HEIGHT-ASSOCIATED VARIATION IN LEAF ANATOMY OF TALL
REDWOODS:
POTENTIAL IMPACTS ON WHOLE-TREE CARBON BALANCE
HUMBOLDT STATE UNIVERSITY
By
Alana Rose Oldham
A Thesis
Presented to
The Faculty of the Department of Biological Sciences
In Partial Fulfillment
Of the Requirements for the Degree
Master of Arts
In Biology
October 2008
HEIGHT-ASSOCIATED VARIATION IN LEAF ANATOMY OF TALL
REDWOODS:
POTENTIAL IMPACTS ON WHOLE-TREE CARBON BALANCE
HUMBOLDT STATE UNIVERSITY
By
Alana Rose Oldham
Approved by the Master’s Thesis Committee:
________________________________________________________________________
Dr. Stephen Sillett, Major Professor
Date
________________________________________________________________________
Dr. Mihai Tomescu, Committee Member
Date
________________________________________________________________________
Dr. Richard Golightly, Committee Member
Date
________________________________________________________________________
Dr. Erik Jules, Committee Member
Date
________________________________________________________________________
Dr. Michael Mesler, Graduate Coordinator
Date
________________________________________________________________________
Chris Hopper, Interim Dean
Date
Research and Graduate Studies
ABSTRACT
HEIGHT-ASSOCIATED VARIATION IN LEAF ANATOMY OF TALL
REDWOODS:
POTENTIAL IMPACTS ON WHOLE-TREE CARBON BALANCE
Alana Rose Oldham
The tallest tree species, coast redwood (Sequoia sempervirens), provides an ideal
model for investigating both the adaptations allowing maximum height growth in plants
and the factors that limit it. Within the crowns of tall redwoods there exists broad
variation in leaf anatomy, much of which is better explained by height-induced hydraulic
constraints than by differences in light environment. We analyzed the anatomy of leaves
and stems collected at 10-m intervals from both the inner and outer crowns in five
redwoods 108 to 113 m tall. Mesophyll porosity, a factor known to limit leaf carbon
fixation rates, strongly decreased with height. Leaf width also decreased with height
while thickness increased, such that leaf cross-sectional area remained constant but the
surface area to volume ratio was minimized at the treetop, again indicative of reduced gas
exchange capacity per unit tissue volume. Likewise, height-associated decreases in leaf
length and xylem cross-sectional area were accompanied by increased investment in
transfusion tissue, and thus a whole-leaf vascular volume that did not significantly
change with height in most trees. Transfusion tracheids became increasingly deformed
iii
with height, which suggests that they may be collapsing under the extreme water stress of
the upper crown and thus acting as a hydraulic buffer that mitigates leaf water stress and
reduces the likelihood of xylem dysfunction. Functional traits such as investment in leaf
thickness and transfusion tissue may serve to improve desiccation tolerance where it is
needed most, but at a presumably high carbon cost. Anatomical changes resulting from
reduced leaf expansion correspond to the previously documented increase in leaf
mass/area ratio and decreases in photosynthetic capacity and internal gas-phase
conductance in redwood. Thus, height-induced hydraulic stress appears to drive a
gradient in leaf anatomy that may override most among-tree developmental variation and
have a profound effect on whole-tree carbon balance as maximum height is approached
in Earth’s tallest plants.
iv
ACKNOWLEDGEMENTS
This study would not have been possible without the support and contributions of
many people. I thank my graduate committee members for their input and support on my
proposal and thesis. I am particularly grateful to Dr. Mihai Tomescu for allowing me to
take over a corner of his lab, answering endless questions, and encouragement in the
pursuit of plant anatomy. I would like to thank Dr. Richard Golightly and Dr. George
Koch for invaluable assistance in the carbon analysis aspects of this study. I also am
indebted to Rich Tate, Shauna McDonald, and Serena Ruiz for their histological help. I
am especially thankful to Barry Chin for his support and encouragement throughout the
course of this study. Most of all, I owe thanks to my major professor, Dr. Stephen Sillett,
for offering patient guidance whenever I needed help, leaving me alone when I didn’t,
and introducing me to a world of arboreal research more inspiring than I ever could have
imagined. This work was funded in part by a National Science Foundation Grant awarded
to S.C. Sillett and G.W. Koch (NSF-0445255).
v
TABLE OF CONTENTS
ABSTRACT.......................................................................................................................iii
ACKNOWLEDGEMENTS................................................................................................v
TABLE OF CONTENTS ..................................................................................................vi
LIST OF TABLES ..........................................................................................................viii
LIST OF FIGURES...........................................................................................................ix
INTRODUCTION..............................................................................................................1
METHODS ........................................................................................................................5
Study Site and Species............................................................................................5
Sampling Design.....................................................................................................5
Sample Preparation.................................................................................................6
Image Analysis .......................................................................................................7
Carbon.....................................................................................................................9
Data Analysis .........................................................................................................9
RESULTS.........................................................................................................................11
Leaf Anatomy.......................................................................................................11
Light Environment................................................................................................12
Water Stress .........................................................................................................12
Carbon ..................................................................................................................13
Stem Anatomy.......................................................................................................13
Developmental Variation.......................................................................................14
vi
Table of Contents (continued)
DISCUSSION....................................................................................................................15
The Hydrostatic Gradient Controls Redwood Leaf Anatomy…...........................15
Reduced Leaf Expansion…...................................................................................16
Improved Water Stress Tolerance.........................................................................19
Among-Tree Developmental Variation in Stems and Leaves…………………...24
CONCLUSIONS AND RECCOMENDATIONS ............................................................27
REFERENCES..................................................................................................................29
vii
LIST OF TABLES
Table
Page
1
Leaf variables used in Principal Components Analysis and their relationships to
height with all 5 redwood trees lumped. The mean % change is the % increase
or decrease in an anatomical trait between the bottom and the top of the crown
averaged among all 5 trees……………….............................................................35
2
Leaf variables and their relationships to height in each individual redwood tree,
in all cases the direction of the change was the same as when all 5 trees were
lumped....................................................................................................................36
3
Pearson correlations between 15 leaf anatomical variables listed by the strength
of their loading on PC1..........................................................................................37
4
ANOVA results for variation among the 5 redwood trees on the basis of
individual leaf anatomical variables, df = 4...........................................................38
5
Leaf variables used in Principal Components Analysis and their relationships to
the axes...................................................................................................................39
6
Stem variables used in Nonmetric Multidimensional Scaling and their
relationships to the single axis revealed by that analysis. “Stem” Area = Total
Area - Leaf Base Area............................................................................................40
7
ANOVA results for variation among the 5 redwood trees on the basis of
individual stem anatomical variables, df = 4.........................................................41
8
Timoshenko’s equation for the critical collapse pressure of pipes. Where pcr =
theoretical collapse strength for a round tube; M = elastic modulus (MPa); v =
Poisson ratio; R = tracheid diameter; and t = wall thickness. The high elastic
modulus used (M = 800 MPa) is based on a value that was proven successful for
Podocarpus and may provide an overestimation of the pressure required to induce
collapse in redwood (Brodribb & Holbrook 2005). The Poisson ratio for lignin
(0.28) was used (Innes 1995), while R was taken from the tracheids measured in
this study and t was simply set to 1 because the cell wall thickness of transfusion
tracheids is unknown……………….………………………………….…………42
viii
LIST OF FIGURES
Figure
Page
1
Leaf mesoporosity decreases with height in 5 tall Sequoia sempervirens trees…43
2
The area of the transfusion tissue increases with height in Sequoia
sempervirens……………………………………………………………..………44
3
Measures of light availability increased exponentially with height in the 5
redwood crowns………………………………………………………………….45
4
Linear correlation between independent variable, height, and PC1 scores for
leaf samples (R2 = 0.72, P < 0.0001)……………………...……….…………….46
5
Redwood leaf cross-sections collected at 48.5 m and 110 m show a clear
reduction in leaf expansion with height …………………………………………47
6
The air chambers subtending the stomata shrink in size as leaf mesoporosity is
reduced with height………………………………………………………………48
7
Transfusion tracheids collected at 48.5 m and 110 m. The tracheids from 110 m
look deformed in comparison to those from 48.5 m……………………………..49
8
A conceptual model showing the influence of height-associated anatomical
variation on whole-tree carbon balance………………………………………….50
ix
INTRODUCTION
As the tallest tree species, coast redwood Sequoia sempervirens D. Don
(Cupressaceae) provides an unparalleled opportunity to investigate the impacts of water
and light availability on structure and growth in an individual plant. The species exhibits
dramatic variation in leaf morphology with height (Koch et al. 2004), but the extent of
corresponding anatomical variation, as well as its causes and tree-level consequences, are
poorly understood.
