Role of Leaf Epidermis in Identification and Differentiation of Grasses in Tribe Chlorideae (Poaceae) from Pakistan

Full Length Research Paper

Role of leaf epidermis in identification and differentiation of grasses in tribe Chlorideae (Poaceae) from Pakistan

Farooq Ahmad1*, Mansoor Hameed1, Muhammad Ashraf1, Mushtaq Ahmad2, Ameer Khan3, Tahira Nawaz1, Khawaja Shafique Ahmad1 and Muhammad Zafar2

1Department of Botany, University of Agriculture Faisalabad, Pakistan. 2Department of Plant sciences, Faculty of Sciences, Quaid-I-Azam University Islamabad, Pakistan. 3Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan.

Accepted 16 February, 2012

In the present investigations, leaf epidermal studies of 5 species belonging to 3 genera of tribe Chlorideae (Poaceae) from salt range of Pakistan were carried out. The investigated species were Chloris barbata Sw, Chloris dolicostachya Lag, Cynodon dactylon Pers, Tetrapogon cenchriformis (A.Rich) Clayton and Tetrapogon villosus Desf. The size of stomatal complex, long cells, shape of subsidiary cells, silica bodies are found to be important in differentiation of different taxa. T. villosus is characterized by large cells having maximum length while T. cenchriformis is differentiated from T. villosus and other taxa by large stomatal complex, while smallest stomatal complex is observed in C. dactylon. Bicelled microhairs with hemispherical distal cell are present only in genus Chloris while found absent in C. dactylon (rarely present on adaxial surface) and Tetrapogon species. The presence of microhairs is not a constant character, as in C. dactylon microhairs may be present or absent on the abaxial side. It may be due to habitat or environmental variations. Saddle shaped silica bodies are found in all the species of the tribe that is a characteristic feature of chloridoid type grasses.

Key word: Epidermis, Chlorideae, Grasses, Pakistan.

INTRODUCTION

Tribe Chlorideae is one of the important tribes of family because of variations in leaf characters that are Poaceae. This tribe is represented by 45 genera taxonomically useful (Barkworth, 1981). The arrangement throughout the tropics. In Pakistan, tribe Chlorideae has 7 of epidermal cells on the adaxial surface of the leaf blade genera with 14 species (Nasir and Ali, 1970, 2002). In the is generally quite different from that of the abaxial present studies from Salt Range of Pakistan 3 genera surface. Most epidermal cells are classified as long cells Tetrapogon, Chloris and Cynodon having 5 species are or short cells. Long cells vary in length and width, wall collected. Tetrapogon and Chloris have two species thickness, and the extent to which the walls are sinuous, each, while one species of Cynodon is present. papillate or pitted. Significant differences in long cells Sometimes there is confusion in identification and exist among genera and even among species of the differentiation of species of the same genus on the basis same genus (Gould, 1969). Leaf epidermal traits i.e. of morphology, so anatomical studies assist in solving epidermal cells, stomata and microhairs have been problem of identification. Leaf epidermis study provides proved valuable in identification and differentiation of valuable data regarding the identification of grasses and different taxa (Stenglein et al., 2003). The shape of silica is recognized as a source of useful taxonomic characters, bodies varies among different grass species from round or oblong to linear, crescent shaped, dumbbell shaped, nodular, sinuous, saddle shaped or cross shaped. Macrohairs also vary greatly in size, shape and wall *Corresponding author. E-mail: [email protected]. thickness (Gould, 1969) and these leaf epidermal 1956 J. Med. Plants Res.

following anatomical characters of both abaxial and adaxial epidermis was studied.

1. Length, width and shape of long cells, whether sinuous, slightly sinuous or non sinuous. 2. Short cells and papillae in intercostal zone are present or absent. 3. Number of rows of long cells between two costal zones. 4. Length and width of stomatal complex and shape of guard and subsidiary cells. 5. Length, width and shape of macrohairs and hooks, their presence or absence. 6. Length, width and shape of silica bodies and short cells and their distribution in costal zone.

A Figure 1. Chloris barbata, Leaf abaxial surface showing A RESULTS hooks. Chloris barbata (Abaxial leaf surface)

Long cells: with sinuous walls, number of rows between two costal zones is 6 to 7, rectangular, conspicuously elongated; Short cells: Cross shaped, present over and between the veins; solitary and sometimes paired; arranged in rows of 2 to 3 cells. Prickle hair: Absent. Unequal angular prickles also present on leaf margins. Micro hair: Bicelled, distal cell is shorter than basal cell and is hemispherical. Macro hair: none seen. Hooks: Present 2 Stomatal complex: Guard cells are dumb bell shaped and subsidiary cells are triangular;

Figure 2. Chloris barbata Leaf adaxial surface showing Silica bodies: saddle shaped, with 2 to 3 layers (Figure angular prickles. 1).

