leaf N : P ratio and l for the
tropical tree and liana seedlings employed in this study,
when grown in a common soil environment. The relation-
ships observed between the leaf N : P ratio and l were
qualitatively consistent with a theoretical prediction, sug-
gesting that the plant N : P ratio should increase at low l,
reach a maximum, and then decrease as l continues to
Table 1 Leaf N : C, P : C and N : P ratios for three tropical tree species grown at high or low soil water content (SWC) and with or without
added fertilizer
Treatment
Tectona grandis Swietenia macrophylla Platymiscium pinnatum
Leaf N : C Leaf P : C Leaf N : P Leaf N : C Leaf P : C Leaf N : P Leaf N : C Leaf P : C Leaf N : P
Unfertilized,
high SWC
28.5 (0.9) 3.65 (0.42) 7.9 (0.8) 30.1 (2.2) 2.54 (0.42) 12.0 (1.4) 75.4 (9.7) 4.67 (0.58) 16.2 (1.8)
Unfertilized,
low SWC
47.3 (7.2) 3.88 (0.38) 12.2 (1.7) 37.5 (1.9) 2.33 (0.28) 16.2 (1.5) 77.0 (14.9) 4.20 (1.57) 19.4 (4.6)
Fertilized,
high SWC
45.2 (5.4) 4.56 (0.24) 9.9 (1.0) 48.5 (3.1) 2.70 (0.35) 18.2 (2.6) 78.3 (12.4) 5.25 (0.97) 15.2 (2.8)
Fertilized,
low SWC
64.9 (3.3) 4.20 (0.67) 15.7 (2.3) 51.7 (3.2) 2.20 (0.39) 24.0 (3.1) 86.9 (13.1) 4.37 (0.53) 19.9 (1.5)
Values are the means for five plants in each treatment, with one standard deviation given in parentheses.
(a)
(b)
Fig. 3 Leaf N : P ratios of three tropical tree species plotted against
the relative growth rate for unfertilized (a) and fertilized (b) soil.
Open symbols, plants grown at low soil water content; filled sym-
bols, plants grown at high soil water content.
(a)
(b)
Fig. 4 Leaf N : P ratios of three tropical tree species plotted against
the relative growth rate for plants grown at high (a) and low (b) soil
water contents. Open symbols, plants grown in unfertilized soil;
filled symbols, plants grown in fertilized soil.
New
Phytologist Research 775
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
increase (A?gren, 2004). Data from our second experiment
suggest that the leaf N : P ratio can increase or decrease
depending on the soil environment, but that species-level
associations between the leaf N : P ratio and l are generally
maintained (Figs 3,4). In addition, we observed a positive
correlation between the leaf N : P ratio and WUE for the
combined dataset including all species and soil treatments,
suggesting that the relationship between these two parame-
ters may be general.
Support for the C : N : P model reviewed in the Intro-
duction section was previously presented for a temperate
tree species, Betula pendula (A?gren, 2004). In that example,
variation in l occurred within a single species as a result of
a varying nutrient supply rate. The data presented here sug-
gest that a similar relationship may also hold for multiple
tropical species growing in a homogeneous soil environ-
ment when the variation in l is mainly caused by species
identity. Thus, variation in l may partly explain the large
within-site variation among tropical tree species in leaf
N : P ratios observed in tropical rainforests (Santiago &
Wright, 2007; Townsend et al., 2007; Ha?ttenschwiler
et al., 2008).
Equations 6?8 predict the C : N : P stoichiometry as a
function of l for situations in which the nutrient or nutri-
ents concerned are limiting growth (A?gren, 2004). Thus,
Eqn 8 predicts a hump-shaped relationship between the
plant N : P ratio and l when nitrogen and phosphorus
simultaneously limit growth. It is unlikely that this assump-
tion would have been met for all species in our experiments.
For example, Fig. 2b suggests that the leaf P : C ratio tends
to decrease with increasing l for the coniferous species in
the first experiment, which suggests that phosphorus is not
limiting growth in these plants. Thus, the relationship
between the leaf P : C ratio and l appears to differ for
angiosperm vs coniferous species. Nevertheless, for plants
grown in a given soil treatment, the leaf N : P ratio still
appeared to vary as a function of l in a manner qualitatively
consistent with that predicted by Eqn 8. Additional data
will be necessary to further test the generality of this obser-
vation.
