Pure Appl. Biol., 10(3):726-737, September, 2021 http://dx.doi.org/10.19045/bspab.2021.100074

Research Article

Leaf anatomy of species growing in different soils of Punjab Pakistan

Aamir Mumtaz1*, Aisha Taseen1 and Amina Ameer1 1. Government Post Graduate College Mianwali-Pakistan *Corresponding author’s email: [email protected] Citation AamirMumtaz, Aisha Taseen and Amina Ameer. anatomy of Cyperus species growing in different soils of Punjab Pakistan. Pure and Applied Biology. Vol. 10, Issue 3, pp726-737. http://dx.doi.org/10.19045/bspab.2021.100074 Received: 21/09/2020 Revised: 20/11/2020 Accepted: 00/11/2020 Online First: 01/12/2020 Abstract also called the Sedge-family is the third-largest monocot family. They have a cosmopolitan distribution with a wide variety of habitats such as steep slopes, high altitude, extreme aridity, extreme salinity, swamps, marshes, and moist areas. In this study, the transverse sections of of different species of the genus Cyperus were collected from different regions of Punjab including Faisalabad, Chiniot, Jarhanwala, Sahianwala, KalarKahar, Khabeki Lake, Pakkana, and Balloki. The soil samples and leaf sections were analyzed for the adaptations in leaf anatomy in different soils of Punjab. This analysis demonstrated diverse modifications in the leaf area; thickness of vascular bundles; altered and metaxylem cell area; sclerenchyma, collenchyma, and thickness; and variations in upper and lower epidermal cell area and the number of stomata on both sides of the , etc. based on the soil structure and composition in different regions of Punjab. The modifications were proportional to the accelerated survival and growth rate of the species in that region. Sedges are present in different environment such as degraded soil, hyper-saline waters, dry land salinity; however, many are related to Lakes or soils. The anatomical structures of various parts are seen as an adaptation to the ecological habitat of certain . It was therefore believed that indigenous species of the Cyperaceous family may have evolved specific leaf anatomy which has permitted them to live under severe environmental conditions such as drought and salinity. Keywords: Cyperaceae; Cyperus; Leaf; Punjab; Pakistan; Soil Introduction vascular plants, this is the second largest Among the top ten families of angiosperms, family with the number of genera above 70. Cyperaceae is the third-largest monocot Also, it is the second-largest family of order family comprising of 4000-5000 species . Moreover, it also occupies the second worldwide. All these species are categorized position among the families of C4 plants with into more than 70 genera after a detailed 1500 species that make up 20% of the total C4 analysis of features such as dorsoventrally and plants. But owing to the varying nature of C4 laterally flattened pleomorphic trimerous plants, these C4 species of Cyperaceae are also gynoecium, and spirally arranged legumes, diverse in their anatomical and biochemical etc., through ontogenetically and anatomical characteristics [2]. techniques of analysis [1]. Among the

Bolan Society for Pure and Applied Biology 726 Mumtaz et al.

