Foliar anatomy and micromorphology of Festuca L. and its taxonomic applications

Introduction

The genus Festuca L. contains an estimated 450 species (Clayton and Renvoize, 1986) and is a large and ancient group and one of the main evolutionary lines in the tribe Poeae which occur in polar, temperate, and alpine regions of both hemispheres (Tzvelev, 1976).

More than a century, foliar anatomy has been used in the taxonomy of the Festuca. Hackel (1882) published the first monograph of the genus Festuca relating to Europea He illustrated characteristics of 37 taxa using block diagrams. Metcalfe (1960) gave diagnostic generic characters and detailed information on the anatomy of several species of Festuca. Tzvelev (1976) in U.S.S.R. and Alexeev (1982) of North America made considerable use of leaf cross section as a taxonomic tool. In flora European Markgraf-Dannenberg (1980) using leaf cross section characters such as the number of veins and the distribution of sclerenchyma succeeded in identifying the species in this genus. The available literature from the other parts of the world dealing with anatomical study of Festuca supported such an assumption (Holmen, 1964; Badoux 1971; Borrill, 1972; Frederickson, 1977; Connor, 1960; Howarth, 1924, 1925; Snait-Yeves, 1925; Aiken et al., 1984, 1985, 1995; Sawicki et al., 2001; Aryavand and Panahi, 2003; Namaganda and Lye, 2008, 2009; Zarinkamar, 2008). However, anatomical study of the genus Festuca is insufficient for the species growing wild in Iran.

The present study considers anatomy and micromorphology of eight Festuca species occurring in Iran with the aim of providing some basic anatomical data for the country, and its taxonomic applications.

Materials and methods

Micromorphological studies were performed on eight populations of eight Festuca species, namely F. sulcata, F. arundinacea Schreb, F. valesiaca s.l. Schleich. ex Gaudin,
F. gigantea (L.). Vill. F. elwendiana Markgr.-Dann., F. heterophylla Lam., F. akhanii Tsvelev and F. drymeia Mertens et Koch (Table 1). The specimens were diagnosed using Flora Iranica (Bor, 1970), Flora Orientalis (Boisser, 1875), Flora of Iran, Flora of Iraq (Bor, 1968, 1970), the illustrated Flora of Golestan National Park, Iran (Akhanii, 2005) and Grasses of the Soviet Union (Tzvelev, 1976). The voucher specimens are deposited in the Herbarium of TARI (Table 1).

Table 1: Voucher specimen of Festuca specimens. Abbreviations: TARI, Research Institute of Forests and Rangelands Herbarium.

Methods

Anatomical analyses of leaves were done on permanent slides, prepared by the standard method for light microscopy (Strittmatter, 1973). Cross-sections of the external leaves from vegetative shoots were cut on a Reichert sliding microtome and by hand cutting. Sections were prepared from the middle region between one quarter and one half of the total length of blade, cleared in sodium hypochlorite and stained with carmine-vest (1% w/v in 50% ethanol) and methyl green (1% w/v, aqueous) then mounted in gelatin.

Epidermal surface were studied with Scanning Electron Microscope (SEM) for which the samples was covered by gold.

All morpho-anatomical measurements were done and the data processed in the statistical package. For each of the quantitative characters, 65 leaf samples were obtained from different individuals belonging to each of the eight populations analyzed. 37 quantitative and 40 qualitative anatomical characters were statistically analyzed in superficial view and on transversal section.

Statistical analyses

In order to group the species studied based on similarities in their anatomical features as well as micromorphological characteristics, different clustering methods of Between Groups, Single Linkage and WARD (minimum spherical cluster method) were used. To obtain value of characters, PCA (principle component analysis) was employed.

Results

Festuca L. is divided into two groups based on lamina width: fine-leaved and broad-leaved. In the fine-leaved, abaxial leaf surface was coated by silica and there were single prickles on veins and leaf margin. In abaxial surface of F. akhanii, trichomes were distributed with different orientations and some outgrowths (Figure 1, A and B). In
F. elwendiana, trichomes had different sizes (Figure 1, C). In F. heterophylla, prickles were only seen on veins and leaf margin of abaxial surface (Figure 1, E and F). There were ribs and furrows on adaxial surface which stomata frequently presented in furrows (Figure 1, D and J). The ribs were pubescent (Figure 1, E).

In the broad-leaved species, external surface and stomata were covered by silica (Figure 2, A and C). Trichomes were present on leaf margins (Figure 2, F). In F. drymeia there were no ribs and furrows and stomata were distributed on both sides of leaf surfaces.
In F. gigantea and F. arundinacea there were ribs and furrows and stomata were frequently present on furrows.

Figure 1: Superficial view of the fine-leaved species. A and B: F. akhanii (223X), (2000X); C andD:
F. elwendiana (239X), (3155X); E and F: F. heterophylla (110X), (116X); J and H: F. valesiaca (1673X), (972X); I: F. sulcata (839X).

Figure 2: superficial view of broad-leaved. A: F. gigantea; B and C: F. drymeia; D and F: F. arundinacea.