Negative correlations between the leaf N : P ratio and l
have been observed previously among some vascular plant
species (Gu?sewell, 2004; Niklas et al., 2005; Niklas, 2006;
Matzek & Vitousek, 2009). These results are consistent
with our observations for species at all but the lowest l val-
ues, which occurred in the coniferous species (Fig. 2). Data
for the angiosperm species in Figs 2?4 are still suggestive of
a hump-shaped relationship between the leaf N : P ratio
and l, but for the overwhelming majority of the range of l
observed, the relationship between the leaf N : P ratio and
l was negative. Thus, careful experimentation will be
required to determine whether the strongly hump-shaped
relationship observed in Fig. 2, when both coniferous and
angiosperm species are considered, is a general pattern
among terrestrial plants, or whether conifers behave qualita-
tively differently from angiosperms with respect to leaf
C : N : P stoichiometry. Variation between these two
groups in the leaf N : P ratio has also been observed in
large, multispecies comparisons over broad spatial scales
(McGroddy et al., 2004; Reich & Oleksyn, 2004; Wright
et al., 2005).
The only potentially N2-fixing species in our experiments
was P. pinnatum. Thus, Fig. 2 only contains data for non-
N2-fixing species. Consistent with the ecophysiology of
legumes generally (McKey, 1994), P. pinnatum had the
highest leaf N : C ratios observed in the study (Table 1). It
also had the lowest mean l value in the second experiment
(Figs 3,4), suggesting a different relationship between the
leaf N : C ratio and l than in the non-N2-fixing species.
Interestingly, however, although the leaf N : C ratio of
P. pinnatum was markedly higher than that of the other
two species in the second experiment, its leaf N : P ratio
was similar to that of S. macrophylla, a non-N2-fixing spe-
cies with a similar l value (Figs 3, 4). This observation is
consistent with data compiled from several tropical rainfor-
ests (Townsend et al., 2007), where leguminous trees tend
to have higher leaf nitrogen concentrations than do nonle-
gumes, but do not have markedly different leaf N : P ratios.
Fig. 5 Leaf N : P ratios plotted against whole-plant water-use effi-
ciency for all plants in this study, including those grown under
different soil conditions. Open symbols, coniferous species; filled
symbols, angiosperm tree species; semi-filled symbols, angiosperm
liana species.
776 Research
New
Phytologist
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
It has been suggested recently that nitrogen fixation by
legumes in tropical forests could increase phosphorus acqui-
sition by enabling the production of nitrogen-rich extracel-
lular phosphatase enzymes (Houlton et al., 2008); this is
one of a variety of possible interactions between nitrogen
and phosphorus acquisition that might contribute towards
the regulation of the N : P ratios in these species.
The slow-release fertilizer employed for the second exper-
iment had an N : P mass ratio of less than two. However,
rather than decreasing in response to fertilizer addition, the
leaf N : P ratio either increased (T. grandis and S. macro-
phylla) or showed little response (P. pinnatum), as shown in
Table 1 and Fig. 4. This probably reflected the difference
in buffering capacity of the soil for nitrate vs phosphate,
forms of nitrogen and phosphorus likely to be important
for absorption by roots. Nitrate does not adsorb to soil par-
ticles, whereas phosphate is strongly adsorbed. As a result,
nitrate released from the fertilizer would probably be in the
soil solution, available for transport to root surfaces by dif-
fusion and mass flow. By contrast, the buffer power (B) for
phosphate can be on the order of 102?103 (Barber, 1995),
where B is defined as dS ?dSl, with S being the concentration
of exchangeable, or labile, solute per unit volume of soil,
and Sl the liquid concentration of the solute in the soil solu-
tion. Thus, 102?103 lmol phosphate would have to be
added to 1 l of soil to increase the phosphate concentration
of the soil solution by 1 lmol l)1. Compared with the situ-
ation for nitrate, this indicates that the phosphate concen-
tration of the soil solution would change relatively little
with phosphate addition to the soil.
