International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(2) pp. xxx-xxx, March, 2011 Available online http://www.interesjournals.org/IRJPS Copyright © 2011 International Research Journals Full length Research Paper

Comparative analysis of stomatal characters in eight

wild atlas pistachio populations (Pistacia atlantica

Desf.; Anacardiaceae)

Safia Belhadj,1* Arezki Derridj,2 Alfonso Moriana,3 Maria Del Carmen Gijon,4 Jean-Phillipe Mevy5 and Thierry Gauquelin 5

* 1 Département d’Agropastoralisme, Université « Ziane Achour » de Djelfa, B.P.3117 Poste Ain Chih, Djelfa 17000,

Algeria. 2 Laboratoire de Biosystématique Végétale, Faculté des Sciences Biologiques et Agronomiques, Université “Mouloud Mammeri”, Tizi-Ouzou 15000, Algeria. E-mail: aderridj@yahoo.fr. 3 EUITA, University of Seville, Crta de Utrera Km, 1, 41013 Sevilla, Spain. E-mail: amoriana@us.es. 4 CMA El Chaparillo, Consejería de Agricultura, Junta de Castilla-La

Mancha, Crta. de Porzuna Km 3.5, 13071 Ciudad Real, Spain. E-mail: cgijon@jccm.es. 5 Institut Méditerranéen d'Ecologie et de Paléoécologie. UMR CNRS 6116, Université de Provence, Case 421 – Av. Escadrille Normandie

Niemen, 13397, Marseille Cedex 20, France. E-mail: Jean-Philippe.Mevy@univ-provence.fr, thierry.gauquelin@univ-provence.fr.

Accepted 21 March, 2011

Little is known about the morphological variations or differences in stomatal characteristics of Pistacia species. How these variations may be related to the environment is also unknown. This paper describes the diverse stomatal morphology recorded among the Algerian representatives of Pistacia atlantica Desf. (Atlas pistachio). Leaf Samples of eight Algerian sites of Pistacia atlantica were characterised according to their stomatal type, distribution and position in the epidermis, shape, size and density by the mean of light microscopy and scanning electron microscopy. The leaves are amphistomatic in all the sites. However the abaxial stomatal densities are much higher. The mean number of stomata per square millimetre

was 30.5 on the adaxial face and 308.5 on the abaxial face. The stomata were elliptical and

slightly sunkun in the epidermis, of the actinocytic or anomocytic types with a mean length of 29.1µm and a mean width of 19.5µm. However, the variations in the aperture of stomata did not reveal a clear pattern in response to the ecological gradient tested, suggesting that it is a genotypic character. Only, the stomatal density showed agreement with the environmental conditions for the areas where the trees were grown. Especially up to 900 m in altitude ecological conditions abaxial leaf stomata density decreases significantly compared to that of lowland habitats. The best discriminating traits recorded in this study were the stomata size and the stomatal density. These characteristics may afford an initial screening method for classifying P. atlantica in terms of drought resistance. Keywords: environment, physiology, Pistacia atlantica, SEM, size, shape, stomata.

INTRODUCTION In North Africa, more than 100,000 hectares are lost to desertification processes every year (Le Houérou, 1995; Gauquelin et al., 1999). The Sahara and the semi-arid areas of North Africa, along with its steppe-land, are *Corresponding author: Tel: (213) 27900203(04). Fax: (213) 27900201. E-mail: belhadjsafia@yahoo.fr.

home to flora of great interest (FAO, 2007). The preservation of local floral diversity is required if reforestation programmes are to be improved and appropriate ecotypes selected for different areas. The genus Pistacia ,including P. lentiscus L., P. terebinthus L., P. atlantica Desf. and P. vera L., is widely present in North Africa however Pistacia atlantica is the most common (Zohary, 1952; Quézel and Santa, 1963,

Monjauze, 1980). These trees are important in forest regions because they provide protection from wind and water erosion and contribute to soil stability and conservation (Esmail-Pour, 2001). On the basis of leaf and fruit characteristics and of eco-geographical distribution, P. atlantica is subdivided into four varieties: (1) latifolia, (2) kurdica, (3) kabulica and (4) atlantica, with the latter being the one occurring in Algeria (Zohary, 1952; Alyafi, 1979; Monjauze, 1980). Pistacia atlantica (Atlas pistachio) is an endemic perennial woody plant in North Africa (Ozenda, 1983; White, 1986; Quézel and Médail, 2003). It used to be widely distributed across arid and semi-arid regions; it occurs as isolated scattered trees or in dense populations at a range of altitude from less than 200m up to 1200m (Monjauze, 1980; Ozenda, 1983; Quézel and Médail, 2003). Atlas pistachio is one of the main constructive species of arid and semi-arid Mediterranean forest ecosystems and plays a very important role in retaining ecological stability preventing desertification. It is a drought-resistant plant able to grow in harsh conditions in which few tree species can be grown and established (Mirzaie-Nodoushan and Arefi, 2001); its large rooting system renders it suitable for reforestation programmes in the semi- arid and arid areas. Nowadays, intense, germplasm erosion is underway due to human activities and intensive grazing, within its wide range of distribution and especially in Algeria (Quézel and Santa, 1963; Monjauze, 1980; Belhadj, 1999, 2001, 2002; Quézel and Médail, 2003). Actually, this species is being conserved and propagated in its potential area however; population differences in morphological and physiological properties have not been well established in this species. Despite the numerous studies on the genus, limited data are available on the physiological characteristics of this species underlying its drought resistance mechanisms. Using morphological characters, such as those related to leaves and fruits (Belhadj et al., 2008), to leaf epidermis (Belhadj et al., 2007a) and to pollen grains (Belhadj et al., 2007b) we showed that the studied Atlas pistachio populations could be divided into different groups. Leaf micromorphology and especially the stomata characteristics could potentially be used as discriminating characters as well. To our knowledge, few investigations have been performed on the stomatal characteristics of Pistacia species. Although great contributions have been made to the description of leaf micromorphology (Alyafi, 1979; Lin et al., 1984; El-Oqlah, 1996; Çaglar and Tekin, 1999; Özeker and Misirli, 2001; Ait-Said et al. 2004; Kadi-Bennane et al., 2005; Smail-Saadoun, 2005), stomata of only a few species have been examined with the light microscopy and fewer with the scanning electron microscopy. Atlas pistachio is not well known systematically and ecologically, and it is quite difficult to make distinction between P. atlantica varieties by the classical leaf morphological traits, since several specimens may have intermediary characters (Zohary,

