Trees
DOI 10.1007/s00468-017-1589-4
ORIGINAL ARTICLE
Within-crown acclimation of leaf-level physiological
and morphological parameters in young loblolly pine stands
Marco A. Yáñez1 • Thomas R. Fox2 • John R. Seiler2 • Fernando Guerra1
Ricardo M. Baettig1 • Francisco Zamudio1 • Javier E. Gyenge3
Received: 10 January 2017 / Accepted: 21 July 2017
Ó Springer-Verlag GmbH Germany 2017
Abstract
Key message Light plays a minor role in the within-
crown acclimation of physiological and morphological
parameters in small loblolly pine trees.
Abstract Whether the plasticity of foliage within the crown
is driven by light or height is still not well known, espe-
cially for small trees. The present study was conducted in
two loblolly pine (Pinus taeda L.) stands growing on two
sites, during the third year after planting. The following
traits were investigated in the lower and upper crowns of
the same individuals twice per season (spring, summer, and
autumn): photosynthesis (Asat), stomatal conductance (gs),
intrinsic water use efficiency (WUEint), nitrogen concen-
tration ***(N), specific leaf area (SLA), photosynthetic
nitrogen use efficiency (PNUE), fascicle diameter, and
needles per fascicle. The trees were open-grown at the
beginning of the study; thus, any vertical gradient of the
traits was not assumed to be driven by light. Overall, Asat,
gs, PNUE, and SLA tended to be higher in the lower than in
the upper crown, whereas N, fascicle diameter, and needles
per fascicle were higher in the upper than in the lower
crown. However, although all traits were affected by crown
Communicated by G. Wieser.
& Marco A. Yáñez
marcoyanez@utalca.cl
1
Centro Tecnológico del Álamo (Poplar Technology Center),
Universidad de Talca, 2 Norte 685, P.O. Box 747, Talca,
Chile
2 shall and Monserud 2003; Shiraki et al. 2016; Woodruff
Department of Forest Resources and Environmental
Conservation, Virginia Tech, 228 Cheatham Hall (0324),
Blacksburg, VA 2406, USA
3
AER Tandil, Gral. Rodrı́guez 370, 7000 Tandil,
Buenos Aires, Argentina
•
position, the magnitude of this effect differed by season.
The fact that crown position affected most of the traits in
spring and summer, but not in autumn, suggest that height
might be the main driver of foliage acclimation. However,
considering that crown position affected SLA in autumn
but not the photosynthetic rate or N concentration, it is
possible that foliar acclimation of these traits was mainly
driven by light in this season.
Keywords Loblolly pine  Gas exchange  Foliage
morphology  Foliage acclimation
Introduction
Accounting for the variability of leaf-level physiological
parameters within-tree crowns using multi-layered process-
based models has improved estimates of carbon assimila-
tion at the stand level (De Pury and Farquhar 1997; Ells-
worth and Reich 1993; Wang et al. 2016). These models
account for the within-crown vertical variation (spatial
variation) of certain functional traits and provide infor-
mation on how these traits are related to photosynthetic
capacity. Understanding how crown acclimates at the
species level and at different stages of stand development is
essential for further improvements of these models for use
in forest plantations (Kenzo et al. 2015). There is a con-
tinuous debate about the degree to which height and light
drive these vertical gradients within the crown (Ambrose
et al. 2009; Cavaleri et al. 2010; Kenzo et al. 2015; Mar-
et al. 2004; Zhang et al. 2012b). Most of the studies on this
topic have been conducted using tall trees, in which height
and light gradients are more marked. However, less
understanding exists about how these factors might affect
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the physiological and morphological plasticity of small
trees.
Some studies have addressed the within-crown varia-
tion of traits such as Asat, gs, foliar N partitioning, and
SLA as influenced by irradiance. Ambient light within the
crown can vary due to self-shading (within the crown)
and shading from adjacent trees in the stand (Wyka et al.
2012). Light is absorbed as it passes through the canopy,
which decreases the photosynthetic capacity in the lower
crown (Lewis et al. 2000). As stands develop and crowns
expand, light heterogeneity increases within the crown
(Chmura and Tjoelker 2008). Typically, the fully exposed
foliage in the upper canopy acclimates to high levels of
irradiance and has a higher N concentration, more pal-
isade tissue and mesophyll cells, and a higher number of
stomata per unit of leaf area, but lower SLA, light-har-
vesting efficiency, and chlorophyll content relative to the
more shaded leaves in the lower crown (Gough et al.
2004; Kazda et al. 2000; Maier et al. 2002; Niinemets
2007; Niinemets et al. 2015; Palardy 2008; Tang et al.
2003; Zhang and Allen 1996). Nitrogen and SLA are key
functional traits used in process-based models. N in
foliage is mostly present in the form of proteins and
metabolites involved in photosynthesis. Therefore, within
the crown, N is preferentially allocated to regions where
carbon assimilation is higher, which generally coincide
with higher irradiance levels (Buckley et al. 2013). This
maximizes the return in carbon assimilation per unit cost
of N. Another mechanism that leaves use to acclimate to
differences in irradiance is the alteration of SLA, which
may have an even greater impact on carbon gain than the
re-distribution of N (Evans and Poorter 2001).
Recent studies have shown that height is more strongly
related to the vertical gradient of functional traits than light
(Cavaleri et al. 2010; Ishii et al., 2008; Meinzer et al. 2008;
Shiraki et al. 2016; Woodruff et al. 2004). Therefore, it
might be questionable to use the terms ‘‘sun leaves’’ and
‘‘shade leaves’’ when referring to the upper and lower
foliage, respectively (Oldham et al. 2010). The position of
the foliage within the crown determines the length of the
water column and the magnitude of the resistance to water
flow (Marshall and Monserud 2003). The increase in xylem
water potential in response to gravity is 0.01 MPa m-1
(Woodruff et al. 2004), which alone may explain reduc-
tions in turgor pressure, water potential, and leaf dry mass
per area (LMA, inverse of SLA) with height (Cavaleri et al.
2010; Meinzer et al. 2008; Zhang et al. 2012a, b).
Nonetheless, studying open-grown ponderosa pine (Pinus
ponderosa Laws.), Hubbard et al. (2002) found that height
alone could not explain the vertical gradient in leaf gas
exchange or leaf-specific hydraulic conductance. However,
Cavaleri et al. (2010) found that height was more strongly
related to LMA than light in tropical species, and the effect
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of light was constrained only to a specific level of diffuse
transmittance.
Height and irradiance are interdependent factors that
affect leaf water potential (Zhang et al. 2012b). Thus, leaf
water stress may be the key factor that affects leaf accli-
mation (Zhang et al. 2012a). The acclimation of functional
traits within the crown is dynamic and changes in response
to environmental drivers that vary during the year such as
the vapor pressure deficit (VPD), temperature, soil mois-
ture content, and light intensity. Consequently, physiolog-
ical and morphological traits acclimate during the growing
season as the leaves elongate. Vertical gradients of leaf
water potential are expected to be steeper in drier months,
which may induce seasonal acclimation. Similarly, the
light intensity and quality varies seasonally affecting the
photosynthetic capacity. Although the seasonal variation in
gas-exchange parameters has been studied in different
Mediterranean species (Broeckx et al. 2014; Limousin
et al. 2010; Misson et al. 2010) and pine species (Aspinwall
et al. 2011; Tyree et al. 2009), little is known about the
seasonal variation of these parameters when different
crown layers are compared.
In small, open-grown trees, hydraulic tension and the
light gradient might be insufficient to drive within-crown
acclimation, which remains unresolved. This study focuses
on loblolly pine, which exhibits considerable phenotypic
plasticity in response to environmental conditions and
management practices (Fox et al. 2007). We investigated
the seasonal and within-crown variations of gas exchange,
N concentration, PNUE, and morphological parameters of
foliage in the lower and upper crowns of 3-year-old
loblolly pine stands at two sites in the United States. We
hypothesized that in young stands prior to canopy closure,
i.e., not affected by light gradients in the outer crown, the
within-crown differences in physiological and morpho-
logical attributes are not driven by light.
Methods
Study sites, tree selection, and sampling
within the crown
Two field trials established in 2009 in the southern United
States, and were part of a larger project designed to assess
the interactions between loblolly pine genotypes, silvicul-
tural intensity, planting density, and site. The two sites
were included in this study, because they represent con-
trasting areas where loblolly pine is planted, and the pre-
vious studies have shown different growth and
physiological responses of the species (Yáñez et al.
2015, 2017). Details of the study sites, experimental
design, and management were presented by Yáñez et al.
Trees

