- Email: [email protected]
Morpho-anatomical differentiation of the balkan populations of the species Teucrium flavum L. (Lamiaceae)
ARTICLE IN PRESS
Flora 201 (2006) 108–119 www.elsevier.de/flora
Morpho-anatomical differentiation of the balkan populations of the species Teucrium flavum L. (Lamiaceae)$ Branislava Lakusˇ ic´a,, Dmitar Lakusˇ ic´b, Radisˇ a Jancˇic´a, Branka Stevanovic´b a
Faculty of Pharmacy, Institute of Botany, University of Belgrade, Vojvode Stepe 450, 1100 Belgrade, Serbia and Montenegro Faculty of Biology, Institute of Botany and Botanical Garden ) Jevremovac* , University of Belgrade, Takovska 43, 11000 Belgrade, Serbia and Montenegro b
Received 3 January 2005; accepted 26 May 2005
Abstract Ecological plasticity, i.e., inter-population differentiation of the species Teucrium flavum was analyzed on the basis of morpho-anatomical variability of its five populations from the maquis (Cisto-Ericetea and Cisto-Micromerietea), rocky grounds (Festuco-Brometea) and rocky crevices (Asplenietea rupestris) in the Eumediterranean and subMediterranean region. Univariate statistic analysis included 22 quantitative characters related to the leaf and stem anatomy and morphology. In order to establish the variability and significance of morpho-anatomical differentiation, principal component analyses (PCA), multivariate analyses of variances (MANOVA), discriminant components analysis (DCA) and clustering, according to the UPMGA method based on Mahalanobius’ distances, have been done. The morpho-anatomical analysis of plants from the five distant populations confirmed that the species T. flavum belongs to malacophyllous xeromorphic species. It was established that the plants from all the five populations analyzed are distinguished by stable conservative xeromorphic characteristics. There is a difference between the pronounced xeromorphic plants belonging to Eumediterranean populations and the subxeromorphic subMediterranean ones. r 2005 Elsevier GmbH. All rights reserved. Keywords: Teucrium flavum; Evergreen shrub; Morphology; Anatomy; Leaf; Indumentum
Introduction The Mediterranean species Teucrium flavum L. belongs to Sect. Chamaedrys (Miller) Schreber (Tutin and Wood, $ This paper represents a part of the Ph.D. thesis of B. Lakusˇ ic´, entitled ‘‘Morphological Variability and Ecological Differentiation of Species of Genus Teucrium L. (Lamiaceae) in Yugoslavia’’ defended at the Faculty of Biology, University of Belgrade, in the year 2000. Corresponding author. E-mail addresses: [email protected] (B. Lakusˇ ic´), [email protected] (D. Lakusˇ ic´), [email protected] (B. Stevanovic´).
0367-2530/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2005.05.001
1972), which includes about 30 taxa, at the species and subspecies rank, distributed mostly in the Mediterranean Basin. The species range extends over the entire Mediterranean Basin, from the eastern coasts of Spain across France, Italy, Croatia, Serbia and Montenegro, Albania and Greece, to Turkey in the east, and in the north of Africa in Algeria, Tunisia and Morocco (Meusel et al., 1978) (Fig. 1). T. flavum is an evergreen, branchy, semi-woody shrub, often over 50 cm tall, that belongs to the life form of suffruticose chamaephytes. By such a habit T. flavum markedly differs from other representatives of the sect.
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
109
Fig. 1. Map of distribution of Teucrium flavum L.
Table 1.
Ecological characteristics of the habitats of the analyzed populations Budva (Montenegro)
Lusˇ tica (Montenegro)
Peloponnesus (Greece)
Canyon of Cijevna (Montenegro)
Canyon of Moracˇa (Montenegro)
Adriatic province of Submediterranean region Rocky grounds (Festuco-Brometea)
Adriatic province of Submediterranean region Rocky grounds (Festuco-Brometea) Limestone 200 m 10–16 1C
Biogeography
Aegean province of Adriatic province Adriatic province of Eumediterranean of Eumediterranean Eumediterranean region region region
Vegetation
Rocky crevices (Asplenietea rupestris) Limestone 40 m 15–16 1C
Maquis (CistoEricetea)
Maquis (CistoMicromerietea)
Limestone 80 m 15–16 1C
Limestone 17–18.5 1C
Limestone 80 m 10–16 1C
7–8 1C
7–8 1C
8.5–10.5 1C
1–5 1C
1–5 1C
1300–2300 mm
1300–2300 mm
350–600 mm
1500–3100 mm
1500–3100 mm
Substratum Altitude Average annual temperature Average January temperature Annual precipitation
Chamaedrys which might indicate the relict character of the species and its belonging to the ancient Mediterranean flora (Tutin and Wood, 1972). On the basis of investigation of flowers and inflorescenses of the genus Teucrium, Ka¨stner (1978) introduced a new classification of the genus, in which the species T. flavum is included in Sect. Pollium Schreber. In the Balkan Peninsula T. flavum is a strictly calciphilous plant that inhabits limestone and dolomite, at altitudes between 0 and 200 m. It is an important element of maquis and garrigue vegetation as well as of their degradation forms in the Mediterranean and subMediterranean floristic region (Lakusˇ ic´, 2000). The aim of the present study was to establish whether there exists a morpho-anatomical differentiation between Eumediterranean and sub-Mediterranean populations of the xeromorphic species T. flavum, bearing in mind the environmental, particularly climatic differences between their habitats. Three out of five popula-
tions analyzed here developed under conditions of the Eumediterranean climate, on the coasts of the Adriatic and Aegean Sea. Another two populations inhabit subMediterranean locations (the Montenegro hinterland, in the canyons of Cijevna and Moracˇa rivers), characterized by the perhumid-sub-Mediterranean Adriatic climate (Table 1).
Material and methods Plant material and morpho-anatomical analysis A morpho-anatomical analysis was done on plant samples from five populations of the species T. flavum growing in the Adriatic (Montenegro) and Aegean (Greece) part of the Mediterranean Basin. The collected plant material was either placed in a herbarium or fixed
ARTICLE IN PRESS 110
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
in 50% alcohol and deposited, respectively in the Herbarium of the Institute of Botany and Botanical Garden ‘‘Jevremovac’’, Faculty of Biology, University of Belgrade (BEOU) and Herbarium of the Institute of Botany, Faculty of Pharmacy, University of Belgrade (HFF). Voucher specimens: 1. Budva (Montenegro – Eumediterranean): rocky vegetation (class. Asplenietea rupestris), at the altitude of 40 m (Lakusˇic´, D., 25 May 1995, HFF – Fix.no. Bu 01-02). 2. Lusˇ tica – Stari Krasˇ ic´i (Montenegro – Eumediterranean): maquis (class. Cisto-Ericetea), limestone, at the altitude of 80 m (Lakusˇic´, D. & B. 2225/96, 27 July 1996, BEOU, HFF – Fix.no. TFS). 3. Peloponnesus (Greece, Peloponnesus – Eumediteranean: maquis (Stevanovic´, V., May 1995, BEOU – Fix.no. TF). 4. Canyon Cijevna, village Dinosˇ a (Montenegro – subMediterranean): rocky vegetation (class. FestucoBrometea), limestone (Lakusˇic´, B., Jancˇic´, R., Slavkovska, V., 09 July 1997, HFF – Fix.no. 11). 5. Canyon of Moracˇa, Dromir (Montenegro – subMediterranean): rocks above the river Moracˇa (class. Festuco-Brometea) (Lakusˇic´, B., Slavkovska, V., Jancˇic´, R. 12 July 1997, HFF – Fix.no. 67). Anatomical analyses of leaves and stems were done on permanent slides, prepared by the standard method for light microscopy. Cross-sections of the leaves (150 samples) and stems (50 samples) were cut on a Reichert sliding microtome (up to 10 mm thick). The sections were cleared in Parazone and thoroughly washed before staining in safranin (1% w/v in 50% ethanol) and alcian blue (1% w/v, aqueous). Epidermal peels (150 samples), for surface structures and stomata analyses, were prepared using Jeffrey’s solution (10% nitric acid and 10% chromic acid, 1: 1) and stained in safranin and alcian blue. All slides were mounted in Canada balsam after dehydration. Density and type of the leaf and stem hairs, as well as the paradermal aspect of epidermal cells, were also studied with SEM (JOEL JSM-6460), for which the samples were covered by gold. All morpho-anatomical measurements were done with the Image Analyzer System Ozaria 2001 and the data processed in the statistical package Statistica 4.5 for Windows. For each of the quantitative characters, 30 leaf samples and 10 stem samples were obtained from different individuals belonging to each of the five populations analyzed. Twenty-two quantitative characters of the statistical analysis were grouped in three categories: I, Leaf anatomy characters (13); II, Leaf shape characters (4) and III, Stem anatomy characters (5).
