specially cut thin glasses. The mounted leaves were photographed and the photographs en-
hanced so that the determination of the vena- tion’s
pattern could
be easily
performed. Drawings of the smaller veins, veinlets and are-
oles were made using a Wild M 5 stereoscopic microscope and a Wild M 20 binocular micro-
scope, both with drawing tubes. Microphoto- graphs were taken with a Zeiss Photomicroscope
II. Drawings of each epidermis were made after removing the other epidermis and most of the
mesophyllic tissue. Means of measurements of anatomical features were based on five measures
per leaf in five leaves of each of the three levels of the ten trees.
The estimation of leaf volume, mesophyll vol- ume, proportion of mesophyll in the leaf and
proportion of spongy and palisade parenchyma were made with data obtained, by stereological
procedures, from
drawings of
photographed transverse sections of paraffin-embedded mate-
rial, stained in safranin-fast green and mounted in balsam. The sampling of tissue blocks to be
used for stereological measurements of leaf anatomy and the estimation of the mentioned
characters was made following the recommenda- tions of Kubı´nova´ 1993. Data were obtained
from five transverse sections per leaf in five leaves of each of the three levels of the ten trees.
Leaf material for scanning electron microscopy SEM was fixed in FAA. Samples were dehy-
drated in a series of alcohol of increasing strength to absolute alcohol and in a series of
alcohol – acetone to pure acetone, treated in a Polaroid critical point dryer and coated with a
film of gold with a sputter coater Pelco 91000. Both epidermis of each sample were examined
with a JEOL 35 SEM operated at 7 KV.
The relation between the various tissues of the petioles at the different level in the plants was
determined by drawings of the cross-section at the basal and distal end of the petioles, using
UTHSCSA Image Tool University of Texas Health Science Center, San Antonio, TX. Free
hand sections were made on five petioles of each of the three levels of the ten trees.
The evaluation of the data was carried out by means of ANOVA and the mean values of the
treatments were compared using the Student – Newman – Keuls’ test.
3. Results
Table 1 shows the data of the environmental conditions registered during the growth period
1997 – 1998 in the sampling zone. Air tempera- ture outside the shaded understorey reached
higher values, up to a maximum of 42°C in Jan- uary 1998. Air humidity in the lower protected
strata reached a minimum of 36 and attained 100 every night, but in the upper sun and wind
exposed zone, values ranged from 18 up to a maximum of 84.
Fig. 1 represents the average monthly mea- sures of daily solar radiation as well as the maxi-
mum hourly records in the upper canopy of the E. angustifolia trees. During spring and summer
time, solar radiation is very high and the values climb up to 927 calorie cm
2
day in December. Light attenuation at the lower level reaches 90
and at the medium level ranges between 60 and 80.
Leaf water content decreased through the grow- ing period at all levels in the plants, but while in
spring the higher values were those of half-ex-
Fig. 1. Mean daily solar radiation cal. cm
2
day and maxi- mum hourly solar radiation cal. cm
2
h registered during the period April 1997 to March 1998 in the upper canopy of the E.
angustifolia trees.
M .G
. Klich
En 6
ironmental and
Experimental
Botany
44 2000
171 –
183
174
Table 1 Environmental conditions of the sampling area during the growth period 1997–1998 of E. angustifolia
a
AH lower height AT °C lower height
AH upper height SWC 20, 40 and 60 cm
Rain mm min, max
max, min, mean min, max
depth 30
81 4.5
33.6 27.8
9.3 August
13.3 1.5
7.4 53
100 September
17.0 30
73 26.3
16.5 15.9
51 100
14.3 1.7
8.0 28.0
17.6 14.1
19.7 17.3
4.8 11.1
41 100
30 69
October 28
63 16.0
20.6 8.1
14.3 November
37 100
33.8 30.9
29.0 52
100 30.0
25 61
23.9 10.1
17.3 December
16.1 15.9
13.7 3.5
7.0 11.3
11.8 25.4
11.4 18.4
36 100
18 60
January 21
69 47.5
11.4 14.0
11.0 February
22.3 10.5
16.4 56
100 35
80 22.5
21.6 6.9
14.2 March
48 100
10.0 14.3
15.3 34
84 48
100 18.1
6.5 11.9
19.5 April
13.9 17.4
18.3
a
Average monthly soil water content SWC at different depth; average maximum, minimum and mean monthly air temperature AT °C; average minimum and maximum monthly air humidity AH at lower 1 m and upper 5 m height; and monthly rain mm.
