Leaf variations in Elaeagnus angustifolia related to ... - Semantic Scholar

Environmental and Experimental Botany 44 (2000) 171 – 183 www.elsevier.com/locate/envexpbot

Leaf variations in Elaeagnus angustifolia related to environmental heterogeneity Marıá Guadalupe Klich * Departamento de Agronomıá, Centro de Recursos Naturales Reno6ables de la Zona Semia´rida (CERZOS), Uni6ersidad Nacional del Sur, C.C. 738, 8000 -Bahıá Blanca, Argentina Received 3 August 1999; received in revised form 6 April 2000; accepted 10 April 2000

Abstract Elaeagnus angustifolia (Russian olive) is a Eurasian tree that has become naturalized and has invaded zones along watercourses in many arid and semiarid regions of the world. These habitats are characterized by vertical environmental gradients, thus trees must develop some plasticity to adapt to the wide range of site conditions. This study was undertaken to test the hypothesis that variations in leaf anatomy and morphology of E. angustifolia reflect their adaptability to the differences in the microclimate that occur within the canopy of single trees. Foliar architecture, blade and petiole epidermal and internal anatomy were examined in leaves at different canopy positions and related to environmental conditions. Upper sun-leaves are exposed to higher solar irradiance and lower air humidity and are smaller, more slender and thicker than the lower, half-exposed and shade-leaves. Color varies between the leaves at different levels, from silvery grey-green in the upper strata, to dark green in the lower one. Bicolor is more evident in half-exposed and shaded leaves. When compared with the lower half-exposed and shade-leaves, the upper leaves of E. angustifolia have a greater areole density, a higher mesophyll proportion and stomatal density. Trichomes are multicellular, pedestalled, stellate-branched or peltate and their form and density can be associated with leaf color and appearance. The slender petioles of the upper leaves have proportionally more epidermis, collenchyma and phloem and less parenchyma and xylem than those of lower leaves, when observed in transverse sections. Foliar morphological and anatomical variability in E. angustifolia may be considered an adaptive advantage that enables leaves to develop and function in habitats marked by strong variations of solar radiation, air temperature and humidity. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Leaf heterogeneity; Leaf architecture; Phenotypic plasticity; Water stress; Xeromorphy; Canopy microclimate

1. Introduction Developmental responses to small-scale environmental heterogeneity can be important for * Corresponding author. Fax: +54-291-4541224. E-mail address: [email protected] (M.G. Klich).

plant adaptation (Novoplansky, 1996). Leaves are the plant organs most exposed to aerial conditions and the changes in their characters have been interpreted as adaptations to specific environments (Fahn and Cutler, 1992). Variations in the morphological and anatomical features of leaves developed at different levels in the plants have

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M.G. Klich / En6ironmental and Experimental Botany 44 (2000) 171–183

been reported for many species and related especially 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, forming monotypic stands along the watercourses in the Rıó 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 MensualiSodi, 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 differences were correlated with internal adaptations. I tested the hypothesis that variations in developmental 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 originated from a stand of E. angustifolia growing at the margin of the Rıó 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 obtained from the Meteorological Station at Fray Luis Beltrań, which is 30 km away from the study site. Soils are alluvial and occasionally subjected 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 automatic data recorder (KADEC-U, Kona System, Sapporo, Japan). Soil water content was determined 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 included measurements of leaf morphology and venation 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 successive 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 enhanced so that the determination of the venation’s pattern could be easily performed. Drawings of the smaller veins, veinlets and areoles were made using a Wild M 5 stereoscopic microscope and a Wild M 20 binocular microscope, both with drawing tubes. Microphotographs 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 volume, 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 material, 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 recommendations of Kubıńova´ (1993). Data were obtained from five transverse sections per leaf in five leaves of each of the three levels of the ten trees.

Fig. 1. Mean daily solar radiation (cal. cm2/day) and maximum hourly solar radiation (cal. cm2/h) registered during the period April 1997 to March 1998 in the upper canopy of the E. angustifolia trees.

