Introduction

Cyperaceae is one of the largest monocotyledonous sedge families, because it has a specialized group of vascular plants [1]. Cyperus is a large genus with over 600 species is found all over the world [2]. The sedge family has a reputation for being taxonomically challenging, as evidenced by the use of anatomical features of the vegetative organs for taxonomic purposes and the presence of species having both C₃ and C₄ photosynthetic pathways [3]. In India, the dominant species in garden land and wetland are C. rotundus and C. difformis, respectively. C. difformis is a sedge that ranks among Holm’s world’s worst weeds. It is a troublesome weed, especially in rice and sugarcane fields. It is the dominant one in direct-seeded rice and occurs in high plant densities, forming thick mats of vegetation in the crop at initial stage, resulting in 12-50% yield loss in rice crops. C. rotundus is a perennial pestiferous weed, mostly occurs in irrigated uplands, that causes significant yield loss in most crops due to its prolific tuber production and underground rhizome. It also has allelopathic properties, which might affect the growth and development of the crop. The effective control of these weeds is important for obtaining higher agricultural productivity. The first step in determining the best control approaches is to examine the morphology and anatomical features of plant leaves and stems. Understanding the morphology and anatomical features of plant leaves and the stem is of prior importance for realizing the best weed control.

Metcalfe [3], Govindarajalu [4], Rad and Sonboli [5] and Silva et al. [6] investigated the anatomy of certain Cyperus species as well as other Cyperaceae genera. Anatomical findings can reveal information on a plant cell type, amount and arrangement, as well as its intercellular structure [7] [8]. The leaf and stem anatomical traits of C. rotundus and C. difformis are yet to be determined. Considering the structural importance for their management, the leaves and stems of both species were anatomically characterized in this study identify to diagnostic features and determine possible phenotypic relationships between C. rotundus and C. difformis.

Novelty statement

Cyperus rotundus and C. difformis are members of Cyperaceae family which are well-known problematic weeds in the agricultural ecosystem. The result shows that anatomical features of the leaf and stem of the studied species, such as the presence of bulliform cells, major vascular bundles and their positions about air cavities, the presence of minor vascular bundles in the leaf, the morphology of leaf blade, stem ground tissues and air cavities, stomatal density, interveinal distance, and kranz tissue provides valuable anatomical features that aid in taxonomic delimitation. It should also be mentioned that when all of these features are employed together rather than a single character, the resolution of these traits for species identification is higher. The findings also provide reliable information regarding the Cyperus genus, which includes both C₃ and C₄ plant species.

Materials and Methods

The sample of C. difformis and C. rotundus specimens were obtained from the Department of Agronomy, Tamil Nadu Agricultural University, Coimbatore, India from the Wetland Farms and Eastern Block Farms, respectively. The plant was thoroughly cleaned under running water to remove soil and other debris. For this experiment, fully developed leaf and stem segments were collected. The samples were immersed in Formalin-Aceto-Alcohol (FAA), from which sections were made. A sterile blade was used to cut the leaves into thin hand sections. Numerous temporary and permanent sections were made and washed with water. Lactic acid (50%) was used to clear the perfect sections. After that, the sections were stained with safranine (0.1%). Glycerol (10%) was used to mount the sections in the slides. Under compound microscope, the mounted semi-permanent slides were examined and photographed. The qualitative characteristics of the sample were compared in which phenotypic similarity was identified. The terminologies used to describe the anatomy of leaves were adopted from Metcalfe [3], Bruhl [9] and Bugg et al. [10].

Stomatal density was calculated as the number of stomata per unit area. Interstomatal distance (average distance between stomata along longitudinal leaf axis) and interveinal distance (distance between vein centers) were measured. A compound microscope was used to observe and capture images of sections. Selected images were imported into ImageJ (1.53e) software (image analyzing software) and all quantitative characters were measured using the software’s calibrated micrometer scale. Data from 25 measurements (n=25) were gathered and mean values were reported.

Results

Visual identification of C. rotundus and C. difformis is not difficult, because the inflorescence of the two species differs. C. difformis has dense, globose, umbellate heads with yellowish-brown or pale-brown inflorescence (Fig. 1A). The inflorescence of C. rotundus is a compact umbel of spikes that are purplish to red-brown and have a simple and slightly compound appearance (Fig. 1B).