I. Leaf anatomy characters: (1) Height of adaxial epidermal cells; (2) thickness of palisade tissue; (3) thickness of spongy tissue; (4) height of abaxial epidermal cells; (5) number of palisade layers; (6) surface area of adaxial epidermal cells; (7) surface area of abaxial epidermal cells; (8) surface area of abaxial stomata; (9) number of abaxial stomata; (10) number of adaxial glandular hairs; (11) number of abaxial glandular hairs; (12) number of adaxial non-glandular hairs; (13) number of abaxial non-glandular hair. II. Leaf shape characters: (14) Leaf length; (15) distance between the largest leaf width point and the leaf top; (16) the largest width of the leaf; (17) leaf surface area. III. Stem anatomy characters: (18) Stem diameter; (19) stem diagonal; (20) stem cortex thickness; (21) thickness of the stem vascular cylinder; (22) stem pith diameter.
Statistical analysis For each of the quantitative characters a univariate statistic analysis was done on the basis of the following parameters: average value, minimum, maximum, standard deviation and standard error. The significance of differences between the populations studied was established by multivariate analyses of variances (MANOVA). The general structure of the sample variability were established by Principal Component Analysis (PCA). For checking the hypothesis that the analyzed sample was composed of discrete groups, which are morphologically differentiated one from the other, a Discriminant Component Analysis (DCA) was done. Overall differences between the compared groups are presented by Mahalanobius’ distances, which are used for clustering on the basis of UPGMA method.
Results Leaf shape and anatomy The leaves of T. flavum are elliptically oval, being the widest at the basal part, and rounded at the tip. The leaf margin is obtusely dentate. The leaf stalk is long. It should be pointed out that the leaf shape was always the same in all the populations studied. In general, in all the populations studied the leaf length was between 12 and 27 mm, whereas the leaf width ranged between 11 and 21 mm. The leaf surface area varied between 80 and 370 mm2. The leaf indumentum of all the plants studied was composed of glandular and non-glandular hairs
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Fig. 2. Teucrium flavum A, indumentum of glandular (g) and non-glandular (h) hairs on the lower leaf side. (SEM) B, peltate (p) and capitate type I (k) hairs. C, capitate hair type III (SEM). D, Paradermal view, peltate (p) and capitate type I (k) hairs.
(Fig. 2A). The glandular hairs were peltate and capitate. Peltate hairs (Fig. 2B, D) consisted of a short unicellular stalk and a multicellular secretory head with large subcuticular space. Capitate hairs were small in size and of three types on the basis of their structure: Type I consisted of a short unicellular stalk and a rather large secretory head of four cells with a small subcuticular space (Fig. 2B, D); Type II consisted of a short unicellular stalk and a secretory head of one or two cells; Type III consisted of a two-cellular stalk and a secretory head of one cell (Fig. 2C). The non-glandular hairs are unicellular, basally widened, sharp on the top (Fig. 3B) or multicellular, mostly having two to three cells, uniseriate, branchless, the top cell having a sharp edge, straight or bended (Fig. 3C). More or less wartlike cuticular structures are observed on the surface of the non-glandular hairs (Fig. 3D).
111
The glandular hairs are at the bottom of the complex arrangement of the leaf indumentum, leaning against the epidermal cells, while between them there are dispersed, long non-glandular hairs that form its upper layer. Basal cells of the glandular hairs are sunk under the level of epidermal cells, and their large heads (composed of several secretory cells) are in contact, leveling with the epidermis or being a bit above thus partially covering the leaf blade (Fig. 3A). The indumentum is particularly well developed on the abaxial leaf side (Fig. 3A), which is a common feature of plants from all five populations. Therefore, the abaxial leaf side is grayish-green in color. The plants from the Eumediterranean populations, namely from the localities near Budva and Krasˇ ic´i, as well as from Peloponnesus in particular, are characterized by conspicuously thick indumentum (Fig. 4). In contrast, the adaxial leaf side is always covered with sparse hairs rendering a dark green color to this side (Fig. 3E and Fig. 4). The cuticle on the leaf adaxial epidermis is thicker than that of the abaxial epidermis in plants of all the five populations studied. Besides, the outer epidermal cell walls are thickened, particularly in the adaxial epidermis occupying 2/3 of the cell lumen. The plants of the subMediterranean populations (from the gorges of Cijevna and Moracˇa) are characterized by larger adaxial epidermal cells in respect to those of the plants of Eumediterranean populations (Fig. 5). Anticlinal walls of the adaxial and abaxial epidermal cells are almost straight in the plants from the localities near Budva and Krasˇ ic´i (Fig. 6B), or slightly undulate, as in the plants from Peloponnesus and from the Moracˇa and Cijevna canyons (Fig. 6A). On the outer periclinal walls of the abaxial epidermal cells larger or smaller cuticle wrinkles are formed. The leaves are always hypostomatic; the stomata are more or less raised (Fig. 7C) above the epidermal cells, of diacytic and anomocytic type (Fig. 7A). Their frequency varies from 129 to 300 per mm2. The highest number of stomata are present in the plants from Krasˇ ic´i (186–300 per mm2), and the lowest in those from the Moracˇa canyon (129–186 per mm2) (Fig. 8). The stomata are usually encircled by the glandular hairs (either peltate or capitate), thus being partially covered with the subcuticular spaces of the secretory cells (Fig. 7B–D). Leaf thickness (Fig. 9) of all plants studied ranged between 210 and 440 mm, being the thickest in the plants from the canyon of Cijevna (267–440 mm). The mesophyll is clearly differentiated into palisade parenchyma and spongy parenchyma; the ratio of these two photosynthetically functional mesophyll tissues is generally 1.4:1, except in the plants from the locality near Budva, being 2:1 (Fig. 9). The palisade parenchyma, just below the upper epidermis, consists of 2–3 rows of elongated and densely arranged cells, which gradually shorten going towards
ARTICLE IN PRESS 112
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Fig. 3. Teucrium flavum A, indumentum on the lower leaf side (SEM). B, three-cellular hair. C, bicellular hair. D, wart-like structures on the surface of the non-glandular hairs (SEM). E, indumentum on peeled upper epidermis.
the spongy parenchyma. The anticlinal walls of the palisade cells are almost always undulate. The cells of spongy parenchyma are usually irregularly shaped, but with relatively small intercellular spaces (Fig. 10A, B). Even in the leaves of the plants from Peloponnesus, which are smaller and less thick, the palisade parenchyma is the thinnest but still densely packed into 2–3 layers, which is always the case in the plants of all the populations studied. In the leaf cross-section, a large number of vascular bundles, situated in the central region of the mesophyll, and only one main vascular bundle, developed in the leaf midrib, are observed. Large bundle sheath cells enclose the vascular bundles.
Stem shape and anatomy T. flavum is an evergreen, branchy, semi-ligneous shrub. In general, the resemblance of the external
morphology of the stem of all different populations is obvious. In all the populations studied, the stem height was between 40 and 70 cm, and a basal ligneous part is clearly developed and reaches the length of up to 30 cm. However, slight differences could be observed between the population groups growing in very different ecological conditions. In the individuals from Eumediterranean populations the height of the shrubs usually varies in between 40 and 45 cm. The internodia are very short and dense, so that the leaves overlap each other. Contrary to them, the shrubs of the sub-Mediterranean populations reach a height of over 60 cm, having longer internodia; therefore their leaves mostly do not overlap. The herbaceous stem of T. flavum specimens is more or less square-shaped (in the cross section), firm, particularly in the plants from the localities near Budva and Peloponnesus, as well as from the Cijevna canyon (Fig. 11A). Seldom, as in the plants from the localities from the Moracˇa canyon it is slightly rounded (Fig. 11B). The stem is well covered with epidermis, and the thick cuticle, permeated by wax deposits and glandular (peltate and capitate) and non-glandular hairs, is arranged in a similar way as in the leaves. Scattered stomata are slightly raised above the level of the surrounding epidermal cells. The stem cortex is differentiated into two to three layered subepidermal supporting tissues, collenchyma, and several layers of thin-walled parenchymatous cells with prominent intercellular spaces. The collenchyma is discontinuously distributed in the peripheral part of the stem, forming thick projecting strands particularly in the stem corners. The vascular tissue commonly forms a cylinder between the stem cortex and the pith, consisting of collateral vascular bundles separated by interfascicular parenchyma. Conspicuous cups of sclerenchymatous fibres are present above the phloem, being particularly large below the stem corners. These fibres are more developed in the vascular cylinder of plants from the localities of Moracˇa and Cijevna canyons as well as in those from Peloponnesus. However, in the stem of plants from the other two populations there are o nly 2–3 sclerenchyma cells above the phloem elements. The correlation coefficient of the cortex and the whole stem diameter ranges from 0.226 (Krasˇ ic´i) to 0.343 (Cijevna).
