- Email: [email protected]
The wax layer and its morphological variability in four European Salix species
Flora 199, 320–326 (2004) http://www.elsevier.de/flora
The wax layer and its morphological variability in four European Salix species Dominik Tomaszewski Institute of Dendrology, Polish Academy of Sciences, ul. Parkowa 5, Kórnik PL-62-035, Poland Submitted: Aug 15, 2003, in revised form: Nov 17, 2003 · Accepted: Feb 4, 2004
Summary The structure of the wax layer on leaves was analysed in four species of the genus Salix (S. alba, S. fragilis, S. triandra and S. pentandra). SEM observations showed that peculiar conical structures (named here ‘conicoids’) are present on the lower surface of the leaves in three species. In S. fragilis and S. triandra, conicoids are similar in size, whereas in S. alba, they are several times larger. In S. pentandra no such structures were found and the cuticle was covered only with an amorphous wax film. The observed differences between species can be used as identifying features in this group of plants. A comparison of conicoids on successive leaves on the shoot revealed differences and shed some light on the process of their formation. Key words: Salix, SEM, epicuticular wax, leaf surface, morphology
Introduction Epicuticular waxes are a very broad group of hydrophobic substances occurring on the surface of plant organs. Both their morphological structure and chemical composition are extremely variable. The epicuticular wax layer is always present in terrestrial plants but it can be formed in different ways. Plant waxes have many functions such as: (1) protecting the plant against penetration by pathogens; (2) decreasing the loss of solutes by reducing water retention on leaf surfaces; (3) reducing the loss of water caused by transpiration ; (4) diminishing the risk of injury due to excessive radiation by reflecting and attenuating sunlight; etc. It is noteworthy that the wax layer forms the external covering of the plant, so it is the first barrier against all factors that come into contact with the plant: insects, fungi, bacteria, solar radiation, etc. This is linked with the significance of this layer for protection of the plant against pests and diseases, especially insects and fungi. The chemical composition and structure of epicuticular waxes mediate the plant’s attractiveness to pests.
Morphology and chemical composition of the wax layer depend not only on the plant taxon but also on geographic location, environmental conditions, and stage of development (e.g. Wilkinson 1980; Safou et al. 1988; Prasad & Gülz 1990; Gülz et al. 1991; Gülz & Boor 1992; Gülz & Müller 1992; Gülz 1994). The usefulness of characteristic features of the wax layer in taxonomy has been confirmed many times (e.g. Tulloch 1976; Barthlott & Theisen 1995; Theisen & Barthlott 1996; Uzunova 1999; Wissemann 2000; Heinrichs et al. 2000). In this project, the morphological variability of the wax layer was investigated in some Salix species. The genus Salix L., together with Populus L. and Chosenia Nakai, forms the family Salicaceae. Salix is represented in the family by many more species (300–500) than the other two genera. Salix species were grouped by Skvortsov (1968) into three subgenera: Salix, Chamaetia and Vetrix. This paper is concerned with four species of the subgenus Salix, which are the most frequent in Europe: i.e. S. alba L., S. fragilis L., S. triandra L. and S. pentandra L. Preliminary observations
* Corresponding author: Dominik Tomaszewski, Institute of Dendrology, Polish Academy of Sciences, ul. Parkowa 5, Kórnik PL-62-035, Poland, e-mail: [email protected] 320
FLORA (2004) 199
0367-2530/04/199/04-320 $ 30.00/0
A
B
C
D
Fig. 1. Wax structures (‘conicoids’) on the abaxial leaf surface: A, B. Salix triandra, C. S. alba, D. S. fragilis. They are composed of apically convergent wax filaments. [A, B – Silesia, Eastern Sudeten, Golden Mts., 300 m, 17. 10. 1995, leg. Piotr Kosi´nski, KOR 41851; C – Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002; D – Grand Poland, between Błaz˙ejewko and Bnin, 10. 10. 2001]
revealed the existence of variability of wax structures on the lower (abaxial) leaf surface. The main objective of this study was to determine if this variability is related to leaf age and taxonomic classification. The morphological structure of the epicuticular wax layer was analysed on leaves of these species at successive stages of leaf development. In addition, the usefulness of features of the wax layer for species identification was assessed. Another objective was to determine the optimum conditions for collecting comparative material, which could be important for the methodology of research on the chemical composition of epicuticular waxes in this genus. So far, these questions have been discussed in only a few publications (Paiero et al. 1984; Paiero et al. 1985; Martini & Paiero 1988 ; Hietala et al. 1997; Cameron et al. 2002).