Table 2 Comparison of leaf water content LWC of E. angustifolia
leaves developed at different heights in a tree
a
ML LL
UL 74.11b
October 70.20a
75.90b December
74.54b 73.27b
68.99a 57.90a
57.54a February
62.07b 51.10a
55.81b 51.13a
April
a
LL, lower shade leaves B1 m height; ML, medium leaves 1–3 m height; UL, upper sun leaves \5 m height. Values
are the mean for 30 measurements. For each row, values which have the same letter are not significantly different at the
0.05 level, as determined by Student–Newman–Keuls’ test.
the tree level increases but its course is generally straight. The angles of divergence of secondary
veins were found to be always acute, although they are wider in the upper leaves. The relative
thickness of secondary veins is moderate and their course is mainly curved. The intersecondary veins
are simple and the intercostal areas are irregular. The pattern of tertiary veins is randomly reticu-
late. The higher order of venation is quaternary and a looped marginal ultimate venation is ob-
served. The areoles are imperfect, pentagonal to polygonal and randomly arranged. In the upper
leaves, the density of areoles is higher 17 areoles mm
2
than in the medium and lower ones 15 and 14 areolesmm
2
, respectively. Veinlets are mainly branched. One to three veinlets generally enter
each areole, but areoles lacking terminal veinlets are also common.
The thickness of the foliar blades increases with the height and the degree of sun exposure, while
their area decreases, so that the mean volume of the leaves at the evaluated levels has no significant
differences Table 4.
Leaves are bifacial, with a biseriate palisade in the two inferior levels, but a third poorly orga-
nized stratum is observed in the upper leaves Fig. 2b,d,f. Spongy mesophyll cells are round or elon-
gate, not lobed and randomly oriented. The pro- portion of mesophyll tissue is higher in the upper
leaves 81.86 than in the medium 74.93 and lower 73.97 leaves, but the ratio between pal-
isade and spongy parenchyma remains constant average 60 and 40, respectively at all leaf
levels.
Leaves are hypostomatous, with anomocytic stomata Fig. 3. Stomata are at the level of the
neighboring ordinary epidermal cells. The pentag- onal or hexagonal epidermal cells are smaller in
the lower than upper epidermis. Adaxial epider- mis is thinner in the upper leaves but the outer
tangential walls are thicker than in the lower leveled leaves. Stomata density is higher in the
upper leaves, but there are no significant differ- ences in their length and width between the three
analyzed leaf levels Table 3. The abaxial surface of the leaves is always more pubescent than the
adaxial
one. Trichomes
are multicellular,
pedestalled, stellate-branched or peltate, and their posed and shade-leaves, in April these were the
leaves that bore the lower values Table 2. The leaves of E. angustifolia are annually decid-
uous, however, many of the upper leaves may persist when winters are wetter and warmer than
normal. New leaves are produced during late win- ter and early spring, along with the abscission of
the old winter persistent leaves. Fully expanded leaves are simple and symmetrical, with entire
margins. Upper leaves of E. angustifolia are in- clined, especially at midday, while the medium
and lower leaves are nearly perpendicularly ori- ented to the incident light. Upper leaves upfold
through the midvein. Table 3 shows the foliar features of the differently leveled leaves. By their
size they are classified as microphyll, although some of the medium leaves are mesophyll. The
form of the whole lamina is ovate in the inferior levels and oblong to elliptic in the upper level; the
form of the base and the apex varies between the leaves in the inferior levels but is always acute in
the upper levels Fig. 2a,c,d. The lamina texture is chartaceous but the color varies; adaxial sur-
faces of leaves are dark green in the shaded inferior branches, light green in the half exposed
and silvery grey – green in the sun exposed canopy. The abaxial surface always presents a lighter
color, a feature that is especially evident in the two lower levels of leaves. The type of venation is
pinnate eucamptodromous Fig. 2a,c,d. The size of the primary vein, calculated as the percent of
vein width to leaf width, is larger as the height of
form and density can be associated with leaf level, color
and appearance.
Lower leaves
have branched hairs and present a woolly appearance
abaxially Fig. 4a,b. The abaxial epidermis of the
Table 3 Comparison of morphological and anatomical features of E. angustifolia leaves developed at different heights in a tree
a
Medium leaves Lower shade leaves
Upper sun leaves 1–3 m height
B1 m height \5 m height
Microphyll from 7.5 to 18.7 Lamina area one side cm
2
Microphyll from 4.2 to 10.5 Microphyll-mesophyll from
4.6 to 23.7 Lamina length
Upto 10 cm. Upto 10 cm.