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Leaf material for scanning electron microscopy (SEM) was fixed in FAA. Samples were dehydrated 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 temperature outside the shaded understorey reached higher values, up to a maximum of 42°C in January 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 measures of daily solar radiation as well as the maximum 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 cm2/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 growing period at all levels in the plants, but while in spring the higher values were those of half-ex-

33.6 26.3 17.6 33.8 16.1 7.0 11.4 10.0 13.9

27.8 16.5 14.1 30.9 15.9 11.3 14.0 14.3 17.4

9.3 15.9 19.7 29.0 13.7 11.8 11.0 15.3 18.3

13.3 14.3 17.3 20.6 23.9 25.4 22.3 21.6 18.1

1.5 1.7 4.8 8.1 10.1 11.4 10.5 6.9 6.5

7.4 8.0 11.1 14.3 17.3 18.4 16.4 14.2 11.9

AT (°C) (lower height) (max, min, mean) 53 51 41 37 52 36 56 48 48

100 100 100 100 100 100 100 100 100

% AH (lower height) (min, max)

30 30 30 28 25 18 21 35 34

81 73 69 63 61 60 69 80 84

% AH (upper height) (min, max)

4.5 17.0 28.0 16.0 30.0 3.5 47.5 22.5 19.5

Rain (mm)

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).

August September October November December January February March April

% SWC (20, 40 and 60 cm depth)

Table 1 Environmental conditions of the sampling area during the growth period (1997–1998) of E. angustifolia a

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M.G. Klich / En6ironmental and Experimental Botany 44 (2000) 171–183 Table 2 Comparison of leaf water content (LWC%) of E. angustifolia leaves developed at different heights in a treea

October December February April

LL

ML

UL

75.90b 74.54b 62.07b 51.13a

74.11b 73.27b 57.90a 51.10a

70.20a 68.99a 57.54a 55.81b

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.

posed and shade-leaves, in April these were the leaves that bore the lower values (Table 2). The leaves of E. angustifolia are annually deciduous, however, many of the upper leaves may persist when winters are wetter and warmer than normal. New leaves are produced during late winter 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 inclined, especially at midday, while the medium and lower leaves are nearly perpendicularly oriented 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 surfaces 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

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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 reticulate. The higher order of venation is quaternary and a looped marginal ultimate venation is observed. The areoles are imperfect, pentagonal to polygonal and randomly arranged. In the upper leaves, the density of areoles is higher (17 areoles/ mm2) than in the medium and lower ones (15 and 14 areoles/mm2, 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 organized stratum is observed in the upper leaves (Fig. 2b,d,f). Spongy mesophyll cells are round or elongate, not lobed and randomly oriented. The proportion 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 palisade 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 pentagonal or hexagonal epidermal cells are smaller in the lower than upper epidermis. Adaxial epidermis 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 differences 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

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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 treea

Lamina area (one side) cm2 Lamina length Shape: whole lamina Shape: base only Form: whole lamina (l/w: length/width) Form: base only Form: apex only Margin Texture Appearance Attachment (petiole) Type of venation Size of primary vein % vein width/leaf width Course of primary vein Angle of divergence of secondary veins Variation in angle of divergence of secondary veins Relative thickness of secondary veins Course of secondary veins Pattern of tertiary veins Adaxial epidermal thickness (mm) Adaxial epidermal outer wall thickness (mm) Abaxial epidermal thickness (mm) Abaxial epidermal outer wall thickness (mm) Stomata/mm2 Guard cell length (mm) Guard cell width (mm) Abaxial indumentum height (mm) a

Lower shade leaves (B1 m height)

Medium leaves (1–3 m height)

Upper sun leaves (\5 m height)

Microphyll-mesophyll from 4.6 to 23.7 Upto 10 cm. Symmetrical Symmetrical Ovate: ovate (l/w: 1.5:1) narrow-ovate (l/w: 2:1) lanceolate (l/w: 3:1) Acute cuneate rounded to cordate Acute, obtuse or obtuse mucronate Entire Chartaceous Adaxial dark green (notable bicolor) Normal Pinnate eucamptodromous Moderate (1.25–2%) to stout (2–4%) Straight (some slightly curved) Acute: narrow (B45°) to moderate (45–65°) Lower and upper secondary veins more obtuse than middle sets Moderate

Microphyll from 7.5 to 18.7

Microphyll from 4.2 to 10.5

Upto 10 cm. Symmetrical Generally symmetrical Ovate: lanceolate (l/w:3:1)

Upto 10 cm. Symmetrical Symmetrical Oblong: narrow-oblong (l/w:3:1) Elliptic: narrow-elliptic (l/w:3:1) Acute normal

Curved Random reticulate 43.16b

Acute cuneate obtuse decurrent Acute or obtuse

Acute

Entire Chartaceous Adaxial light green (notable bicolor) Normal Pinnate eucamptodromous Stout (2–4%)

Entire Chartaceous Silvery grey–green (bicolor)

Acute: narrow (B45°) to moderate (45–65°) Uniform

Moderate

Acute: moderate (45–65°) to wide (65–80°) Lower and upper secondary veins more obtuse than middle sets Moderate

Curved Random reticulate 42.90b

Straight to curved Random reticulate 29.37a

Normal Pinnate eucamptodromous Stout (2–4%) to massive (\4%) Straight (some slightly curved) Straight

5.36a

5.02a

9.09b

18.18a

20.31a

16.99a

2.72a

2.49a

3.01a

247a 16.96a 10.10a 327b

270b 16.94a 10.07a 341b

312c 17.02a 10.11a 257a

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.