Material and methods Material for this study consisted of leaves of four Salix species (S. alba L. (subsp. alba), S. fragilis L., S. triandra L. and S. pentandra L.) collected from individuals growing in Poland, mainly in the Grand Poland District (western part of the country). The material was checked critically by Prof. Jerzy Zieli´nski from the Institute of Dendrology (Polish Academy of Sciences) in Kórnik. Leaf samples were coated with gold at the Department of Animal Anatomy, Agricultural University of Pozna´n, and viewed with a Hitachi S3000N SEM belonging to the Institute of Plant Protection in Pozna´n. Dried leaves (usually recently collected) were used, as the morphology of the studied structures does not change substantially during drying at room temperature. The structures are very durable, which is evidenced by their low solubility in the standard organic solvents used for wax extraction. FLORA (2004) 199
321
A
B
C
D
Fig. 2. Scanning electron micrographs of abaxial leaf surfaces. A comparison between: A. Salix pentandra, B. S. alba, C. S. fragilis & D. S. triandra. The most distinct is S. pentandra, where the wax layer is present as an amorphous film. S. alba has very large conicoids with a loose filamentous structure, and the smallest differences exist between S. triandra and S. fragilis. Magnification for all the micrographs is the same. [A – Grand Poland, Dolsk, 19. 07. 2002; B, C, D – Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002] Samples from the mature blades were collected to compare the wax structures on successive leaves. For observations of the development of wax structures, successive leaves from the terminal part of the shoot were compared. Additionally, always a fragment of the blade of a very young leaf was dissected for analysis, and the remaining part was left on the shrub. After a few days, another fragment was dissected from the undamaged half of the leaf. Both fragments were taken at half the length of the leaf blade.
Results and discussion The investigations confirmed the existence of substantial differences between the analysed species in the structure of the wax layer. In this group of species, the most distinct is S. pentandra, where the wax layer is present on both the upper and the lower surface of the leaves only as an amorphous film. In the other species, 322
FLORA (2004) 199
the upper surface of the leaf blade is also covered with a uniform wax film, but the lower surface is characterized by the presence of specific wax structures. The structures cannot be assigned to any of the types distinguished in the classification proposed by Barthlott et al. (1998). They are visible in micrographs found in a paper by Martini & Paiero (1988), but those authors did not discuss this feature. Paiero et al. (1985) briefly described the leaf underside as follows: ‘abaxial epidermis covered with a thick, discontinuous wax deposit in the form of vertical scales’. However, this description is not precise enough. “Conicoids” is the name proposed here for this unique type of wax structure in Salix species, because of their conical shape. The analysis showed that conicoids are formed in the species in different ways, but the general plan of formation is the same. They are composed of apically convergent wax filaments (Fig. 1).
Fig. 3. The hypothetical scheme of development of conicoids. Initially the cuticle is covered with a homogenous, continuous wax layer (A), which is getting thicker from the base by accumulating new material. The increase from the base is accompanied by horizontal expansion due to leaf development (B & C). The expansion causes the splitting of the wax layer into patches. D – mature conicoids.