Multivariate analysis of the morpho-anatomical characters By PCA it could be shown that the structural variability of the populations studied is extremely complex, since the first three axes comprise only 47.58% of total variability. By MANOVA statistically significant differences between all populations were
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
113
Fig. 4. Box and whisker plots of basic statistic parameters of indumentum.
Fig. 5. Box and whisker plots of basic statistic parameters of epidermis.
established. It should be pointed out that the most important characters in structural differentiation are those related to leaf shape, stem anatomy, indumentum, and leaf anatomy features in this order of significance (Table 2). DCA of the populations studied of T. flavum has shown that the Eumediterranean (Peloponnesus, Krasˇ ic´i, Budva) and sub-Mediterranean (Moracˇa, Cijevna) populations represent two morphologically almost completely separate groups. On the first two
axes, the populations from Peloponnesus and Budva stand completely separated from the sub-Mediterranean populations. The population from Krasˇ ic´i shows (Fig. 12) transitional characteristics between the Peloponnesus and Montenegro sub-Mediterranean populations, as it was the case also in the PCA analysis. Morpho-anatomical separation between Mediterranean and sub-Mediterranean populations is clearly observed also on the basis of overall Mahalanobious’ distances (Fig. 13).
ARTICLE IN PRESS 114
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Discussion
Fig. 6. Teucrium flavum paradermal view A, undulate anticlinal walls of the upper epidermal cells of plants from Moracˇa. B, straight anticlinal walls of the upper epidermal cells of plants from Budva.
Fig. 7. Teucrium flavum A, anomocytic (a) and diacytic (d) stomata. B, stomata encircling by the glandular hairs or glandular hairs encircling stomata. (SEM). C, highly raised stomata and cuticular striae. (SEM). D, peltate hair around the stomata (SEM).
The analyzed populations of the species T. flavum inhabit the Mediterranean area on ‘‘terra-rossa’’ (red soil) substrate, which develops on porous limestone. The species grows within the maquis-garrigue vegetation of different stages of progression and regression, under various Mediterranean bio-climate conditions – perhumid Mediterranean Adriatic, arid Mediterranean Aegean and perhumid sub-Mediterranean Adriatic climate. The resemblance of plants from different populations is obvious, even at first sight, due to the similar general habit, i.e., of quite a similar external morphology of the stem and evergreen leaves. It is rather important to stress this uniformity of the species T. flavum that contrastes to the extreme morphological heterogeneity that is present within the whole genus Teucrium. A detailed growthform analysis of Teucrium have shown significant morphological differences between shrubs, semi-shrubs and perennial and annual herbs, especially in regard to the extension and volume of lignification, as well as with respect to the existence or the lack of sclerenchyma elements in the cortex. Some of these features can be used taxonomically on the level of sections and species groups (Ka¨stner, 1978, 1979, 1981, 1986). The macromorphological similarity of the stems and evergreen leaves of T. flavum populations might be assumed as a strong indication of structural stability and some kind of morpho-anatomical conservatism of this ancient Mediterranean xerophyte. However, this physiognomic uniformity obviously is combined with ecoanatomical differentiations of leaves and stems of T. flavum populations which thrive both in sites with summers not particularly dry, and in those with a pronounced summer drought. In general, the evergreen leaves of T. flavum are xeromorphic, moderately to significantly thick, tough but not flabby neither rigid nor hard. In the Mediterranean vegetation there is a large number of xerophytes with such leaves, commonly known as malakophyllous, notably within the genera of Cistus, Rosmarinus, Thymus, etc. (Breckle, 2002; Kummerow, 1973). The prominent xeromorphic features of the T. flavum leaves are: simple form and relatively small size, dense indumentum, especially on the lower side where a high number of stomata are also located. The smallest leaves, having a reduced external surface and mesophyll thickness, are found in plants from the Eumediterranean, Peloponnesian locality. In contrast, the thickest leaves with the largest lamina surface area have the plants from the sub-Mediterranean locality in the canyon of Cijevna. A dense and complex indumentum, composed of glandular and non-glandular hairs, covers the lower leaf side, shielding stomata and thus advantageously
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
115
Fig. 8. Box and whisker plots of basic statistic parameters of stomata.
Fig. 9. Box and whisker plots of basic statistic parameters of mesophyll.
reducing transpiration loss. Stomata and glandular hairs are very closely related, meaning that the stomata are surrounded by glandular hairs or glandular hairs are surrounded by stomata. In any case, stomata are partially or totally flanged by the large subcuticular swellings of peltate hairs and/or secretory cells of the capitate hairs. Since the protrusions of capitate secretory cells have only a small storing space, there is a continuous evaporation of essential oils (Werker et al., 1985a, b). This renders the air near the leaf surface more condensed thus providing a higher boundary layer
resistance to gas diffusion. Regardless of the small quantity of essential oils (0.1–0.2%), which characterizes the specimens of T. flavum, even their minimal presence in the external secretory structures may be efficient in reducing both transpiration and overheating (C´orovic´ et al., 1969; Todorovic´ and Stevanovic´, 1994). Otherwise, it is well known that all species of the genus Teucrium are distinguished by only a low quantity of essential oils (Kovacˇevic´ et al., 2001; Petricˇic´ et al., 1993). Beside essential oils, some other terpenoids, as well as flavonoids and/or phenylcarbonic acids might be
ARTICLE IN PRESS 116
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Fig. 10. Cross section of the leaf: A, from Krasˇ ic´i. B, from Peloponnesus.
Fig. 11. Cross section of the stem: A, square-shaped stem. B, rounded stem.
produced by these glandular hairs (Wollenweber, 1984). Terpene exudates are the resins that cover the surface of the plant above-ground parts, while the flavonoids are incorporated either into the resins or into the coating the leaf surfaces of many plants from the semi-arid regions
(Kelsey et al., 1984; Wollenweber, 1984). It has been reported previously that the resin content in T. flavum leaves is about 1.03%, consisting of terpenes, another phenolic compounds, and free flavonoids (Kovacˇevic´ et al., 1998). All these mostly aromatic volatile compounds
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Table 2.
117
Analysis of variances on the level of individual characters
Dependent variable
Mean sq. effect
Mean sq. error
F(df1,2) 4,45
p-level
Height of adaxial epidermal cells Thickness of palisade tissue Thickness of spongy tissue Height of abaxial epidermal cells Number of palisade layers Surface area of adaxial epidermal cells Surface area of abaxial epidermal cells Surface area of abaxial stomata Number of abaxial stomata Number of adaxial glandular hairs Number of abaxial glandular hairs Number of non-glandular adaxial hairs Number of non-glandular abaxial hairs Leaf length Distance between the largest leaf width point and the leaf top Largest width of the leaf Leaf surface area Stem diameter Stem diagonal Stem cortex thickness Thickness of stem vascular cylinder Stem pith diameter
134 2773 3143 20 0 2,180,681 130,681 33,954 285 6 45 30 54 75 48 35 353 1,838,400 1,726,035 2125 81,305 118,351
14.7 269.9 397.5 6.0 .1 118111.4 48122.2 9273.7 27.3 2.3 11.7 1.5 2.1 7.1 4.7 3.3 27.5 134840.0 55039.8 7129.5 5851.5 8893.8
9.09521 10.27664 7.90878 3.38172 2.36066 18.46292 2.71561 3.66135 10.47165 2.57299 3.86175 20.55275 25.43458 10.52627 10.19968 10.47934 12.84811 13.63394 31.35979 .29809 13.89465 13.30719
.000018 .000005 .000065 .016757 .067463 .000000 .041445 .011518 .000004 .050396 .008824 .000000 .000000 .000004 .000006 .000004 .000000 .000000 .000000 .877662 .000000 .000000
Fig. 13. Mahalnobius distances between the analyzed populations of Teucrium flavum.