(2015). Briefly, one trial was located on the North Carolina

Coastal Plain (NC), at the Bladen Lakes State Forest
(34°490 49.6300 N, 78°350 18.5200 W), while the other trial was
located on the Virginia Piedmont (VA) at the Reynolds
Homestead Forest Resources Research Center
(36°380 35.3200 N, 80°090 18.8400 W). The soil at the VA site
was a well-drained Fairview Series (fine, kaolinitic, mesic
Typic Kanhapludults), whereas the soil at the NC site was a
poorly drained Rains series (fine-loamy, siliceous, semi-
active, thermic Typic Paleaquults). The annual average
temperature and precipitation at VA is 13 °C and
1159.3 mm, respectively. The annual average temperature
and precipitation at NC is 16.9 °C and 1170.7 mm,
respectively (obtained from NOAA online weather data
http://sercc.com/nowdata.html). The present study was
carried out in 2012 (third growing season since planting).
Figure 1 shows the monthly patterns for temperature and
precipitation in 2012.
This study was conducted on a subsample of trees
selected at each site (36 trees per site). The sampled trees
were planted at an initial density of 1235 trees ha-1
(3.66 m between rows and 2.21 m within rows). We
assumed that at this planting density and stage of stand
development (the trees were open-grown), intraspecific
competition for light would be minimal. Thus, the foliage
of both the upper and lower crowns was exposed to similar
irradiance during the day. Therefore, we discarded any
vertical gradient of irradiance at the crown positions sam-
pled. Figure 2 shows a view of the stands during the study.
Fig. 2 View of the sites on the Virginia Piedmont (upper panel) and
North Carolina Coastal Plain (lower panel) in spring during the third
50 250 growing season
Precipitation
Max Temperature NC
40 Average Temperature
Min Temperature 200
Precipitation (mm)