Cyperaceae is also called the sedge-family of is saline while Faisalabad has saline-sodic soil angiosperms because of the genus Cyperus. [8]. L.containing almost 600 species that can grow Studies have identified that the leaf anatomy in all types of temperate and tropical zones. of most of the members of the Cyperaceae These are perennial/annual aquatic plants that family shows midrib with solid bulliform can also grow up to 0.5 cm in water [3]. cells, abaxial epidermal cells shorter than the Because of differences in the development of adaxial epidermal cells, and double-layered anatomical features, the genus can be bundle sheath and collateral vascular bundle subdivided into two sub-genera i.e., C4 type with outer parenchymatous layer and fibrous photosynthetic Cyperus with chlorocyperoid sclerified inner layer. Cyperaceae family also anatomy of kranz cells and C3 type shows phytolith (silica bodies) in the Photosynthetic Cyperus with eucyperoid epidermis. Some of the species also have anatomy in the absence of kranz cells [4]. Paracytic stomata with two subsidiary cells Cyperaceae family has a cosmopolitan adjacent to the aperture [9]. distribution. In Pakistan, the species are The composition and structure of soil affect distributed largely throughout from the plains the growth of various parts of plants. Plants of Sindh to Punjab, sandy mountains of modify their anatomical features to sustain the Baluchistan, and green valleys of Kashmir, challenging environments for survival and Gilgit Baltistan and Khyber Pakhtunkhwa [5]. efficient growth. We designed this study to It has been reported that over 22 genera and identify the anatomical changes in the leaf of more than 179 species are growing throughout Cyperaceae species growing in different soil the country [6]. Punjab province lies 27°42’ to regions of Punjab, Pakistan. The study aimed 34°02’ North latitude and 69°18’ to 75°23’ to determine the impact of soil structure on the east longitudes on the globe and is bordered on leaf anatomy of selected species of West by Khyber Pakhtunkhwa, on South by Cyperaceae family in the selected areas of Sindh, and on North by Baluchistan. It Punjab. After a detailed analysis of anatomical occupies 20.63 million hectares of the tropical structures, our study found that the species zone and spreads 616km from East to West diversity of Cyperus in different regions of and 1078km from North to South (Table 1). Punjab was based on the changes in leaf Punjab is divided into many districts and anatomy of the plants due to differences in the regions which have different texture and soil texture and composition. composition of the soil [7]. For example, Materials and Methods Chakwal district has a soil structure For this study, detailed surveys were carried comprising mostly of sandstone, limestone out throughout the Punjab region including and shale rocks. So, it is weak and calcareous major cities such as Faisalabad, Rawalpindi, and also has gravels, stones, and rocks, etc. Murree, Sargodha, Sahianwala, Khushab, The consistency of soil in this district ranges and Lahore, Mianwali, Jhang, Nowshera and from sandy loam to loam with a pH of 7.7-7.8. some smaller districts. During these surveys, Similarly, the Sahianwala of Punjab has three soil samples from different ecological zones different soil types i.e., wetland, dry saline and pertaining to the studied areas were obtained highly saline soil. Murree has two different and were subjected to laboratory analysis. soil structures comprising of reddish/purple Soil samples underwent chemical analysis; in sandstones and greyish sandstones divided addition, electrical conductivity studies for into upper and lower regions. Likewise, the various ions such as Na+, K+, Ca2+, Cl- were soil of Chiniot is sandy and that of KalarKahar also performed.

727 Pure Appl. Biol., 10(3):726-737, September, 2021 http://dx.doi.org/10.19045/bspab.2021.100074

Soil samples acquired from the Sahianwala was used for material fixation. Long term region were categorized as three different fixation of tissues was done using Acetic types: 1) Dry saline soil 2) Highly saline soil Alcohol solution (25% acetic acid with 75% 3) Wetland. Conductivity meters were ethyl alcohol). employed for the analysis of electrical Slides of the transverse section were prepared conductivity. The anatomical parameters using a double-stained standard technique. were assessed using a compound microscope, The formula mentioned below was used to a calibrated stage micrometer together with calculate the area of different cells and tissues an ocular micrometer. (this formula is a modification of area of Formalin Acetic Acid (FAA) solution circle i.e. πr2). comprising of 50% ethyl alcohol, 35% Area= Maximum length x Maximum width x 22 distilled water, 10% acetic acid, 5% formalin, 28

Table 1. Habitats descriptions of collecting sites LOCATIONS Latitude Longitude Altitude(Feet) Sahiwanla 31⁰38'20.93" 73⁰14'04.10" 633 Faisalabad 31⁰27'17.47" 73⁰45'06.66" 619 Sargodha 32⁰07'10.33" 73⁰33'42.63" 592 Murree 33⁰55'21 .53" 73⁰23'30.28" 5909 Rawalpindi 33⁰34'24.30" 73⁰05'45.46" 1669 Lahore 31⁰31'57.27" 74⁰2' 45.31" 712 Mianwali 32⁰33'35.75" 71⁰33'08.19" 698 Khushab 33⁰53'34.04" 73⁰20'20.84" 213 Nowshera 32⁰33'44.31" 72⁰09'33.00" 2852 Jhang 31⁰16'41.05" 72⁰19'54.03" 519