Fig. 12. Discriminant analysis of the basic components (DCA).
and free flavonoids, along with the dense covering of the leaves by hairs, might have an important role in the adaptation of T. flavum not only to the arid and semiarid conditions of its habitats (Kelsey et al., 1984), but also in its protection from the intense light and UV radiation (Tomas-Barberan and Wollenweber, 1990). Similar micromorphological and phytochemical characteristics were established in the Apennine populations of the species T. flavum (Maleci and Servettaz, 1991; Maleci et al., 1995). Furthermore, it could be assumed that the waxy, thick and wrinkled cuticle as well as the sparse indumentum
ARTICLE IN PRESS 118
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
on the upper leaf side consisting of short and protruding non-glandular hairs and of all the types of glandular hairs mentioned, especially in plants from the Eumediterranean populations, also have an important influence on the spectral features of the leaves. Such structural adaptations increase leaf reflectance, thus reducing solar inception, heat load and therefore water deficit. This has been reported for leaves of Mediterranean species from the genus Cistus (Gausman and Quisenberry, 1990). It is worth mentioning that the more dense indumentum of leaves from the Peloponnesian population must be regarded as a favorable adaptive modification which helps in protecting the mesophyll from excessive water loss and intense radiation in this particular hostile, perarid Mediterranean environment. More or less conspicuous xeromorphic characteristics of T. flavum, from different populations, are represented by a relatively high number of stomata, ranging from 129 to 257 per mm2 in the plants from the subMediterranean sites to 143–300 per mm2 in those from the Adriatic coast and the Peloponnesus. A similar high number of stomata was established also in the Apennine populations of T. flavum, in which the number of stomata varies from 168 to 258 per mm2 (Ka¨stner, 1979). It is also a typical xeromorphism that the dense, complex indumentum on the lower leaf side protects stomata which, on the other side are somewhat raised above the level of the epidermal cells. Smaller and thick-walled epidermal cells on the upper leaf side are a pronounced xeromorphic feature of the Eumediterranean populations of the taxon, compared with the two sub-Mediterranean ones. The anticlinal walls of both adaxial and abaxial epidermal cells of plant leaves from all populations studied are straight to wavy, which is described as an other xeromorphic feature (Fahn and Cutler, 1992). A ratio between 1.4:1 and 2:1 of 2–4 layered palisade parenchyma to 2–3 layered spongy parenchyma is characteristic for plants from all the five populations. The number of palisade layers is increased in the plants from Eumediterranean populations, particularly in those from the localities near Budva and Krasˇ ic´i, but also in those from Peloponnesus. Moreover, the palisade cells of these plants are narrower and more compact. In addition, in leaves of these groups of plants of spongy cells can be found, which elongate in such a way that they resemble palisade cells. A partial differentiation into an isolateral mesophyll is asumed to be a strongly xeromorphic character. Again, similar characteristics of the palisade and spongy tissues were established also in the Apennine populations of T. flavum (Ka¨stner, 1979). The stems of the studied T. flavum populations are also protected by a thick cuticle with waxy layers and with a dense indumentum of the same structural pattern as that of the leaves. The stem hairs are more protruding and less intertwined than those on the leaves. The ratio
of the stem cortex to stem diameter ranges from 0.226 to 0.343, which is within the usual values found in xeromorphic stems (Fahn and Cutler, 1992). All the data obtained, particularly those subjected to a comparative multivariant analysis of morpho-anatomical characteristics of the five populations from three different climate variants, have shown that the species T. flavum has maintained quite a stable, conservative morpho-anatomical structure. Such a substantial similarity between different populations is also characteristic of other ancient Mediterranean plants since their adaptive structures evolved, in the first place, as the consequence of severe summer drought and high temperatures (Margaris, 1981). In all the localities, both eu- and sub-Mediterranean ones (arid-Mediterranean and perhumid-sub-Mediteranean), the plant populations of which have been studied, more or less pronounced, longer or shorter intervals of summer drought stress prevail. Inter-population differences refer to small variations in leaf size, indumentum density, number of stomata, thickness of cuticle and outer epidermal cell walls, as well as to the number of the mesophyll tissues layers. While all these anatomical features are of the same pattern, they are clearly more expressed in the Eumediterranean than in the subMediterranean populations.
Acknowledgements The authors are grateful to Prof. Dr. Vladimir Stevanovic´, Institute of Botany and Botanical garden ‘‘Jevremovac’’ University of Belgrade for useful advice and valuable comments and to the Ministry for Science, Technology and Development of Serbia (Project Nos. 1568 and 1505) for financial support.
References Breckle, S.W., 2002. Walter’s Vegetation of the Earth. Springer, Berlin. C´orovic´, M., Stejepanovic´, L., Nikolic´, R., Pavlovic´, S., Zˇivanovic´, P., 1969. Comparative investigation of the level of osmotic values, transpiration and quantity of essential oil in some species of Labiatae family. Glasnik Botanicˇkog zavoda i basˇ te Univerziteta u Beogradu 4, 19–27. Fahn, A., Cutler, F.D., 1992. Xerophytes. Encyclopedia of Plant Anatomy. Borntraeger, Berlin. Gausman, H.W., Quisenberry, J.E., 1990. Spectrophotometric detection of plant leaf stress. In: Katterman, F. (Ed.), Environmental Injury to Plants. Academic Press, San Diego, pp. 257–280. Ka¨stner, A., 1978. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. I. Die Infloreszenzen und Blu¨ten. Flora 167, 485–514.
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Ka¨stner, A., 1979. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. II. Anatomie der Sprosse und Bla¨tter. Flora 168, 431–467. Ka¨stner, A., 1981. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. III. Wuchsformen und Verbreitung von Arten der Sektionen Teucropis und Teucrium. Flora 171, 466–519. Ka¨stner, A., 1986. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. V. Wuchsformen und Verbreitung von Arten der Verwandtschaftskreise von T. marum und T. chamaedrys sect. Chamaedrys. Flora 178, 111–138. Kelsey, R.G., Reynolds, W.G., Rodriguez, E., 1984. The chemistry of biologically active constituents secreted and stored in plant glandular trichomes. In: Rodriguez, E., Healey, L.P., Mehta, I. (Eds.), Biology and Chemistry of Plant Trichomes. Plenum Press, New York, London, pp. 187–243. Kovacˇevic´, N., Ristic´, M., okovic´, D., Vukovic´, Z., Lakusˇ ic´, B., 1998. Herb exudates of Teucrium L. species. Arch. Pharm. 6, 944–945. Kovacˇevic´, N., Lakusˇ ic´, B., Ristic´, M., 2001. Composition of the essential oils of seven Teucrium species from Serbia and Montenegro. J. Essential Oil Res. 13, 163–165. Kummerow, J., 1973. Comparative anatomy of sclerophyllus of Mediterranean climatic areas. In: Di Castri, F., Mooney, A.H. (Eds.), Mediterranean Type Ecosystems – Origine and Strukture. Springer, Berlin, Heidelberg, New York. Lakusˇ ic´, B., 2000. Morphological variability and ecological differentiation of species of genus Teucrium L. (Lamiaceae) in Yugoslavia. Unpublished D. Phil. Thesis, Fac. Biol., University in Belgrade. Maleci, L.B., Servettaz, O., 1991. Morphology and distribution of trichomes in Italian species of Teucrium sect. Chamaedrys (Labiatae) – a taxonomical evaluation. Plant Syst. Evol. 174, 83–91. Maleci, L.B., Pinetti, A., Servettaz, O., 1995. Micromorphological and phytochemical characteristics of the two
119
subspecies of Teucrium flavum L. (Labiatae) from the Italian flora. Flora 190, 237–242. Margaris, N.S., 1981. Adaptive strategies in plants dominating Mediterranean-type ecosystems. In: Di Castri, F., Goodall, D.W., Specht, L.R. (Eds.), Mediterranean-type Shrublands. Elsevier, Amsterdam, pp. 309–315. Meusel, H., Ja¨ger, E., Rauschert, S., Weinert, E., 1978. Vergleichende Chorologie der Zentraleuropa¨ischen Flora, vol. 2. Fischer, Jena. Petricˇic´, J., Blazˇevic´, N., Plazibat, M., 1993. Essential oil of Teucrium flavum L. from Croatia. J. Essential Oil Res. 6, 625–627. Todorovic´, B., Stevanovic´, B., 1994. Adaptive characteristics of the endemic species Satureja horvatii Sˇilic´ (Lamiaceae) in mountain-Mediterranean and Mediterranean habitats. Bot. J. Linnean Soc. 114, 367–376. Tomas-Barberan, F.A., Wollenweber, E., 1990. Flavonoid aglycones from the leaf surfaces of some Labiatae species. Plant Syst. Evol. 173, 109–118. Tutin, T.G., Wood, D., 1972. Teucrium L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea, Vol. 3. Cambridge University Press, Cambridge, pp. 129–135. Werker, E., Ravid, U., Putievsky, E., 1985a. Structure of glandular hairs and indetification of the main components of their secreted material in some species of the Labiatae. Israel J. Bot. 34, 31–45. Werker, E., Ravid, U., Putievsky, E., 1985b. Glandular hairs and their secretions in the vegetative and reproductive organs of Salvia sclarea and S. dominica. Israel J. Bot. 34, 239–252. Wollenweber, E., 1984. The systematic implication of flavonoids secreted by plants. In: Rodriguez, E., Healey, L.P., Mehta, I. (Eds.), Biology and Chemistry of Plant Trichomes. Plenum Press, New York, London, pp. 53–69.