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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 composed of hairs totally branched or stellate with cells arranged radially and joined near the trichome 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 indumentum 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, although 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 enlargement of the proportion of epidermis, collenchyma and phloem while the relative amount of parenchyma and xylem diminishes (Table 5). Petioles are slender in the upper leaves. In transverse sections through the basal end, they exhibit


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Table 4 Comparison of leaf dimensions and tissue compositions of E. angustifolia leaves developed at different heights in a treea LL Thickness (mm) (without hairs) Area (mm2) Volume (mm3) % Mesophyll % Palisade % Spongy

ML 0.326a

1250.08b 407.53a 73.97a 60.23a 39.77a

0.382b 1132.00a,b 432.42a 74.93a 58.40a 41.60a

UL 0.474c 955.91a 450.82a 81.86b 62.62a 37.38a

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.

an open vascular arc and it becomes incurved to the distal end. Though the vascular strand nearly forms a cylinder in the petioles of the upper leaves, the vascular arc remains slightly open (Fig. 5). Trichomes on petiole epidermis are in accord with those of the corresponding leaf.

4. Discussion The environmental measurements made during the study demonstrate the great variation in the habitat conditions to which the leaves at different levels are exposed. As the area is included in a semiarid to arid region, summer temperature outside the shaded understorey reaches much higher values than under the canopy. Differences are also found in air humidity; while in the lower strata it reaches saturation every night, at the upper exposed canopy the values are always lower. The high solar radiation is greatly attenuated by the foliage in such a way that the lower leaves can really be considered as growing in the shade. As there is an exponential relationship between light absorption and cumulative foliage area, the light gradient across the canopy should be greater for higher values of incident irradiance (Niinemets, 1996b). If the climatic data are considered in a general regional sense, it can be assumed that the scarcity of soil water resources and/or the high evaporative demand of the atmosphere during the warmer seasons induce stress situations. The survival of

Fig. 3. Drawings of abaxial (a,c,e) and adaxial (b,d,f) epidermis (amidst trichomes) of E. agustifolia. (a,b) Lower shade leaves; (c,d) medium half sun-exposed leaves; (e,f) upper sun-leaves. (Bar: 10 mm).


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Fig. 4. Scanning electron micrographs of the epidermis of E. angustifolia. (a,b,c) Lower shade leaves; (d,e,f) medium half sun-exposed leaves; (g,h,i) upper sun-leaves. (a,b,d,e,g,h) Abaxial epidermis; (c,f,i) adaxial epidermis.

the plants to adverse conditions requires longand short-term plasticity responses and trees may develop stress avoidance mechanisms by an adequate canopy architecture (Save´ et al., 1995), root development (Fernańdez et al., 1988) or leaf heterogeneity (Niinemets, 1996a). When light is the limiting factor, since trees have the potential to reach unshaded overstorey, the adaptability of crown architecture may be an important determinant of competitive strength than foliar plasticity (Niinemets, 1996b). However, when trees develop at different strata, the canopy of the same plant is exposed to diverse natural environments. Thus, the leaf morphoanatomical plasticity with respect to the combined conditions of incident irradiance, air temperature and humidity may be one of the

Table 5 Comparison of petiole tissue composition (%) of E. angustifolia leaves developed at different heights in a treea

Collenchyma Phloem Parenchyma Xylem Epidermis

LL

ML

UL

10.04a 16.44a 54.14c 9.42c 9.94a

16.97b 16.30a 48.58b 8.82b 9.32a

23.90c 18.75b 35.90a 7.20a 14.30b

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 100 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.