Chloris barbata (Adaxial leaf surface) characters are of great value in grass systematic and characterization of broad groups within the grasses, Long cells: Adaxial intercostal long cells are with particularly subfamilies and tribes (Ahmad, et al., 2011). sinuous walls, 42.5 to 53.25 μm long and 8.75 – 8.80 μm Leaf epidermal features are important to clarify taxonomic wide. Numbers of rows of long cells between two costal relationships in different taxa (Davila and Clark, 1990; Cai zones are 5 to 8 with sinuous walls. and Wang, 1994; Mejia – Saules and Bisbey, 2003; Ogie- Odia et al., 2010). So the aim of the present Short cells: Short cells are with sinuous walls, 10 to 17.5 investigations is to carry out leaf epidermal studies to μm long and 6.25 to 10 μm wide. identify and differentiate grass species in tribe chlorideae. Prickle hair: Present. Micro hair: none seen. Macro hair: none seen. MATERIALS AND METHODS Hooks: Present

Fresh leaves of five species of grasses were used directly for Stomatal complex: Guard cells are dumb bell shaped anatomical studies. Leaf samples were prepared according to the and subsidiary cells are triangular. modified method of Cotton (1974) who followed Clark (1960) Silica bodies: Saddle shaped (Figure 2). technique. The fresh leaves were placed in a tube filled with 88% lactic acid in water bath for about 50 to 60 min. The abaxial and adaxial epidermis from middle portion of the leaf was removed, along with the mesophyll cells by using scalpel blade. The Chloris dolicostachya (Abaxial leaf surface) epidermis was placed on the slide and mounted in clean 88% lactic acid. The micro photographs of the mounted materials were taken Long cells: Abaxial intercostal long cells are with thin by using a camera mounted on Leica light microscope. The sinuous walls, 60 to 65 μm long and 10 to 12.5 μm wide. Ahmad et al. 1957

Micro hair: Microhairs are bi-celled; distal cell is shorter than basal cell. Distal cell is hemispherical in shape and with rounded apices while basal cell is with rounded base, 21.25 to 23.75 μm long and 7.5 to 8.75 μm wide. Macro hair: none seen. Hooks: none seen Stomatal complex: Stomatal complex is 15.25 to 20 μm long and 15.25 to 17.5 μm wide, guard cells are dumb bell shaped and subsidiary cells are triangular to high dome shaped. Silica bodies: Silica bodies are saddle shaped, 12.5 to 3 20 μm long and 11.25 to 15 μm wide (Figure 3).

Figure 3. Chloris dolicostachya, Leaf abaxial surface. Chloris dolicostachya (Adaxial leaf surface)

Long cells: Adaxial intercostal long cells are with sinuous walls, 32.5 to 55 μm long and 6.25 to 10 μm wide. Numbers of rows of long cells between two costal zones are 5 to 7. Number of stomatal rows between two costal zones are 1 to 2. Short cells: Short cells are 12.5 to 16.25 μm long and 8.75 to 10 μm wide.

Prickle hair: Prickles are 25 to 30 μm long and 8.75 to 10 μm wide. Micro hair: Microhairs are bicelled and both cells are 4 almost equal or distal cell is shorter than basal cell and distal cell is hemispherical. Macro hair: Absent Figure 4. Chloris dolicostachya, Leaf adaxial surface. Hooks: Absent Stomatal complex: Stomatal complex is 15 to 17.5 μm long and 12.5 to 13.75 μm wide. Silica bodies: Silica bodies are saddle shaped, 7.5 – 8.0 μm wide horizontally and 11 – 12.25 μm wide vertically (Figure 4).