Flora 201 (2006) 108–119 www.elsevier.de/flora
Morpho-anatomical differentiation of the balkan populations of the species Teucrium flavum L. (Lamiaceae)$ Branislava Lakusˇ ic´a,, Dmitar Lakusˇ ic´b, Radisˇ a Jancˇic´a, Branka Stevanovic´b a
Faculty of Pharmacy, Institute of Botany, University of Belgrade, Vojvode Stepe 450, 1100 Belgrade, Serbia and Montenegro Faculty of Biology, Institute of Botany and Botanical Garden ) Jevremovac* , University of Belgrade, Takovska 43, 11000 Belgrade, Serbia and Montenegro b
Received 3 January 2005; accepted 26 May 2005
Abstract Ecological plasticity, i.e., inter-population differentiation of the species Teucrium flavum was analyzed on the basis of morpho-anatomical variability of its five populations from the maquis (Cisto-Ericetea and Cisto-Micromerietea), rocky grounds (Festuco-Brometea) and rocky crevices (Asplenietea rupestris) in the Eumediterranean and subMediterranean region. Univariate statistic analysis included 22 quantitative characters related to the leaf and stem anatomy and morphology. In order to establish the variability and significance of morpho-anatomical differentiation, principal component analyses (PCA), multivariate analyses of variances (MANOVA), discriminant components analysis (DCA) and clustering, according to the UPMGA method based on Mahalanobius’ distances, have been done. The morpho-anatomical analysis of plants from the five distant populations confirmed that the species T. flavum belongs to malacophyllous xeromorphic species. It was established that the plants from all the five populations analyzed are distinguished by stable conservative xeromorphic characteristics. There is a difference between the pronounced xeromorphic plants belonging to Eumediterranean populations and the subxeromorphic subMediterranean ones. r 2005 Elsevier GmbH. All rights reserved. Keywords: Teucrium flavum; Evergreen shrub; Morphology; Anatomy; Leaf; Indumentum
Introduction The Mediterranean species Teucrium flavum L. belongs to Sect. Chamaedrys (Miller) Schreber (Tutin and Wood, $ This paper represents a part of the Ph.D. thesis of B. Lakusˇ ic´, entitled ‘‘Morphological Variability and Ecological Differentiation of Species of Genus Teucrium L. (Lamiaceae) in Yugoslavia’’ defended at the Faculty of Biology, University of Belgrade, in the year 2000. Corresponding author. E-mail addresses: [email protected] (B. Lakusˇ ic´), [email protected] (D. Lakusˇ ic´), [email protected] (B. Stevanovic´).
0367-2530/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2005.05.001
1972), which includes about 30 taxa, at the species and subspecies rank, distributed mostly in the Mediterranean Basin. The species range extends over the entire Mediterranean Basin, from the eastern coasts of Spain across France, Italy, Croatia, Serbia and Montenegro, Albania and Greece, to Turkey in the east, and in the north of Africa in Algeria, Tunisia and Morocco (Meusel et al., 1978) (Fig. 1). T. flavum is an evergreen, branchy, semi-woody shrub, often over 50 cm tall, that belongs to the life form of suffruticose chamaephytes. By such a habit T. flavum markedly differs from other representatives of the sect.
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
109
Fig. 1. Map of distribution of Teucrium flavum L.
Table 1.
Ecological characteristics of the habitats of the analyzed populations Budva (Montenegro)
Lusˇ tica (Montenegro)
Peloponnesus (Greece)
Canyon of Cijevna (Montenegro)
Canyon of Moracˇa (Montenegro)
Adriatic province of Submediterranean region Rocky grounds (Festuco-Brometea)
Adriatic province of Submediterranean region Rocky grounds (Festuco-Brometea) Limestone 200 m 10–16 1C
Biogeography
Aegean province of Adriatic province Adriatic province of Eumediterranean of Eumediterranean Eumediterranean region region region
Vegetation
Rocky crevices (Asplenietea rupestris) Limestone 40 m 15–16 1C
Maquis (CistoEricetea)
Maquis (CistoMicromerietea)
Limestone 80 m 15–16 1C
Limestone 17–18.5 1C
Limestone 80 m 10–16 1C
7–8 1C
7–8 1C
8.5–10.5 1C
1–5 1C
1–5 1C
1300–2300 mm
1300–2300 mm
350–600 mm
1500–3100 mm
1500–3100 mm
Substratum Altitude Average annual temperature Average January temperature Annual precipitation
Chamaedrys which might indicate the relict character of the species and its belonging to the ancient Mediterranean flora (Tutin and Wood, 1972). On the basis of investigation of flowers and inflorescenses of the genus Teucrium, Ka¨stner (1978) introduced a new classification of the genus, in which the species T. flavum is included in Sect. Pollium Schreber. In the Balkan Peninsula T. flavum is a strictly calciphilous plant that inhabits limestone and dolomite, at altitudes between 0 and 200 m. It is an important element of maquis and garrigue vegetation as well as of their degradation forms in the Mediterranean and subMediterranean floristic region (Lakusˇ ic´, 2000). The aim of the present study was to establish whether there exists a morpho-anatomical differentiation between Eumediterranean and sub-Mediterranean populations of the xeromorphic species T. flavum, bearing in mind the environmental, particularly climatic differences between their habitats. Three out of five popula-
tions analyzed here developed under conditions of the Eumediterranean climate, on the coasts of the Adriatic and Aegean Sea. Another two populations inhabit subMediterranean locations (the Montenegro hinterland, in the canyons of Cijevna and Moracˇa rivers), characterized by the perhumid-sub-Mediterranean Adriatic climate (Table 1).
Material and methods Plant material and morpho-anatomical analysis A morpho-anatomical analysis was done on plant samples from five populations of the species T. flavum growing in the Adriatic (Montenegro) and Aegean (Greece) part of the Mediterranean Basin. The collected plant material was either placed in a herbarium or fixed
ARTICLE IN PRESS 110
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
in 50% alcohol and deposited, respectively in the Herbarium of the Institute of Botany and Botanical Garden ‘‘Jevremovac’’, Faculty of Biology, University of Belgrade (BEOU) and Herbarium of the Institute of Botany, Faculty of Pharmacy, University of Belgrade (HFF). Voucher specimens: 1. Budva (Montenegro – Eumediterranean): rocky vegetation (class. Asplenietea rupestris), at the altitude of 40 m (Lakusˇic´, D., 25 May 1995, HFF – Fix.no. Bu 01-02). 2. Lusˇ tica – Stari Krasˇ ic´i (Montenegro – Eumediterranean): maquis (class. Cisto-Ericetea), limestone, at the altitude of 80 m (Lakusˇic´, D. & B. 2225/96, 27 July 1996, BEOU, HFF – Fix.no. TFS). 3. Peloponnesus (Greece, Peloponnesus – Eumediteranean: maquis (Stevanovic´, V., May 1995, BEOU – Fix.no. TF). 4. Canyon Cijevna, village Dinosˇ a (Montenegro – subMediterranean): rocky vegetation (class. FestucoBrometea), limestone (Lakusˇic´, B., Jancˇic´, R., Slavkovska, V., 09 July 1997, HFF – Fix.no. 11). 5. Canyon of Moracˇa, Dromir (Montenegro – subMediterranean): rocks above the river Moracˇa (class. Festuco-Brometea) (Lakusˇic´, B., Slavkovska, V., Jancˇic´, R. 12 July 1997, HFF – Fix.no. 67). Anatomical analyses of leaves and stems were done on permanent slides, prepared by the standard method for light microscopy. Cross-sections of the leaves (150 samples) and stems (50 samples) were cut on a Reichert sliding microtome (up to 10 mm thick). The sections were cleared in Parazone and thoroughly washed before staining in safranin (1% w/v in 50% ethanol) and alcian blue (1% w/v, aqueous). Epidermal peels (150 samples), for surface structures and stomata analyses, were prepared using Jeffrey’s solution (10% nitric acid and 10% chromic acid, 1: 1) and stained in safranin and alcian blue. All slides were mounted in Canada balsam after dehydration. Density and type of the leaf and stem hairs, as well as the paradermal aspect of epidermal cells, were also studied with SEM (JOEL JSM-6460), for which the samples were covered by gold. All morpho-anatomical measurements were done with the Image Analyzer System Ozaria 2001 and the data processed in the statistical package Statistica 4.5 for Windows. For each of the quantitative characters, 30 leaf samples and 10 stem samples were obtained from different individuals belonging to each of the five populations analyzed. Twenty-two quantitative characters of the statistical analysis were grouped in three categories: I, Leaf anatomy characters (13); II, Leaf shape characters (4) and III, Stem anatomy characters (5).