Anatomical features of leaf

Both C. rotundus and C. difformis come under hypostomatous, with more number of stomata found only on the lower surface of leaves (Fig 1(C-J)). Leaves have epidermal cells on their upper surface. Stomatal density varied widely with C. difformis had the highest mean value (19 mm^-2) whereas in C. rotundus with the lowest mean value (6 mm^-2). Inter-stomatal distance differed amongst the species similarly. The mean value of C. rotundus and C. difformis was 148.7 μm and 46.8 μm. A cross section of the leaf showed that Kranz tissue, which appears as specialized chlorenchymatous leaf bundle sheaths (Fig. 2E), is found in C. rotundus species but not in C. difformis. In interveinal distance, the mean value of C. difformis and C. rotundus was 370.7 μm and 93.1 μm (Table 1).

            The major morphological difference between the species was that C. rotundus had prickle hairs on its leaf blades, but absent in C. difformis. Fig. 2 (A-D) and 2 (F-H) shows the transverse section of C. rotundus and C. difformis leaf, respectively. In terms of the midrib of the leaf foliage, both species have V-shaped flange in trans-section, air cavities and bulliform cells. C. rotundus has a flanged V-shaped foliage leaf in trans-section, as well as air cavities and bulliform cells on the adaxial side of the midrib. Bulliform cells are single-layered and morphologically dissimilar from epidermal cells in both species. However, the total number of bulliform cells in the leaf differed in both species. C. difformis had less bulliform cells (5 nos.)  than C. rotundus (Table 1).

Regarding vascular bundles, both species had major vascular bundles in alternative positions about relation to air cavities. Major vascular bundles were closer to the adaxial surface in C. rotundus, whereas they are closer to the abaxial surface in C. difformis. C. rotundus had minor vascular bundles that were closer to the adaxial surface. In C. difformis, minor vascular bundles were absent. Based on the minor and major vascular bundles, the total number of vascular bundles in leaves has differed in both the species. C. rotundus had an average of 61 numbers of both major and minor vascular bundles and C. difformis had an average of 12.6 numbers of major vascular bundles only (Table 1).

Anatomical features of stem

The transverse section of C. rotundus stem is shown in Fig. 3 (A-C). In the transverse section, the stem of C. rotundus was triangular in shape, sides that were almost flat to slightly concave and grooved with rounded corners. Ground tissue was spongy, breaking down to generate a few big v-shaped cavities. It had thin-walled parenchyma and arenchyma cells which was made up of a large number of collateral and closed vascular bundles. Totally 28.4 vascular bundles were present in the C. rotundus stem (Table 1). The vascular bundles present in the outermost circle were smaller in size and embedded in assimilating tissue, and also numerous vascular bundles were distributed in ground tissue. The remaining vascular bundles were larger and scattered in central ground tissue.

The transverse section of C. difformis is shown in Fig. 3 (D-F). The stem of C. difformis had a star-like shape with three sharp edges. At the periphery, there was a single layer of epidermal cells followed by large air cavities. Various vascular bundles alternately exist between the air cavities (air cavities followed by vascular bundles). C. difformis had an average of 30 vascular bundles and 28 air cavities in the stem (Table 1).  The spongy tissue in the leaves, roots or stems of aquatic plants that possesses air channels and voids are known as air cavities. Large air cavities present in parenchyma give buoyancy to the plants and allow them to float in water. Each edge had one vascular bundle. The rest of the portion had ground tissue of parenchymatous cells. Both species lacks epidermal hairs (also known as trichomes) and vacuoles.

Discussion

The occurrence of kranz tissue, which is associated with C₄ photosynthesis, is a key feature in categorizing Cyperus species [11]. This is due to the presence of this structure in C₄ metabolism allows for the spatial dissociation of the photosynthetic enzymes, such as phosphoenolpyruvate-carboxylase (Pepcase), which acts within the mesophyll tissues and Ribulose-1,5-bisphosphate carboxylase (Rubisco) which acts within the vascular bundles [12] [13]. The number of vascular bundles also varied between the species [14] [15] and C. rotundus had more vascular bundles than C. difformis (Table 1). In the present study, C. difformis had no Kranz tissue, implies that it lacks a C₃ photosynthetic pathway [16] [17] [18]. Both species constantly have single-layered epidermal cells and bulliform cells [10] [19]. But the number of bulliform cells varied between species (Table 1). C. rotundus had more bulliform cells than C. difformis.