The leaf N : P ratio increased in response to declining
soil water content in all three species in the second experi-
ment (Fig. 3). This pattern can be at least partly explained
by differential changes in the mobility of nitrate vs phos-
phate in response to declining soil water content. The effec-
tive diffusion coefficient (De) for a solute in soil can be
calculated as De = Dlflh ?B, where Dl is the diffusion coeffi-
cient of the solute in free solution, fl is the impedance factor
and h is the volumetric water content of the soil (Tinker &
Nye, 2000). For an anion such as nitrate, that is generally
not adsorbed to soil particles, the quotient h ?B will tend
towards unity, such that De = Dlfl. The ratio of the effective
diffusion coefficients for nitrate and phosphate (DeN ?DeP)
can thus be described as DeN ?DeP = DlNflN ? (DlPflPh ?BP),
where subscripts ?N? and ?P? refer to nitrate and phosphate,
respectively. If fl is assumed to vary similarly as a function
of h for both anions (Barber, 1995), it cancels from the
right side of the equation. Then, to the extent that
DlN ? (DlP ?BP) can be reasonably assumed to be constant,
DeN ?DeP will vary as a function of 1 ?h. Thus, as h declines,
DeN ?DeP will increase, and the mobility of nitrate relative
to that of phosphate will increase, making nitrate relatively
more available for absorption by roots. These consider-
ations, combined with the results in Fig. 3, suggest that it
may be useful to take the variation in soil water content into
account when interpreting the variation in leaf N : P ratios
among tropical woody plants.
When the data from the two experiments in this study
were combined, we observed a positive correlation between
the leaf N : P ratio and WUE. This result is consistent with
previous observations for seedlings of a tropical pioneer
tree, Ficus insipida (Cernusak et al., 2007). If data from that
study and the present study are combined, the relationship
between the leaf N : P ratio and WUE is even stronger,
with WUE explaining 47% of the variation in the leaf
N : P ratio (R2 = 0.47, P < 0.001, n = 178). As pointed
out in the Introduction section, we suggest that this rela-
tionship results from correlations between plant nitrogen
concentration and WUE (Guehl et al., 1995; Livingston
et al., 1999; Raven et al., 2004; Ripullone et al., 2004;
Cernusak et al., 2007), and between plant transpiration and
the transport of phosphorus to root surfaces by mass flow
(Barber, 1995; Tinker & Nye, 2000; Cernusak et al., 2007;
Scholz et al., 2007; Cramer et al., 2008, 2009). The corre-
lation shown in Fig. 5 includes data from a range of species
and soil conditions, including fertilizer addition, manipula-
tion of soil water content and variable mixtures of rice husks
with soil (40% by volume in the first experiment vs 20% in
the second). Thus, the relationship between the leaf N : P
ratio and WUE may be general, but more data are needed
to test this hypothesis. If supported, the idea could have
important implications for ecosystem analysis, because it
links plant C : N : P stoichiometry with plant transpira-
tion, and therefore integrates carbon, nutrient and hydro-
logical cycles.
Although phosphorus is generally considered to be rela-
tively immobile in soils, some phosphorus-containing mole-
cules may be more amenable to transport by mass flow than
others. Soil organic phosphorus can play an important role
in the phosphorus nutrition of forests growing on strongly
weathered soils (Tiessen et al., 1994; Johnson et al., 2003;
Turner et al., 2007), which are common in the tropics. Soil
organic phosphorus occurs in a diverse range of compounds
that differ markedly in their physical and chemical behavior
in soils. For example, many simple phosphate monoesters
(e.g. sugar phosphates and mononucleotides) and phos-
phate diesters (e.g. nucleic acids and phospholipids) are
generally weakly adsorbed to soil particles (Condron et al.,
2005). Mass flow of the soil solution caused by plant tran-
spiration may therefore play an important role in transport-
ing these molecules towards root surfaces, where
extracellular phosphatase enzymes can catalyze the hydroly-
sis of ester-bonded phosphorus to release phosphate, mak-
ing it available for absorption into the root. The physical
and chemical characteristics of the soil matrix interacting
with phosphorus-containing molecules will also probably
play an important role in determining the extent of phos-
phorus transport by mass flow. Many more data are needed
New
Phytologist Research 777
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
to adequately assess the role of transpiration in the modula-
tion of phosphorus uptake in terrestrial plants in general
(Cramer et al., 2009), and this is particularly true for tropi-
cal woody plants.
We conclude that the variation in the leaf N : P ratios of
tropical tree and liana seedlings can be associated with the
variation in physiological processes (l and WUE) and in
the external soil environment (soil water content and fertil-
izer addition). This suggests both internal and external con-
trols over leaf N : P ratios of tropical trees and lianas, as
recognized previously for other terrestrial plant species
(Gu?sewell, 2004; A?gren, 2008). We observed a hump-
shaped relationship between the leaf N : P ratio and l for
seedlings grown in a common soil environment, where the
variation in l was mainly associated with species? identity.
