been reported for many species and related espe- cially to the amount of sun exposure or water
availability. Kaufmann and Troendle, 1981; Niinemets and Kull, 1994; Smith et al., 1997.
Elaeagnus angustifolia L. Russian olive is a Eurasian tree that has become naturalized, form-
ing monotypic stands along the watercourses in the Rı´o Negro valleys of Argentina. This species
is known for its capacity to grow over a wide range of environmental conditions. For example,
seedlings are tolerant of shade and mature trees can
live exposed
to high
light intensities
Shafroth et al., 1995; Lucchesini and Mensuali- Sodi, 1996. E. angustifolia can displace native
woody species and has been so successful in colonising disturbed areas and old fields, that its
use is prohibited in some areas Dawson, 1990. The ability of E. angustifolia to establish, grow
and invade new areas has led to investigations of the conditions that might favor its spread
Shafroth et al., 1995.
Visiting the invaded zone, I noticed that within clustered individuals of E. angustifolia there were
variations in form and color between leaves growing at different levels in a tree. I evaluated
the environmental
heterogeneity within
the canopy of trees growing along the Rio Negro
watercourse in this dry-region. Anatomical and morphological studies were performed in order
to prove if the externally observed leaf differ- ences were correlated with internal adaptations. I
tested the hypothesis that variations in develop- mental responses of the E. angustifolia leaves to
spatial heterogeneity are related to the ecological strategies of this invasive species.
2. Material and methods
The study was conducted during three growth periods 1995 – 1998, using leaf samples origi-
nated from a stand of E. angustifolia growing at the margin of the Rı´o Negro, Argentina 39°30
S, 65°30 W in an area where the expansion of this species has been notable in the last 20 years.
The climate is temperate semiarid to cold arid. The average temperature during the coldest
month July is 6.83°C and during the hottest month January is 23.02°C. The average annual
precipitation is 300 mm, most falling during the spring and autumn. Average relative air humidity
ranges from 48 January to 70 June. The average annual evapotranspiration is \ 800 mm,
with a negative water balance throughout the year. The former regional climatic data were ob-
tained from the Meteorological Station at Fray Luis Beltra´n, which is 30 km away from the
study site. Soils are alluvial and occasionally sub- jected to flooding. During the study, the local
data of ambient air temperature and humidity were recorded with an hygrothermograph and
those of solar radiation and rain with an auto- matic data recorder KADEC-U, Kona System,
Sapporo, Japan. Soil water content was deter- mined monthly by gravimetry.
Undamaged leaves of ten well-developed trees 8 – 9 m height were collected from the upper
sun-exposed crown from 5 m up, the medium half sun-exposed branches between 1 and 3 m
height and the lower shaded crown B 1 m height.
Leaf water content LWC during the growing period was determined by collecting : 100 g of
leaf material at each level of the ten trees. The fresh weight was determined in recently cut
leaves and the dry weight after heating them at 60°C for 48 h. Sampling was made in October
after the beginning of the growing period, in December, in February and finally in April, just
before the initiation of the cold latency period. Leaf blade size was determined with a portable
area meter LI-COR, LI 3000A from ten leaves of each level of the ten trees.
Foliar architecture was defined according to Hickey Hickey, 1974; Dilcher, 1974 and in-
cluded measurements of leaf morphology and ve- nation patterns. Cleared leaves — five leaves of
each level of the ten trees — were obtained by boiling formol-acetic acid-alcohol FAA-fixed
leaves in 5 sodium hydroxide, decolorized with 10 sodium hypochlorite, cleared in saturated
chloral hydrate, rinsed thoroughly in water, dehydrated through an alcohol series and stained
in saturated safranin in alcohol 50. After suc- cessive steps in an alcohol series to xylene, each
leaf was mounted with Canada balsam between
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