Cynodon dactylon (Abaxial leaf surface)

Long cells: Abaxial intercostal long cells are with thin sinuous walls, 39.5 to 52.5 μm long and 10 to 15 μm wide while interstomatal cells are with, deeply sinuous walls and with concave ends. Numbers of rows of long cells between two costal zones are 6 to 10. Numbers of stomatal rows between two costal zones are 1 to 3. Short cells: Short cells are with sinuous walls, 15 to 17.5 Figure 5. Cynodon dactylon, leaf abaxial surface, showing saddle shaped silica bodies, and long cells with μm long and 6.25 to 10 μm wide. Rounded bodies (cork deep sinuous walls cells) are present between the short cells. Prickle hair: Prickles are pointed at the tip, 40 to 42.5 μm long and 10 to 13 μm wide. Micro hair: Absent. Numbers of rows of long cells between two costal zones Macro hair: Absent are 5 to 9 Numbers of stomatal rows between two costal Hooks: Absent zones are 2 to 3. Stomatal complex: Stomatal complex is 15 to 16.25 μm Short cells: Short cells are with sinuous walls, 22.5 to 25 long, 11.25 to 12.5 μm wide, guard cells are dumb bell μm long and 12.5 to 13.25 μm wide. shaped, and subsidiary cells are low to high dome Prickle hair: Present. shaped. 1958 J. Med. Plants Res.

Stomatal complex: Guard cells are dumb bell shaped, subsidiary cells are triangular or high dome shaped 23.75 to 25 μm long and 12.5 to 17.5 μm wide. Silica bodies: Silica bodies are saddle shaped, with 1 to 3 layers, 6.25 to 10 μm long and 6.25 to 7.5 μm wide (Figure 6).

Tetrapogon cenchriformis(Abaxial leaf surface)

Long cells: Abaxial intercostal long cells are with 6 irregular sinuous walls, 31.5 to 55 μm long and 10 to 12.5 μm wide. Number of rows of long cells between two

Figure 6. Cynodon dactylon, leaf adaxial surface costal zones are 4 to 8 and number of stomatal rows showing pointed prickles in coastal zone. between two costal zones are 2. Long cells in costal zone are also with sinuous walls, present over the veins with silica bodies, 36.25 to 37.5 μm long and 8.75 to 9.37 μm wide. Short cells: Short cells are with sinuous walls, 16 to 18.75 μm long and 8 to 10 μm wide. Rounded bodies or cork cells are present between silica bodies and short cells, 6.25 to 8.75 μm long and 6.25 to 8.75 wide. Prickle hair: Angular prickles are present at the margins, 32 to 35 μm long and 8 to 10 μm wide. Micro hair: Absent. Macro hair: Absent Hooks: Absent Stomatal complex: Stomatal complex is 20 to 27.5 μm long and 20 to 22.5 μm wide, guard cells are dumb bell shaped, 5 μm wide and subsidiary cells are high dome 7 shaped, 6.25 μm wide. Silica bodies: Silica bodies are saddle shaped and Figure 7. Tetrapogon cenchriformis, leaf abaxial surface. dumb bell shaped. Saddle shaped silica bodies are 9 to 10 μm wide horizontally and vertical diameter 10 to 11.25 μm wide while dumb bell shaped silica bodies are 15 to 17.5 μm long and 7.5 to 8.75 μm wide (Figure 7). Silica bodies: Silica bodies are saddle shaped, 7.0 to 7.5 μm horizontally and vertical diameter is 10 to 11.25 μm and silica bodies are 7.5 to 10 μm long and 6.5 to 7.5 Tetrapogon cenchriformis (Adaxial leaf surface) μm wide (Figure 5). Long cells: Adaxial intercostal long cells are with sinuous walls, 37.5 – 72.5 μm long and 10 – 11.25 μm Cynodon dactylon:(Adaxial leaf surface) wide. Number of rows of long cells between two costal zones ars 6 – 7. Number of stomatal rows between two Long cells: Adaxial intercostal long cells are with thin costal zones are 2 – 3. sinuous walls, 22.5 to 37.5 μm long and 7.5 to 10 μm wide. Numbers of rows of long cells between two costal Short cells: Short cells are with sinuous walls, 16 – zones are 8 to 11. Numbers of stomatal rows between 18.75 μm long and 8 – 10 μm wide. Rounded bodies or two costal zones are 2 to 3. cork cells are present between silica bodies and short Short cells: Short cells are with sinuous walls, 16 to cells, 6.25 – 8.75 μm long and 6.25 – 8.75 wide. 18.75 μm long and 8 to 10 μm wide. Rounded bodies or Prickle hair: Angular prickles are present at the margins, cork cells are present between silica bodies and short 32 – 35 μm long and 8 – 10 μm wide. cells, 6.25 to 8.75 μm long and 6.25 to 8.75 wide. Micro hair: Microhairs are bicelled, basal and distal cells Prickle hair: Prickles are 12.5 to 20 μm long and 6.25 to are almost equal, distal cell is rounded at the tip, basal 7.5 μm wide. cell having a rounded structure, 22.5 –26.75 μm long and Micro hair: Absent. 12.5 μm wide. Macro hair: Absent Macro hair: Macrohairs are rounded at the base, 8.0 – Hooks: Absent 15 μm long and 7.5 – 12.5 μm wide; Ahmad et al. 1959