The height of the selected trees ranged from 1.68 to 3.35 m

Temperature (°C)

30
150 at the site in NC and from 2.62 to 4.42 m at the site in VA.
20 The selected trees were free from disease, mechanical
100
10 damage, forking, and dieback.
50
0
Leaf physiological and morphological parameters
-10 0
50 250
VA Leaf-level physiology was repeatedly measured on each
40 selected tree. From March 2012 to November 2012, leaf-
200
Precipitation (mm)
Temperature (°C)

30 level gas exchange was measured every 6–8 weeks (a total

150 of 6 dates per site in the months of March, May, June,
20
August, October, and November). The trees at both sites
100
10 were measured within a 2-week period for each sampling
50 date. The measurements were conducted on branches that
0
were fully exposed to the sun and were randomly selected
-10 0 in the lower and upper third of the crown. A composite
r r t
Jan Feb Ma Ap May Jun Jul Aug Sep Oc Nov Dec sample of three fascicles per crown position was sampled,
and each fascicle was collected from different branches.
Fig. 1 Monthly precipitation and average maximum, minimum, and
mean air temperature for the sites on the North Carolina Coastal Plain For proper selection of sun-exposed fascicles at both crown
(lower panel) and Virginia Piedmont (upper panel) in 2012 positions, the measurements through June were made on

123

the first flush of 1-year-old foliage; subsequently, the first
fully expanded flush of current year foliage was measured.
We measured the light-saturated photosynthetic rate (Asat,
lmol m-2 s-1, at photosynthetically active radiation
(PAR) of 1600 lmol m-2 s-1) and stomatal conductance
(gs, mmol m-2 s-1) between 9 a.m. and 3 p.m. using a
portable photosynthesis system (LI-6400, LiCor Inc., Lin-
coln, NE, USA). The derived intrinsic water use efficiency
(WUEint) was calculated as Asat/gs. The fascicles collected
from each crown position were placed in the 3 9 2 cm
chamber immediately after detachment (\30 s). The
chamber air temperature and relative humidity were
maintained at values similar to ambient conditions. For this
reason, during the study period, we varied the block tem-
perature in the chamber from 5 to 32 °C at VA and from 9
to 34 °C at NC. The carbon dioxide concentration was set
to 385 lmol mol-1. Additional measurements of leaf
morphology (fascicle diameter and number of needles per
fascicle) were used to calculate leaf area according to the
formula specified by Ginn et al. (1991): leaf area per fas-
cicle = 3.14159(d)(l) ? (n)(d)(l), where d is the fascicle
diameter, l is the needle length, and n is the number of
needles per fascicle. In addition, the mean number of
needles per fascicle was calculated for each tree, position,
and sampling date.
Beginning with the May sampling date, the fascicles
collected for physiological assessment were oven dried,
weighed, and ball-milled to a fine powder. Nitrogen con-
centration (N) was measured by dry combustion using a
VARIO MAX CN Analyzer (Elementar, Hanau, Germany).
Specific leaf area (SLA, cm2 g-1) was determined for the
foliage samples collected in August, October, and
November, and was calculated as the sum of the leaf area
for the three fascicle samples divided by the dry weight.
Subsequently, nitrogen use efficiency (PNUE,
lmol mol-1 s-1) was determined as PNUE = (Asat 9
SLA) 9 28/N, where 28 is the conversion factor of grams
to mole for N.
Statistical analysis
Analysis of variance per site was performed using PROC
MIXED in SAS version 9.4 (SAS Institute, Cary, NC,
USA). Individual tree information for both crown positions
was averaged by season; therefore, March–May, June–
August, and October–November corresponded to spring,
summer, and autumn, respectively. To account for both
spatial and seasonal variations of each trait, a model
including the explanatory variables season, position (lower
and upper crowns), and the interaction among these factors
was applied to each trait. The model did not include ran-
dom factors. Because the measurements were conducted on
the same individuals in different seasons (possible
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temporal covariance) and at two positions within the crown
(possible within-crown spatial covariance), the variance–
covariance structures were modeled as doubly repeated
measurements, using the unstructured matrix for both
temporal and spatial covariances. Preliminary analyses
using the Akaike (AIC) and Bayesian (BIC) information
criteria showed that these were the best models to account
for the different variance–covariance structures at each
site. Differences were considered significant at an alpha
level of 0.05.
Results
The statistical approach used in this study enabled us to
detect small differences in the physiological and morpho-
logical traits assessed. Table 1 shows the means per crown
position across the growing season and the analysis of
variance results obtained for each physiological and mor-
phological trait per study site. At this stage of stand
development, and at both sites, all traits except WUEint at
VA were affected by crown position either as a main effect
or as an effect that differed by season (season by crown
position interaction) (P \ 0.05). Figure 3 shows the sea-
sonal trend of each physiological and morphological trait
per site and crown position. As can be observed, when the
interaction term was significant, it was codirectional except
for WUEint at NC. At both sites, the seasonal patterns for
Asat and gs were similar. These variables tended to increase
in summer (Fig. 3a, b). At NC, Asat and gs were signifi-
cantly higher in the lower than in the upper crown only in
spring, whereas these differences persisted until summer at
VA. At both sites, the higher gs during the summer months,
more than Asat, decreased WUEint in this season (Fig. 3c).
At NC, the differences in WUEint between crown positions
were small and also differed by date (Fig. 3c). At this site,
WUEint was higher in the lower than in the upper crown in
spring and then shifted in summer and autumn, whereas at
VA, no effect of crown position was detected on WUEint. N
concentration was higher in the upper than in the lower
crown, and these differences were greater at NC than at
VA. At both sites, N in the lower crown was maintained at
a fairly stable concentration (1.2%) (Fig. 3d). N exhibited a
slight increase from spring to summer followed by a
decrease in autumn. At VA, no differences in N between
crown positions were detected in autumn. At both sites,
PNUE was significantly higher in the lower than in the
upper crown in both summer and autumn, whereas SLA
was significantly higher in the lower than in the upper
crown only in autumn (Fig. 3e, f). At both sites, the
number of needles per fascicle and fascicle diameter was
consistently higher in the upper than in lower crown during
the growing season (Fig. 3g, h).
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Table 1 Upper and lower crown least-square means and P values nitrogen use efficiency (PNUE), specific leaf area (SLA), number of
based on the analysis of variance results for light-saturated photo- needles per fascicle, and fascicle diameter per site (NC North
synthetic rate (Asat), stomatal conductance (gs), intrinsic water use Carolina Coastal Plain, VA Virginia Piedmont)
efficiency (WUEint), nitrogen concentration (N), photosynthetic
Site Crown Asat gs WUEint N PNUE SLA Needles per Fascicle
position fascicle diameter