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Fig. 5. Transverse sections through the basal (a,c,e) and distal (b,d,f) end of the petioles of E. angustifolia. (a,b) Lower shade leaves; (c,d) medium half sun-exposed leaves; (e,f) upper sun-leaves. (External white: epidermis; squared: collenchyma; internal white: parenchyma; dashed: xylem; dotted: phloem). (Bar: 150 mm).

features that confers the species success in their permanence and even in their competition with other species. The changes in the dimensions — especially the width — of the leaves of E. angustifolia, can be considered to have a functional significance related to the different natural conditions in which they developed. The shaded as well as the half-exposed branches, with wider ovate leaves, develop in an environment of low irradiance, relatively high air humidity content and are protected from the wind. The upper narrow oblong or elliptic leaves are exposed to high light, low air humidity and dry winds. The reduction of leaf dimension generates an increase in convective heat dissipation, that is important to counteract the negative effects of overheating and high transpiration rates (Gates, 1980) such as those to which the upper leaves of E. angustifolia are exposed.

Upper leaves of E. angustifolia are inclined, especially at midday, while the medium and lower leaves are nearly perpendicularly oriented to the incident light. This characteristic may be related to the high values of irradiation registered in the region and to which the upper leaves are exposed, for it has been determined that high light has a detrimental impact on photosynthetic performance (Smith et al., 1997) and leaf movements tending to avoid full exposition partially counteract these harmful effects. Furthermore, during the higher irradiation periods, upper leaves upfold through the midvein. Lower shade leaves, on the other hand, must capture as much light as possible and their orientation helps to fulfill this requirement. Although narrow blades and inclined leaves contribute to the avoidance of mutual shading between leaves (Yamada and Suzuki, 1996), the solar radiation reaching the


under storey of E. angustifolia clusters is nevertheless greatly attenuated. Bicolor is a common phenomena among species that occupy shaded habitats; the leaf side that faces away from the sun is lighter in color than the leaf surface facing towards the sun. The lighter surface may act as a reflective surface and enhance the participation of the spongy mesophyll in leaf photosynthesis. Smith et al. (1997) suggest that the presence in shaded environments of thin, horizontal, bicolor laminar leaves that have stomata only in the abaxial epidermis may have evolved as a result of a selective pressure to enhance light capture while avoiding the detrimental effect of exposing the stomata directly to sunlight and minimizing transpirational water-loss. In E. angustifolia the difference in color between both leaf surfaces is especially evident in the two lower levels, but it also occurs in the upper level. As sun leaves have a silvery grey – green adaxial face and a brilliant silver colored abaxial one and as they upfold through the medium vein under the higher solar radiation conditions, they mainly expose this highly reflective surface. Though there are some variations in the gross morphology of the leaves of E. angustifolia at different levels in a tree, the pattern of major venation is the same in all of them, which is logical, as the latter is a character considered of systematic value (Dilcher, 1974). Increased vein density is positively correlated with water stress in the habitat (Pyykko¨, 1966) and the higher areole density in the upper leaves of E. angustifolia may be a response to the dryer habitat to which they are submitted. The proportionally greater primary vein size of upper leaves means an additional mechanical advantage under those stress environmental conditions. The use of only the size and morphology of the leaf as indicators for xeromorphy or plasticity is insufficient and anatomical features, such as the volume and organization of the mesophyll tissue, should be taken into consideration (Fahn and Cutler, 1992). The greater thickness of the upper sun E. angustifolia leaves is related to a higher proportion of mesophyllic tissue. These characteristics are mentioned as structural mechanisms that increase photosynthesis per unit leaf area and

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enable a greater water-use efficiency. Leaves in the upper part of the canopy or sun leaves have higher rates of carbon assimilation and water loss and are thus, physiologically more active (Boardman, 1977). Leaves exposed to high irradiation conditions or sun leaves generally develop a well-defined palisade parenchyma and those that grow under low irradiation condition or shade leaves, are thinner and present a less defined palisade layer than the former (Vogelmann, 1993). The tissue distribution in the mesophyll remains constant among the studied E. angustifolia leaves, but the average diameter and length of palisade cells are smaller in the upper ones in which an incomplete third palisade stratum is a common feature. A higher number of palisade cells in the mesophyll volume may imply an increase in photosynthetic efficiency. The columnar cells of the palisade tissue, beside their contribution in the CO2 exchange, may have also an optical function (Vogelman, 1989). The directionality of light can affect light gradients within leaves. Vogelman and Martin (1993) stated that palisade appears to facilitate the penetration of directional but not diffuse light. This is particularly important in leaves exposed to direct sunlight, where the irradiance is highly collimated. The epidermis and cuticle strengthen the leaf. The epidermal thickness of the upper, more-exposed leaves of E. angustifolia is smaller when compared with the other leaves, but has a proportionally greater outer tangential wall (measured as wall plus cuticle) and this feature has been related to increased aridity and/or insolation (Pyykko¨, 1966; Fahn and Cutler, 1992). No differences were found in the size of stomata but their density increase from the lower to the upper leaves of E. angustifolia. Size and density of stomata have been widely studied and related to many environmental factors (Salibury, 1927; Shields, 1950; Klich et al., 1996a,b). Small stomata in large numbers seemed to be characteristics of xeromorphic leaves (Shields, 1950); however, Meidner and Mamsfield (1968) found a great stomatal frequency variation among mesophytes, so that the efficiency of stomata in regulating water loss could not be directly related to their size and frequency.