Fig. 4. The inner structure of the conicoid of S. alba revealed after rinsing the material in chloroform. It can be observed that the conicoid is composed of filaments divided into thinner filaments in the lower, younger parts of the structure. [Grand Poland, between Błaz˙ejewko and Jeziory Wielkie, 10. 10. 2001]
S. alba has very large conicoids with a loose filamentous structure. The smallest differences exist between S. triandra and S. fragilis, but both species can be distinguished on the basis of the formation of their wax layer. Although the maximum diameter of the conicoids is more or less the same in both taxa (ca. 11–14 µm), in S. fragilis these structures are looser and more often are split into smaller parts. Furthermore, there are no “splinters”, which are very characteristic for S. triandra, i.e. small platelets or appendices of another shape, which are often arranged on the upper surface of conicoids, on the lateral walls and even on their base (Fig. 2). The above-mentioned wax structures did not appear on leaf margins or major veins. In the light of the results presented above as well as preliminary observations in other groups of Salix species and published data (Martini & Paiero 1988), it is surprising that S. pentandra lacks conicoids. This may be linked with the fact that this species is relatively primitive among the willows, especially among the studied group (Szafer 1959).
accumulating new material. The increase from the base, i.e. from the cuticle, lasts throughout the conicoid’s growth and determines the height of these structures, but it is accompanied by horizontal expansion due to leaf development (Figs. 3 B & C). The expansion causes the splitting of the originally continuous wax layer into patches, and later leads to the fragmentation of the already partly formed conicoids. The common feature of the discussed structures is the fact that the thick filaments are linked with one another in their apical parts, which is the result of the above-described process: it is a remnant of the earliest stage of growth, when the wax, later building the top of the conicoids, constituted an approximately homogenous layer. That part of the cuticle from which the material for the formed conicoid was extruded, was gradually extended, but the filaments originating from them remained connected at the oldest, upper part. This hypothesis is also indirectly confirmed by the image of conicoids of S. alba after rinsing the material in chloroform (Fig. 4). The micrograph shows the inner structure of the conicoid. It is composed of filaments divided into thinner filaments in the lower, younger parts of the conicoid.
Development of conicoids A hypothetical scheme of development of conicoids can be summarized as follows. Initially the cuticle is covered with a homogenous, continuous wax layer (Fig. 3 A), which is getting thicker from the base by
Wax layer on successive leaves on the shoot The structure of the wax layer depends on leaf position on the shoot. On the oldest 3–4 leaves, called cataFLORA (2004) 199
323
A
B
C
D
Fig. 5. The wax layer on successive leaves on a shoot of Salix alba: A. the first leaf (the oldest one), B. the 2nd, C. the 3rd, D. the 4th. Conicoids are present even on the oldest leaf, but are smaller there and their structure is not the same as on younger leaves. [Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002]
phylls, its development stops at a very early stage of growth. On the successive leaves, conicoids have a structure and size typical for the particular given species. On the youngest leaves, which are not fully developed yet, the process of wax formation is ongoing, so its structure is not the same as in fully developed leaves. This means that only leaves from the middle part of the shoot should be used for comparisons between species. Changes in the structure of the wax layer on successive leaves are presented for two species. In S. alba, conicoids are present even on the oldest leaf (cataphyll), but are smaller there and their structure is not the same as on adjacent, younger leaves (Fig. 5). In S. triandra, well-developed wax structures may be formed on the oldest leaf but the wax layer is uneven there, forming ‘islands’. At the centre of an island, typical conicoids can be seen, but at the edges of the islands they are much 324
FLORA (2004) 199
smaller and gradually pass into amorphous wax film between the islands. The proportion of leaf area covered by conicoids on successive leaves gradually increases. Starting from the third leaf, the whole adaxial surface is covered by conicoids (Fig. 6). However, the last leaves (the youngest ones) in the terminal part of the shoot, i.e. those developed in early autumn, show some regression. Their blades sometimes are totally or partially free of conicoids.
Acknowledgements The work was supported by the State Committee for Scientific Research (project no. 6P04C 075 21). I am grateful to Magdalena Gawlak for her excellent technical assistance and Prof. Jerzy Zieli´nski for checking the herbarium material.