NC Lower 5.21 (0.08) 114.7 (2.1) 0.048 (0.0006) 1.24 (0.01) 165.4 (3.5) 150.8 (2.1) 3.04 (0.01) 1.23 (0.01)
Upper 5.11 (0.09) 111.9 (2.1) 0.047 (0.0006) 1.46 (0.01) 138.5 (3.5) 141.6 (2.0) 3.39 (0.04) 1.47 (0.01)
VA Lower 5.34 (0.07) 95.2 (2.0) 0.058 (0.0007) 1.29 (0.01) 168.9 (4.0) 151.2 (2.4) 3.04 (0.01) 1.16 (0.01)
Upper 5.02 (0.08) 90.6 (2.3) 0.057 (0.0007) 1.35 (0.01) 143.3 (3.9) 140.1 (2.2) 3.44 (0.04) 1.41 (0.01)
Source of variation
NC Season \0.0001 \0.0001 \0.0001 \0.0001 \0.0001 \0.0001 0.1157 0.0008
Pos. 0.2470 0.1367 0.4626 \0.0001 \0.0001 \0.0001 \0.0001 \0.0001
Season 9 Pos. \0.0001 0.0397 \0.0001 \0.0001 0.5098 0.0210 0.0730 0.0003
VA Season \0.0001 \0.0001 \0.0001 \0.0001 \.3424 0.5450 0.0573 0.0020
Pos. 0.0003 0.0216 0.5664 0.0002 \0.0001 0.0017 \0.0001 \0.0001
Season 9 Pos. 0.0038 0.0020 0.2595 0.0001 0.0259 0.0925 0.4859 0.0061

Values in parentheses are standard errors

The effects are season and crown position (Pos.)