1952, 1987; Yaltirik, 1967). Pistacia atlantica is known to have a higher adaxial leaf stomatal density than P. khinjuk Stocks or P. vera (Çaglar and Tekin, 1999; Özeker and Misirli, 2001), and a lower abaxial one compared to P. terebinthus. No conclusions regarding the relationship with drought resistance or ecosystem type have been drawn. Stomatal characteristics may therefore be good descriptors of the environmental conditions reigning in different ecosystems. The aim of this study was (i), to access the impact of contrasting local climatic conditions on P. atlantica leaf stomata traits with emphasis on the physiological mechanisms involved (ii), to discriminate phenotypic or genotypic traits that could serve to some extent as references toward infra-specific classification of P. atlantica. MATERIALS AND METHODS Plant material: Pistacia atlantica leaves used for this study were collected from eight different sites around Algeria. Specimens were selected such as to cover a wide climatic variation, and each site is characterized by its Mediterranean bioclimatic type using Q3, the classical Emberger’s quotient (Table 1). For each site, the following voucher specimens were kept in the herbarium of the University of Provence, Marseille (France): Guerrara, (Mar-2007-PA-G); Berriane, (Mar-2007-PA-Be); Brezina, (Mar-2007-PA-Br); Elhamel, (Mar-2007-PA-E); Messaad, (Mar-2007-PA-M); Ain Oussara, (Mar-2007-PA-Ao); Aflou, (Mar-2007-PA-Af) and finally Oued Safene, (Mar-2007-PA-Os). Other leaf samples were kept as well in the herbarium of the University of Djelfa (Algeria). Light microscopy: In each area, five randomly selected samples of fully expanded leaflets were taken from different leaves, from five healthy trees (a total of 200 leaflets). The samples were allowed to dry under laboratory environmental conditions until use. After several months they were remoisten in distilled water for 15 to 30 minutes and, when dry, the leaf impression technique was applied to view stomata (Banon et al., 2004). A thin layer of clear nail varnish was painted onto both adaxial and abaxial leaf surfaces and left for 5 to 10 min. A strip of transparent sticky tape (sellotape) was placed over the dried varnish and pressure applied to obtain an imprint. The sellotape with its imprint was peeled from the leaflets and placed onto a glass microscope slide. Replicas were examined under an optical microscope (Leica D.M.L.S, Germany). The length and width of ten stomata per leaf were measured on the abaxial face (a total of 2000 measurements). In addition, the stomata in ten adaxial and ten abaxial areas of each leaf were counted (a total of 4000 measurements). Scanning electron microscopy: Others leaves were soaked in distilled water or either cleaned with ethanol (90%) in order to remove external particles and dust then, standard procedure was followed for SEM (Belhadj et al., 2007a). Two specimens from each site were examined. A section of 5 mm2 of the dry leaf surface (both adaxial and abaxial surfaces) was fixed on a labelled stub. The samples were coated with carbon and scanned in a JEOL. JSM-6360 LV-Japan Microscope. Micromorphological observations included stomata shape and type description, distribution and position in the epidermis. SEM pictures were digitally recorded in different magnifications. Stomatal terminology was based on the classification proposed by Metcalfe and Chalk (1950), Baranova (1972, 1983, 1987, 1992), Wilkinson (1979) as well as the studies of Prabhakar (2004) and Carpenter (2005). Statistical analysis: The data were subjected to ANOVA and the mean separation was made with the Tukey (HSD) test to determine whether the differences among and within populations were

Table 1. Main climatic features of the selected experimental sites.