A
B
C
Fig. 6. The wax layer on successive leaves on a shoot of Salix triandra. The proportion of leaf area covered by conicoids on successive leaves gradually increases. A. the first leaf (the oldest one), B. the 2nd, C. the 3rd. [Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002]
References Barthlott, W.; Neinhuis, C.; Cutler, D.; Ditsch, F.; Meusel, I.; Theisen, I. & Wilhelmi, H. (1998): Classification and terminology of plant epicuticular waxes. – Bot. J. Linn. Soc. 126: 237–260. Barthlott, W. & Theisen, I. (1995): Epicuticular wax ultrastructure and classification of Ranunculiflorae. – Plant Syst. Evol., Suppl. 9: 39–45. Cameron, K. D.; Teece, M. A.; Bevilacqua, E. & Smart, L. B. (2002): Diversity of cuticular wax among Salix species and Populus species hybrids. – Phytochemistry 60: 715–725. Gülz, P. G. (1994): Epicuticular leaf waxes in the evolution of the plant kingdom. – J. Plant Physiol. 143: 453–464. Gülz, P. G. & Boor, G. (1992): Seasonal variations in epicuticular wax ultrastructures of Quercus robur leaves. – Z. Naturforsch. Sect. C 47: 807–814. Gülz, P. G. & Müller, E. (1992): Seasonal variation in the composition of epicuticular waxes of Quercus robur leaves. – Z. Naturforsch. Sect. C 47: 800–806. Gülz, P. G.; Müller, E. & Prasad, R. B. N. (1991): Devel-
opmental and seasonal variations in the epicuticular waxes of Tilia tomentosa leaves. – Phytochemistry 30: 769–773. Heinrichs, J.; Anton, H.; Gradstein, S. R.; Mues, R. & Holz, I. (2000): Surface wax, a new taxonomic feature in Plagiochilaceae. – Plant Syst. Evol. 225: 225–233. Hietala, T.; Mozes, N.; Genet, M. J.; Rosenqvist, H. & Laakso, S. (1997): Surface lipids and their distribution on willow (Salix) leaves: a combinated chemical, morphological and physicochemical study. – Colloids Surf. B Biointerfaces 8: 205–215. Martini, F. & Paiero, P. (1988): I salici d’Italia. – Edizioni Lint, Trieste. Paiero, P.; Mariani, P. & Marzi, M. (1984): Preliminary observations on microfeatures of leaf surface in Salix species. – G. Bot. Ital. 118: 84–85. Paiero, P.; Mariani, P. & Urso, T. (1985): Formazione di depositi cerosi in foglie di Salix L. – G. Bot. Ital. Suppl. 119: 112. Prasad; R. B. N. & Gülz, P. G. (1990): Developmental and seasonal variations in the epicuticular waxes of beech leaves (Fagus sylvatica L.). – Z. Naturforsch. Sect. C 45: 805–812. FLORA (2004) 199
325
Safou, O.; Saint Martin, M. & Rouane, P. (1988): Stomates et cires dans le genre Quercus. – C. R. Acad. Sci., Ser. III. Sci. Vie/Life Sci. 307: 701–707. Skvortsov, A. K. (1968). Ivy SSSR. Sistematicheskij i geograficheskij obzor. – Nauka, Moskva. Szafer, W. (1959): Rodowody drzew w swietle ewolucji. – Szczecinskie Towarzystwo Naukowe, Szczecin. Theisen, I. & Barthlott, W. (1996): Die Mikromorphologie epicuticularer Wachse bei den Caprifoliaceae. – Fedd. Repert. 107: 31–35. Tulloch, A. P. (1976): Chemistry of waxes of higher plants.
326
FLORA (2004) 199
In: Kolattukudy, P. E. (ed.): Chemistry and biochemistry of natural waxes. – Elsevier, Amsterdam. 235–287. Uzunova, K. (1999): A comparative study of leaf epidermis in European Corylaceae. – Fedd. Repert. 110: 209–218. Wilkinson, R. E. (1980): Ecotypic variation of Tamarix pentandra epicuticular wax and possible relationship with herbicide sensitivity. – Weed Sci. 28: 110–113. Wissemann, V. (2000): Epicuticular wax morphology and the taxonomy of Rosa (section Caninae, subsection Rubiginosae). – Plant Syst. Evol. 221: 107–112.