I. Leaf anatomy characters: (1) Height of adaxial epidermal cells; (2) thickness of palisade tissue; (3) thickness of spongy tissue; (4) height of abaxial epidermal cells; (5) number of palisade layers; (6) surface area of adaxial epidermal cells; (7) surface area of abaxial epidermal cells; (8) surface area of abaxial stomata; (9) number of abaxial stomata; (10) number of adaxial glandular hairs; (11) number of abaxial glandular hairs; (12) number of adaxial non-glandular hairs; (13) number of abaxial non-glandular hair. II. Leaf shape characters: (14) Leaf length; (15) distance between the largest leaf width point and the leaf top; (16) the largest width of the leaf; (17) leaf surface area. III. Stem anatomy characters: (18) Stem diameter; (19) stem diagonal; (20) stem cortex thickness; (21) thickness of the stem vascular cylinder; (22) stem pith diameter.
Statistical analysis For each of the quantitative characters a univariate statistic analysis was done on the basis of the following parameters: average value, minimum, maximum, standard deviation and standard error. The significance of differences between the populations studied was established by multivariate analyses of variances (MANOVA). The general structure of the sample variability were established by Principal Component Analysis (PCA). For checking the hypothesis that the analyzed sample was composed of discrete groups, which are morphologically differentiated one from the other, a Discriminant Component Analysis (DCA) was done. Overall differences between the compared groups are presented by Mahalanobius’ distances, which are used for clustering on the basis of UPGMA method.
Results Leaf shape and anatomy The leaves of T. flavum are elliptically oval, being the widest at the basal part, and rounded at the tip. The leaf margin is obtusely dentate. The leaf stalk is long. It should be pointed out that the leaf shape was always the same in all the populations studied. In general, in all the populations studied the leaf length was between 12 and 27 mm, whereas the leaf width ranged between 11 and 21 mm. The leaf surface area varied between 80 and 370 mm2. The leaf indumentum of all the plants studied was composed of glandular and non-glandular hairs
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Fig. 2. Teucrium flavum A, indumentum of glandular (g) and non-glandular (h) hairs on the lower leaf side. (SEM) B, peltate (p) and capitate type I (k) hairs. C, capitate hair type III (SEM). D, Paradermal view, peltate (p) and capitate type I (k) hairs.
(Fig. 2A). The glandular hairs were peltate and capitate. Peltate hairs (Fig. 2B, D) consisted of a short unicellular stalk and a multicellular secretory head with large subcuticular space. Capitate hairs were small in size and of three types on the basis of their structure: Type I consisted of a short unicellular stalk and a rather large secretory head of four cells with a small subcuticular space (Fig. 2B, D); Type II consisted of a short unicellular stalk and a secretory head of one or two cells; Type III consisted of a two-cellular stalk and a secretory head of one cell (Fig. 2C). The non-glandular hairs are unicellular, basally widened, sharp on the top (Fig. 3B) or multicellular, mostly having two to three cells, uniseriate, branchless, the top cell having a sharp edge, straight or bended (Fig. 3C). More or less wartlike cuticular structures are observed on the surface of the non-glandular hairs (Fig. 3D).
111
The glandular hairs are at the bottom of the complex arrangement of the leaf indumentum, leaning against the epidermal cells, while between them there are dispersed, long non-glandular hairs that form its upper layer. Basal cells of the glandular hairs are sunk under the level of epidermal cells, and their large heads (composed of several secretory cells) are in contact, leveling with the epidermis or being a bit above thus partially covering the leaf blade (Fig. 3A). The indumentum is particularly well developed on the abaxial leaf side (Fig. 3A), which is a common feature of plants from all five populations. Therefore, the abaxial leaf side is grayish-green in color. The plants from the Eumediterranean populations, namely from the localities near Budva and Krasˇ ic´i, as well as from Peloponnesus in particular, are characterized by conspicuously thick indumentum (Fig. 4). In contrast, the adaxial leaf side is always covered with sparse hairs rendering a dark green color to this side (Fig. 3E and Fig. 4). The cuticle on the leaf adaxial epidermis is thicker than that of the abaxial epidermis in plants of all the five populations studied. Besides, the outer epidermal cell walls are thickened, particularly in the adaxial epidermis occupying 2/3 of the cell lumen. The plants of the subMediterranean populations (from the gorges of Cijevna and Moracˇa) are characterized by larger adaxial epidermal cells in respect to those of the plants of Eumediterranean populations (Fig. 5). Anticlinal walls of the adaxial and abaxial epidermal cells are almost straight in the plants from the localities near Budva and Krasˇ ic´i (Fig. 6B), or slightly undulate, as in the plants from Peloponnesus and from the Moracˇa and Cijevna canyons (Fig. 6A). On the outer periclinal walls of the abaxial epidermal cells larger or smaller cuticle wrinkles are formed. The leaves are always hypostomatic; the stomata are more or less raised (Fig. 7C) above the epidermal cells, of diacytic and anomocytic type (Fig. 7A). Their frequency varies from 129 to 300 per mm2. The highest number of stomata are present in the plants from Krasˇ ic´i (186–300 per mm2), and the lowest in those from the Moracˇa canyon (129–186 per mm2) (Fig. 8). The stomata are usually encircled by the glandular hairs (either peltate or capitate), thus being partially covered with the subcuticular spaces of the secretory cells (Fig. 7B–D). Leaf thickness (Fig. 9) of all plants studied ranged between 210 and 440 mm, being the thickest in the plants from the canyon of Cijevna (267–440 mm). The mesophyll is clearly differentiated into palisade parenchyma and spongy parenchyma; the ratio of these two photosynthetically functional mesophyll tissues is generally 1.4:1, except in the plants from the locality near Budva, being 2:1 (Fig. 9). The palisade parenchyma, just below the upper epidermis, consists of 2–3 rows of elongated and densely arranged cells, which gradually shorten going towards
ARTICLE IN PRESS 112
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Fig. 3. Teucrium flavum A, indumentum on the lower leaf side (SEM). B, three-cellular hair. C, bicellular hair. D, wart-like structures on the surface of the non-glandular hairs (SEM). E, indumentum on peeled upper epidermis.
the spongy parenchyma. The anticlinal walls of the palisade cells are almost always undulate. The cells of spongy parenchyma are usually irregularly shaped, but with relatively small intercellular spaces (Fig. 10A, B). Even in the leaves of the plants from Peloponnesus, which are smaller and less thick, the palisade parenchyma is the thinnest but still densely packed into 2–3 layers, which is always the case in the plants of all the populations studied. In the leaf cross-section, a large number of vascular bundles, situated in the central region of the mesophyll, and only one main vascular bundle, developed in the leaf midrib, are observed. Large bundle sheath cells enclose the vascular bundles.
Stem shape and anatomy T. flavum is an evergreen, branchy, semi-ligneous shrub. In general, the resemblance of the external
morphology of the stem of all different populations is obvious. In all the populations studied, the stem height was between 40 and 70 cm, and a basal ligneous part is clearly developed and reaches the length of up to 30 cm. However, slight differences could be observed between the population groups growing in very different ecological conditions. In the individuals from Eumediterranean populations the height of the shrubs usually varies in between 40 and 45 cm. The internodia are very short and dense, so that the leaves overlap each other. Contrary to them, the shrubs of the sub-Mediterranean populations reach a height of over 60 cm, having longer internodia; therefore their leaves mostly do not overlap. The herbaceous stem of T. flavum specimens is more or less square-shaped (in the cross section), firm, particularly in the plants from the localities near Budva and Peloponnesus, as well as from the Cijevna canyon (Fig. 11A). Seldom, as in the plants from the localities from the Moracˇa canyon it is slightly rounded (Fig. 11B). The stem is well covered with epidermis, and the thick cuticle, permeated by wax deposits and glandular (peltate and capitate) and non-glandular hairs, is arranged in a similar way as in the leaves. Scattered stomata are slightly raised above the level of the surrounding epidermal cells. The stem cortex is differentiated into two to three layered subepidermal supporting tissues, collenchyma, and several layers of thin-walled parenchymatous cells with prominent intercellular spaces. The collenchyma is discontinuously distributed in the peripheral part of the stem, forming thick projecting strands particularly in the stem corners. The vascular tissue commonly forms a cylinder between the stem cortex and the pith, consisting of collateral vascular bundles separated by interfascicular parenchyma. Conspicuous cups of sclerenchymatous fibres are present above the phloem, being particularly large below the stem corners. These fibres are more developed in the vascular cylinder of plants from the localities of Moracˇa and Cijevna canyons as well as in those from Peloponnesus. However, in the stem of plants from the other two populations there are o nly 2–3 sclerenchyma cells above the phloem elements. The correlation coefficient of the cortex and the whole stem diameter ranges from 0.226 (Krasˇ ic´i) to 0.343 (Cijevna).