Sites Latitude Longitude Altitude (m)

M. (ºC)

m. (ºC)

An. Rain (mm)

Q3 Climate type

Guerrara (G) 32º48’N 4º29’E 107 42.5 4.7 67.2 6 Saharian mild Berriane (Be) 32º51’N 3º46’E 764 36 2.3 119.7 12.2 Saharian fresh Brezina (Br) 33º6’N 1º15’E 900 35.1 0.3 230.2 22.5 Arid fresh Elhamel (E) 35º9’N 4º4’E 730 38.7 4 247.9 24.5 Arid mild Messaad (M) 34º11’N 3º36’E 780 35.4 1.3 250.8 25.1 Arid fresh Ain Oussara (Ao) 35º25’N 2º50’E 820 35.8 0.5 238.2 25.9 Arid fresh Aflou (Af) 34º9’N 2º5’E 930 33.4 -0.05 342 35 Semi arid cold Oued Safene (Os) 34º55’N 0º48’E 1100 32.7 1.8 462.9 51.2 Semi arid fresh

Source of climatic data: National Meteorology Office of Algeria. Q3: Emberger quotient, M: Mean of the maxima temperatures of the warmest month, m: Mean of the minima temperatures of the coldest month.

significant or not using Statistix analytical software (version 1.0, 1996). RESULTS Stomata distribution and position in the epidermis: The leaves were amphistomatic in all the sites; the stomata were present both on the abaxial (Figures 1A, 1B) and the adaxial face (Figures 2A-D, 2F). On the adaxial face, they were located exclusively along and near the midribs, while on the abaxial surface they were widely spread and sunken (Berriane, Messaad, Aflou and Elhamel) slightly sunken below the epidermis (Guerrara, Brezina, Elhamel, Ain Oussara and Oued Safene) to level (Guerrara and Brezina) with the epidermis (Table 2; Figures 1B, 1D, 2C-E, 3). Stomata type, shape and size: Four different stomata types were recorded in our study for P. atlantica leaves (published data in Belhadj et al., 2007a). The most frequently observed were the actinocytic (Figure 1C) and the anomocytic types. Nonetheless, the laterocytic and paracytic types were recorded as well. In the actinocytic type, the stomata were surrounded by a circle of radiating cells while in the anomocytic type, the stoma were surrounded by a limited number of cells that are indistinguishable in size, shape, or form from those of the remainder of the epidermis (Metcalfe and Chalk 1950, Baranova 1983). The guard cells were surrounded by a ring of subsidiary cells in the laterocytic type while the paracytic type was characterized by one or two lateral subsidiary cells oriented parallel to the guard cells (Baranova 1983, Carpenter 2005). Two to four types were recorded within a same population in this study. In Aflou (Af) site only the actinocytic and the anomocytic types were recorded. The paracytic type was not recorded in the Elhamel (E) and Guerrara (G) sites whereas in the Berriane (Be), Brezina (Br), Messaad (M), Ain oussara (Ao) and Oued safene (Os) sites the four types were recorded (Table 2).

The different sites varied significantly in the dimensions of their stomata on the abaxial face (Table 2). Their dimensions varied between 23.9 µm (E site) to 32.6 µm (Os site) in length with a mean value of 29.1 µm. For the width, the values were lower than those recorded for the length. The mean value was about 19.5 µm and the least value (15.7 µm) was recorded for the E site whereas the highest (21.2 µm) for the Os site. The stomata were longer than larger in all the sites with a mean ratio L/W around 1.52. The highest value (1.56) was recorded in the E site whereas the lowest value (1.46) was recorded in the Ao and Af sites. The stomata were elliptical in all the sites except for the Ao and Be sites where they appeared somehow circular (Table 2, Figures 3C, 3F). Variation among and within populations expressed by the coefficient of variation was more or less high for the stomatal dimensions. The coefficients of variation were high among populations especially for the width (18.6%) and the length (17.9%). The variation within populations was important both in length and width as well for the ratio. The highest values were recorded, for the length in M (17.6%) and Be (17.2%) sites whereas for the width, the E (18.9%) and Be (18.1%) sites had the highest variation. The lowest values were recorded in G site both for length and width (respectively, 11% and 12%). Concerning the ratio, the E site had the highest value of variation (20.9%); the lowest (14.7%) was recorded in the G site. The statistical analysis showed highly significant differences among and within populations both in length and width except for Br site (Table 2). The Os site had the longest stomata, followed by G site (no significant differences with the latter). Messaad site was near the previous group but significantly lower than Os. Aflou and Br sites (no significant differences between both of them) were lower than the first ones but without significant difference with M. Bellow 30 µm was Ao site though without significant difference with Af and Br. Finally, Be and E sites which are significantly the lowest. Variation in width was less appreciable. The widest stomata (>20 µm)

Table 2. Stomata characteristics of P. atlantica leaves from different sampling sites. Data are means ± SD, range (Min-Max) and coefficient of variation (%).

Site

Stomata length (L) (µm)

Stomata width (W) (µm)

L/W ratio

stomatal density (no.mm

-2 )

Stomata position in the epidermis

Abaxial Adaxial Guerrara (G) 31.5***ab ± 3.7

22.0-43.0 (11.0) 20.6***ab ± 2.5 12.0-30.0 (12.0)

1.55*ab ± 0.23 1.0-2.33 (14.7)

318.5***bc ± 47.3 194.6-457.6 (14.9)

44.7***ab ± 17.2 0.0-94.7 (38.5)

Level with to slightly sunken

Berriane (Be) 24.8***e ± 4.3 14.0-37.0 (17.2)

16.6***c ± 3.0 10.0-27.0 (18.1)

1.52NSabc ± 0.24

0.93-2.31 (16.2) 329.6***ab ± 58.6 78.9-583.9 (17.8)

41.9***b ± 19.4 0.0-99.9 (46.3)

Sunken

Brézina (Br) 29.9NScd ± 3.8

20.0-40.0 (12.8) 19.7NS

b ± 3.0 10.0-30.0 (15.1)

1.54NSabc ± 0.23

1.07-2.50 (15.03) 281***f ± 41.3 194.6-431.3 (14.7)