The wax layer and its morphological variability in four European Salix species Dominik Tomaszewski Institute of Dendrology, Polish Academy of Sciences, ul. Parkowa 5, Kórnik PL-62-035, Poland Submitted: Aug 15, 2003, in revised form: Nov 17, 2003 · Accepted: Feb 4, 2004
Summary The structure of the wax layer on leaves was analysed in four species of the genus Salix (S. alba, S. fragilis, S. triandra and S. pentandra). SEM observations showed that peculiar conical structures (named here ‘conicoids’) are present on the lower surface of the leaves in three species. In S. fragilis and S. triandra, conicoids are similar in size, whereas in S. alba, they are several times larger. In S. pentandra no such structures were found and the cuticle was covered only with an amorphous wax film. The observed differences between species can be used as identifying features in this group of plants. A comparison of conicoids on successive leaves on the shoot revealed differences and shed some light on the process of their formation. Key words: Salix, SEM, epicuticular wax, leaf surface, morphology
Introduction Epicuticular waxes are a very broad group of hydrophobic substances occurring on the surface of plant organs. Both their morphological structure and chemical composition are extremely variable. The epicuticular wax layer is always present in terrestrial plants but it can be formed in different ways. Plant waxes have many functions such as: (1) protecting the plant against penetration by pathogens; (2) decreasing the loss of solutes by reducing water retention on leaf surfaces; (3) reducing the loss of water caused by transpiration ; (4) diminishing the risk of injury due to excessive radiation by reflecting and attenuating sunlight; etc. It is noteworthy that the wax layer forms the external covering of the plant, so it is the first barrier against all factors that come into contact with the plant: insects, fungi, bacteria, solar radiation, etc. This is linked with the significance of this layer for protection of the plant against pests and diseases, especially insects and fungi. The chemical composition and structure of epicuticular waxes mediate the plant’s attractiveness to pests.
Morphology and chemical composition of the wax layer depend not only on the plant taxon but also on geographic location, environmental conditions, and stage of development (e.g. Wilkinson 1980; Safou et al. 1988; Prasad & Gülz 1990; Gülz et al. 1991; Gülz & Boor 1992; Gülz & Müller 1992; Gülz 1994). The usefulness of characteristic features of the wax layer in taxonomy has been confirmed many times (e.g. Tulloch 1976; Barthlott & Theisen 1995; Theisen & Barthlott 1996; Uzunova 1999; Wissemann 2000; Heinrichs et al. 2000). In this project, the morphological variability of the wax layer was investigated in some Salix species. The genus Salix L., together with Populus L. and Chosenia Nakai, forms the family Salicaceae. Salix is represented in the family by many more species (300–500) than the other two genera. Salix species were grouped by Skvortsov (1968) into three subgenera: Salix, Chamaetia and Vetrix. This paper is concerned with four species of the subgenus Salix, which are the most frequent in Europe: i.e. S. alba L., S. fragilis L., S. triandra L. and S. pentandra L. Preliminary observations
* Corresponding author: Dominik Tomaszewski, Institute of Dendrology, Polish Academy of Sciences, ul. Parkowa 5, Kórnik PL-62-035, Poland, e-mail: [email protected] 320
FLORA (2004) 199
0367-2530/04/199/04-320 $ 30.00/0
A
B
C
D
Fig. 1. Wax structures (‘conicoids’) on the abaxial leaf surface: A, B. Salix triandra, C. S. alba, D. S. fragilis. They are composed of apically convergent wax filaments. [A, B – Silesia, Eastern Sudeten, Golden Mts., 300 m, 17. 10. 1995, leg. Piotr Kosi´nski, KOR 41851; C – Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002; D – Grand Poland, between Błaz˙ejewko and Bnin, 10. 10. 2001]
revealed the existence of variability of wax structures on the lower (abaxial) leaf surface. The main objective of this study was to determine if this variability is related to leaf age and taxonomic classification. The morphological structure of the epicuticular wax layer was analysed on leaves of these species at successive stages of leaf development. In addition, the usefulness of features of the wax layer for species identification was assessed. Another objective was to determine the optimum conditions for collecting comparative material, which could be important for the methodology of research on the chemical composition of epicuticular waxes in this genus. So far, these questions have been discussed in only a few publications (Paiero et al. 1984; Paiero et al. 1985; Martini & Paiero 1988 ; Hietala et al. 1997; Cameron et al. 2002).