Height increases the influence of gravity on water potential (Ψ), which decreases
by 0.0098 MPa per meter above the ground (Zimmermann 1983). The gravitational
component of pressure potential (hydrostatic tension) interacts with hydraulic path-length
resistance (hydrodynamic tension) to further lower Ψ during active transpiration. Trees
can compensate for this by raising osmotic potential (another component of Ψ) in upper
crown leaves, but this involves carbon-costly solute use and is limited in its effectiveness
(Woodruff et al. 2004). A fundamental factor limiting maximum tree height may thus be
a reduction in photosynthetic efficiency caused by lower water potentials at the treetop.
According to the hydraulic limitation hypothesis, the increase in leaf-level water stress as
trees grow taller leads to decreased photosynthesis and carbon uptake as a direct result of
reduced stomatal aperture and early closure (Ryan & Yoder 1997). Thus, there is a
delicate balance between maintaining photosynthesis and avoiding xylem cavitation due
to highly negative Ψ at the tops of tall trees (Tyree & Sperry 1988).
1
2
Not only is turgor required for CO2 assimilation through keeping the stomata
open, it also drives leaf expansion (Cosgrove 1993, 2000). In redwood, a decreased ratio
of surface area:volume and decreased intercellular space are visually apparent between
the large, laterally expanded lower leaves and the short, awl-like upper leaves (see
Results). These differences in leaf structure are primarily driven by the height-associated
reduction in turgor pressure rather than the gradient of light availability (Jennings 2002,
Boyer & Silk 2004, Ishii et al. 2008, Koch et al. 2004, Mullin et al. 2009, Woodruff et al.
2004, Woodruff et al. 2008, Zwieniecki et al. 2004a, 2004b). In fact, branches cut from
the upper crown of a tall S. sempervirens tree showed lateral leaf expansion like that of
lower crown leaves when grown in a high light environment with unlimited water (Koch
et al. 2004). This contrasts with the classical view that within-crown foliar variation,
especially leaf expansion, is driven by light acclimation with broad ‘shade leaves’ in the
lower crown and thick, narrow ‘sun leaves’ in the upper crown (Bond et al. 1999,
Ellsworth & Reich 1993, Han et al. 2003, Niinemets & Kull 1995, Niinemets et al.
1998).
In addition to degree of expansion, vascular architecture also changes with height;
the total tracheid area of needles (xylem plus transfusion tissue) in the upper crown is
more than double that of those from the lower crown (Jennings 2002), which indicates a
substantial investment in high-carbon tissue in an area of limited photosynthetic capacity.
Variation in redwood leaf anatomy also includes more frequent occurrence of stomata on
the adaxial leaf surface with increasing height as well as increased leaf mass: area ratio
(LMA) and thus higher tissue densities with increasing height (Jennings 2002, Koch et al.
3
2004, Ishii et al. 2008, Ambrose et al. 2009). Potential impacts of these changes on
whole-tree carbon balance are unknown, but such tissue investments likely provide
functional advantages, perhaps by partially mitigating the effects of low water potential.
As redwood height increases, foliar mitochondrial respiration rate rises, photosynthesis
declines, and stomata close earlier in the day (Koch et al. 2004, Ishii et al. 2008, Mullin
et al. 2009). Any or all of these physiological impacts on carbon balance may be
controlled or reflected by leaf-level anatomical variation.
In explanation of the height-associated increase in LMA, it has been proposed that
leaf mesoporosity may decrease with height in S. sempervirens (Jennings 2002).
Mesoporosity, defined here as the proportion of a leaf cross-section devoted to air space,
is an index of relative tissue density or “sponginess” and so has a strong influence on leaf
and shoot mass as well as gas exchange capacity. The volume of air in a leaf, including
the chambers beneath stomata, is positively related to the degree of leaf expansion and so
should be closely tied to turgor during leaf development. A loss of intercellular air space
lowers the internal conductance of CO2 by reducing the distance traveled in the gas-phase
and so forcing absorbed gas to pass through more diffusion-resistant mesophyll tissue
before reaching the chloroplasts (Flexas et al. 2008). Thus, low mesoporosity leads to a
limitation on the photosynthetic capacity of leaves (Hanba et al. 1999, Parkhurst 1994).
Approaching existing height gradients within tall redwood crowns as continuous
manipulations in a natural experiment, this study quantified potential impacts of light and
water availability on leaf architecture. The great morphological plasticity of leaves and
deep crowns of tall redwoods permitted exploration of physiological and ecological
circumstances favoring certain leaf designs in conifers. Two primary objectives of this
4
study were 1) to separate the effects of water and light availability on foliar anatomy by
quantifying mesophyll porosity and distribution of leaf and stem vascular tissues within
crowns and 2) to explore potential costs and benefits of leaf anatomical variation in tall
redwoods. The extent, sources, and costs of height-associated variation in redwood leaf
anatomy were used to assess leaf-level impacts on whole-tree carbon balance and test the
hypothesis that: Water status as altered by tree height has a greater impact on leaf
anatomy than does light environment. The relative effects of gradients in Ψ and light
were separated through the use of height-paired samples from the dark inner- and bright
outer-crowns. Data obtained from anatomical analyses of transverse leaf sections were
used to estimate the proportions of mid-leaf cross-sectional area devoted to transfusion
tissue, xylem, and air space in leaves from different heights and degrees of light
availability. Leaf-bearing stem anatomy was also quantified. Focusing on vascular
tissues, surface area, and mesoporosity targeted the leaf-level features most closely
associated with impacts on whole-tree carbon balance and water-stress tolerance. These
measurements from across the broad natural gradients in water potential and light
environment help decouple the effects of light and water availability on anatomy and
carbon allocation as maximum tree height is approached.
METHODS
Study Site and Species
Redwoods are well known as the world’s tallest tree species (up to 115.56 m, S.