Statistical analysis Another species Cyperus glaber in the Head Analysis of variance (ANOVA) via complete Rasool Region also showed modifications in randomized design (CRD) with the two- leaf anatomy in the form of the thick epidermis factor factorial arrangement is used for the (Fig. 1c), largest cortical cell area (Fig. 1f) and analysis of the data. maximum phloem cell area (Fig. 1q) while the Results Cyperus squarossus species in the same region Analysis of modifications in leaf anatomy had maximum aerenchyma cell area when with respect to different texture and compared to other species of the same genera composition of soil in various regions of in Punjab (Fig. 1n). Punjab showed that Cyperus laevigatus The soils of Khabeki Lake showed anatomical growing in the soils of Sahianwala in the dry changes in the leaf of Cyperus maritimus in and saline regions had increased stomatal cell the form of increased leaf area (Fig. 1a), area in the upper and lower epidermis of the thickest epidermis (Fig 1c), minimum upper leaf (Fig. 1g, 1h) while the leaves also had epidermal cell area, the highest number of thickest sclerenchyma layer and thinnest stomata on the upper epidermis (Fig. 1j), aerenchyma as compared to other Cyperus maximum aerenchyma cell area (Fig. 1n)and species in the surrounding areas (Fig. 1l, 1n).

728 Mumtaz et al. minimum stomatal cell area on lower upper epidermal stomatal cell area (Fig. 1h), epidermis (Fig. 1g). and maximum number of stomata on lower Likewise, the leaf anatomy of Cyperus epidermis (Fig. 1i). compressus growing in the soils of C. nutans growing in Khari Mural also showed KalarKahar showed thinnest epidermis (Fig. modifications in the leaf anatomy in the form 1c), and minimum bulliform cell area (Fig. 1k) of large lower epidermal cell area (Fig. 1d), as modifications in response to soil thickest collenchyma(Fig 1m), thick bulliform composition and structure. cell area(Fig. 1k), minimum number of The analysis of leaf anatomy of C. haspans stomata on lower and upper epidermis(Fig 1i, growing in Chiniot demonstrated minimum 1j), minimum vascular bundle cell area(Fig. phloem cell area (Fig. 1q), and largest 1o), and minimum metaxylem cell area(Fig. bulliform cell area (Fig. 1k). Another species 1p).Cyperus esculuntus samples collected Cyperus difformis collected from the soils of from Pakkana region showed overall Jarhanwala in Punjab had maximum upper minimum epidermal cell area (Fig 1b), and epidermal cell area (Fig. 1e), maximum minimum lower epidermal cell area (Fig. 1d). vascular bundle cell area (Fig. 1o) and Finally, a single species C.alopecuroides maximum metaxylem cell areas (Fig. 1p). C. Collected from Balloki demonstrated alternifolia collected from botanical garden of maximum epidermal cell area (Fig. 1b) in leaf university of agriculture, Faisalabad, showed anatomy modifications with respect to soil in minimum leaf area (Fig. 1a), and minimum different regions of Punjab. The transverse sclerenchyma thickness (Fig. 1l).Another sections of some species of Cyperaceae in species C. rotundus collected from the same Punjab have been shown in (Fig. 2). university showed large vascular bundle cell The (Table 2) below shows statistical results, area (Fig. 1o), thin collenchyma (Fig. 1m), analysis of leaf anatomy modifications in minimum cortical cell area (Fig. 1f), minimum various species of Cyperus in Punjab.