Material and methods Material for this study consisted of leaves of four Salix species (S. alba L. (subsp. alba), S. fragilis L., S. triandra L. and S. pentandra L.) collected from individuals growing in Poland, mainly in the Grand Poland District (western part of the country). The material was checked critically by Prof. Jerzy Zieli´nski from the Institute of Dendrology (Polish Academy of Sciences) in Kórnik. Leaf samples were coated with gold at the Department of Animal Anatomy, Agricultural University of Pozna´n, and viewed with a Hitachi S3000N SEM belonging to the Institute of Plant Protection in Pozna´n. Dried leaves (usually recently collected) were used, as the morphology of the studied structures does not change substantially during drying at room temperature. The structures are very durable, which is evidenced by their low solubility in the standard organic solvents used for wax extraction. FLORA (2004) 199
321
A
B
C
D
Fig. 2. Scanning electron micrographs of abaxial leaf surfaces. A comparison between: A. Salix pentandra, B. S. alba, C. S. fragilis & D. S. triandra. The most distinct is S. pentandra, where the wax layer is present as an amorphous film. S. alba has very large conicoids with a loose filamentous structure, and the smallest differences exist between S. triandra and S. fragilis. Magnification for all the micrographs is the same. [A – Grand Poland, Dolsk, 19. 07. 2002; B, C, D – Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002] Samples from the mature blades were collected to compare the wax structures on successive leaves. For observations of the development of wax structures, successive leaves from the terminal part of the shoot were compared. Additionally, always a fragment of the blade of a very young leaf was dissected for analysis, and the remaining part was left on the shrub. After a few days, another fragment was dissected from the undamaged half of the leaf. Both fragments were taken at half the length of the leaf blade.
Results and discussion The investigations confirmed the existence of substantial differences between the analysed species in the structure of the wax layer. In this group of species, the most distinct is S. pentandra, where the wax layer is present on both the upper and the lower surface of the leaves only as an amorphous film. In the other species, 322
FLORA (2004) 199
the upper surface of the leaf blade is also covered with a uniform wax film, but the lower surface is characterized by the presence of specific wax structures. The structures cannot be assigned to any of the types distinguished in the classification proposed by Barthlott et al. (1998). They are visible in micrographs found in a paper by Martini & Paiero (1988), but those authors did not discuss this feature. Paiero et al. (1985) briefly described the leaf underside as follows: ‘abaxial epidermis covered with a thick, discontinuous wax deposit in the form of vertical scales’. However, this description is not precise enough. “Conicoids” is the name proposed here for this unique type of wax structure in Salix species, because of their conical shape. The analysis showed that conicoids are formed in the species in different ways, but the general plan of formation is the same. They are composed of apically convergent wax filaments (Fig. 1).
Fig. 3. The hypothetical scheme of development of conicoids. Initially the cuticle is covered with a homogenous, continuous wax layer (A), which is getting thicker from the base by accumulating new material. The increase from the base is accompanied by horizontal expansion due to leaf development (B & C). The expansion causes the splitting of the wax layer into patches. D – mature conicoids.