C. Sillett personal communication). Individuals can live over 2000 years, are among the
largest organisms (>1000 m3), and are characterized by thick fire-resistant bark, extreme
resistance to wood decay, and inedibility to most herbivores (Sawyer et al. 2000). The
species extends along the coastal fog-belt from Monterey County, California to extreme
southwestern Oregon (Sawyer et al. 2000). Historically occurring in an almost unbroken
band of nearly pure redwood forest, old-growth redwood forests are now confined to
public reserves as a result of industrial logging since European settlement.
The largest remaining old-growth redwood forest occurs on the alluvial flats of
Bull Creek in Humboldt Redwoods State Park (40.3˚ N, 124.0˚ W). Five trees 108 to 113
m tall were selected for detailed study. These trees have been the focus of ongoing
research (Sillett et al. 2009).
Sampling Design
Leaves were collected from both inner and outer crown positions at ~10-m height
intervals up to 110 m (N = 12-16 samples per tree). Within each sample 10 leaves and 2
green stem segments from the centers of second-year and mature first-year annual shoots
were selected, excluding those with any visible physical damage. Microscope slides for
anatomical analyses were then prepared (N = 171). Hemispherical photographs taken
5
6
directly above each sample with a digital camera on a self-leveling mount were used to
calculate the light availability, including measurements of direct site factor, indirect site
factor, total site factor, and % sky (canopy openness) via WinScanopy (Régents
Instruments).
Sample Preparation
The mid-leaf (central 2 mm) and stem tissues were removed with a razor, fixed in
formalin propionic acid, and dehydrated with isopropyl alcohol before being embedded in
Paraplast. Each leaf and stem was transversely sectioned at 10μm thickness with a
microtome and mounted on glass slides. Sections were stained with Weigert’s Iron
Hemotoxylin, Bismark Brown, Phloxine, and Fast Green-Orange G, using a modification
of the Sam Stain procedure in order to differentiate all tissue types (D. K. Walker
unpublished). Beginning in the top left corner of the slide, the first complete section
from each leaf and stem was selected. In some cases sections with a missing or torn area
were used when the missing portions could be accurately drawn in place based on
remaining tissue. Each section was photographed with a Cannon PowerShot digital
camera mounted on a compound microscope. Each leaf was photographed twice, once to
capture the whole cross section and again at higher magnification focusing on the
vascular tissue. Photographs of a slide micrometer were also taken for scale calibration.
7
Image Analysis
Photographs of all leaves and stems were analyzed using the NIH software
ImageJ. First, thickness of upper and lower epidermis and hypodermis as well as the area
of resin ducts in leaf cross-sections were measured. Each image was converted to 32-bit
grey scale and manually thresholded to the point where the histogram began to grow
steep. Using the “wand” tool, the entire cross-section was then selected, and the image
was cleared outside the sample to remove any artifacts from the slide. This resulted in an
isolated binary image of the section allowing for automated measurement of the area,
perimeter, width, thickness, and circularity (as a 0-1 index) of the leaf.
To create an index of mesoporosity, any cellular (not empty) space that was not
already black was filled black, including the vascular bundle, transfusion tissue, resin
ducts, and any pale mesophyll cell lumens. The “create selection” function was then used
to select all the now black cellular material in the section, excluding all intercellular (air)
space within the mesophyll. This cellular area was then subtracted from the total crosssectional area to quantify the amount of air space in the leaf section. The proportion of
total area comprised of this empty space was used as an index of leaf mesoporosity.
In higher magnification photographs of the vascular system, phloem area, xylem
area, thickness, and width, as well as transfusion tissue area were measured by handselecting the boundaries of each tissue type using the “lasso” tool. Xylem tracheids were
counted and thicknesses of 3 cell walls were measured to obtain an average. The xylem
tissue was then made binary and smoothed twice to allow “wand” selection of the
8
smallest, the largest, and a typical cell lumen for area measurements. The original image
was then cleared of all but the transfusion tracheids. Cell walls of each tracheid (N =
5499) were individually traced in black using the “paintbrush” tool adjusted to match the
wall thickness. This image was then converted to 32-bit grey scale and thresholded until
all cell lumens were shown in white and all cell walls were still black. After smoothing
four times the image was reset to binary and inverted so cell lumens appeared black on a
white background, which permitted use of the “analyze particles” function to
simultaneously obtain all transfusion tracheid areas and circularity values (a 0-1 index) as
well as count cells. To avoid pseudoreplication, two leaves from each sample were
averaged to create a single representation of leaf anatomy at that site (N = 57).
Stem cross-sections were measured to quantify areas of pith, xylem (1 or 2 years
of growth), phloem, and cortex. Because redwoods have decurrent leaf bases, there is no
clear boundary between stem cortex and leaf mesophyll. All tissue to the inside of the
depressions between leaf bases and all stem tissue between this border and the phloem
was considered cortex. The image was then converted to binary so that the section, leaf
bases included, could be selected with the “wand” and its total area and perimeter were
measured. Stem area was subtracted from total area including leaf base area. A leaf base
mesoporosity index was then calculated following methods used for the leaf crosssections.
9
Carbon
Carbon content of leaf bearing first-year shoots was measured by Northern
Arizona University’s Colorado Plateau Stable Isotope Laboratory with an elemental
analyzer. Each sample, which contained ~10 mg of tissue, was pulverized, encapsulated
in tin, and combusted (CE Instruments NC 2100) at 1000°C. The resultant CO2 was
purified and its carbon content was quantified by mass spectrometry (Delta Plus XL,
ThermoQuest Finnigan) in continuous-flow mode.
Data Analysis
Principal components analysis, an indirect ordination method suitable for
parametric data, was used to illuminate the dominant patterns of variation among leaf
anatomical variables. This process reduces the dimensionality of normally distributed
multivariate data to a smaller number of orthogonal axes (principal components) that
represent the strongest patterns of linear covariation in the primary data matrix.
Correlation coefficients were used to create the cross-products matrix and the solution
was not rotated. Multivariate data were considered to fit a normal distribution when all
variables in the primary matrix had absolute skewness values < 1 and absolute kurtosis
values < 3 (mean leaf variable skewness = 0.345 and kurtosis = 0.077). The relationships
of the resulting principal components to height, light, morphology, and elemental
composition were assessed by linear regression of sample scores against these
independent variables. Anatomical variation among leaves of different trees was
10
analyzed with multiple response permutation procedures (MRPP). Among-tree
differences in ordination scores were assessed with one-way ANOVA. The MannWhitney U test was used to compare the leaves of the inner and outer tree crowns. To
fully remove height from this analysis, treetops as well as the lowermost inner-crown
samples were excluded so that only height-paired inner and outer crown leaves were
considered.
Nonmetric Multidimensional Scaling (NMS), an indirect ordination technique
suitable for non-parametric data, was used to uncover the strongest trends in stem
anatomical covariation. This approach was chosen over principal components analysis
because the multivariate stem data did not fit the criteria for normality. The “slow and
thorough” auto-pilot mode run with the Euclidean distance measure suggested a onedimensional solution (axis 1 P = 0.004). NMS ordination was then re-run over 250
iterations, limiting the results to a single dimension and using the best start configuration
from the autopilot run. The relationships of the resulting axis to height, light, stem
morphology, and elemental composition were assessed by linear regression against
variables in a secondary matrix. Anatomical variation among the stems of different trees
was evaluated with MRPP using Euclidean distance, and among-tree differences in
ordination scores were assessed with one-way ANOVA. The program PC-ORD (McCune
& Mefford 1999) was used for all multivariate analyses, and NCSS (Hintze 2002) was
used for all univariate analyses.