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Pubescence is another characteristic that confers stress resistance (Ehleringer, 1980; Klich et al., 1996b) and the presence of hairs on the aerial parts of the plants is regarded as an adaptation to arid conditions (Fahn, 1986). Hairs may affect transpiration by influencing the water diffusion boundary as an indumentum decreases air movements on the leaf surface, creates a zone of still air and reduces diffusion of water vapor from the leaf interior to the atmosphere. An indirect influence of trichomes on plant water economy is through temperature regulation (Ehleringer, 1980). The microroughness caused by anatomical characteristics, as epidermal cell surface contours and dense trichome layers, substantially increases leaf reflectance and reduces radiation absorption which results in the reduction of leaf temperature and consequently of leaf transpiration rates (Premachandra et al., 1991, McWhorter, 1993; Karabourniotis et al., 1995; Klich et al., 1997). The abaxial epidermis of all the leaves of E. angustifolia is densely covered by hairs and although the indumentum height of the lower leaves is greater than that of the upper leaves, the trichomes of the latter, with their overlapping flattened shields, may constitute a stronger barrier to gas diffusion. The lower leaves of E. angustifolia are the most sensitive to environmental changes and when soil water content decreases or when these leaves become exposed because of the removal of neighboring plants, the trees shed the leaves of the inferior branches (personal observations, unpublished data). Lower strata are also the first to shed their leaves at the end of the growth period, initiated by a decrease in their water content. The higher proportion of epidermis and collenchyma of the upper leaf petioles is not unexpected, as Pyykko¨ (1966) already confirmed a correlation between strong development of mechanical tissue and habitat aridity. Leaf variability in E. angustifolia can be considered an adaptive advantage of both upper and lower leaves in habitats marked by strong variations of sun radiation, air humidity and temperature and wind exposition. Upper leaves show many xeromorphic characters that enables the trees to maintain their canopy foliage even under

the unfavorable conditions — high solar radiation, high temperature, low humidity — during the summer. Lower leaves show many traits of shaded leaves and allow the plant to compete for space also in the understorey. These data confirm the hypothesis that variations in developmental responses of E. angustifolia leaves to spatial heterogeneity are related to its ecological strategies. This invasive species relies, at least partially, on its foliar plasticity to overcome the environmental gradient between the lower and upper parts of a developed tree and even to compete against other species when it grows in places where spatial environment heterogeneity can easily manifest, as in a river valley of a semiarid region. Acknowledgements I would like to thank Marıá Elena Garcıá and Lucrecia Gallego for their technical assistance and their help with the drawings; Federico and Luis Fresser for their help with field temperature and humidity measurements and the technical assistance of the Electron Microscopy Laboratory of the Centro Regional de Investigaciones Ba´sicas y Aplicadas de Bahıá Blanca (CRIBABB), Argentina. This research was supported by a grant from the Universidad Nacional del Sur, Bahıá Blanca, Argentina References Boardman, N.K., 1977. Comparative photosynthesis of sun and shade plants. Annu. Rev. Plant Phys. 28, 355 – 377. Dawson, J.O., 1990. Interactions among actinorhizal and associated plant species. In: Schwintzer, C.R., Tjepkema, J.D. (Eds.), The Biology of Frankia and Actinorhizal Plants. Academic Press, New York, pp. 299 – 316. Dilcher, D.L., 1974. Approaches to the identification of angiosperm leaf remains. Bot. Rev. 40, 1 – 157. Ehleringer, J., 1980. Leaf morphology and reflectance in relation to water and temperature stress. In: Turner, N.C., Kramer, P.J. (Eds.), Adaptations of plants to water and high temperature stress. Wiley, New York, NY, pp. 295– 308. Fahn, A., 1986. Structural and functional properties of trichomes of xeromorphic leaves. Ann. Bot. (Lond.) 57, 631 – 637.

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