Further experiments are needed to test the generality of this
pattern. Modification of the external soil environment
caused the leaf N : P ratio to shift, whereas the underlying
variation associated with l was generally maintained. Thus,
for the assessment of the nutrient status among sites based
on the leaf N : P ratios, it would be prudent to sample the
same species or suite of species at each site. Finally, the posi-
tive correlation observed between the leaf N : P ratio and
whole-plant WUE implies that the C : N : P stoichiometry
may be linked to hydraulic processes in terrestrial plants.
Acknowledgements
We thank Jorge Aranda, Milton Garcia, Aurelio Virgo, Lisa
Petheram, Carlos Martinez, Tania Romero and Aneth Sar-
miento for technical assistance in carrying out the experi-
ments. Lucas A. Cernusak was supported by a Tupper
Postdoctoral Fellowship from the Smithsonian Tropical
Research Institute and by an Australian Postdoctoral
Fellowship from the Australian Research Council.
References
Aerts R, Chapin FS. 2000. The mineral nutrition of wild plants revisited:
a re-evaluation of processes and patterns. Advances in Ecological Research
30: 1?67.
A?gren GI. 2004. The C : N : P stoichiometry of autotrophs ? theory and
observations. Ecology Letters 7: 185?191.
A?gren GI. 2008. Stoichiometry and nutrition of plant growth in natural
communities. Annual Review of Ecology, Evolution and Systematics 39:
153?170.
Barber SA. 1995. Soil nutrient bioavailability: a mechanistic approach. New
York, NY, USA: John Wiley & Sons.
Blackman VH. 1919. The compound interest law and plant growth.
Annals of Botany 33: 353?360.
Cernusak LA, Winter K, Aranda J, Turner BL, Marshall JD. 2007. Tran-
spiration efficiency of a tropical pioneer tree (Ficus insipida) in relation
to soil fertility. Journal of Experimental Botany 58: 3549?3566.
Cernusak LA, Winter K, Aranda J, Turner BL. 2008. Conifers, angio-
sperm trees, and lianas: growth, whole-plant water and nitrogen use effi-
ciency, and stable isotope composition (d13C and d18O) of seedlings
grown in a tropical environment. Plant Physiology 148: 642?659.
Cernusak LA, Winter K, Turner BL. 2009. Physiological and isotopic
(d13C and d18O) responses of three tropical tree species to water and
nutrient availability. Plant, Cell & Environment 32: 1441?1455.
Condron LM, Turner BL, Cade-Menun BJ 2005. The chemistry and
dynamics of soil organic phosphorus. In: Sims JT, Sharpley AN, eds.
Phosphorus: agriculture and the environment. Madison, WI, USA: ASA-
CSSA-SSSA, 87?121.
Craine JM, Morrow C, Stock WD. 2008. Nutrient concentration ratios
and co-limitation in South African grasslands. New Phytologist 179:
829?836.
Cramer MD, Hoffmann V, Verboom GA. 2008. Nutrient availability
moderates transpiration in Ehrharta calycina. New Phytologist 179:
1048?1057.
Cramer MD, Hawkins HJ, Verboom GA. 2009. The importance of nutri-
tional regulation of plant water flux. Oecologia 161: 15?24.
Dyckmans J, Flessa H. 2001. Influence of tree internal N status on uptake
and translocation of C and N in beech: a dual 13C and 15N labeling
approach. Tree Physiology 21: 395?401.
Guehl JM, Fort C, Ferhi A. 1995. Differential response of leaf conduc-
tance, carbon isotope discrimination and water use efficiency to nitrogen
deficiency in maritime pine and pedunculate oak plants. New Phytologist
131: 149?157.
Gu?sewell S. 2004. N : P ratios in terrestrial plants: variation and func-
tional significance. New Phytologist 164: 243?266.
Gu?sewell S, Koerselman M. 2002. Variation in nitrogen and phosphorus
concentrations of wetland plants. Perspectives in Plant Ecology, Evolution
and Systematics 5: 37?61.
Ha?ttenschwiler S, Aeschlimann B, Couteaux MM, Roy J, Bonal D. 2008.
High variation in foliage and leaf litter chemistry among 45 tree species
of a neotropical rainforest community. New Phytologist 179: 165?175.
Houlton BZ, Wang YP, Vitousek PM, Field CB. 2008. A unifying frame-
work for dinitrogen fixation in the terrestrial biosphere. Nature 454:
327?330.
Johnson AH, Frizano J, Vann DR. 2003. Biogeochemical implications of
labile phosphorus in forest soils determined by the Hedley fractionation
procedure. Oecologia 135: 487?499.
Kerkhoff AJ, Fagan WF, Elser JJ, Enquist BJ. 2006. Phylogenetic and
growth form variation in the scaling of nitrogen and phosphorus in the
seed plants. American Naturalist 168: E103?E122.