42.6NSb ± 12.6

0.0-78.9 (29.7) Level with to slightly sunken

Elhamel (E) 23.9***e ± 4.0 13.0-35.0 (16.8)

15.7***c ± 2.9 8.0-28.0 (18.9)

1.56***a ± 0.33 0.75-2.87 (20.9)

307***cd ± 51.1 205.1-541.8 (16.7)

7.4NSe ± 8.2

0.0-36.8 (110.6) Slightly sunken to sunken

Messad (M) 30.9***bc ± 5.4 18.0-47.0 (17.6)

20.8***a ± 3.3 12.0-32.0 (15.9)

1.50***abc ± 0.25 0.90-2.34 (16.6)

338.1***a ± 83.6 10.5-589.1 (24.8)

6.6NSe ± 7.7

0.0-36.8 (116.7) Sunken

Ain oussara (Ao) 29.1***d ± 3.4 22.0-43.0 (11.5)

20.4***ab ± 3.3 14.0-35.0 (16.1)

1.46NSc ± 0.23

0.88-2.28 (16.1) 309.9***cd ± 47.8 31.6-431.3 (15.4)

16.3**d ± 12.4 0.0-57.9 (76.5)

Slightly sunken

Aflou (Af) 30.2***cd ± 4.2 22.0-44.0 (13.9)

20.8***a ± 2.7 12.0-30.0 (12.8)

1.46NSbc ± 0.22

1.00-2.53 (14.8) 283.5***ef ± 49.7 157.8-473.4 (17.6)

35.4***c ± 14.7 0.0-78.9 (41.6)

Sunken

Oued safène (Os) 32.6***a ± 5.3 20.0-46.0 (16.4)

21.2***a ± 3.4 14.0-31.0 (16.2)

1.55**a ± 0.24 1.07-2.25 (15.3)

299.8***de ± 67.2 168.3-478.7 (22.4)

49.3***a ± 18.9 0.0-105.2 (38.7)

Slightly sunken

Mean 29.1*** ± 5.23 13.0-47.0 (17.9)

19.5*** ± 3.62 8.0-35.0 (18.6)

1.52*** ± 0.25 0.75-2.87 (16.5)

308.5*** ± 60.33 10.5-589.1 (19.6)

30.5*** ± 21.9 0.0-105.2 (71.9)

Slightly sunken

a,b,c, d, e Mean separation within columns, by Tukey test (p<0.001). Values with same letters are not significantly different. . *: significant at p<0.05; **: significant at p<0.01; ***: significant at p<0.001; NS: not significant

were noticed in the Os, M, Af, G and Ao sites (no significant difference), followed by those of Br (no significant difference to Ao and G, although different to the other members of the preceding group). The narrowest stomata were found in the Be and E sites (no significant difference between the two). Stomatal density: Stomatal densities differed statistically among and within sites. The variations were less apreciable in the adaxial face, with no significant differences in Br, E and M sites (Table 2). Sites M (338.1 no/mm2) and Be (329.6

no/mm2) showed the highest abaxial stomatal densities. The lowest values were recorded in the Br (281 no/mm2) and Af (283.5 no/mm2) sites. The mean value was about 308.5 no/mm2. Concerning the adaxial stomatal densities, the values varied from 6.6 no/mm2 in the M site to 49.3 no/mm2 in the Os site whereas the mean value was about 30.5 no/mm2 (Table 2). Variations among and within populations for the stomatal densities were high especially for the adaxial ones. The coefficient of variation varied from 14.7% (Br) to 24.8% (M), with a mean value of 19.6% in the

abaxial face. From 29.7% (Br) to 116.7% (M) and a mean value of 71.9% in the adaxial face (Table 2).

The statistical analysis showed highly significant differences among the populations for the stomatal densities. Thought the populations were separated onto three groups for the abaxial density and onto four groups for the adaxial one (Table 2). In the abaxial face, the number of stomata varied significantly among the populations with the M site being the first followed by Be site but not significant from each other.

Figure 1. SEM photographs of stomata distribution in leaves of P. atlantica on the abaxial leaf side, from the Aflou site. (A) Stomata appearance and density (x 400), (B) stomata appearance at 25% leaf plane inclination (x 650), (C, D) higher magnification of stomata, (C) Actinocytic type (x 1200) and (D) sunkun stoma (x 1100), respectively.

These were followed by the G site (no significant difference with the Be site, but significantly different to the M site) and the Ao and E sites (both of them significantly different from Be and M sites but no significant from the G site). The Os and Af sites (significantly not different from each other) were near the Br site (no significant difference) which was significantly different from the rest (Table 2). In the abaxial face, sites Os and G were the first (no significant difference between the two) followed by the Be and the Br sites (no significant difference between the two and the previous). The Af site was near the Ao site (both of them significantly different from each other and from the previous sites). Finally, the E and M sites were not different from each other but significantly different from the rest of the sites (Table 2). DISCUSSION The results described in this report deal with the effect of such ecological parameters as temperature, precipitation and altitude (Table 1) on the micromorphological features on P. atlantica leaf stomata. The over all leaf stomata distribution was not affected by the local climatic conditions investigated. Indeed, our observations showed that the leaves were