Multivariate analysis of the morpho-anatomical characters By PCA it could be shown that the structural variability of the populations studied is extremely complex, since the first three axes comprise only 47.58% of total variability. By MANOVA statistically significant differences between all populations were
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
113
Fig. 4. Box and whisker plots of basic statistic parameters of indumentum.
Fig. 5. Box and whisker plots of basic statistic parameters of epidermis.
established. It should be pointed out that the most important characters in structural differentiation are those related to leaf shape, stem anatomy, indumentum, and leaf anatomy features in this order of significance (Table 2). DCA of the populations studied of T. flavum has shown that the Eumediterranean (Peloponnesus, Krasˇ ic´i, Budva) and sub-Mediterranean (Moracˇa, Cijevna) populations represent two morphologically almost completely separate groups. On the first two
axes, the populations from Peloponnesus and Budva stand completely separated from the sub-Mediterranean populations. The population from Krasˇ ic´i shows (Fig. 12) transitional characteristics between the Peloponnesus and Montenegro sub-Mediterranean populations, as it was the case also in the PCA analysis. Morpho-anatomical separation between Mediterranean and sub-Mediterranean populations is clearly observed also on the basis of overall Mahalanobious’ distances (Fig. 13).
ARTICLE IN PRESS 114
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Discussion
Fig. 6. Teucrium flavum paradermal view A, undulate anticlinal walls of the upper epidermal cells of plants from Moracˇa. B, straight anticlinal walls of the upper epidermal cells of plants from Budva.
Fig. 7. Teucrium flavum A, anomocytic (a) and diacytic (d) stomata. B, stomata encircling by the glandular hairs or glandular hairs encircling stomata. (SEM). C, highly raised stomata and cuticular striae. (SEM). D, peltate hair around the stomata (SEM).
The analyzed populations of the species T. flavum inhabit the Mediterranean area on ‘‘terra-rossa’’ (red soil) substrate, which develops on porous limestone. The species grows within the maquis-garrigue vegetation of different stages of progression and regression, under various Mediterranean bio-climate conditions – perhumid Mediterranean Adriatic, arid Mediterranean Aegean and perhumid sub-Mediterranean Adriatic climate. The resemblance of plants from different populations is obvious, even at first sight, due to the similar general habit, i.e., of quite a similar external morphology of the stem and evergreen leaves. It is rather important to stress this uniformity of the species T. flavum that contrastes to the extreme morphological heterogeneity that is present within the whole genus Teucrium. A detailed growthform analysis of Teucrium have shown significant morphological differences between shrubs, semi-shrubs and perennial and annual herbs, especially in regard to the extension and volume of lignification, as well as with respect to the existence or the lack of sclerenchyma elements in the cortex. Some of these features can be used taxonomically on the level of sections and species groups (Ka¨stner, 1978, 1979, 1981, 1986). The macromorphological similarity of the stems and evergreen leaves of T. flavum populations might be assumed as a strong indication of structural stability and some kind of morpho-anatomical conservatism of this ancient Mediterranean xerophyte. However, this physiognomic uniformity obviously is combined with ecoanatomical differentiations of leaves and stems of T. flavum populations which thrive both in sites with summers not particularly dry, and in those with a pronounced summer drought. In general, the evergreen leaves of T. flavum are xeromorphic, moderately to significantly thick, tough but not flabby neither rigid nor hard. In the Mediterranean vegetation there is a large number of xerophytes with such leaves, commonly known as malakophyllous, notably within the genera of Cistus, Rosmarinus, Thymus, etc. (Breckle, 2002; Kummerow, 1973). The prominent xeromorphic features of the T. flavum leaves are: simple form and relatively small size, dense indumentum, especially on the lower side where a high number of stomata are also located. The smallest leaves, having a reduced external surface and mesophyll thickness, are found in plants from the Eumediterranean, Peloponnesian locality. In contrast, the thickest leaves with the largest lamina surface area have the plants from the sub-Mediterranean locality in the canyon of Cijevna. A dense and complex indumentum, composed of glandular and non-glandular hairs, covers the lower leaf side, shielding stomata and thus advantageously
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
115
Fig. 8. Box and whisker plots of basic statistic parameters of stomata.
Fig. 9. Box and whisker plots of basic statistic parameters of mesophyll.
reducing transpiration loss. Stomata and glandular hairs are very closely related, meaning that the stomata are surrounded by glandular hairs or glandular hairs are surrounded by stomata. In any case, stomata are partially or totally flanged by the large subcuticular swellings of peltate hairs and/or secretory cells of the capitate hairs. Since the protrusions of capitate secretory cells have only a small storing space, there is a continuous evaporation of essential oils (Werker et al., 1985a, b). This renders the air near the leaf surface more condensed thus providing a higher boundary layer
resistance to gas diffusion. Regardless of the small quantity of essential oils (0.1–0.2%), which characterizes the specimens of T. flavum, even their minimal presence in the external secretory structures may be efficient in reducing both transpiration and overheating (C´orovic´ et al., 1969; Todorovic´ and Stevanovic´, 1994). Otherwise, it is well known that all species of the genus Teucrium are distinguished by only a low quantity of essential oils (Kovacˇevic´ et al., 2001; Petricˇic´ et al., 1993). Beside essential oils, some other terpenoids, as well as flavonoids and/or phenylcarbonic acids might be
ARTICLE IN PRESS 116
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Fig. 10. Cross section of the leaf: A, from Krasˇ ic´i. B, from Peloponnesus.
Fig. 11. Cross section of the stem: A, square-shaped stem. B, rounded stem.
produced by these glandular hairs (Wollenweber, 1984). Terpene exudates are the resins that cover the surface of the plant above-ground parts, while the flavonoids are incorporated either into the resins or into the coating the leaf surfaces of many plants from the semi-arid regions
(Kelsey et al., 1984; Wollenweber, 1984). It has been reported previously that the resin content in T. flavum leaves is about 1.03%, consisting of terpenes, another phenolic compounds, and free flavonoids (Kovacˇevic´ et al., 1998). All these mostly aromatic volatile compounds
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Table 2.
117
Analysis of variances on the level of individual characters
Dependent variable
Mean sq. effect
Mean sq. error
F(df1,2) 4,45
p-level
Height of adaxial epidermal cells Thickness of palisade tissue Thickness of spongy tissue Height of abaxial epidermal cells Number of palisade layers Surface area of adaxial epidermal cells Surface area of abaxial epidermal cells Surface area of abaxial stomata Number of abaxial stomata Number of adaxial glandular hairs Number of abaxial glandular hairs Number of non-glandular adaxial hairs Number of non-glandular abaxial hairs Leaf length Distance between the largest leaf width point and the leaf top Largest width of the leaf Leaf surface area Stem diameter Stem diagonal Stem cortex thickness Thickness of stem vascular cylinder Stem pith diameter
134 2773 3143 20 0 2,180,681 130,681 33,954 285 6 45 30 54 75 48 35 353 1,838,400 1,726,035 2125 81,305 118,351
14.7 269.9 397.5 6.0 .1 118111.4 48122.2 9273.7 27.3 2.3 11.7 1.5 2.1 7.1 4.7 3.3 27.5 134840.0 55039.8 7129.5 5851.5 8893.8
9.09521 10.27664 7.90878 3.38172 2.36066 18.46292 2.71561 3.66135 10.47165 2.57299 3.86175 20.55275 25.43458 10.52627 10.19968 10.47934 12.84811 13.63394 31.35979 .29809 13.89465 13.30719
.000018 .000005 .000065 .016757 .067463 .000000 .041445 .011518 .000004 .050396 .008824 .000000 .000000 .000004 .000006 .000004 .000000 .000000 .000000 .877662 .000000 .000000
Fig. 13. Mahalnobius distances between the analyzed populations of Teucrium flavum.