Interveinal distances, which are expressed as the average distance between vein centers, is another attribute explored in this study (Table 1). It is widely assumed that closer vein spacing allows more efficient photosynthates transport between cells [20]. Takeda et al. [21] and Li and Jones [22] determined that a species is C₄ if its leaf interveinal distance is less than 130 μm and higher in C_3. Here, C. difformis had interveinal distances of 370.7 μm which was more than 130 μm indicates it is as a C₃ species. The other species had an interveinal distance of 93.1 μm, which was less than 130 μm and may be classified as C₄ species [23].

The presence of large air cavities in the transverse section of C. difformis stem indicated that it is a semi-aquatic weed that grows under submergence conditions. Sorrell [24] and Silveira et al. [25] reported the presence of aerenchyma in the root cortex of C. alopecuroidesis as an important anatomical trait of aquatic plants that facilitates their growth and survival in anoxic conditions.

This study showed that a combination of anatomical features can be used as criteria to categorize plant species according to their photosynthetic pathway. In this case, the combination of features, such as the presence of kranz tissue and interveinal distance, was shown to give a credible basis for assessing the photosynthetic pathways of the studied Cyperus species. The findings support the idea of accurately predicted anatomical data alone may be used to determine the species photosynthetic pathway of Cyperaceae, as suggested by [17]. The anatomical feature from this study reveals that C. difformis possess C₃ whereas C. rotundus possess C₄ photosynthetic pathways respectively.

Conclusion

Anatomical features of leaf and stem of the studied species, found the presence of bulliform cells, major vascular bundles and their positions about air cavities, the presence of minor vascular bundles in the leaf, the morphology of leaf blade, stem ground tissues and air cavities, stomatal density, interveinal distance, and kranz tissue presence provided valuable anatomical features that aid in taxonomic delimitation. When all of these features are employed together rather than a single character, the resolution of these traits for species identification is higher. The findings also result inreliable information regarding the Cyperus genus, which includes both C₃ and C₄ plant species.

Acknowledgment

The first author is grateful to the DST- INPSIRE, Govt. of India for providing Fellowship and financial support. The authors are thankful to the Tamil Nadu Agricultural University and the Department of Agronomy for giving the laboratory facilities and for instrument facilities to carry out the study.

Author Contributions

This work was carried out in collaboration among all authors. All authors read and approved the final manuscript.

Conflict of Interest

Authors have declared that no conflict of interest.

Data Availability

Data presented in this study will be available on a fair request to the corresponding author

Ethics Approval

Not applicable in this paper

References

[1]. Muasya AM, DA Simpson, MW Chase, A Culham (1998). An assessment of the suprageneric phylogeny in Cyperaceae using rbcL DNA sequences. Plant Syst and Evolution 211:257-271.

[2]. Boulos L (2005). Flora of Egypt, Volume Four Monocotyledons (Alismataceae-Orchidaceae). Al-Hadara Publishing, Cairo, Egypt.

[3]. Metcalfe CR (1971). Anatomy of the monocotyledons. Vs. Cyperaceae. Clarendon Press, Oxford, U.K.

[4].Govindarajalu E (1974). The systematic anatomy of south Indian Cyperaceae: Cyperus L. subgen. Juncellus, Cyperus subgen. ariscus and Lipocarpha R. Bot J Linn Soc 68: 235-266.

[5]. Rad MA, A Sonboli (2008). Leaf and stem anatomy of Cyperus subgenus Cyperus in Iran. Rostaniha 9:6-22.

[6]. Silva AL, MVS Alves, AI Coan (2014). Importance of anatomical leaf features for the characterization of three species of Mapania (Mapanioideae, Cyperaceae) from the Amazon forest, Brasil. Acta Amazonica 44:447-456.

[7]. Atanackovic V, V Randjelovic, RIH Ibrahim (2012). Morphological andanatomical observations on Ranunculus lingua L. under flooding conditions in Vlasina Lake (SE Serbia). Biologica Nyssana 3:97-103.

[8]. Peng Y, Z Zhou, R Tong, X Hu, K Du (2017). Anatomy and ultrastructure adaptations to soil flooding of two full-sib poplar clones differing in flood[1]tolerance. Flora 233:90-98.

[9]. Bruhl JJ (1995). Sedge genera of the world: relationships and new classification of the Cyperaceae. Aust Syst Bot 8:125-305.