Upto 10 cm. Symmetrical
Symmetrical Shape: whole lamina
Symmetrical Symmetrical
Generally symmetrical Shape: base only
Symmetrical Oblong: narrow-oblong
Ovate: ovate lw: 1.5:1 Ovate: lanceolate lw:3:1
Form: whole lamina lw: lw:3:1 Elliptic:
lengthwidth narrow-ovate lw: 2:1
narrow-elliptic lw:3:1 lanceolate lw: 3:1
Acute normal Acute cuneate rounded to
Acute cuneate obtuse Form: base only
decurrent cordate
Form: apex only Acute
Acute or obtuse Acute, obtuse or obtuse
mucronate Entire
Entire Margin
Entire Texture
Chartaceous Chartaceous
Chartaceous Silvery grey–green bicolor
Appearance Adaxial light green notable
Adaxial dark green notable bicolor
bicolor Attachment petiole
Normal Normal
Normal Pinnate eucamptodromous
Type of venation Pinnate eucamptodromous
Pinnate eucamptodromous Moderate 1.25–2 to stout
Stout 2–4 to massive Size of primary vein vein
Stout 2–4 2–4
\4 widthleaf width
Course of primary vein Straight some slightly
Straight some slightly curved Straight
curved Acute: narrow B45° to
Angle of divergence of Acute: moderate 45–65° to
Acute: narrow B45° to wide 65–80°
moderate 45–65° secondary veins
moderate 45–65° Lower and upper secondary
Uniform Variation in angle of
Lower and upper secondary veins more obtuse than middle
divergence of secondary veins more obtuse than
veins sets
middle sets Moderate
Moderate Moderate
Relative thickness of secondary veins
Curved Course of secondary veins
Straight to curved Curved
Pattern of tertiary veins Random reticulate
Random reticulate Random reticulate
42.90b 29.37a
43.16b Adaxial epidermal thickness
mm Adaxial epidermal outer wall
5.02a 5.36a
9.09b thickness mm
18.18a Abaxial epidermal thickness
20.31a 16.99a
mm 2.49a
2.72a 3.01a
Abaxial epidermal outer wall thickness mm
312c 247a
Stomatamm
2
270b Guard cell length mm
16.94a 16.96a
17.02a Guard cell width mm
10.10a 10.07a
10.11a 327b
Abaxial indumentum height 341b
257a mm
a
Morphological and data are the result of measurements conducted on 100 leaves per leaf type. Venation patterns were determined from measurements conducted on 50 leaves per height. Anatomical values are the mean for 250 measurements. For each
row, values which have the same letter are not significantly different at the 0.05 level as determined by Student–Newman–Keuls’ test.
Fig. 2. Cleared leaves of E. angustifolia and drawings of transverse sections. a,b Lower shade leaves; c,d medium half sun-exposed leaves; e,f upper sun-leaves. For a,c,e, bar: 1 cm; for b,d,f, bar: 50 mm.
light green leaves from half exposed branches have several hair layers, the external ones com-
posed of hairs totally branched or stellate with cells arranged radially and joined near the tri-
chome stalk and the internal layer formed by peltate hairs with the cells of the shield partially
joined. The rays of adjacent trichomes interlock and form a dense cover over the abaxial leaf
surface Fig. 4d,e. The silvery highly sun exposed leaves from the upper branches have only peltate
hairs that, in the abaxial leaf surface, are arranged to form two or three layers of their flattened
multicellular shields Fig. 4g,h. Abaxial indumen- tum height is greater in the lower leaves Table 3.
All abaxial epidermal cells remain covered by the indumentum. The same type of hairs are found in
the respective adaxial epidermis of each type of analyzed leaf, but they are too sparse and, al-
though their rays have a greater length and spread, they do not form a canopy and many
epidermal cells remain exposed, especially in the two lower levels of leaves Fig. 4c,f,i.
When the tissue distribution of the petioles of the differently leveled leaves is compared, it is
found that when height increases there is an en- largement of the proportion of epidermis, col-
lenchyma and phloem while the relative amount of parenchyma and xylem diminishes Table 5.
Petioles are slender in the upper leaves. In trans- verse sections through the basal end, they exhibit
Table 4 Comparison of leaf dimensions and tissue compositions of E.
angustifolia leaves developed at different heights in a tree
a
ML UL
LL 0.326a
0.382b Thickness mm
0.474c without hairs
1132.00a,b Area mm
2
955.91a 1250.08b
Volume mm
3
407.53a 432.42a
450.82a Mesophyll
73.97a 74.93a
81.86b 58.40a
60.23a 62.62a
Palisade 41.60a
Spongy 37.38a
39.77a
a
LL, lower shade leaves B1 m height; ML, medium leaves 1–3 m height; UL, upper sun leaves \5 m height. Values
are the mean for 250 measurements. For each row, values which have the same letter are not significantly different at the
0.05 level as determined by Student–Newman–Keuls’ test.
4. Discussion