RESULTS
Leaf Anatomy
There was a strong linear relationship to height in 13 leaf anatomical traits when
data from all five trees were pooled (Table 1). On an individual tree basis most, but not
all, of those leaf attributes were still significantly related to height (Table 2). Many leaf
variables were more tightly correlated with each other than with height (Table 3). Leaf
mesoporosity decreased by >130% from the bottoms to the tops of the tree crowns
(Figure 1). The circularity of redwood leaf cross-sections more than doubled with height
due to the increase in thickness accompanying decreased leaf width (Table 1). Leaf
thickness and width changed at similar enough rates that the cross-sectional area of
leaves was nearly height-constant in most trees and did not decrease significantly with
height when trees were pooled (Table 2). Average transfusion tissue cross-sectional area
increased almost 300% between the lowermost branches and the tree tops (Figure 2).
Transfusion tracheids had mean lumen areas ~ 3.5 times larger than those of the xylem
tracheids in the leaf vein. In addition, the maximum area of transfusion tracheid lumens
increased by approximately 250% with height. While vascular volume decreased slightly
overall (R2 = 0.15, P < 0.0031), it did not change significantly with height in 3 of the 5
study trees in spite of the drastic shortening of leaves. Also, leaf surface area decreased
strongly with height (R2 = 0.68, P < 0.0001) so that the ratio of vascular volume: leaf
surface area increased along the height gradient (R2 = 0.51, P < 0.0001). Variations in
11
12
epidermal thickness, resin duct area, minimum transfusion tracheid size, xylem lumen
area, and cell wall thickness were uncorrelated with height and did not vary significantly
along either of the first two structural dimensions revealed by principal components
analysis (see below).
There were noteworthy differences among trees in 9 of the 15 leaf anatomy
variables (Table 4). Overall there was more within-tree leaf anatomical homogeneity
than expected by chance (MRPP: A= 0.1236, t = -5.485, P = 0.0002). This separation of
trees remained significant when individuals were removed from the analysis suggesting
that the MRPP results were not caused by a single unusual tree.
Light Environment
Light availability increased exponentially with height in the redwood crowns
(Figure 3). The outer tree crown was much brighter than the inner crown when heights
were paired (for percent sky: t = 4.6622, df = 48, P = 0.0001). None of the leaf
anatomical variables differed significantly between inner and outer tree crowns despite
strikingly different light environments in these two crown positions. This lack of
response to horizontal crown placement was true for the crowns as a whole as well as for
upper and lower crowns individually.
Water Stress
Principal components analysis (PCA) revealed two significant dimensions that
explained 81.4 % of the total variation among 15 anatomical variables (Table 5). The
13
primary axis (PC1) explained 62.3 % (P = 0.001) of the variation in leaf anatomy (Table
5). Ordination scores along PC1 were more strongly correlated with height than any of
variables describing light environment, so PC1 was interpreted as a gradient of increasing
water stress (Figure 2). PC1 scores did not vary significantly among trees.
Carbon
The percent of leaf mass composed of carbon increased with both height (R2 =
0.60, P < 0.0001) and transfusion tissue cross-sectional area (R2 = 0.54, P < 0.0001).
Stem Anatomy
NMS of 10 anatomical variables uncovered a one-dimensional solution
explaining 99.8% of the variation in the stem cross-sections (final stress after 71
iterations = 2.24805, instability < 0.00001, Table 6). Stem scores along the NMS axis
were more strongly correlated with the shoot mass-to-area (SMA) relationship than to
any other independent variable (R2 = 0.27, P < 0.0001). Stem scores along the NMS axis
were also correlated with height (R2 = 0.21, P = 0.0004). The variables with the strongest
loading on this axis were those most directly reflecting the size of the stem cross section
such as perimeter. The NMS axis thus represented a gradient in stem size that increases
slightly with height. There were strong linear relationships between ordination scores and
all 10 stem features when data from the five trees were pooled, probably because these
variables are all related to overall stem size (Table 6). As with leaves, none of the stem
14
anatomical variables differed significantly between the inner and outer tree crowns.
Again, the lack of response to horizontal crown placement was true for the crowns as a
whole as well as for the upper and lower crowns individually.
Developmental Variation
The secondary PCA axis (PC2) explained 19.0 % (P = 0.001) of the remaining
variation in leaf anatomy (Table 5). Leaf cross-sectional area, a feature unrelated to
height because of the simultaneous decrease in width and increase in thickness of leaves,
was the anatomical trait best expressed by PC2 (R2 = 0.67, P < 0.0001). Unlike PC1, leaf
scores on PC2 differed among the 5 trees (ANOVA: df = 4, F = 5.94, P = 0.0005). For
these reasons PC2 was considered to characterize a size-related developmental gradient
independent of height-induced water stress. Supporting this conclusion, leaf scores on
PC2 were also significantly correlated (R2 = 0.16, P = 0.002) with stem scores along the
stem-size axis revealed by NMS. Although there was some apparent variation among
trees along the NMS axis, these differences were not statistically significant (ANOVA: df
= 4, F = 2.38, P = 0.0636). In the original 10-dimesional data set there was more withintree stem anatomical homogeneity than expected by chance (MRPP: A= 0.0648, t = 2.2868, P = 0.0298). The separation among trees was no longer detectable if either of two
trees were removed from the MRPP analysis, this suggests that the stem anatomical
differences among trees most likely due to one or two individuals with strong internal
consistency. These small but significant differences among trees were based on “stem”
area, cortex area, pith area, and cellular area (Table 7).
DISCUSSION
A key to understanding the physiological implications of great height lies in
understanding the extent to which leaf shape, the amount of vascular tissues, and degree
of mesoporosity change with crown position. Illuminating the source of leaf-level
anatomical change in the tallest trees may help resolve the debate on the mechanisms
limiting tree height. The anatomical variables related to height in S. sempervirens can be
divided into the following two categories: 1) direct results of hydrostatic limitations on
leaf expansion, and 2) functional traits improving water-stress tolerance. These sets of
responses to hydraulic constraints may impact whole-tree carbon balance in a variety of
ways.
The Hydrostatic Gradient Controls Redwood Leaf Anatomy
The increase in hydrostatic tension as indexed by height explained nearly 70% of
the variation in key anatomical traits of redwood leaves. Light environment failed to
correlate significantly with any anatomical variable including leaf width, length, and
thickness. Nor were there any differences between the foliage of dark inner crowns and
bright outer crowns when height was removed as a factor. These results indicate a lack of
anatomical responsiveness to light in the presence of a strong Ψ gradient in S.
sempervirens leaves.
Even in the lower crown, with its long branches and high variability in light
environment, significant anatomical differences could not be detected in relation to
15
16
horizontal crown position. The absence of an anatomical response to light in the lower
crown appears to contradict recent findings of light-determined morphological and
physiological variation below 70 m in S. sempervirens crowns, including some of the
same individual trees in this study (Ishii et al. 2008, Mullin et al. 2009). However, this
discrepancy may simply imply that in the lower crown of very tall trees hydrostatic
limitation is already beginning to drive anatomical structure while light is still the
primary factor on a macroscopic and shoot-performance scale.