Leaf cross section: Fine-leaved of Festuca species were ≤ 2 mm wide because they were more or less tightly rolled or folded, often with leaf margin overlapping. A previous study on fine-leaved of Festuca species, demonstrated that some characters as number of ribs and furrows in adaxial surface, number of vascular bundles and number of sclerenchyma bundles could distinguished species (Eslami et al.,1387).

Broad-leaved species included F. arundinacea, F. gigantea and F. drymeia. Epidermal cells were different in size and type. Leaves were or appeared to be, ≤4 mm wide.

The characters are suitable for distinguishing the species as follows: Adaxial surface in F. drymeia is smooth (Figure 2, D, E and F), whereasin F. arundinacea (Figure 3, G, H and I) and F. gigantea (Figure 3, A, B and C) are observed as rounded rib over each vein. Bulliform cells occupy ¼ of the leaf thickness on the adaxial epidermal cells or at the base of the ribs (Figure 3, F). In the above-mentioned species, minute sclerenchyma tissues were observed opposite middle and as large vascular bundles. Sometimes sclerenchyma girders were extended from vascular bundles to both adaxial and abaxial epidermis (Figure 3, D, E, F, G, H and I).

Figure 3: Transversal section of broad-leaved species. A and B: C, F. gigantea; D, E and F: F. drymeia,G, H and I: F. arundinacea.

Table 2: Useful anatomical characters for distinguish species. Relation the sclerenchyma tissue of Midrib and Adaxial epidermal: 1, attachment; 2, apart. Shape furrow: 1; V-shape between 45°; 2, V-shape between 90° and 180°. Epidermal cell shape: 1, oblong; 2, oblong- elliptic. Stomata type: 1, present, 2, absent.

Different clustering methods like Between Groups, Single Linkage and WARD produced similar results whereas Between Groups possessed the highest correlation. In analyses, two clusters were observed. The first cluster was composed of F. sulcata,
F. valesiaca, F. elwendiana, F. heterophylla and F. akhanii. The species of F. arundinacea, F. gigantea and F. drymeia were positioned in cluster two (Figure 4).

Figure 4: Ward method clustering of the Festuca species studied

The PCA data demonstrated the most important characters for anatomy as follow: width and thickness of leaf cross section, shape and number of veins and arrangement of vascular bundles. Moreover, the position and degree of scleranchyma development, number of ribs in adaxial surface, density of crystal, observation of stomata in abaxial surface, form of epidermal cells and number of bulliform cells could be also valuable for taxonomic application.

Figure 5: PCA plot of the Festuca species studied

The PCA plot suggested that the position of F. akhanii was between the fined and broad-leaved species of Festuca (Figure 5).

Discussion

The anatomical characters in cross section of leaf are important in identification of the taxa. Broad-leaved species such as F. arundinacea from the subgenus Schedorous,
F. drymeia from the subgenus Drymanthele and F. gigantea from the subgenus Drymoneates have linear leaves with 2mm width and sheaths that divided into two groups based on ribs and furrows but in F. drymeia there are not ribs and furrows on both surface linear and have 2 mm width.

In general, bulliform cells are dispersed between veins on the adaxial surface. Papilla or prickle are scattered on both surfaces. There is one extension of sclerenchyma tissue from the abaxial to adaxial surface in midrib and the middlesized veins, this structure is actually absent in small veins. These species hasve no auricle in leaf sheath.

These furrows on the adaxial surface of F. arundinacea are distinguished from smooth adaxial surface of F. gigantea.

Stomata are present on both sides but more dense on adaxial furrows, and prickles are just present on the leaf margin. Another significant anatomical feature of F. arundinacea is the prensence of a big vascular bundle on both sides of midrib.

F. gigantea and F. arundinacea are morphologically distinguished from other broad-leaved species by the presence of auricle leaf sheath.

Fine-leaved species includes F. elwendiana, F. valesiaca, F. sulcata, F. akhanii and
F. heterophylla from the subgenus Festuca. 

The species of F. akhanii is distinguished by sclerenchyma strand, presence of furrows on both sides of the midrib and papilla, while other species of the subgenus Festuca has more than two furrows on adaxial surface and no sclerenchyma strand.

Another important and stable anatomical character is the number of sclerenchyma tissues on the adaxial surface which is useful character to distinguish other species of the subgenus Festuca. There are three groups of sclerenchyma tissues with different layers in
F. valesiaca, F. sulcata and F. elwendiana on abaxial surface. The sclerenchyma tissues are completely separated in F. valesiaca and F. sulcata sometimes tends to continue in
F. elwendiana. F. elwendiana is morphologically characterized by the presence of variable sized of trichomes on both leaf surfaces, pubescence sheathes and glabrous ovary.
F. sulcata is morphologically and anatomically similar to F. valesiaca which is distinguished by group of four bulliform cells, complexes with wavy walls and furrows on adaxial surface near midrib.

There are five to seven sclerenchyma tissue groups with different layers in
F. heterophylla. Sclerenchyma tissueforms discrete strands opposite to the large vascular bundles.