amphistomatic, this is in accordance with the studies of Lin et al. (1984) and Özeker and Misirli (2001). Amphistomaty is a common feature in xeric habitats (Fahn, 1967; Yiotis et al., 2006), and is regarded as adaptation designed to raise maximum leaf conductance to CO2 (Bondada and Oosterhuis, 2000). Concerning the adaxial leaf surface, the stomata were located only near the major and minor veins in accordance with the studies of Lin et al. (1984) while they were spread all over the whole surface as described by Özeker and Misirli (2001). Besides in our study, the stomata were slightly sunken in the most of the sites whereas they were level with the epidermis in Alyafi’s (1979) and Lin et al. (1984) studies. In most mesomorphic plants the stomata are level with or very slightly sunken below or raised above the rest of the epidermis, but in some xeromorphic plants they are often sunken to a considerable degree (Stace, 1965). Our results provide evidence that stomata position according to epidermis surface level reflects adaptations to the native habitats where the plant grows. Investigating stomata size and shape we came to the conclusion that the stomata were longer than wide with a constant ratio L/W along the ecological gradient tested. Hence the lack of variation in stomata aperture suggests that this trait is rather genotypic than adaptative. To this

Figure 2. SEM photographs of stomata distribution in leaves of P. atlantica on the adaxial leaf side. (A, B) stomata along the midrib, (A) Berriane (x 300), (B) Brezina (x 400); (C, D, E, F) higher magnifications of stomata, (C) Berriane (x 1100), (D) Elhamel (x 900), (E) Berriane (x 1600), (F) Berriane (x 950).

stand point, the stomata size recorded in this study are not similar to those reported by other authors for the same species: they were shorter and wider for Özeker and Mirisli (2001), longer and wider for Ait-Said et al. (2004) (Table 3). With regard to the stomata shape, they were described as elliptical or orbicular depending to the local conditions. From the resurrection plant Reaumuria soongorica (Pall.) Maxim it has been shown that upon rewatering, stomata were ring-shaped (Liu et al., 2007) suggesting that elliptical and orbicular forms may be considered as xeromorphic characters. Stomatal characters are widely used in the characterisation of plants belonging to different ecotypes.

These characteristics correlate negatively with one another in most of the species studied so far. No strict relationship exists between stomatal size or density and the regular availability of water. Nonetheless, adaxial and abaxial leaf stomatal characters are commonly related as reported in Eucalyptus spp. (Ngugi et al., 2004) and in Glycine max L. (soybean) (Gitz et al., 2005). In addition other environmental factors such as soil salinity, CO2 concentration, frost and disease tolerance can affect these leaf characteristics (Roselli et al., 1989; Botti et al., 1998; Sharma et al., 2001; Lawson et al., 2002). Several authors have used stomatal size and density to characterise lines growing at different sites, usually with

Table 3. Stomata characteristics of Pistacia atlantica. Comparison of data from different sources.

respect to drought resistance (XueJun and XinShi, 2000; Balok and Hilaire, 2002), but also with the amount of shade (Gamage et al., 2003) or the NO2 content of the air (Siegwolf et al., 2001). In the natural dry lands environment, trees are frequently subjected to seasonal water stress, which limits their growth and development. Trees differ markedly in their ability to withstand desiccation and have evolved complex mechanisms to cope with various environmental stresses including drought. Plants respond to

drought through changes in morphological, physiological, biochemical and metabolic processes (Elfadl and Luukkanen, 2006). A careful examination of the climatic conditions of the investigated sites (Table 1) showed that precipitations increase with elevation while maximum temperature decreases. Along this gradient, up to 900 m of altitude, abaxial leaf stomata density decreases significantly compared to the data from lowland sites (Table 2). In mountainous conditions, short-wave solar

radiation often increases as well as the overall incident sunlight beam. As a result, plant transpiration increases with altitude as demonstrated by Gale (1972). This is in accordance with several authors who stated that leaf stomata density increases from low- to upland sites (Kofidis et al., 2007; Yang et al., 2007). Especially, soil-water gradient was shown as the main ecological factor discriminating leaf stomata density along with elevation (Yang et al., 2007).

Author Abaxial Stomata length (µm)

Abaxial Stomata width (µm)

Abaxial Stomatal density (no.mm

-2)

Adaxial Stomatal density (no.mm

-2)

Stomata type Stomata position

Origin

Alyafi (1979) / / / / / Level with the epidermis

/

Lin et al. (1984)

/ / 569 ± 142 84 ± 24 Actinocytic Level with the epidermis

California (USA)

Özeker and Misirli (2001)

18.9 36.3 81.78 439.8 / / Manisa Yunt mountain (Turkey)

Ait-Said et al. (2004)

42.4 40.4 24.3

34.9 32.7 14.9

282.9 303.4 427.4

0 0 0

/ / Ain oussara (Algeria) Messaad (Algeria) Taissa (Algeria)

Kadi-Bennane et al. (2005)

/ / 282.9 303.4 427.4

0 0 0

Anomocytic perigenous Anomocytic mesoperigenous Anisocytic mesoperigenous Paracytic mesoperigenous Paracytic mesogenous

/ Ain oussara (Algeria) Messaad (Algeria) Taissa (Algeria)

Smail-Saadoun (2005)

/ / / 0 Anomocytic perigenous Anomocytic mesoperigenous Anisocytic mesoperigenous Paracytic mesoperigenous Paracytic mesogenous

/ Algeria

Our results (mean values)

29.1 ± 5.2 19.5 ± 3.6 308.5 ± 60.3 30.5 ± 21.9 Actinocytic Anomocytic Paracytic Laterocytic

Level with to slightly sunken in the epidermis

Algeria

Figure 3. SEM photographs showing different shape and position in the epidermis of stomata of the studied P. atlantica populations, on the abaxial leaf side (x 1400). (A) Brezina, (B) Guerrara, (C) Berriane, (D) Elhamel, (E) Messaad, (F) Ain oussara.