These findings add to a growing body of evidence invalidating the general
application of the terms “sun” and “shade” leaves within the crowns of the tallest trees
(Ishii et al. 2008, Koch et al. 2004, Meinzer et al. 2008, Mullin et al. 2009). Additionally,
the similarity between inner and outer crown leaves suggests that horizontal path-length,
and by extension hydrodynamic tension, has no significant effect on internal development
of redwood leaves at the branch level. Even in the most water-rich portions of the crown,
hydrostatic tension holds more influence over leaf anatomy than does the need to
maximize light interception through expansion.
Reduced Leaf Expansion
A casual glance at the foliage of tall redwoods reveals treetop leaves that are on
average 4 times shorter and less than half as wide as those leaves growing from 50-60 m
in the lower crown (Figure 5.). This height-associated reduction in leaf expansion has
been well documented in S. sempervirens (Burgess & Dawson 2007, Ishii et al. 2008,
17
Jennings 2002, Koch et al. 2004, Mullin et al. 2009). The degree of final leaf expansion
and thus leaf size within individual trees is primarily determined by site-specific
environmental conditions influencing water availability and transpiration rates (Koch et
al. 2004, Woodruff et al. 2004, Zwieniecki et al. 2004b, Zwieniecki et al. 2006). In tall
trees, especially those standing within intact forests, the dominant factor controlling
water availability is the gravity-driven gradient in water potential. During development,
the redwood leaf tip is propelled away from its base by tissue production along with
sufficient turgor to breach the yield threshold of cell walls and allow cellular expansion
(Cosgrove 1993, 2000). Length in single-veined leaves is a function of the xylempressure threshold for stomatal closure at the tip of the leaf (Zwieniecki et al. 2006). If
the longitudinal expansion of a redwood leaf is regulated by its ability to maintain turgor
in the most distal portion of the vein, then Ψ should determine optimal leaf length for a
given hydraulic conductivity. Considering that xylem cross-sectional area diminishes
with height, short leaves in the upper crown come as no surprise. The potential width of
single-veined leaves is related to the radial hydraulic resistances of both the vein and
mesophyll (Zwieniecki et al. 2004a). In combination with the need for turgor sufficient to
drive cellular growth, this explains why lateral expansion is also likely to be controlled
by Ψ in tall trees, overriding adaptations to exploit light. Indeed, leaf size across taxa
correlates with climate, and smaller leaves are associated with more xeric or harsh
environments rather than light availability (Ackerly et al. 2000, Parkhurst & Loucks
1972, Thoday 1931, Westoby & Wright 2006, Wright et al. 2004, 2005, Wright &
Westoby 2002).
18
Reduced leaf expansion has implications for leaf performance and, hence, wholetree carbon balance (Koch et al. 2004, Niinemets 1999, Parkhurst 1994, Vanderklein et
al. 2007). A simultaneous decrease in both length and width of leaves results in a sharp
decline in leaf surface area with height, which partially explains previously reported
increases in leaf mass:area (LMA) and shoot mass:area (SMA) with height in S.
sempervirens (Burgess & Dawson 2007, Ishii et al. 2008, Jennings 2002, Koch et al.
2004, Mullin et al. 2009). High LMA is associated with a reduction in mass-based
photosynthetic capacity (Ishii et al. 2008, Niinemets 1999, Wright et al. 2004). Averaged
across species, a 10-fold decrease in LMA generates a 21-fold increase in photosynthetic
capacity (Wright et al. 2004). This decrease is caused not only by reduction in light
interception from low surface area but also by loss of mesoporosity as leaf mass:area
increases (Hanba et al. 1999, Niinemets & Kull 1998, Parkhurst 1994).
Another direct result of hydrostatic constraints on leaf expansion is the reduction
in mesoporosity with height, which also contributes to the increase in LMA and SMA. A
loss of intercellular air space lowers the internal conductance of CO2 and so limits the
ability of a leaf to assimilate carbon (Flexas et al. 2008). Accordingly, the previously unquantified reduction in mesoporosity with height in S. sempervirens crowns corresponds
with recent measurements of impaired internal CO2 conductance and lower maximum
photosynthetic rate in treetops of this species (Ambrose et al. 2009, Ishii et al. 2008,
Mullin et al. 2009). Moreover, a reduction in the volume of sub-stomatal chambers (the
air pockets between stomata and the mesophyll) means that less CO2 is stored within the
leaf when stomata close, further limiting the time of active photosynthesis (Figure 6.).
19
Thus the two direct results of hydrostatic constraints on leaf expansion, decreases in both
leaf surface area and mesoporosity, have the potential to impact whole-tree carbon
balance by further limiting the capacity for carbon uptake in regions of the crown already
impacted by early stomatal closure.
Improved Water-Stress Tolerance
In addition to constrained leaf expansion, there is another set of anatomical
changes with height representing a general increase in functional traits associated with
improved water-stress tolerance. In tall S. sempervirens trees both transfusion tissue
cross-sectional area and leaf thickness increase drastically between the lowermost
branches and the tree tops. Although they are not direct results of low turgor pressure,
these traits can be seen as investments in foliar survival and photosynthetic maximization
in the face of hydraulic limitations and risks. These functional traits may help mitigate
the effects of the hydrostatic gradient on localized water-stress at a presumably high cost
in terms of carbon allocation to individual leaves.
Characteristic of gymnosperms, transfusion tissue is involved in bi-directional
radial transport between the leaf vein and mesophyll and has been associated with water
storage, xylem protection, low radial resistance, solute retrieval, and increased surface
contact between the vein and mesophyll (Brodribb & Holbrook 2005, Canny 1993, Esau
1977, Thoday 1931, Zwieniecki et al. 2004). Like all tracheids, those in the transfusion
tissue possess secondarily thickened lignified cell walls. Lignin synthesis requires 58%
more glucose per gram than does cellulose (Chung and Barnes 1977) signifying a
20
significant step-up in carbon investment as lignified tissue volume is increased. Its
presumably high expense implies that transfusion tissue provides functional advantages
of increasing importance with height and significant enough to justify allocating that
carbon to the leaf instead of growth elsewhere.
While the cross-sectional area and number of tracheids in the transfusion tissue is
greater in upper crown leaves, xylem cross-sectional area and tracheid numbers are
reduced with height. The height-associated rise in the total area devoted to vascular tissue
(Jennings 2002) is therefore entirely due to increased investment in transfusion tissue.
Because of this increase in vascular cross-sectional area, the total vascular volume of S.
sempervirens leaves was not related to height in most of the study trees in spite of the
drastic shortening of leaves. Consequently, the ratio of hydraulic capacity to leaf surface
area multiplies strongly with height, with great potential for improving the water-stress
tolerance of individual leaves as height increases.
Hydraulic capacity is directly related to vascular area; higher capacity leaves are
capable of keeping stomata open longer when detached from the water column (Brodribb
et al. 2005). Stomatal closure occurs at or near the turgor loss point for leaves (Brodribb
& Holbrook 2003, Woodruff et al. 2004). Turgor of a leaf is related to its hydraulic
capacity. Assuming equal conductivity, higher capacity leaves should maintain turgor
longer under water stress (Brodribb & Holbrook 2003, Brodribb et al. 2003) and so
maximize the time that stomata are open. Hydraulic capacity is positively correlated with
photosynthetic rate primarily during times of water-stress (Brodribb et al. 2002)
suggesting that it is even more important in the upper crown. On a whole-tree level,
21
capacity to store water is positively linked to annual net carbon assimilation (McDowell
et al. 2005). Thus, hydraulic capacity of leaves should impact carbon yield in a similar
manner.