Fig. 12. Discriminant analysis of the basic components (DCA).
and free flavonoids, along with the dense covering of the leaves by hairs, might have an important role in the adaptation of T. flavum not only to the arid and semiarid conditions of its habitats (Kelsey et al., 1984), but also in its protection from the intense light and UV radiation (Tomas-Barberan and Wollenweber, 1990). Similar micromorphological and phytochemical characteristics were established in the Apennine populations of the species T. flavum (Maleci and Servettaz, 1991; Maleci et al., 1995). Furthermore, it could be assumed that the waxy, thick and wrinkled cuticle as well as the sparse indumentum
ARTICLE IN PRESS 118
B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
on the upper leaf side consisting of short and protruding non-glandular hairs and of all the types of glandular hairs mentioned, especially in plants from the Eumediterranean populations, also have an important influence on the spectral features of the leaves. Such structural adaptations increase leaf reflectance, thus reducing solar inception, heat load and therefore water deficit. This has been reported for leaves of Mediterranean species from the genus Cistus (Gausman and Quisenberry, 1990). It is worth mentioning that the more dense indumentum of leaves from the Peloponnesian population must be regarded as a favorable adaptive modification which helps in protecting the mesophyll from excessive water loss and intense radiation in this particular hostile, perarid Mediterranean environment. More or less conspicuous xeromorphic characteristics of T. flavum, from different populations, are represented by a relatively high number of stomata, ranging from 129 to 257 per mm2 in the plants from the subMediterranean sites to 143–300 per mm2 in those from the Adriatic coast and the Peloponnesus. A similar high number of stomata was established also in the Apennine populations of T. flavum, in which the number of stomata varies from 168 to 258 per mm2 (Ka¨stner, 1979). It is also a typical xeromorphism that the dense, complex indumentum on the lower leaf side protects stomata which, on the other side are somewhat raised above the level of the epidermal cells. Smaller and thick-walled epidermal cells on the upper leaf side are a pronounced xeromorphic feature of the Eumediterranean populations of the taxon, compared with the two sub-Mediterranean ones. The anticlinal walls of both adaxial and abaxial epidermal cells of plant leaves from all populations studied are straight to wavy, which is described as an other xeromorphic feature (Fahn and Cutler, 1992). A ratio between 1.4:1 and 2:1 of 2–4 layered palisade parenchyma to 2–3 layered spongy parenchyma is characteristic for plants from all the five populations. The number of palisade layers is increased in the plants from Eumediterranean populations, particularly in those from the localities near Budva and Krasˇ ic´i, but also in those from Peloponnesus. Moreover, the palisade cells of these plants are narrower and more compact. In addition, in leaves of these groups of plants of spongy cells can be found, which elongate in such a way that they resemble palisade cells. A partial differentiation into an isolateral mesophyll is asumed to be a strongly xeromorphic character. Again, similar characteristics of the palisade and spongy tissues were established also in the Apennine populations of T. flavum (Ka¨stner, 1979). The stems of the studied T. flavum populations are also protected by a thick cuticle with waxy layers and with a dense indumentum of the same structural pattern as that of the leaves. The stem hairs are more protruding and less intertwined than those on the leaves. The ratio
of the stem cortex to stem diameter ranges from 0.226 to 0.343, which is within the usual values found in xeromorphic stems (Fahn and Cutler, 1992). All the data obtained, particularly those subjected to a comparative multivariant analysis of morpho-anatomical characteristics of the five populations from three different climate variants, have shown that the species T. flavum has maintained quite a stable, conservative morpho-anatomical structure. Such a substantial similarity between different populations is also characteristic of other ancient Mediterranean plants since their adaptive structures evolved, in the first place, as the consequence of severe summer drought and high temperatures (Margaris, 1981). In all the localities, both eu- and sub-Mediterranean ones (arid-Mediterranean and perhumid-sub-Mediteranean), the plant populations of which have been studied, more or less pronounced, longer or shorter intervals of summer drought stress prevail. Inter-population differences refer to small variations in leaf size, indumentum density, number of stomata, thickness of cuticle and outer epidermal cell walls, as well as to the number of the mesophyll tissues layers. While all these anatomical features are of the same pattern, they are clearly more expressed in the Eumediterranean than in the subMediterranean populations.
Acknowledgements The authors are grateful to Prof. Dr. Vladimir Stevanovic´, Institute of Botany and Botanical garden ‘‘Jevremovac’’ University of Belgrade for useful advice and valuable comments and to the Ministry for Science, Technology and Development of Serbia (Project Nos. 1568 and 1505) for financial support.
References Breckle, S.W., 2002. Walter’s Vegetation of the Earth. Springer, Berlin. C´orovic´, M., Stejepanovic´, L., Nikolic´, R., Pavlovic´, S., Zˇivanovic´, P., 1969. Comparative investigation of the level of osmotic values, transpiration and quantity of essential oil in some species of Labiatae family. Glasnik Botanicˇkog zavoda i basˇ te Univerziteta u Beogradu 4, 19–27. Fahn, A., Cutler, F.D., 1992. Xerophytes. Encyclopedia of Plant Anatomy. Borntraeger, Berlin. Gausman, H.W., Quisenberry, J.E., 1990. Spectrophotometric detection of plant leaf stress. In: Katterman, F. (Ed.), Environmental Injury to Plants. Academic Press, San Diego, pp. 257–280. Ka¨stner, A., 1978. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. I. Die Infloreszenzen und Blu¨ten. Flora 167, 485–514.
ARTICLE IN PRESS B. Lakusˇ ic´ et al. / Flora 201 (2006) 108–119
Ka¨stner, A., 1979. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. II. Anatomie der Sprosse und Bla¨tter. Flora 168, 431–467. Ka¨stner, A., 1981. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. III. Wuchsformen und Verbreitung von Arten der Sektionen Teucropis und Teucrium. Flora 171, 466–519. Ka¨stner, A., 1986. Beitra¨ge zur Wuchsformenanalyse und systematischen Gliederung von Teucrium L. V. Wuchsformen und Verbreitung von Arten der Verwandtschaftskreise von T. marum und T. chamaedrys sect. Chamaedrys. Flora 178, 111–138. Kelsey, R.G., Reynolds, W.G., Rodriguez, E., 1984. The chemistry of biologically active constituents secreted and stored in plant glandular trichomes. In: Rodriguez, E., Healey, L.P., Mehta, I. (Eds.), Biology and Chemistry of Plant Trichomes. Plenum Press, New York, London, pp. 187–243. Kovacˇevic´, N., Ristic´, M., okovic´, D., Vukovic´, Z., Lakusˇ ic´, B., 1998. Herb exudates of Teucrium L. species. Arch. Pharm. 6, 944–945. Kovacˇevic´, N., Lakusˇ ic´, B., Ristic´, M., 2001. Composition of the essential oils of seven Teucrium species from Serbia and Montenegro. J. Essential Oil Res. 13, 163–165. Kummerow, J., 1973. Comparative anatomy of sclerophyllus of Mediterranean climatic areas. In: Di Castri, F., Mooney, A.H. (Eds.), Mediterranean Type Ecosystems – Origine and Strukture. Springer, Berlin, Heidelberg, New York. Lakusˇ ic´, B., 2000. Morphological variability and ecological differentiation of species of genus Teucrium L. (Lamiaceae) in Yugoslavia. Unpublished D. Phil. Thesis, Fac. Biol., University in Belgrade. Maleci, L.B., Servettaz, O., 1991. Morphology and distribution of trichomes in Italian species of Teucrium sect. Chamaedrys (Labiatae) – a taxonomical evaluation. Plant Syst. Evol. 174, 83–91. Maleci, L.B., Pinetti, A., Servettaz, O., 1995. Micromorphological and phytochemical characteristics of the two
119
subspecies of Teucrium flavum L. (Labiatae) from the Italian flora. Flora 190, 237–242. Margaris, N.S., 1981. Adaptive strategies in plants dominating Mediterranean-type ecosystems. In: Di Castri, F., Goodall, D.W., Specht, L.R. (Eds.), Mediterranean-type Shrublands. Elsevier, Amsterdam, pp. 309–315. Meusel, H., Ja¨ger, E., Rauschert, S., Weinert, E., 1978. Vergleichende Chorologie der Zentraleuropa¨ischen Flora, vol. 2. Fischer, Jena. Petricˇic´, J., Blazˇevic´, N., Plazibat, M., 1993. Essential oil of Teucrium flavum L. from Croatia. J. Essential Oil Res. 6, 625–627. Todorovic´, B., Stevanovic´, B., 1994. Adaptive characteristics of the endemic species Satureja horvatii Sˇilic´ (Lamiaceae) in mountain-Mediterranean and Mediterranean habitats. Bot. J. Linnean Soc. 114, 367–376. Tomas-Barberan, F.A., Wollenweber, E., 1990. Flavonoid aglycones from the leaf surfaces of some Labiatae species. Plant Syst. Evol. 173, 109–118. Tutin, T.G., Wood, D., 1972. Teucrium L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea, Vol. 3. Cambridge University Press, Cambridge, pp. 129–135. Werker, E., Ravid, U., Putievsky, E., 1985a. Structure of glandular hairs and indetification of the main components of their secreted material in some species of the Labiatae. Israel J. Bot. 34, 31–45. Werker, E., Ravid, U., Putievsky, E., 1985b. Glandular hairs and their secretions in the vegetative and reproductive organs of Salvia sclarea and S. dominica. Israel J. Bot. 34, 239–252. Wollenweber, E., 1984. The systematic implication of flavonoids secreted by plants. In: Rodriguez, E., Healey, L.P., Mehta, I. (Eds.), Biology and Chemistry of Plant Trichomes. Plenum Press, New York, London, pp. 53–69.