Discussion For instance, more N is allocated to leaves grown under

Tree canopies permanently acclimate to changing temporal
and spatial conditions, which influences physiological
processes (Sellin and Kupper 2004). In this study, the
seasonal variation in physiological and morphological
parameters at two crown positions in young loblolly pine
stands located at two contrasting sites was examined under
the assumptions that these trees were open-grown and there
was no intense intraspecific competition for light. Under
these conditions, we hypothesized that within-crown dif-
ferences in physiological and morphological attributes
during the early stages of stand development are not driven
by light. Our major findings partially support this hypoth-
esis and also suggest that height might play a more
important role in the foliage acclimation of small trees
compared with light.
There is controversy surrounding the extent to which
light intensity or height drives foliage acclimation within
the crown (Cavaleri et al. 2010; Kenzo et al. 2015; Mar-
shall and Monserud 2003; Shiraki et al. 2016; Woodruff
et al. 2004; Zhang et al. 2012b). Some studies highlight
how the acclimation of leaf physiological processes and
morphology within the crown is driven specifically by light
(Larbi et al. 2015; Le Roux et al. 2001; Niinemets et al.
2015). By definition, photosynthesis is the conversion of
light energy into carbon compounds from carbon dioxide
and water, and the dependence of this process on irradiance
is typically analyzed using A/light curves (Palardy 2008).
Moreover, irradiance induces changes in key traits that
determine the carboxylation efficiency, such as N and SLA.
high light where carbon assimilation is higher (Buckley
et al. 2013). SLA is also related to irradiance as it describes
the efficiency of light capture by leaves relative to biomass
construction (Marshall and Monserud 2003). In our study,
the within-crown differences in the physiological and
morphological traits are likely not attributable to light
gradients as the main cause due to the following reasons:
(1) tree height ranged from 1.6 to 4.4 m at the beginning of
the growing season (spring), and the trees were open-
grown, (2) according to the within-tree sampling strategy,
only foliage that was fully exposed to the sun was selected
from each crown position, and (3) for the gas-exchange
measurements, PAR was controlled within the chamber. If
any light gradient affected within-crown acclimation, we
expected this to occur toward the end of the growing sea-
son, facilitated by a lower solar angle and shading by taller
trees, which was not the case. By contrast, at both sites, the
gas-exchange parameters were most sensitive to the effect
of crown position in spring. Similarly, traits such as fas-
cicle diameter and number of needles per fascicle are
determined in spring when the foliage develops. Our results
disagree with the findings of McGarvey et al. (2004), who
conducted a similar study. These authors did not find dif-
ferences in photosynthetic rates between canopy layers,
although their trees were taller than those in the current
study and were planted at a higher planting density. Sim-
ilarly, Hubbard et al. (2002) did not find differences in leaf
gas-exchange or leaf-specific hydraulic conductance
between the lower and upper crowns of four open-grown
ponderosa pine (P. ponderosa Laws.) trees. It is possible