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Figure1. Statistical Data of leaf anatomy parameters of different species of Cyperus growing in Punjab, Pakistan

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Cyperusnutans Cyperusrotundus

Cyperussquarrosus Cyperusiria

Cyperusalopecuroides Cyperuscompressus

Cyperuslaevigatushighly saline Cyperusglaber

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Scirpusmaritimus C. alternifolia

Cyperuslaevigatus Dry saline Figure 2. TS of the leaf of some species of family Cyperaceae from Punjab

Discussion stomata morphology to keep the With a diverse morphology and cosmopolitan evapotranspiration process continued even in distribution, Cyperaceae is among the core short supply of water [11]. The soil of eudicots in angiosperms having a wide Sahianwala region of Punjab is dry and saline variety of habitats such as damp marshes, in nature with climate conditions similar to temperate forests, steep slopes, old fields and drought. Our study found that the C. even the arctic tundra. These varieties of laevigatus species growing in Sahianwala habitats require specific modifications in the region have increase cell area of the stomata anatomy of various parts of plants to survive in the upper and lower epidermis of the leaf. the harsh climate and soil conditions of that This modification involving the ability to region [10]. In this study, we focused on the regulate the stomata opening serves to leaf anatomy modifications due to different control the loss of water in the dry saline soil soil conditions of various regions of Punjab, and hence helps in the survival of the species Pakistan. We observed distinct leaf anatomy in that area. Other changes in leaf anatomy changes in species growing in different involve increase in sclerenchyma thickness districts of Punjab. and decrease in aerenchyma thickness to Researchers have demonstrated that in areas limit further loss of water. with limited water supply, plants modify their

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Table2.Leaf anatomy parameters of different species of Cyperus

Stem Anatomical

C. C. iria

Parameters C.L wet

C. C. glaber

C. C. longus

C. C. nutans

C. C. haspans

C. C. rotundus

C. C. difformis

C. C. esculuntus

C. C. squarossus

C. C. alternifolia

C.L dry saline

C. C. compressus

C. C. alopecuroides

C. C. L highly saline Scirpusmaritimus

Leaf area (µm2)

818490

64301.84 83664.85 125433.9 230490.4 191293.4 94641.44 81962.21 61965.24 329460.9

295987.70 306565.80

260902.456

299773.3717 127679.9092 105074.6423

Epidermis thickness

0.6

7.06 0.63 30.5 8.32 6.93 8.32 10.4

13.86 26.34 26.34 11.09 12.68 29.12 353.2 (µm) 194.13

Epidermis cell area

353.2

688.30 434.71 525.28 1793.2 217.35 163.01 217.35 108.67

(µm2) 1394.71 2372.82 2517.73 2626.41 1992.45 3115.46 3350.94

Lower Epidermis cell area

688.30 434.71 398.49 615.84 815.09 3441.5 815.09 543.39 579.66 163.03 869.43 (µm2) 1394.71 2372.82 2282.26 1702.64 3115.46

Upper Epidermis Cell Area

344.15 398.49 2553.9 869.43 326.03 2064.9 271.69

2 3006.79 1557.73 2644.52 2264.14 1702.64 2209.81 1938.11 1919.99 (µm ) 16301.88

Cortical Cell Area (µm2)

475.90 294.60 942.74 326.33 843.02 498.56 339.93 353.52 380.72 135.97 738.78

5130.69 1065.11 1110.44 1722.31 1529.69

Lower

Epidermis

Stomatal Cell 951.8

299.13 299.13 870.22 865.69 398.85 380.72 552.95 920.08 285.54 Area (µm2) 1137.63

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Upper

Stomatal Cell

2 294.6 462.3 Area (µm ) 516.69 516.69 290.07 729.71 521.22 217.55 262.88 376.19 278.74

Lower

Epidermis

Stomatal 52 62 98 84 85

93.33 96.66 55.66 33.33 88.33 88.66 Number 292.07

Upper

Epidermis

36

124

Stomatal 90.66 86.33 77.33 Number 121.33 120.66 105.33 109.33 121.33 102.66 105.33

Bulliform cell

area (µm2)

108.77

2674.12 1495.69 2506.42 1486.63

Sclerenchym

a Thickness

72.1 31.2 (µm) 24.96 12.48 12.48

Collenchyma

Thickness

83.2 41.6 72.8 (µm) 45.76 87.36 69.33 66.56 90.13 29.12 34.66 63.78

Aerenchyma

Cell Area 2

(µm ) 8747.56 13030.7 4623.06 8158.35

21483.67 25789.46 13252.79 10914.07 12763.29 12273.79 25834.79

Vascular Bundle Cell 2

2642.4 3761.9 4419.1

Area (µm ) 3095.64 6087.03 4192.48 3353.99 2374.98 2379.52 2515.49 1767.64 2152.89 4804.36 2402.18 2855.42 1808.43