μm long and 16.25 – 17.5 μm wide, guard cells are dumb bell shaped, 5 μm wide and subsidiary cells are high dome shaped, 6.25 μm wide. Silica bodies: Silica bodies saddle shaped, 8.5 - 10 μm wide horizontally and 13.75 - 15 μm wide vertically. Short cells with sinuous walls, 10 – 11.25 μm long and 8.75 – 10 μm wide. Prickles are abundant in the costal zone, 41 – 42.5 μm long and 12 – 16.25 μm wide (Figure 8).

Tetrapogon villosus(Abaxial leaf surface) 8 Long cells: Abaxial intercostal long cells are with

Figure 8. Tetrapogon cenchriformis, leaf adaxial sinuous walls, 42.5 to 92.5 μm long and 10 to 13.75 μm surface, showing pointed prickles rounded at the base. wide. Numbers of rows of long cells between two costal zones are 2 to 3. Number of stomatal rows between two costal zones is 1. Short cells: Short cells are with sinuous walls, 20 to 27.5 μm long and 7.5 to 11.25 μm wide. Silica bodies and short cells are arranged in a row Prickle hair: Angular prickles are at the margins of costal zone, obliquely projected into pointed tip, 87.5 to 95 μm long and 17.5 to 20 μm wide. Micro hair: Absent Macro hair: Absent Hooks: Absent Stomatal complex: Stomatal complex is 17.5 to 18.75 μm long and 15 to 17.5 μm wide, guard cells are dumb bell shaped and subsidiary cells are high dome shaped. Guard cells are 3.75 to 7.5 μm wide and subsidiary cells are 6.25 to 7.5 μm wide. Microhairs, macrohairs and 9 hooks are absent. Silica bodies: Silica bodies are saddle shaped, 12.5 to Figure 9.Tetrapogon villosus, leaf abaxial surface. 13.75 μm long and 7 to 8.75 μm wide. Number of rows of silica bodies are 1 to 4 (Figure 9).

Tetrapogon villosus (Adaxial leaf surface)

Long cells: Adaxial intercostal long cells are with sinuous walls, 62.5 to 65 μm long and 12.5 to 16.25 μm wide. Numbers of rows of long cells between two costal zones are 4 to 5. Number of stomatal rows between two costal zones is one. Short cells: Short cells are with sinuous walls, 25 to 27.5 μm long and 7.0 to 8.75 μm wide. Prickle hair: Angular prickles are at the margins of costal zone, obliquely projected into pointed tip, 87.5 to 95 μm long and 17.5 to 20 μm wide. 10 Micro hair: Absent A Macro hair: Absent Hooks: Absent Figure 10. Tetrapogon villosus, leaf adaxial surface. Stomatal complex: Stomatal complex is 20 to 22 μm long and 15 to 16.25 μm wide, guard cells are dumb bell shaped, and subsidiary cells are high dome shaped. Silica bodies: Silica bodies are saddle shaped, 12.5 Hooks: Absent to13.75 μm long horizontally and 10 to 16.25 μm wide Stomatal complex: Stomatal complex is 18.75 - 19.37 vertically (Figure 10). 1960 J. Med. Plants Res.