Redwoods possess the unusual ability to uptake fog water directly through their
leaves in such quantities as to reverse xylem flow (Burgess & Dawson 2004). Before
trunk-level sensors can detect this flow reversal, not only the whole branch, but also all
the leaves must first fill with water (Burgess & Dawson 2004). This implies that a leaf’s
vascular capacity determines its ability to act as a local reservoir for fog water and
maximize the impact of summer morning fog events on its own Ψ and photosynthetic
output. Because it is made up of tracheids larger than those in the xylem, transfusion
tissue almost certainly has a higher lumen:cell wall volume ratio, which would promote
high vascular capacity with minimal carbon investment in lignified walls.
The most striking difference between transfusion and xylem tracheids is cell size.
Such a difference, coupled with the effects of larger vascular area, may underlie the
functional significance of the anatomical variation in vascular tissue with height. In S.
sempervirens, transfusion tracheids have mean lumen areas much larger than those of the
xylem tracheids in the leaf vein. As tracheid diameter increases, resistance decreases,
resulting in greater hydraulic efficiency in vascular tissue with larger cells (Pittermann et
al. 2005, Pittermann et al. 2006, Sperry et al. 2006, Westoby & Wright 2006).
Additionally, since water travels more efficiently through tracheids than mesophyll cells,
increased transfusion tissue volume should boost leaf-level hydraulic conductivity and
decrease radial resistance (Brodribb et al. 2007). When Ψ is equal, lateral expansion
22
capability in a leaf is related to radial resistance, so transfusion tissue may help to
maximize leaf width expansion in the upper crown (Zwieniecki et al. 2004). The
hydraulic conductivity of leaves is also strongly related to their photosynthetic maxima
(Brodribb et al. 2002, Brodribb et al. 2007, Hubbard et al. 1999). Size-associated
hydraulic efficiency of cells is a trade-off that increases vulnerability to cavitation under
tension due to the decreased surface contact between the tracheid walls and the water
column (Pittermann et al. 2006).
Larger diameter cells are biomechanically less able to withstand tension compared
to smaller cells. Using Timoshenko’s equation for the theoretical buckling pressure of
pipes with published structural values for lignin and assuming equal cell wall thickness,
one can estimate that the average xylem tracheid in a redwood leaf vein is ~ 6.5 times
more resistant to tension-induced deformation than a transfusion tracheid (Table 8).
Transfusion tracheids in redwood leaves become less circular with height and
occasionally seem to be collapsed in upper leaves of S. sempervirens (Figure 7).
Although a bit surprising, this observation of hydraulically-linked tracheid
distortion is not entirely without precedent. Accessory transfusion tracheids have been
observed deforming under water stress and then returning to their normal shapes when
the stress is removed in Podocarpus (Brodribb & Holbrook 2005). In this case, the
collapsible vascular tissue was hypothesized to provide a Ψ buffer, temporarily relieving
tension in the xylem long enough for the stomata to close before an embolism occurs in
the vein (Brodribb & Holbrook 2005). Likewise, transfusion tracheids in excised Pinus
leaves collapse or distort during desiccation while xylem tracheids do not change shape
23
(Parker 1952). Temporary leaf cavitation is a mechanism that may help regulate stem
hydraulic conditions in Pseudotsuga (Woodruff et al. 2007). Similarly, trees may
sacrifice highly vulnerable twigs to improve the water-status of adjacent branches during
drought events (Tyree & Sperry 1988, Zimmerman 1983). In addition, the maximum
cross-sectional area of tracheid lumens in redwood transfusion tissue increased with
height, possibly signifying that size is being utilized to enhance cell collapsibility. This
supports the notion that transfusion tissue is semi-sacrificial and serves a protective
function for the vein likely decreasing leaf (and thus branch) mortality during times of
extreme water stress.
In addition to investments in transfusion tissue, the thickness of S. sempervirens
leaves also increased substantially with height. Not only does added tissue have
construction costs and contribute to rising LMA, but it is also linked to observations of
increased respiratory demands with height (Mullin et al. 2009). However, leaf thickness
is a functional trait related to improved water stress tolerance and photosynthetic yield.
The circularity of redwood leaf cross-sections rose with height due to the increase in
thickness accompanying the hydraulic constraints on leaf width. A more circular leaf
midsection means that the surface area to volume ratio, and hence evaporative demand, is
being minimized. It is for this reason that thick leaves are considered more xeromorphic
than thin ones (England & Attiwill 2006, Westoby & Wright 2006, Wright et al. 2005).
Although thicker leaves generally have lower mesoporosity, they can have more surface
area of their mesophyll contacting intercellular space thus improving the rate of gasphase CO2 conductance per unit air space (Hanba et al. 1999), which may help explain
24
why thick leaves also have increased area-based photosynthetic capacity (Niinemets
1999, Oguchi et al. 2003). Moreover, in redwoods the leaf thickness increase
corresponded with the height-related decrease in width in such a way that the crosssectional area of leaves was nearly height-constant. This implies that thickness may be an
area-preserving compensation mechanism for the loss of width and so potentially a relict
of developmental constraints on cellular proliferation and growth (Flemming 2006,
Horiguchi et al. 2006, Tsukaya 2003, 2006).
Any or all of these possible leaf-trait functions could amplify CO2 fixation ability
and water stress tolerance in the upper crown, providing a carbon-level justification for
the observed investments in transfusion tissue and leaf thickness. An optimally
performing leaf should maximize its ratio of net photosynthesis to water loss (Parkhurst
& Loucks 1972). Redwood leaves, with their plastic anatomy, seem to do just that. From
a leaf economics perspective, it is probable that the benefit of increased hydraulic
tolerance outweighs its expense in the upper crown, even given the locally limited carbon
fixation capacity. Indeed, these may be some of the very functional traits that allow S.
sempervirens to reach such great heights.
Among-Tree Developmental Variation in Stems and Leaves
The anatomy of redwood stems, like that of the leaves was related to the Ψ
gradient associated with height. However in the case of stems, that association explained
only 20.6%, in spite of a NMS ordination that retained a shocking 99.8% of the variation
25
in stem anatomy on a single axis. This axis described the overall size of the stems and
appeared to vary among trees and so seems to be primarily a developmental gradient. The
positive relationship of this axis to SMA means that while the cross-sectional area of
stems increases, the reduction in leaf area (a component of SMA in redwoods) with
height outweighs changes in stem size. However, this may also suggest that the stem-size
gradient may be functional in maximizing light interception efficiency (i.e. actually
lowering SMA) as leaf area shrinks. Other variables in the primary matrix that would
have revealed changing investments in tissue type, for example cortex area, were also
well explained by the NMS axis, probably because they too are area measurements and
so also related to stem size. Even so, there were significant anatomical differences among
trees, especially in those ‘tissue type’ variables. Such developmental variation is likely
based on genotypic differences between individual trees and may be additionally
reflective of age-related changes in gene expression.
The stem NMS axis was also correlated with PC2, which described leaf
anatomical variation unrelated to height. PC2 was similarly representative of organ size
and explained the variation in leaf features such as cross-sectional area and the total
amount of cellular area (non-air space) and so was also considered to represent a
developmental gradient. The leaf PCA axis scores likewise varied significantly among
trees as did the majority of the anatomical traits measured.