[10]. Bugg C, C Smith, N Blackstock, D Simpson, PA Asthon (2013). Consistent and variable leaf anatomical characters in Carex (Cyperaceae). Bot J Linn Soc 172: 34 – 46.

[11]. Larridon I, M Reynders, W Huygh, K Bauters, K Van de Putte, AM  Muasya, P Boeckx, DA Simpson, A Vrijdaghs, P Goetghebeur (2011). Affinities in C₃ Cyperus lineages (Cyperaceae) were revealed using molecular phylogenetic data and carbon isotope analysis. Bot J of the Linn Soc 167(1):19-46.

[12]. Lange OL (2012). Physiological plant ecology II: Water relations and carbon assimilation, Vol. 12, pp: 115-123. Springer Science & Business Media.

[13]. Antonielli M, S Pasqualini, P Batini, L Ederli, A Massacci, F Loreto (2002). Physiological and anatomical characterization of Phragmites australis leaves. Aquat Bot 72(1):55-66.

[14]. Mallick T, A Ghosh (2018). Comparative study of foliage leaf and bract leaf anatomy of six species of Cyperaceae from West Bengal. Mod Phytomorph 12:106–116.

[15]. Yasser AA, MAG Ahmed (2017). Anatomical investigation of three emergent Cyperus species growing naturally on the canal banks of the Nile delta, Egypt. J of Sci Agric 1:294-299.

[16]. Li, MR, MB Jones, M Alves (1999). C₃ and C₄ photosynthesis in Cyperus (Cyperaceae) in temperate eastern North America. Can J Bot 77:209 – 218.

[17]. Bruhl JJ, KL Wilson (2007). Towards a comprehensive survey of C₃ and C₄ photosynthetic pathways in Cyperaceae. Aliso 23:99 – 148.

[18]. Martins S, M Alves (2009). Anatomical features of species of Cyperaceae from northeastern Brazil. Brittonia 61:189–200.

[19]. Dhyani S (2017). Morphological and micromorphological features of leaf and stem of Cyperus rotundus L. and Cyperus procerus Rottb – A comparative analysis. Int. J. Res. Ayurveda Pharm 8:46-51.

[20]. Monson RK, GE Edwards, MS Ku (1984). C₃-C₄ intermediate photosynthesis in plants. Bioscience 34(9):563-574.

[21]. Takeda T, O Ueno, W Agata (1980). The occurrence of C₄ species in the genus Rhynchospora and its significance in Kranz anatomy of the Cyperaceae. The Bot magazine 93(1):55-65.

[22]. Li, MR, MB Jones (1994). Kranzkette, a unique C₄ anatomy occurring in Cyperus japonica leaves.  Photosynthetica 30:117-131.

[23]. Ayeni OB, MA Jimoh, SA Saheed (2015). Leaf anatomical characters about the C₃ and C₄ photosynthetic pathway in Cyperus (Cyperaceae). Nordic J of Bot 000:1-6.

[24]. Sorrell BK (1999). Effect of external oxygen demand on radial oxygen loss by Juncus roots in titanium citrate solutions. Plant Cell and Environ 22:1583-1587.

[25]. Silveira MJ, VC Harthman, TS Michelan, LA Souza (2016). Anatomical development of roots of native and non-native submerged aquatic macrophytes in different sediment types. Aquat Bot 133:24-27.

[26]. Zarrinkamar F, A Jalili, B Hamzehee, Y Asri, JC Hodgson, K Thompson, S Shaw (2002). Foliar anatomy of Carex in Arasbaran, NW Iran. Iranian J of Bot 9:261-270.

Table 1: Significant anatomical features of the studied Cyperus species

Fig. 1: Morphological and Microscopic view of upper and lower leaf surface of Cyperus difformis and C. rotundus

Fig. 2: Transverse section of C. rotundus  and C. difformis leaf (bulliform cells (BC), air cavities (AC), adaxial epidermal cell (adec), abaxial epidermal cell (abec), transverse septum between air cavities (TS), major vascular bundle (MVB), minor vascular bundle (NVB), vascular bundles (VB), phloem (PH), metaxylem (MX), xylem cavities (XC), kranz sheath (KS)

Fig. 3: Transverse section of C. rotundus and C. difformis stem (air cavities (AC), epidermis (E), vascular bundles (VB), fibre tissue (FI), bundle sheath (BS), ground tissue (GT), phloem (PH), metaxylem (MX), xylem cavities (C))