Our findings on the reduction of stomata density disagree with the above-mentioned data suggesting that this leaf trait is not solely driven by precipitations. Hence it is likely that the edaphic environment would have played a key role and this raises the question of the physiological mechanisms involved in stomata density changes. Because low soil-water retention capacity results in a decrease of transpiration rate, we may argue that this change might in turn impair the mobile flux of abscisic acid and kinetin which are known to increase plant stomata density (Wang et al., 1997; Franks and Farquhar, 2001). The stomata type recorded in this study were numerous. From 764 m of altitude and for minimum temperature up to 0°C, four stomatal types were recorded. However, for minimum temperature below 0°C only two stomatal types were shown. This is in accordance with the previous studies (Kadi-Bennane et al., 2005; Smail-Saadoune, 2005) in which several types were recorded for P. atlantica from three different populations whereas Lin et al. (1984) reported only a single type (Table 3). In addition, in our study, the laterocytic type was recorded;

this may be due to the adaptation of the species to the harsh environmental conditions where it grows by differentiating a new type of stomata. Plants occurring in Mediterranean type climates are subjected to heat and drought stress during the summer, most of them has developed morphological and physiological mechanisms which allow them to adapt and survive. These mechanisms mainly comprise deeply developing stomata and accumulation of secondary metabolites (Bosabalidis and Kofidis, 2002). Drought is the environmental condition that most affects leaf characteristics (XueJun and XinShi, 2000). Drought stressed olive plants for instance in order to save water, undergo anatomical alterations in their leaves that, principally comprise an increase of the stomatal density. With respect to stomatal size, significant reductions of the length and width values were estimated in the drought stressed plants as well as increased stomata density on the lower leaf surface, which contributes to a better control of transpiration (Bosabalidis and Kofidis, 2002) whereas large stomata seem to be a feature for plants living in humid conditions (Hetherington and Woodward, 2003). In literature report,

with the exception of more advanced development of palisade tissue, none of the characteristics commonly associated with xeromorphic leaves were found in the Pistacia species (Lin et al., 1984) which has been referred to as xerophytes. We’ve showed in our previous study (Belhadj et al., 2007a) that the covering hairs are always associated with stomata, and located along the midrib in the adaxial leaf face where stomata exclusively occur. In addition to that, these stomata are always covered with wax particles, and the epidermis was strongly winkled, especially around the stomata. CONCLUSION Significant differences in stomata parameters among populations were detected in this study. Stomata size on the abaxial surface and stomata density on the adaxial surface were the best discriminating traits. Oued Safene (Os) site had the biggest stomata while Elhamel (E) site had the smallest ones. The Messad (M) site registered the highest stomatal density on the abaxial leaf surface and the lowest on the adaxial surface one. However, stomatal densities were the best characteristics related to environmental conditions whereas their sizes were rather a genotypic trait. These results along with our previous observations on the morphological study and the micromorphological as well as the palynological studies (Belhadj et al., 2007a, b; Belhadj et al., 2008) provided evidence that these variations may reflect phenotypic and genotypic adaptations to native habitats. Such knowledge might provide a basis for the selection of the appropriate plant material for local reforestation programmes and/or for the agronomical production, as this species is used as rootstock for P. vera. ACKNOWLEDGMENTS We thank the laboratorio Regional de Sanidad Vegetal de la Junta de Castilla la Mancha, for providing laboratory facilities, the staff of the Electron microscope unit of the Laboratoire des Mécanismes et des Transferts en Géologie (LMTG) and the staff of the Ecolab UMR 5245 CNRS/UPS/INP, Toulouse, for their technical assistance and co-operation. Also, Dr. Kevin J. Carpenter from the University of California-Davis, U.S.A. and Dr. Odile Décamps from the University of Toulouse, France for their help in stomatal type identification. REFERENCES Ait-Said S, Kadi-Bennane S, Smail-Saadoune N (2004). Phenological

adaptation of Atlas Pistachio (Pistacia atlantica Desf. ssp. atlantica) in three microclimatic zones of Algeria as measured by the stomatal dimensions. Book of abstracts. First world congress of Agroforestry. 27 June- 2 July 2004. Orlando, Florida, USA.

Alyafi J (1979). Approches systématique et écologique du genre

Pistacia L. dans la région Méditerranéenne. Thèse de Doctorat 3eme

cycle. Université d’Aix – Marseille. France, 123 p. Balok CA, St. Hilaire R (2002). Drought responses among seven south-

western landscape tree taxa. J. Amer. Soc. Hort. Sc. 127:211-218. Banon A, Fernandez JA, Franco JA, Torrecillas A, Alarcon JJ, Sanchez-

Blanco MJ (2004). Effects of water stress and night temperature preconditioning on water relations and morphological and anatomical changes of Lotus creticus plants. Sci. Hort. 101:333-342.

Baranova M (1972). Systematic anatomy of the leaf epidermis in the Magnoliaceae and some related families. Taxon. 21:447-469.

Baranova M (1983). On the laterocytic stomatotype in Angiosperms. Brittonia. 35(1):93-102.