Remarkably, those leaf features directly linked to turgor limitation with height
(leaf width, perimeter, length, mesoporosity, and transfusion tracheid circularity) did not
vary significantly among redwood trees while traits related to water stress tolerance and
26
organ size did. The total cellular area in a leaf cross-section, the variable most strongly
loaded on PC2, was also the most different among trees (Table 4). Evidence of
hydrostatic constraints overriding tree individuality supports the suggestion that genetic
variation may have more control on structural development where the impacts of highly
negative Ψ are less directly influential (Fabre et al. 2007). A possible height-related
change from primarily genetic to increasingly hydraulic influence on the development of
some tree features fits with recent observations of greater among-tree variability in lowercrown and short-tree S. sempervirens stems (Mullin et al. 2009).
Only one tree exhibited correlations between size features such as leaf and stem
cross-sectional areas and height. On the cellular-level, this tree generally had stronger
responses and tighter correlations to height than the other trees in this study (Table 2,
Tree 5). A stronger response to water stress is especially intriguing in light of recent
evidence that this particular tree is ~30-50% older than the other trees in this study
(Sillett et al. 2009). Previous research on other tree species has not shown age-control of
either growth-rates or carbon assimilation when cuttings from old trees are grown
alongside those from young trees (Mencuccini et al. 2005). However, this age-related
work focused on “old” trees significantly younger than those discussed here. The
potential for tree-age enhancing anatomical susceptibility to the forces of hydrostatic
tension has fascinating implications for age-based constraints on height growth, possibly
explaining age-related investments in structural complexity within the existing heightspan of the crown.
CONCLUSIONS AND RECOMMENDATIONS
As redwoods grow in height their leaves provide diminishing returns to the tree.
Height-associated declines in Ψ drive leaf anatomical gradients impacting whole-tree
carbon balance while light has little influence on the foliar anatomy of tall S.
sempervirens trees. The carbon invested per g of leaf tissue increases while the
mitochondrial respiration rate rises (Mullin et al. 2009), the photosynthetic rate falls
(Ishii et al. 2008), and stomata close earlier in the day at greater heights (Koch et al.
2004). These physiological processes limiting carbon-uptake efficiency are likely to be
heavily influenced by hydraulic constraints on leaf expansion leading to increased LMA
and reduced mesoporosity with height. Highly negative Ψ also compels investments in
water-stress tolerance that may promote organ survival and maintain photosynthetic
activity but cost the tree in terms of carbon allocation and respiratory demand. The
synergistic effect of these tissue-level changes is likely to be a gradual slowing in height
growth (Figure 8). Regardless of developmental variation among trees, hydraulic
constraints on leaf anatomy appear to underlie the complex set of factors determining the
fundamental limits to tree height.
This research was conducted in parallel with several other complementary studies
allowing access to whole-tree samples. The anatomical information was used in
conjunction with building a structural, dendrochronological, physiological,
morphological, and microclimatic database for these trees. The addition of leaf and shoot
cross-section tissue distribution data helps complete the multi-scale picture of how
27
28
biophysical and ecological factors act jointly to limit tree height and determine growth
patterns.
Investigations into S. sempervirens anatomy raise many questions about the
response of leaves to their hydraulic environment. Additional scrutiny of the role of
transfusion tissue in water-status regulation of conifer leaves is warranted. The in situ
freezing of leaves at various stages of water stress for cryostatic sectioning as well as
experimental induction of tracheid collapse in lower-crown leaves and inquiries into
refilling potential would confirm evidence of a protective function for this tissue.
Quantification of leaf anatomy must be extended to other tall conifer species to assess the
degree to which leaf structure is as a rule hydraulically determined and to uncover traits
individual species may possess to support height growth. Furthermore, carbon availability
may not be the dominant factor limiting tree height; diminished turgor attributed to the
effects of gravity may be a sufficient explanation. Research into the ability of trees to
store and access carbon in the trunk, leaf growth responses to amplified CO2, and studies
of leaf-level contributions to height limitation in shorter conifers in arid climates may
help illuminate the causes of declining height growth with increasing tree height. Lastly,
an examination of tree-to-tree foliar variability, especially as it might relate to age, would
provide insight into the roles of tree age and environment in genetic expression. Such
questions inspire further research on the smallest parts of the tallest trees.
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35
Table 1. Leaf variables used in Principal Components Analysis and their relationships to
height with all 5 redwood trees lumped. The mean % change is the % increase or
decrease in an anatomical trait between the bottom and the top of the crown averaged
among all 5 trees.
Response Variable
Leaf Area
Leaf Cellular Area
Leaf Circularity
Leaf Perimeter
Leaf Width
Leaf Thickness
Mesoporosity
Xylem Area
Xylem Width
# Of Xylem Tracheids
Phloem Area
Transfusion Tissue Area
Maximum T. T. Lumen Area*
Mean T. T. Circularity*
Leaf Length
* T. T. = Transfusion tracheid
R2
0.05
0.01
0.74
0.63
0.69
0.61
0.54
0.44
0.45
0.47
0.30
0.42
0.31
0.59
0.65
P
0.0864
0.5258
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
% change
+171
-79
-115
+78
-134
-196
-131
-166
-281
+292
+248
-24
-303
Table 2. Leaf variables and their relationships to height in each individual redwood tree, in all cases the direction of the
change was the same as when all 5 trees were lumped.
Tree 1
Tree 2
R2
P
Leaf Circularity
0.91
< 0.0001
328
0.76
0.0005
Leaf Perimeter
Leaf Width
0.84
0.86
< 0.0001
< 0.0001
71
99
0.67
0.80
0.0020
0.0002
% change
R2
P
Tree 3
R2
P
123
0.70
0.0027
62
93
0.24
0.34
0.1471
0.0778
% change
Tree 4
R2
P
74
0.73
0.0016
56
0.76
0.72
% change
Tree 5
R2
P
100
0.87
< 0.0001
228
0.0010
0.0019
61
112
0.82
0.84
< 0.0001
< 0.0001
157
215
% change
% change
Leaf Thickness
0.79
< 0.0001
72
0.76
0.0005
112
0.71
0.0021
41
0.64
0.0052
72
0.54
0.0042
97
Mesoporosity
0.80
< 0.0001
120
0.85
< 0.0001
153
0.34
0.0764
45
0.35
0.0704
-
0.62
0.0014
193
Xylem Area
0.57
0.0027
171
0.69
0.0016
137
0.16
0.2592
-
0.30
0.1047
-
0.87
< 0.0001
430
Xylem Width
0.56
0.0034
98
0.83
0.0001
109
0.16
0.2483
-
0.36
0.0650
-
0.93
< 0.0001
220
# Of Xylem Tracheids
0.73
0.0002
145
0.66
0.0023
100
0.18
0.2212
-
0.51
0.0203
156
0.87
< 0.0001
352
Phloem Area
0.27
0.0667
-
0.76
0.0005
248
0.03
0.6394
-
0.02
0.6930
-
0.85
< 0.0001
453
Transfusion Tissue Area
0.59
0.0022
296
0.74
0.0007
319
0.37
0.0602
-
0.55
0.0137
500
0.68
0.0005
147
Max T. T. Lumen Area
0.49
0.0081
316
0.59
0.0055
251
0.18
0.2216
-
0.62
0.0067
286
0.41
0.0180
209
Mean T. T. Circularity
0.89
< 0.0001
21
0.48
0.0189
28
0.28
0.1122
-
0.56
0.0127
30
0.78
0.0001
24
Leaf Length
0.82
< 0.0001
325
0.86
< 0.0001
346
0.69
0.0027
158
0.79
0.0006
295
0.72
0.0002
392
36
Table 3. Pearson correlations between 15 leaf anatomical variables listed by the strength of their loading on PC1.