Kitayama K. 2005. Comment on ??Ecosystem properties and forest decline
in contrasting long-term chronosequences??. Science 308: 633.
Koerselman W, Meuleman AFM. 1996. The vegetation N : P ratio: a new
tool to detect the nature of nutrient limitation. Journal of Applied Ecology
33: 1441?1450.
Livingston NJ, Guy RD, Sun ZJ, Ethier GJ. 1999. The effects of nitrogen
stress on the stable carbon isotope composition, productivity and water
use efficiency of white spruce (Picea glauca (Moench) Voss) seedlings.
Plant, Cell & Environment 22: 281?289.
Matzek V, Vitousek PM. 2009. N : P stoichiometry and protein : RNA
ratios in vascular plants: an evaluation of the growth-rate hypothesis.
Ecology Letters 12: 765?771.
McGroddy ME, Daufresne T, Hedin LO. 2004. Scaling of C : N : P stoi-
chiometry in forests worldwide: implications of terrestrial redfield-type
ratios. Ecology 85: 2390?2401.
McKey DM. 1994. Legumes and nitrogen: the evolutionary ecology of a
nitrogen-demanding lifestyle. In: Sprent JI, McKey DM, eds. Advances
in legume systematics 5: the nitrogen factor. Kew, UK: The Royal Botanic
Gardens, 211?228.
Niklas KJ. 2006. Plant allometry, leaf nitrogen and phosphorus stoichiom-
etry, and interspecific trends in annual growth rates. Annals of Botany 97:
155?163.
Niklas KJ, Owens T, Reich PB, Cobb ED. 2005. Nitrogen ? phosphorus
leaf stoichiometry and the scaling of plant growth. Ecology Letters 8:
636?642.
778 Research
New
Phytologist
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org
Raven JA, Handley LL, Wollenweber B. 2004. Plant nutrition and water
use efficiency. In: Bacon MA, ed. Water use efficiency in plant biology.
Oxford, UK: Blackwell Publishing Ltd, 171?197.
Reich PB, Oleksyn J. 2004. Global patterns of plant leaf N and P in rela-
tion to temperature and latitude. Proceedings of the National Academy of
Sciences, USA 101: 11001?11006.
Ripullone F, Lauteri M, Grassi G, Amato M, Borghetti M. 2004. Varia-
tion in nitrogen supply changes water-use efficiency of Pseudotsuga men-
ziesii and Populus ? euroamericana; a comparison of three approaches to
determine water-use efficiency. Tree Physiology 24: 671?679.
Santiago LS, Wright SJ. 2007. Leaf functional traits of tropical forest
plants in relation to growth form. Functional Ecology 21: 19?27.
Scholz FG, Bucci SJ, Goldstein G,Meinzer FC, Franco AC,Miralles-
Wilhelm F. 2007. Removal of nutrient limitations by long-term fertilization
decreases nocturnal water loss in savanna trees. Tree Physiology 27: 551?559.
Sterner RW, Elser JJ. 2002. Ecological stoichiometry: the biology of elements
from molecules to the biosphere. Princeton, NJ, USA: Princeton University
Press.
Tessier JT, Raynal DJ. 2003. Use of nitrogen to phosphorus ratios in plant
tissue as an indicator of nutrient limitation and nitrogen saturation.
Journal of Applied Ecology 40: 523?534.
Tiessen H, Cuevas E, Chacon P. 1994. The role of soil organic matter in
sustaining soil fertility. Nature 371: 783?785.
Tinker PD, Nye PH. 2000. Solute movement in the rhizosphere. New York,
NY, USA: Oxford University Press.
Townsend AR, Cleveland CC, Asner GP, Bustamante MMC. 2007.
Controls over foliar N : P ratios in tropical rain forests. Ecology 88:
107?118.
Turner BL, Condron LM, Richardson SJ, Peltzer DA, Allison VJ. 2007.
Soil organic phosphorus transformations during pedogenesis. Ecosystems
10: 1166?1181.
Wright IJ, Reich PB, Cornelissen JHC, Falster DS, Garnier E, Hikosaka
K, Lamont BB, Lee W, Oleksyn J, Osada N et al. 2005. Assessing the
generality of global leaf trait relationships. New Phytologist 166: 485?
496.
New
Phytologist Research 779
 The Authors (2009)
Journal compilation  New Phytologist (2009)
New Phytologist (2010) 185: 770?779
www.newphytologist.org