Baranova M (1987). Historical development of the present classification of morphological types of stomata. Bot. Rev. 53:53-79.

Baranova M (1992). Principles of comparative stomatographic studies of flowering plants. Bot. Rev. 58:49-99.

Belhadj S (1999). Pistachio situation in Algeria. FAO.CIHEAM.NUCIS NEWS LETTER. 8:30.

Belhadj S (2001). Les pistacheraies algériennes: Etat actuel et dégradation. In Ak, B.E., (Eds.). 11 GREMPA Seminar on Pistachios and Almonds, September 01-04, 1999. Sanliurfa, Turkey. Cah. Opt. Méd. 56:107-10911.

Belhadj S (2002). Geographical distribution of Pistacia atlantica DESF. in Algeria. Acta Hort. (ISHS). 591:499-503.

Belhadj S, Derridj A, Aigouy T, Gers C, Gauquelin T, Mevy JP (2007a). Comparative morphology of leaf epidermis in eight populations of Atlas pistachio (Pistacia atlantica Desf., Anacardiaceae). Microsc. Res. Tech. 70: 837–846.

Belhadj S, Derridj A, Civeyrel L, Gers C, Aigouy T, Otto T, Gauquelin T (2007b). Pollen morphology and fertility of wild Atlas pistachio (Pistacia atlantica Desf., Anacardiaceae). GRANA. 46: 48-156.

Belhadj S, Derridj A, Auda Y, Gers C, Gauquelin T (2008). Analyse de la variabilité morphologique chez huit populations spontanées de Pistacia atlantica en Algérie. Botany 86 (5) : 520-532.

Bondada BR, Oosterhuis DM (2000). Comparative epidermal ultrastructure of Cotton (Gossypium hirsutum L.) leaf, bract and capsule wall. Ann. Bot. 86:1143-1152.

Bosabalidis AM, Kofidis G (2002). Comparative effects of drought stress on leaf anatomy of two olive cultivars. Plant Sci. 163:375-379.

Botti C, Palzkill D, Muñoz D, Prat L (1998). Morphological and anatomical characterization of six jojoba clones at saline and non-saline sites. Ind. Crops Prod. 9:53-62.

Çaglar S, Tekin H (1999). The stomata density of Pistachio cultivars on different Pistacia rootstocks.Turk. J. Agri. For. 23:1029-1032.

Carpenter KJ (2005). Stomatal architecture and evolution in basal angiosperms. Amer. J. Bot. 92 (10):1595-1615.

Elfadl MA, Luukkanen O (2006). Field studies on the ecological strategies of Prosopis juliflora in dry land ecosystem 1. A leaf gas exchange approach. J. Arid Env. 66:1-15.

El-Oqlah AA (1996). Biosystematic research on the genus Pistacia in

Jordan. In Taxonomy, distribution, conservation and uses of Pistacia genetic resources. Eds. S. Padulosi, T. Caruso and E. Barone. Workshop 29-30 June 1995. Palermo, Italy, pp 12-19.

Esmail-Pour A (2001). Distribution, use and conservation of pistachio in Iran. In Towards a comprehensive documentation and use of Pistacia genetic diversity in Central and West Asia, North Africa and Europe. Eds. S. Padulosi and A. Hadj-Hassan. Workshop 14-17 December 1998. Irbid, Jordan, pp 16-26.

Fahn A (1967). Plant anatomy. First edn. Pergamon Press, Exeter. 534 p.

F.A.O. (2007). Situation des forêts du monde. Rome. 143 p. Franks PJ, Farquhar GD (2001). The effect of exogenous abscisic acid

on stomatal development, stomatal mechanisms and leaf gas exchange in Tradescantia virginiana. Plant Physiology. 125: 935-942.

Gale J (1972). Elevation and transpiration. Some theoretical considerations, with special reference to Mediterranean type climates. Journal of Applied Ecology 9: 691–702.

Gamage HK, Ashton MS, Singhakumara BMP (2003). Leaf structure of Syzygium spp (Myrtaceae) in relation to site affinity within a tropical rain forest. Bot. J. Linn. Soc. 141:365-377.

Gauquelin T, Bertaudiere V, Montes N, Badri W, Asmode JF (1999).

Endangered stands of thuriferous juniper in the western Mediterranean

basin. Ecological status, conservation and management. Biodiversity and Conservation. 8:1479-1498.

Gitz DC, Liu-Gitz L, Britz SJ, Sullivan JH (2005). Ultraviolet-B effects on stomatal density, water-use efficiency, and stable carbon isotope discrimination in four glasshouse-grown soybean. Env. Exp. Bot. 53: 343-355.

Hetherington AM, Woodward FI (2003). The role of stomata in sensing and driving environmental change. Nature. 424: 901-908.

Kadi-Bennane S, Ait-Said S, Smail-Saadoun N (2005). Etude adaptative de trois populations de Pistacia atlantica Desf. ssp. atlantica (Ain oussara – Messaad – Taissa) par le biais du complexe stomatique. In M.M. Oliveira and V. Cordeiro (Eds.). Proceedings of the XIII Meeting of the Mediterranean Research Group for Almond and Pistachio (GREMPA). Mirandela (Portugal), 1-5 June 2003. pp 365-368.

Kofidis G, Bosabalidis AM, Moustakas M (2007). Combined effects of altitude and season on leaf characteristics of Clinopodium vulgare L. (Labiatae). Environmental and Experimental Botany 60: 69–76.