Variable
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
Leaf Width
2
Leaf Circularity
-0.93
1
3
Leaf Perimeter
0.99
-0.91
1
4
Leaf Length
0.86
-0.88
0.84
1
5
# Of Xylem Tracheids
0.80
-0.75
0.79
0.76
1
6
Xylem Area
0.80
-0.72
0.80
0.76
0.93
1
7
Xylem Width
0.79
-0.73
0.78
0.74
0.93
0.95
1
8
Mean T. T. Circularity
0.72
-0.78
0.68
0.75
0.66
0.61
0.58
1
9
Leaf Thickness
-0.75
0.86
-0.67
-0.77
-0.54
-0.53
-0.56
-0.74
1
10
Phloem Area
0.69
-0.59
0.68
0.61
0.84
0.92
0.92
0.49
-0.43
1
11
Transfusion Tissue Area
-0.63
0.75
-0.58
-0.74
-0.52
-0.50
-0.50
-0.64
0.78
-0.35
1
12
Max T. T. Lumen Area
-0.61
0.70
-0.58
-0.69
-0.58
-0.50
-0.50
-0.58
0.65
-0.34
0.79
1
13
Mesoporosity
0.69
-0.64
0.67
0.65
0.41
0.42
0.37
0.63
-0.60
0.28
-0.49
-0.42
1
14
Leaf Area
0.51
-0.26
0.59
0.27
0.51
0.53
0.50
0.11
0.15
0.50
0.07
-0.06
0.22
1
15
Leaf Cellular Area
0.20
0.02
0.28
-0.02
0.34
0.35
0.34
-0.16
0.41
0.37
0.28
0.11
-0.23
0.90
15
1
1
37
38
Table 4. ANOVA results for variation among the 5 redwood trees on the basis of
individual leaf anatomical variables, df = 4.
F
Leaf Area
Leaf Cellular Area
Leaf Circularity
Leaf Perimeter
Leaf Width
Leaf Thickness
Mesoporosity
Xylem Area
Xylem Width
# Of Xylem Tracheids
Phloem Area
Transfusion Tissue Area
Maximum T. T. Lumen Area
Mean T. T. Circularity
Leaf Length
P
3.49
6.04
1.45
0.76
0.78
2.68
1.40
3.04
3.92
4.30
3.99
3.23
3.80
1.13
1.41
0.0134
0.0005
0.2300
0.5579
0.5404
0.0416
0.2464
0.0251
0.0074
0.0044
0.0067
0.0192
0.0088
0.3518
0.2422
39
Table 5. Leaf variables used in Principal Components Analysis and their relationships to
the axes.
PC1
Response Variable
Leaf Area
Leaf Cellular Area
Leaf Circularity
Leaf Perimeter
Leaf Width
Leaf Thickness
Mesoporosity
Xylem Area
Xylem Width
# Of Xylem Tracheids
Phloem Area
Transfusion Tissue Area
Maximum T. T. Lumen Area
Mean T. T. Circularity
Leaf Length
2
R
0.18
0.02
0.88
0.87
0.91
0.61
0.43
0.79
0.77
0.80
0.59
0.53
0.50
0.63
0.84
PC2
P
0.0012
0.3195
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
2
R
0.67
0.89
0.04
0.02
0.00
0.29
0.09
0.10
0.09
0.07
0.16
0.23
0.11
0.08
0.03
P
< 0.0001
< 0.0001
0.1645
0.3143
0.7007
< 0.0001
0.0254
0.0170
0.0217
0.0421
0.0022
0.0002
0.0133
0.0297
0.2412
40
Table 6. Stem variables used in Nonmetric Multidimensional Scaling and their
relationships to the single axis revealed by that analysis. “Stem” Area = Total Area - Leaf
Base Area.
Response Variable
Pith Area
Total Area
Total Perimeter
Cellular Area
Xylem Area
Phloem Area
Cortex Area
"Stem" Area
Leaf Base Area
Air Space
R2
P
0.76
0.96
1.00
0.95
0.25
0.49
0.61
0.56
0.95
0.85
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
41
Table 7. ANOVA results for variation among the 5 redwood trees on the basis of
individual stem anatomical variables, df = 4.
Pith Area
Total Area
Total Perimeter
Cellular Area
Xylem Area
Phloem Area
Cortex Area
"Stem" Area
Leaf Base Area
Air Space
F
3.44
2.55
2.34
2.81
1.50
2.22
7.27
4.31
2.00
1.86
P
0.01432
0.05028
0.06707
0.03456
0.21693
0.07930
<0.00001
0.00435
0.10855
0.13190
42
Table 8. Timoshenko’s equation for the critical collapse pressure of pipes. Where pcr =
theoretical collapse strength for a round tube; M = elastic modulus (MPa); v = Poisson
ratio; R = tracheid diameter; and t = wall thickness. The high elastic modulus used (M =
800 MPa) is based on a value that was proven successful for Podocarpus and may
provide an overestimation of the pressure required to induce collapse in redwood
(Brodribb & Holbrook 2005). The Poisson ratio for lignin (0.28) was used (Innes 1995),
while R was taken from the tracheids measured in this study and t was simply set to 1
because the cell wall thickness of transfusion tracheids is unknown.
43
0.4
2
R = 0.54
0.35
Mesoporosity
0.3
0.25
0.2
0.15
0.1
0.05
0
40
50
60
70
80
90
100
110
Height (m)
Figure 1. Leaf mesoporosity decreases with height in 5 tall Sequoia sempervirens trees.
2
Transfusion Tissue Area (mm )
44
2
R = 0.42
0.03
0.02
0.01
0
40
50
60
70
80
90
100
110
Height (m)
Figure 2. The area of the transfusion tissue increases with height in Sequoia
sempervirens.
Canopy Openness (%)
45
100
80
2
R = 0.91
60
40
20
2
R = 0.76
Indirect Site Factor (%)
0
100
2
R = 0.88
80
60
40
20
2
R = 0.32
0
Direct Site Factor (%)
100
80
2
R = 0.58
60
40
20
2
R = 0.001
0
40
50
60
70
80
90
100 110
Height (m)
Figure 3. Measures of light availability increased exponentially with height in the 5
redwood crowns. Open symbols indicate outer crown sampling locations while closed
symbols indicate sites in the inner crown.
46
1
0.9
R ² = 0.72
PC1 Score
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
40
50
60
70
80
90
100
110
Height (m)
Figure 4. Linear correlation between independent variable, height, and PC1 scores for
leaf samples (R2 = 0.72, P < 0.0001).
47
48.5 m
110 m
Figure 5. Redwood leaf cross-sections collected at 48.5 m and 110 m show a clear
reduction in leaf expansion with height. Scale bar = 0.5 mm.
48
48.5 m
110 m
Figure 6. The air chambers subtending the stomata (designated by arrows) shrink in size
as leaf mesoporosity is reduced with height. Scale bar = 0.1 mm.
49
48.5 m
110 m
Figure 7. Transfusion tracheids collected at 48.5 m and 110 m. The tracheids from 110
m look deformed in comparison to those from 48.5 m. Scale bar = 0.01 mm.
50
Figure 8. A conceptual model showing the influence of height-associated anatomical
variation on whole-tree carbon balance.