Fig. 4. The inner structure of the conicoid of S. alba revealed after rinsing the material in chloroform. It can be observed that the conicoid is composed of filaments divided into thinner filaments in the lower, younger parts of the structure. [Grand Poland, between Błaz˙ejewko and Jeziory Wielkie, 10. 10. 2001]
S. alba has very large conicoids with a loose filamentous structure. The smallest differences exist between S. triandra and S. fragilis, but both species can be distinguished on the basis of the formation of their wax layer. Although the maximum diameter of the conicoids is more or less the same in both taxa (ca. 11–14 µm), in S. fragilis these structures are looser and more often are split into smaller parts. Furthermore, there are no “splinters”, which are very characteristic for S. triandra, i.e. small platelets or appendices of another shape, which are often arranged on the upper surface of conicoids, on the lateral walls and even on their base (Fig. 2). The above-mentioned wax structures did not appear on leaf margins or major veins. In the light of the results presented above as well as preliminary observations in other groups of Salix species and published data (Martini & Paiero 1988), it is surprising that S. pentandra lacks conicoids. This may be linked with the fact that this species is relatively primitive among the willows, especially among the studied group (Szafer 1959).
accumulating new material. The increase from the base, i.e. from the cuticle, lasts throughout the conicoid’s growth and determines the height of these structures, but it is accompanied by horizontal expansion due to leaf development (Figs. 3 B & C). The expansion causes the splitting of the originally continuous wax layer into patches, and later leads to the fragmentation of the already partly formed conicoids. The common feature of the discussed structures is the fact that the thick filaments are linked with one another in their apical parts, which is the result of the above-described process: it is a remnant of the earliest stage of growth, when the wax, later building the top of the conicoids, constituted an approximately homogenous layer. That part of the cuticle from which the material for the formed conicoid was extruded, was gradually extended, but the filaments originating from them remained connected at the oldest, upper part. This hypothesis is also indirectly confirmed by the image of conicoids of S. alba after rinsing the material in chloroform (Fig. 4). The micrograph shows the inner structure of the conicoid. It is composed of filaments divided into thinner filaments in the lower, younger parts of the conicoid.
Development of conicoids A hypothetical scheme of development of conicoids can be summarized as follows. Initially the cuticle is covered with a homogenous, continuous wax layer (Fig. 3 A), which is getting thicker from the base by
Wax layer on successive leaves on the shoot The structure of the wax layer depends on leaf position on the shoot. On the oldest 3–4 leaves, called cataFLORA (2004) 199
323
A
B
C
D
Fig. 5. The wax layer on successive leaves on a shoot of Salix alba: A. the first leaf (the oldest one), B. the 2nd, C. the 3rd, D. the 4th. Conicoids are present even on the oldest leaf, but are smaller there and their structure is not the same as on younger leaves. [Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002]
phylls, its development stops at a very early stage of growth. On the successive leaves, conicoids have a structure and size typical for the particular given species. On the youngest leaves, which are not fully developed yet, the process of wax formation is ongoing, so its structure is not the same as in fully developed leaves. This means that only leaves from the middle part of the shoot should be used for comparisons between species. Changes in the structure of the wax layer on successive leaves are presented for two species. In S. alba, conicoids are present even on the oldest leaf (cataphyll), but are smaller there and their structure is not the same as on adjacent, younger leaves (Fig. 5). In S. triandra, well-developed wax structures may be formed on the oldest leaf but the wax layer is uneven there, forming ‘islands’. At the centre of an island, typical conicoids can be seen, but at the edges of the islands they are much 324
FLORA (2004) 199
smaller and gradually pass into amorphous wax film between the islands. The proportion of leaf area covered by conicoids on successive leaves gradually increases. Starting from the third leaf, the whole adaxial surface is covered by conicoids (Fig. 6). However, the last leaves (the youngest ones) in the terminal part of the shoot, i.e. those developed in early autumn, show some regression. Their blades sometimes are totally or partially free of conicoids.
Acknowledgements The work was supported by the State Committee for Scientific Research (project no. 6P04C 075 21). I am grateful to Magdalena Gawlak for her excellent technical assistance and Prof. Jerzy Zieli´nski for checking the herbarium material.