123

(a)
µ
(b)
(c)
µ
(d)
Fig. 3 Least-squares means for loblolly pine saturated photosynthetic
rate (Asat) (a), stomatal conductance (gs) (b), intrinsic water use
efficiency (WUIint) (c), nitrogen concentration (N) (d), photosynthetic
nitrogen use efficiency (PNUE) (e), specific leaf area (SLA) (f),
number of needles per fascicle (g), and fascicle diameter (h) per site
that the lower number of trees sampled in those studies
compared to ours might have influenced the statistical
power required to detect small differences as was found in
our study. Nevertheless, the results presented by McGarvey
et al. (2004) suggest that N and SLA are more sensitive
than the photosynthetic rate to small gradients in
irradiance.
The effect of crown position on N concentration also
differed by season and site. Overall, the N concentration
was higher in the upper crown than in the lower. At both
sites, N in the lower crown was maintained at a fairly
stable concentration, whereas the trend in the upper crown
followed a seasonal pattern. N in the upper crown increased
slightly from spring to summer and then decreased during
autumn. At VA, no differences in N between crown posi-
tions were detected in autumn. The decrease in N con-
centration in the upper crown in this season may be
associated with nutrient remobilization. We speculated that
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(e)
µ
(f)
(g)
(h)
(NC North Carolina Coastal Plain, VA Virginia Piedmont), crown
position (lower and upper crowns), and season (spring, summer, and
autumn). Asterisk denotes significant differences at an alpha level of
0.05. The sample size was 36 trees per site
remobilization should begin first in the upper crown, as
most of the foliar N is in the form of proteins involved in
carboxylation, a process that should be down-regulated by
lower temperatures and incident light in autumn. Alterna-
tively, the higher investment of N in the light-harvesting
complexes in the lower crown might have allowed better
acclimation of this crown layer to function under higher
exposure to diffuse light, which is more typical in autumn
than in summer. These assertions are supported by the
results reported by Tarvainen et al. (2015) for Norway
spruce. These authors found that the photosynthetic gra-
dient along tree height in the growing season decreased in
the autumn and winter months, mainly because of a
decrease in photosynthesis in the upper crown relative to
the levels measured in the lower crown. In general, the N
concentration of current loblolly pine foliage is high in
spring and decreases toward autumn, and there is a small
increase that coincides with 1-year-old foliage senescence
Trees
(Munger et al. 2003; Zhang and Allen 1996). This trend
partially agrees with our results, although the seasonal
pattern of foliar N will vary by site (Gough et al. 2004;
Munger et al. 2003). At both sites, PNUE was consistently
higher in the lower than in the upper crown, despite that at
VA, neither Asat nor N was affected by crown position in
autumn. In this season, the vertical pattern of PNUE is
likely a direct consequence of vertical variation in SLA.
These results differ from those reported by Chmura and
Tjoelker (2008), who observed a gradient of gas-exchange
and morphological (N and SLA) parameters in young
loblolly pine (during the fourth and fifth growing seasons);
however, a PNUE gradient was not observed. The differ-
ences among these studies may be attributed to the dif-
ferent phases of stand development.
In general, a little information exists on the seasonal
variation in loblolly pine SLA. In the present study, the
effect of crown position was detected in autumn. SLA was
140 and 151 cm2 g-1 for the upper and lower crowns,
respectively (9% higher in the lower crown). These values
are similar to those reported by Tyree et al. (2009) but are
lower than the SLA reported by McGarvey et al. (2004)
(155–187 cm2 g-1 for the lower and upper crowns,
respectively). In closed canopies, the phenotypic plasticity
of loblolly pine is primary expressed as an increase in SLA
from the top to the bottom of the canopy (Ellsworth and
Reich 1993; Gratani 2014). Hence, the fact that SLA was
similar at both crown positions in summer provides evi-
dence that the light intensity was similar at both crown
positions. However, the lower irradiance and zenith in the
autumn months could explain the differences and decrease
in SLA in autumn at both crown positions and why the
decrease was greater in the upper crown. This might be
associated with a higher accumulation of non-structural
compounds (Poorter et al. 2009) because of a lower growth
rate and less demand from other parts of the tree. More-
over, acclimation of trees to diffuse light may likely induce
a lower SLA, as has been shown for other plant species (Li
et al. 2014; Li and Yang 2015). Interestingly, both the
fascicle diameter and number of needles per fascicle were
higher in the upper than in the lower crown, which may be
associated with higher gas-exchange rates on a per fascicle
basis in the upper than in the lower crown (Ingwers et al.
2016). Some studies indicate that the number of needles
per fascicle might be an adaptation to environmental
moisture (Cole et al. 2008; Ingwers et al. 2016), which was
not measured in our study.
Some studies support the hypothesis that the vertical
acclimation of physiological and morphological traits is
driven by height as it constrains vertical water transport,
which induces osmotic and turgor pressure adjustments at
the leaf level (Cavaleri et al. 2010; Ishii et al. 2008;
Meinzer et al. 2008; Oldham et al. 2010; Woodruff et al.
2004; Shiraki et al. 2016; Zhang et al. 2012b). In general,
these types of studies typically involved tall trees and
only one sampling point in time, but does this hypothesis
apply to small trees? Zhang et al. (2012a) found differ-
ences in leaf water potential and several morphological
traits attributed to height in small Robinia pseudoacacia
trees. The fact that crown position affected most of the
traits in spring and summer, but not in autumn, suggests
that height might be the main driver of foliage acclima-
tion. However, considering that crown position affected
SLA in autumn but not the photosynthetic rate or N
concentration, it is possible that foliar acclimation of
these traits was mainly driven by light in this season as
discussed above. Our study showed that the sampling time
may produce different results. In view of the results,
within-crown acclimation may not be explained by only
light or height. The effect of both of these factors likely
differs by season as soil moisture, nutrients, and the
proportion of incident and diffuse light change during the
year.
Our results provide insights into the seasonal and
within-crown variation of leaf-level physiological and
morphological traits of young loblolly pine stands (trees
shorter than 5 m). We found a small but significant effect
of crown position on these traits; however, the magnitude
of this effect differed by season. These results partially
support the hypothesis that within-crown acclimation of
young trees is not driven by light. In this study, the within-
crown acclimation observed during spring and summer
might be better explained by a greater effect of height than
light, whereas the autumn acclimation of some traits might
be more strongly attributed to the effect of light.
Author contribution statement MY is the main author of the
manuscript, leaded the research, data analysis, and writing. TF par-
ticipated in the design of the field test, and helps in the sampling
strategy for this specific study. He also contributed in the edition of
the whole manuscript. He leads the Forest Productivity Cooperative
and the Center for Advance Forestry System; both organizations
provided funds for the field work. JS helped in the sampling strategy
for this specific study and did some field work for collecting the data.
He provided the equipment involved in the study. He also contributed
in the edition of the whole manuscript. FG contributed in the edition
of the whole manuscript as well as suggesting the approaches for the
statistical analysis. RB contributed in the writing of the whole
manuscript. FZ contributed in the edition of the whole manuscript as
well as in the statistical analysis. JG contributed in the edition of the
whole manuscript.
Acknowledgements This research was funded through the NSF
Center for Advanced Forestry Systems (CAFS) and the Forest Pro-
ductivity Cooperative (FPC). Funding for this work was also provided
in part by the Virginia Agricultural Experiment Station and the Pro-
gram McIntire Stennis of the National Institute of Food and Agri-
culture, US Department of Agriculture. We also thank John Peterson
and Samuel Frye for their help in the field work, Timothy Albaugh for
providing the meteorological data.
123