Lawson T, Craigon J, Black CR, Colls JJ, Landon G, Weyers JDB (2002). Impact of elevated CO2 and O3 on gas exchange parameters and epidermal characteristics in potato (Solanum tuberosum L.). J. Exp. Bot. 53:737-746.

Le Houérou HN (1995). Bioclimatologie et biogéographie des steppes arides du Nord de l’Afrique. Diversité Biologique, développement durable et désertification. Opt. Méd. Série B. 10.

Lin TS, Crane JC, Ryugo K, Polito VS, Dejong TM (1984). Comparative study of leaf morphology, photosynthesis, and leaf conductance in selected Pistacia species. J. Amer. Soc. Hort. Sci. 109 (3):325-330.

Liu YB, Wang G, Liu J, Zhao X, Tan HJ, Li XR (2007). Anatomical, morphological and metabolic acclimation in the resurrection plant Reaumuria soongorica during dehydration and rehydration. Journal of Arid Environments 70: 183–194.

Metcalfe CR, Chalk L (1950). Anatomy of the Dicotyledons. Leaves, stem, and wood, in relation to taxonomy with notes on economic uses. Volume I, II edit. Oxford at the Clarendon Press.

Mirzaie-Nodoushan H, Arefi HM (2001). Variability in seed blankness in Pistacia atlantica Desf. in a natural habitat. PGR Newsletter. FAO-Biodiversity. Bull. Ress. Phytogén. 127 :46-48.

Monjauze A (1980). Connaissance du betoum Pistacia atlantica Desf. Biologie et foret. Rev. For. Fr. 4:357-363.

Ngugi MR, Doley D, Hunt MA, Ryan P, Dart P (2004). Physiological responses to water stress in Eucalyptus cloeziana and E. argophloia

seedlings. Trees. 18:381-389. Özeker E, Misirli A (2001). Research on leaf properties and stomata

distribution of some Pistacia spp. Cah. Opt. Méd. 56: 37-241. Ozenda P (1983). Flore du Sahara. Deuxième édition. C.N.R.S. 566 p. Prabhakar M (2004). Structure, delimitation, nomenclature and

classification of stomata. Acta Bot. Sin. 46:242-252. Quézel P, Santa S (1963). Nouvelle flore de l’Algérie et des régions

désertiques méridionales. Tome II. C.N.R.S., Paris. 1087 p. Quézel P, Médail F (2003). Ecologie et biogéographie des forêts du

bassin méditerranéen. Environmental series. Elsevier. 571 p. Roselli G, Benelli G, Morelli D (1989). Relationship between stomatal

density and winter hardiness in olive (Olea europaea L.). J. Hort. Sc. Biotech. 64:199-203.

Sharma RC, Sharma S, Gupta AK (2001). Stomatal characters of Populus ciliata in relation to leaf rust and growth parameters. Phytomorphology. 51:199-205.

Smail-Saadoun N (2005). Types stomatiques du genre Pistacia: Pistacia atlantica Desf. ssp. atlantica et Pistacia lentiscus L. In Oliveira, M.M., Cordeiro, V., (Eds). Proceedings of the XIII Meeting of the Mediterranean Research Group for Almond and Pistachio (GREMPA). Mirandela (Portugal), 1-5 June 2003. pp 365-368.

Siegwolf RTW, Matyssek R, Saurer M, Maurer S, Günthardt-Goerg MS, Schmutz P, Bucher JB (2001). Stable isotope analysis reveals differential effects of soil nitrogen and nitrogen dioxide on the water use efficiency in hybrid poplar leaves. New Phytol. 149:233-246.

Stace CA (1965). Cuticular studies as an aid to plant taxonomy. Bull. Bri. Mus. (Natural History). Bot. 4:1-78.

Wang J, Letham DS, Cornish E, Stevenson KR (1997). Studies of

cytokinin action and metabolism using tobacco plants expressing either the IPT or GUS gene controlled by a chalcone synthase promoter. Aust. J. Plant Physiol. 24: 661-672.

Wilkinson HP (1979). The plant surface (mainly leaf). In Anatomy of the dicotyledons. Eds. C.R. Metcalfe and L. Chalk. Clarendon Press, Oxford, UK. 2nd ed., Vol.1. pp 97-165.

White F (1986). La végétation de l’Afrique. ORSTOM-UNESCO. Pp 165-250.

XueJun D, XinShi Z (2000). Special stomatal distribution in Sabina vulgaris in relation to its survival in a desert environment. Trees. 14: 369-375.

Yaltirik F (1967). Pistacia L. In Flora of Turkey and the east Aegean Islands. Eds P.H. Davis. Edinburgh University Press. 2. pp 542-548.

Yang L, Han M, Zhou G, Li J (2007). The changes in water-use efficiency and stoma density of Leymus chinensis along Northeast China Transect. Acta Ecologica Sinica, 27 (1): 16-24.

Yiotis C, Manetas Y, Psaras GK (2006). Leaf and green stem anatomy of the drought deciduous Mediterranean shrub Calicotome villosa (Poiret) Link. (Leguminosae). Flora. 201:102-107.

Zohary M (1952). A monographical study of the genus Pistacia. Palestine Journ. Bot. J. series. 5 (4):187-228.

Zohary M (1987). Flora Palaestina. Platanaceae to Umbelliferae. 2 :296-300. Second printing.