DISCUSSION REFERENCES

Barkworth ME (1981).Foliar epidermis and taxonomy of North America The studies of leaf epidermis in all species of tribe Stipeae(Gramineae). Syst. Bot., 6(2): 136-152. revealed that all intercostal long cells on abaxial and Cai LB, Wang SJ (1994). Studies on the evolutionary trends and adaxial surface are with sinuous walls, however the walls mechanism of the constituent cells of the leaf epidermis in Poaceae. are thin sinuous in Chloris dolicostachya and Cynodon Acta Biologia Plateau Sinica, 12:13-27. Chaudhary MI, Mumtaz AS, Khan MA (2001). Leaf epidermal anatomy dactylon (Figures 5 and 6). In Tetrapogon cenchriformis of medicinal grasses of Islamabad, Attock and Mirpur (Azad long cells with irregular sinuous walls are observed. Long Kashmir). Pak. J. Biol. Sci., 4 (12): 1466 -1469. cells with maximum length (92.5 µm) are present in Clark J (1960). Preparation of leaf epidermis for topographic study. Tetrapogon villosus. It is found that size of stomatal Stain Technol., Pp. 35–39. Cotton R (1974). Cytotaxonomy of the genus Vulpia. Ph.D Thesis, Univ. complex and shape of subsidiary cells are important Manchester, USA. characters in identification and differentiation of different Davila P, Clark LG (1990). Scanning electron microscopy survey of leaf taxa. The largest stomatal complex is observed in T. epidermis of Sorghastrum (Poaceae) Andropogoneae. Americ. J. cenchriformis that is 20 to 27.5 µm long and 20 to 22.5 Bot., 77: 499-511. Freire SE, Arambarri AM, Bayon ND, Sancho G, Urtubey E, Monti C, µm wide and small in C. dactylon which ranges in length Novoa MC, Colares MN (2005). Epidermal characteristics of toxic from 15 to 16.25 µm long and 11.25 to 12.5 µm wide. plants for cattle from the Salado River basin (Buenos Aires, Peterson et al. (2011) observed low dome shaped or Argentina). Boil. Soc. Argent. Bot., 40 (3 - 4): 1 – 28. triangular stomata in new genus Ellosochloa of subfamily Gould FW (1969). Grass Systematics. Mc. Graw. Hill book company, New York. Pp. 40 – 41. Chloridoideae. Mejia T, Bisbey FA (2003). Silica bodies and hooked papillae in lemmas In the present studies, subsidiary cells are triangular to of Melica species (Gramineae: Pooideae) Bot. J. Linn. Soc.,141: 447 high dome shaped in genus Chloris, low to high dome – 463. shaped in C. dactylon and high dome shaped in Metcalfe CR (1960). Anatomy of the monocotyledons. 1. Gramineae. Clarendon Press, Oxford at the series 13. HMSo, p.389. Tetrapogon species, indicating that stomatal complex Nasir E, Ali SI (1970 – 2002). Flora of Pakistan, National Herbarium, proved to be useful taxonomic feature in this tribe. Agricultural Research Council Islamabad Pp. 1-192. Chaudhary et al. (2001) found that in C. dactylon stomata Ogie-Odia EA, Mokwenye AI, Kekeri O, Timothy O (2010). Comparative are with triangular subsidiary cells, silica bodies are vegetative and foliar epidermal features of three Paspalum L. Species in Edestate, Nigeria. Ozean.J.App.Sci., 3(1): 29-38. saddle shaped, and microhairs with hemispherical distal Peterson PM, Romaschenko K, Barker NP, Linder HP (2011). cells are present, while macrohairs are absent. In the Centropodieae and Ellisochloa, a new tribe and genus in present investigations, C. dactylon showed triangular and Chloridoideae (Poaceae). Taxon., 60 (4):1113-1122. low to high dome shaped subsidiary cells and micro hairs Prat H (1934). Contribution al etude Systematique et histologique de Chloridees. Bull. Soc. Bot. Fr., 81: 475–491. are not observed abaxially (Figure 5) but a few microhairs Prat H (1961). Emploi des characters epidermiques dans la are present adaxially (Figure 6). Freire et al. (2005) also classification des graminees. pp. 99-102 in Recent advances in observed the presence of microhairs in C. dactylon. botany, vol.1.Toronto Univ. Toronto Press Metcalfe (1960) studied 3 species of C. dactylon Stenglein SA, Colares MN, Arambarri AM, Novoa MC, Vizcaino CE, Katinas L (2003). Leaf epidermal microcharacters of the old world collected from different localities and found that silica species of Lotus (Leguminoseae: Loteae) and their systematic bodies are saddle shaped, stomata are with triangular or significance. Aus. J. Bot., 51: 459-469. low dome shaped subsidiary cells, and microhairs were Watson L, Dallwitz MJ, Johnston CR (1986). Grass genera of the world: present in all the species examined. The present findings 728 detailed descriptions from an automated database. Aus. J. Bot., 34: 223-230. are similar to that of Metcalfe except the microhairs which are absent on abaxial side in Cynodon, in the present studies. It may be due to environmental variations as C. dactylon is a wide spreading grass, which varies considerably in habit. Bi-celled microhairs (21.25 to 23.75 µm) with hemispherical distal cell shorter than basal cell are found only in genus Chloris (Figures 1 and 3), while found absent in C. dactylon (rarely present on adaxial surface) and Tetrapogon species. All the species in the tribe have saddle shaped silica bodies as according to Prat (1934, 1961) and Watson and Johnston (1986), the chloridoid type is characterized by globose or club shaped bi-cellular microhairs and saddle shaped silica bodies.