A
B
C
Fig. 6. The wax layer on successive leaves on a shoot of Salix triandra. The proportion of leaf area covered by conicoids on successive leaves gradually increases. A. the first leaf (the oldest one), B. the 2nd, C. the 3rd. [Grand Poland, near Radzewice, at the Warta River, 12. 06. 2002]
References Barthlott, W.; Neinhuis, C.; Cutler, D.; Ditsch, F.; Meusel, I.; Theisen, I. & Wilhelmi, H. (1998): Classification and terminology of plant epicuticular waxes. – Bot. J. Linn. Soc. 126: 237–260. Barthlott, W. & Theisen, I. (1995): Epicuticular wax ultrastructure and classification of Ranunculiflorae. – Plant Syst. Evol., Suppl. 9: 39–45. Cameron, K. D.; Teece, M. A.; Bevilacqua, E. & Smart, L. B. (2002): Diversity of cuticular wax among Salix species and Populus species hybrids. – Phytochemistry 60: 715–725. Gülz, P. G. (1994): Epicuticular leaf waxes in the evolution of the plant kingdom. – J. Plant Physiol. 143: 453–464. Gülz, P. G. & Boor, G. (1992): Seasonal variations in epicuticular wax ultrastructures of Quercus robur leaves. – Z. Naturforsch. Sect. C 47: 807–814. Gülz, P. G. & Müller, E. (1992): Seasonal variation in the composition of epicuticular waxes of Quercus robur leaves. – Z. Naturforsch. Sect. C 47: 800–806. Gülz, P. G.; Müller, E. & Prasad, R. B. N. (1991): Devel-
opmental and seasonal variations in the epicuticular waxes of Tilia tomentosa leaves. – Phytochemistry 30: 769–773. Heinrichs, J.; Anton, H.; Gradstein, S. R.; Mues, R. & Holz, I. (2000): Surface wax, a new taxonomic feature in Plagiochilaceae. – Plant Syst. Evol. 225: 225–233. Hietala, T.; Mozes, N.; Genet, M. J.; Rosenqvist, H. & Laakso, S. (1997): Surface lipids and their distribution on willow (Salix) leaves: a combinated chemical, morphological and physicochemical study. – Colloids Surf. B Biointerfaces 8: 205–215. Martini, F. & Paiero, P. (1988): I salici d’Italia. – Edizioni Lint, Trieste. Paiero, P.; Mariani, P. & Marzi, M. (1984): Preliminary observations on microfeatures of leaf surface in Salix species. – G. Bot. Ital. 118: 84–85. Paiero, P.; Mariani, P. & Urso, T. (1985): Formazione di depositi cerosi in foglie di Salix L. – G. Bot. Ital. Suppl. 119: 112. Prasad; R. B. N. & Gülz, P. G. (1990): Developmental and seasonal variations in the epicuticular waxes of beech leaves (Fagus sylvatica L.). – Z. Naturforsch. Sect. C 45: 805–812. FLORA (2004) 199
325
Safou, O.; Saint Martin, M. & Rouane, P. (1988): Stomates et cires dans le genre Quercus. – C. R. Acad. Sci., Ser. III. Sci. Vie/Life Sci. 307: 701–707. Skvortsov, A. K. (1968). Ivy SSSR. Sistematicheskij i geograficheskij obzor. – Nauka, Moskva. Szafer, W. (1959): Rodowody drzew w swietle ewolucji. – Szczecinskie Towarzystwo Naukowe, Szczecin. Theisen, I. & Barthlott, W. (1996): Die Mikromorphologie epicuticularer Wachse bei den Caprifoliaceae. – Fedd. Repert. 107: 31–35. Tulloch, A. P. (1976): Chemistry of waxes of higher plants.
326
FLORA (2004) 199
In: Kolattukudy, P. E. (ed.): Chemistry and biochemistry of natural waxes. – Elsevier, Amsterdam. 235–287. Uzunova, K. (1999): A comparative study of leaf epidermis in European Corylaceae. – Fedd. Repert. 110: 209–218. Wilkinson, R. E. (1980): Ecotypic variation of Tamarix pentandra epicuticular wax and possible relationship with herbicide sensitivity. – Weed Sci. 28: 110–113. Wissemann, V. (2000): Epicuticular wax morphology and the taxonomy of Rosa (section Caninae, subsection Rubiginosae). – Plant Syst. Evol. 221: 107–112.