Metaxylem Cell Area

2 90.64 90.64 321.8 49.85 489.5 (µm ) 135.97 815.83 135.97 122.37 330.86 108.77 131.44 281.01 244.75 163.16 385.25

Phleom Cell 2

Area (µm ) 49.85 77.05 31.72 462.3 45.32 36.25 462.3 81.58

1314.4 412.45 421.51 915.54 453.24 235.68 299.13 244.75

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Modifications in the are also associated Similarly, another species sample C. rotundus with harsh soil conditions. Cortex is involved in collected from the same university demonstrated the storage of carbohydrates and ultimately in large vascular bundle cell area, minimum cortical maintaining the water-pull by the transpiration cell area, thin collenchyma, minimum upper stream. C. glaber species in the Head Rasool epidermal stomatal cell area, and increased region of Punjab also show leaf anatomy number of stomata on lower epidermis. modifications of larger cortical cell area and Cyperus species collected from the Jarhanwala larger phloem cell area along with thick region showed anatomical changes in the form of epidermis which indicate the modifications in maximum upper epidermal cell area, maximum response to harsh soil conditions of the area. metaxylem cell area and maximum vascular Another species C. squarossus shows increase in bundle cell area. the aerenchyma cell area which shows that there Another region explored in Punjab is Khari Mural is a diversity of species in the Head Rasool where C. nutans which showed leaf region. This diversity can be attributed to modifications in the form of thickest difference in the soil conditions suitable for collenchyma, large lower epidermal cell area, a different types of Cyperus species. minimum number of stomata on the upper and Studies have shown that highly saline soil lower epidermis, thick bulliform cell area, small conditions can also affect the number of leaves, vascular bundle cell area, and minimum leaf area, and plant fresh weight. The growth of metaxylem cell area. the leaf area is an important factor in determining Likewise, C. esculuntus samples collected from the plant’s transpiration, light interception, and the Pakkana region showed leaf anatomy with overall productivity. Other modifications minimum epidermal cell area and minimum observed in the leaf anatomy of the species lower epidermal cell area. The maximum include increased thickness of epidermis and epidermal cell area was shown by C. reduction of upper epidermal cell area, increase alopecuroides collected from the Balloki region in the number of stomata on the upper epidermis, of Punjab. larger aerenchyma cell area and reduced stomatal After analyzing all the soil samples and cell area on lower epidermis. All these comparing the leaf anatomies of different species, modifications can help the plant in preventing we could relate that the modifications in different excessive water loss during transpiration and cells of the leaves in Cyperaceae are the cause absorbing more light to undergo rapid behind the survival and growth of its species in photosynthesis for keeping the plant growth such a variety of soil habitats in Punjab. This is a unaffected despite the high saline soil conditions small-sized study focusing only on the analysis of [12]. the transverse sections of selected species in C. compressus found in the highly saline region Punjab. However, further elaborated studies can of KalarKahar also shows anatomical be planned for the detailed analysis and modifications in the leaf in the form of the comparison of leaf modifications in Cyperus thinnest epidermis and smallest bulliform cell plants with respect to different mineral ion area. While on the other hand, C. haspens in the concentrations, salinity, and moisture in the soil Chiniot district has the largest bulliform cell area [13]. with minimum phloem cell area in its leaves. Conclusion The city of Faisalabad has dry loamy soil with Soil structure and composition can affect the little rain and hence can be thought of as a water- survival and growth of a plant. For this purpose, deficient area for plants. Probably, to meet this the plant undergoes specific modifications to cope water shortage, C. alternifolia collected from the with the challenges of the harsh climate and soil botanical garden of the University of Agriculture, conditions. The Cyperaceae species distributed in Faisalabad, has the smallest leaf area among all various soil regions of Punjab also undergo the species in Punjab. The small leaf area can different leaf anatomy modifications such as prevent the loss of water from transpiration. The changes in leaf area, different thickness of the species also show minimum sclerenchyma upper and lower epidermis, changes in the number thickness in leaves. of stomata on the upper and lower epidermis,