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Ambrose AR, Sillett SC, Dawson TE (2009) Effects of tree height on
branch hydraulics, leaf structure and gas exchange in California
redwoods. Plant Cell Environ 32:743–757
Aspinwall MJ, King JS, McKeand SE, Domec JC (2011) Leaf-level
gas-exchange uniformity and photosynthetic capacity among
loblolly pine (Pinus taeda L.) genotypes of contrasting inherent
genetic variation. Tree Physiol 31:78–91
Broeckx LS, Fichot R, Verlinden MS, Ceulemans R (2014) Seasonal
variations in photosynthesis, intrinsic water-use efficiency and
stable isotope composition of poplar leaves in a short-rotation
plantation. Tree Physiol 34:701–715
Buckley TN, Cescatti A, Farquhar GD (2013) What does optimization
theory actually predict about crown profiles of photosynthetic
capacity when models incorporate greater realism? Plant Cell
Environ 36:1547–1563
Cavaleri MA, Oberbauer SF, Clark DB, Clark DA, Ryan MG (2010)
Height is more important than light in determining leaf
morphology in a tropical forest. Ecology 91:1730–1739
Chmura DJ, Tjoelker MG (2008) Leaf traits in relation to crown
development, light interception and growth of elite families of
loblolly and slash pine. Tree Physiol 28:729–742
Cole KL, Fisher J, Arundel ST, Cannella J, Swift S (2008)
Geographical and climatic limits of needle types of one- and
two-needled pinyon pines. J Biogeogr 35:257–269
De Pury DGG, Farquhar GD (1997) Simple scaling of photosynthesis
from leaves to canopies without the errors of big-leaf models.
Plant Cell Environ 20:537–557
Ellsworth DS, Reich PB (1993) Canopy structure and vertical patterns
of photosynthesis and related leaf traits in a deciduous forest.
Oecologia 96:169–178
Evans JR, Poorter H (2001) Photosynthetic acclimation of plants to
growth irradiance: the relative importance of specific leaf area
and nitrogen partitioning in maximizing carbon gain. Plant Cell
Environ 24:755–767
Fox TR, Jokela EJ, Allen HL (2007) The development of pine
plantation silviculture in the southern United States. J Forest
105:337–347
Ginn SE, Seiler JR, Cazell BH, Kreh RE (1991) Physiological and
growth responses of eight-year-old loblolly pine stands to
thinning. For Sci 37:1030–1040
Gough CM, Seiler JR, Johnsen KH, Sampson DA (2004) Seasonal
photosynthesis in fertilized and nonfertilized loblolly pine. For
Sci 50:1–9
Gratani L (2014) Plant phenotypic plasticity in response to environ-
mental factors. Adv Bot 2014:1–17
Hubbard RM, Bond BJ, Senock RS, Ryan MG (2002) Effects of
branch height on leaf gas exchange, branch hydraulic conduc-
tance and branch sap flux in open-grown ponderosa pine. Tree
Physiol 22:575–581
Ingwers MW, Urban J, McGuire MA, Bhuiyan RA, Teskey RO
(2016) Physiological attributes of three- and four-needle fasci-
cles of loblolly pine (Pinus taeda L.). Trees 30:1923–1933
Ishii HT, Jennings GM, Sillett SC, Koch GW (2008) Hydrostatic
constraints on morphological exploitation of light in tall Sequoia
sempervirens trees. Oecologia 156:751–763
Kazda M, Salzer J, Reiter I (2000) Photosynthetic capacity in relation
to nitrogen in the canopy of a Quercus robur, Fraxinus
123
Trees
angustifolia and Tilia cordata flood plain forest. Tree Physiol
20:1029–1037
Kenzo T, Inoue Y, Yoshimura M, Yamashita M, Tanaka-Oda A, Ichie
T (2015) Height-related changes in leaf photosynthetic traits in
diverse Bornean tropical rain forest trees. Oecologia
177:191–202
Larbi A, Vazquez S, El-Jendoubi H, Msallem M, Abadia J, Abadia A,
Morales F (2015) Canopy light heterogeneity drives leaf
anatomical, eco-physiological, and photosynthetic changes in
olive trees grown in a high-density plantation. Photosynth Res
123:141–155
Le Roux X, Bariac T, Sinoquet H, Genty B, Piel C, Mariotti A,
Girardin C, Richard P (2001) Spatial distribution of leaf water-
use efficiency and carbon isotope discrimination within an
isolated tree crown. Plant Cell Environ 24:1021–1032
Lewis JD, McKane RB, Tingey DT, Beedlow PA (2000) Vertical
gradients in photosynthetic light response within an old-growth
Douglas-fir and western hemlock canopy. Tree Physiol
20:447–456
Li T, Yang Q (2015) Advantages of diffuse light for horticultural
production and perspectives for further research. Front Plant Sci
6:704
Li T, Heuvelink E, Dueck TA, Janse J, Gort G, Marcelis LFM (2014)
Enhancement of crop photosynthesis by diffuse light: quantify-
ing the contributing factors. Ann Bot 114:146–156
Limousin JM, Misson L, Lavoir AV, Martin NK, Rambal S (2010) Do
photosynthetic limitations of evergreen Quercus ilex leaves
change with long-term increased drought severity? Plant Cell
Environ 33:863–875
Maier CA, Johnsen KH, Butnor J, Kress LW, Anderson PH (2002)
Branch growth and gas exchange in 13-year-old loblolly pine
(Pinus taeda) trees in response to elevated carbon dioxide
concentration and fertilization. Tree Physiol 22:1093–1106
Marshall JD, Monserud RA (2003) Foliage height influences
specific leaf area of three conifer species. Can J For Res
33:164–170
McGarvey RC, Martin TA, White TL (2004) Integrating within-
crown variation in net photosynthesis in loblolly and slash pine
families. Tree Physiol 24:1209–1220
Meinzer FC, Bond BJ, Karanian JA (2008) Biophysical constraints on
leaf expansion in a tall conifer. Tree Physiol 28:197–206
Misson L, Limousin JM, Rodriguez R, Letts MG (2010) Leaf
physiological responses to extreme droughts in Mediterranean
Quercus ilex forest. Plant Cell Environ 33:1898–1910
Munger GT, Will RE, Borders BE (2003) Effects of competition
control and annual nitrogen fertilization on gas exchange of
different-aged Pinus taeda. Can J For Res 33:1076–1083
Niinemets U (2007) Photosynthesis and resource distribution through
plant canopies. Plant Cell Environ 30:1052–1071
Niinemets U, Keenan TF, Hallik L (2015) A worldwide analysis of
within-canopy variations in leaf structural, chemical and phys-
iological traits across plant functional types. New Phytol
205:973–993
Oldham AR, Sillett SC, Tomescu AM, Koch GW (2010) The
hydrostatic gradient, not light availability, drives height-related
variation in Sequoia sempervirens (Cupressaceae) leaf anatomy.
Am J Bot 97:1087–1097
Palardy SG (2008) Physiology of woody plants. Academic Press, San
Diego
Poorter H, Niinemets Ü, Poorter L, Wright IJ, Villar R (2009) Causes
and consequences of variation in leaf mass per area (LMA): a
meta-analysis. New Phytol 182:565–588
Sellin A, Kupper P (2004) Within-crown variation in leaf conduc-
tance of Norway spruce: effects of irradiance, vapour pressure
deficit, leaf water status and plant hydraulic constraints. Ann For
Sci 61:419–429
Trees
Shiraki A, Azuma W, Kuroda K, Ishii HR (2016) Physiological and
morphological acclimation to height in cupressoid leaves of
100-year-old Chamaecyparis obtusa. Tree Physiol 36:1–10
Tang Z, Chambers JL, Sword MA, Barnett JP (2003) Seasonal
photosynthesis and water relations of juvenile loblolly pine
relative to stand density and canopy position. Trees Struct Funct
17:424–430
Tarvainen L, Räntfors M, Wallin G (2015) Seasonal and within-
canopy variation in shoot-scale resource-use efficiency trade-offs
in a Norway spruce stand. Plant Cell Environ 38:2487–2496
Tyree MC, Seiler JR, Maier CA, Johnsen KH (2009) Pinus taeda
clones and soil nutrient availability: effects of soil organic matter
incorporation and fertilization on biomass partitioning and leaf
physiology. Tree Physiol 29:1117–1131
Wang X, Zhou H, Han F, Li Y, Hu D (2016) Dynamic spatial patterns
of leaf traits affect total respiration on the crown scale. Sci Rep
6:1–12
Woodruff DR, Bond BJ, Meinzer FC (2004) Does turgor limit growth
in tall trees? Plant Cell Environ 27:229–236
Wyka TP, Oleksyn J, Zytkowiak R, Karolewski P, Jagodzinski AM,
Reich PB (2012) Responses of leaf structure and photosynthetic
properties to intra-canopy light gradients: a common garden test
with four broadleaf deciduous angiosperm and seven evergreen
conifer tree species. Oecologia 170:11–24
Yáñez MA, Fox TR, Seiler JR (2015) Early growth responses of
loblolly pine varieties and families to silvicultural intensity. For
Ecol Manag 356:204–215
Yáñez MA, Seiler JR, Fox TR (2017) Crown physiological responses
of loblolly pine clones and families to silvicultural intensity:
assessing the effect of crown ideotype. For Ecol Manag
398:25–36
Zhang S, Allen HL (1996) Foliar nutrient dynamics of 11-year-old
loblolly pine (Pinus taeda) following nitrogen fertilization. Can J
For Res 26:1426–1439
Zhang Y, Equiza MA, Zheng Q, Tyree MT (2012a) Factors
controlling plasticity of leaf morphology in Robinia pseudoaca-
cia L. II: the impact of water stress on leaf morphology of
seedlings grown in a controlled environment chamber. Ann For
Sci 69:39–47
Zhang Y, Zheng Q, Tyree MT (2012b) Factors controlling plasticity
of leaf morphology in Robinia pseudoacacia L. I: height-
associated variation in leaf structure. Ann For Sci 69:29–37
123