736 Mumtaz et al. variations in the size of vascular bundle cell area, 5. Carolin RC, Jacobs SWL & Vesk M (1977). bulliform cell area, metaxylem, and phloem cell The Ultrastructure of Kranz Cells in the area and finally altered thickness of sclerenchyma, Family Cyperaceae. Bot Gaz 138(4): 413- collenchyma, and aerenchyma in leaves in order to 419. meet the water and light requirements of the plant. 6. Butt MA, Zafar M, Ahmad M, Sultana S, Cyperaceae species with a greater number of Ullah F, Jan G & Naqvi S (2018). Morpho‐ modifications show a higher growth rate even in palynological study of Cyperaceae from unfavorable soil and climate conditions while wetlands of Azad Jammu and Kashmir using plants with moderate or no modifications show SEM and LM.Microsc Res Techniq 81(5): limited growth in those areas. This study showed 458-468. dispersal of selected species of Cyperus genus in 7. Ikram S, Bhatti KH &ParvaizM (2014). different soil regions of Punjab with respect to Ethnobotanical studies of aquatic plants of their leaf anatomy modifications. district Sialkot, Punjab (Pakistan). J Med Authors’ contributions Plants 2(1): 58-63. Conceived and designed the experiments: A 8. Condom N, Kuper M, Marlet S, Valles V Teseen & A Ameer, Performed the experiments: &Kijne J (1999). Salinization, alkalinization A Taseen & A Ameer, Analyzed the data: A and sodification in Punjab (Pakistan): Mumtaz, Contributed materials/ analysis/ tools: characterization of the geochemical and A Mumtaz, Wrote the paper: A Mumtaz. physical processes of degradation. Land Acknowledgment Degrad Dev 10(2): 123-140. The authors of this study acknowledge and 9. Hassan A, Ijaz SS, Lal R, Ali S, Hussain Q, appreciate the efforts of all participants involved. Ansar M &Baloch MS (2016). Depth References distribution of soil organic carbon fractions 1. Besnard G, Muasya AM, Russier F, Roalson in relation to tillage and cropping sequences EH, Salamin N & Christin PA (2009). in some dry lands of Punjab, Pakistan. Land Phylogenomics of C4 Photosynthesis in Degrad Dev 27(4): 1175-1185. Sedges (Cyperaceae): Multiple Appearances 10. Metcalfe CR (1969). Anatomy as an aid to and Genetic Convergence. Mol Biol Evol classifying the cyperaceae. Am J Bot 56(7): 26(8): 1909-1919. 782-790. 2. Blaser HW (1941). Studies in the 11. Ueno O & TakedaT (1992). Photosynthesis Morphology of the Cyperaceae. I. pathways, ecological characteristics, and the Morphology of Flowers. A. Scirpoid geographical distribution of the Cyperaceae Genera. Am J Bot 28(7): 542-551. in Japan. Oecologia 89(2): 195-203. 3. Besnard G, Muasya AM, Russier F, Roalson 12. Xu Z& Zhou G (2008). Responses of leaf EH, Salamin N & Christin PA (2009). stomatal density to water status and its Phylogenomics of C4 Photosynthesis in relationship with photosynthesis in a grass. J Sedges (Cyperaceae): Multiple Appearances Exp Bot 59(12): 3317-3325. and Genetic Convergence. Mol Biol Evol 13. Koester RP, Skoneczka JA, Cary TR, Diers 26(8): 1909-1919. BW & Ainsworth EA (2014). Historical 4. Escudero M, Hipp AL, Waterway MJ & gains in soybean (Glycine max Merr.) seed Valente LM (2012). Diversification rates yield are driven by linear increases in light and chromosome evolution in the most interception, energy conversion, and diverse angiosperm genus of the temperate partitioning efficiencies. J Exp Bot 65(12): zone (Carex, Cyperaceae). Mol Phylogenet 3311-3321. Evol 63(3): 650-655.

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