1 Introduction

Strandlines are created along the high water mark on a range of habitats including coastal vegetated shingle, sand dunes and salt marshes (Lee and Ignaciuk, 1985). They are coastal environments above high water, which is out of reach of the average high tide and it is characterized by the deposition of drift materials, litter, debris and many discarded items left behind by the previous receding tide (Rozema et al., 1982). Plate A shows a view of a strandline environment in Nigeria. They have worldwide distribution with ecological, social and economic values (Williamson, 2005). The vegetation provides essential habitat, spawning sites and food for many diverse species of terrestrial and marine wildlife. They are able to withstand periodic coastal disturbances and they are important on exposed shores, where they act as precursors to sand dunes (Williamson, 2005), as shown in Plate B.

Strandline environment is usually occupied by only few plant species due to the severity of the environment (Rozema et al., 1982) as several environmental factors affect the vegetation, and salinity is possibly the most imperative ecological restriction that determines the species existence (Owen et al., 2001; Peter et al., 2003; Pichler and Oberhuber, 2007), thus plant ability to survive in the habitat is greatly determined by its adaptation to salinity.

Most studies on salinity tolerance in strand plants have focused mainly on morphological and physiological adaptations (Barbour et al., 1985; Flowers and Colmer, 2008) while anatomical adaptations which can also offer some explanations of species abilities to grow on the strandlines, and can be easily recognized and quantified have been largely ignored.

Plate A A view of the strandline environment along Arogbo Ijaw Seaside Beach, Ondo State, Nigeria

Plate B  A view of some plant species at Arogbo Ijaw Seaside Beach, Ondo State, Nigeria Note: DI= discarded item by tide

It is evident that there are big changes in anatomy of plants growing in saline soils (Ediga et al., 2013). Depending on the species, anatomical changes in plants may be either adaptations to salinity stress or signs of damage and disturbance of the normal life processes (Yeo and Flowers, 1980). Salinity affects cell division and expansion processes, reduces the size of apical meristems, cortex and vascular cylinder (Zidan et al., 1990). Also, salinity causes changes in tracheary element density, lumen volume and vessels diameter (Junghans et al., 2006; Escalante-Perez et al., 2009; Chen and Polle, 2010) as well as stomatal density, shape, and size (Abbruzzese et al., 2009). Anatomical changes can affect plant ability to conduct water and nutrients, thus anatomical adaptations are as important as morphological and physiological processes (Younis et al., 2014). Some specific anatomical adaptations to salinity stress include increased succulence by cell expansion, changed number and size of stomata, thickened cuticle, inhibited differentiation and changed diameter and number of xylem vessels, development of tyloses and earlier occurrence of casparian strips and lignification (Flowers et al., 1986).

Plate C Photograph of Diodia maritime

Plate D Kyllinga peruviana growing on the strandline along Nigerian coastline

Buttonweed (Diodia maritima Thonn.) belongs to the family Rubiaceae. It is a perennial prostrate herb usually rooting at nodes, rarely scandent or somewhat tufted. The leaves are ovate and subacute at apex, cuneate into a short petiole at base and not fleshy and glabrous (Hutchinson et al., 1968) as shown in Plate C. Peruvian spikesedge (Kyllinga peruviana Lam.) belonging to the Cyperaceae family. It is rhizomatous sedge with a distinctive spherical, white inflorescence that is densely crowed with very small greenish yellow flowers. The flowers are in spikes or spikelets and the perianth is either reduced or represents only a set of bristles or scales. It often has leafless stems and the leaves are sword shaped, narrow and flat. It propagates itself mainly by the rapid growth of its spreading rhizome as shown in Plate D. Both plants are widely distributed and restricted to strandline habitat in Nigeria where they grow naturally in a short distance from sea on the sandy beaches (Hutchinson et al., 1968).

Both plants were chosen for this study because they are among the few species found growing naturally, abundant, wide spread and restricted to the habitat. I hypothesized that they have some structural anatomical changes for survival under salt stress, which are largely unavailable in scientific literatures. This research is therefore aimed at investigating leaf, stem and root anatomical changes in the coastal halophytes, Diodia maritima and Kyllinga peruviana under salinity stress in order to boost scientific knowledge on some of the adaptation mechanisms of salinity tolerance in strand plants, which will be useful in breeding program to evolve salt-tolerant crops.

2 Materials and Method

2.1 Plant materials

Young rhizomes of Kyllinga peruviana and seedlings of Diodia maritima collected from the coastal sand dune at Arogbo Ijaw Seaside Beach, Ondo State, Nigeria (60 111N latitude, 40 291E Longitude) were used as planting materials. Uniform plants raised from the young rhizomes of Kyllinga peruviana and seedlings of Diodia maritima were grown in individual perforated plastic pots (20 x 26 cm) filled with 2:1 mixture (v/v) of river sand to topsoil.

2.2 Planting soil

The soil used for planting was a 2:1 mixture (v/v) of river sand to topsoil. The sand was collected from river sand deposited at a construction site while top soil was collected from the experimental farm of Plant Science and Biotechnology Department, both at Adekunle Ajasin University, Ondo State, Nigeria (Latitude 70 28’N, Longitude 50 44’ E). Both soils were air-dried, thoroughly mixed at 2:1 mixture (v/v) and filled into the perforated plastic pots. The soil was passed through a 2-mmol/L sieve, and analyzed for the physico-chemical parameters. Particle distribution of the soil was obtained using the rapid method, while pH was measured in 1:1 soil: water suspension. Nitrogen was determined by the modified Kjeldahl method while phosphorus was assayed by Bray’s P1 solution and read on a spectrophotometer. Cations were extracted with 1.0 M acetate solution at pH 7.0; sodium and potassium contents in the extract were determined by flame photometry while calcium and magnesium were obtained by atomic absorption spectrophotometry. Organic carbon was determined by the wet oxidation method while cation exchange capacity (CEC) was by distillation method particle distribution using the rapid method Soil pH was measured in 1:1 soil: water suspension. Nitrogen was determined by the modified Kjeldahl method while phosphorus was assayed by Bray’s P1 solution and read on a spectrophotometer. Cations were extracted with 1.0 M acetate solution at pH 7.0; sodium and potassium contents in the extract were determined by flame photometry while calcium and magnesium were obtained by atomic absorption spectrophotometry. Organic carbon was determined by the wet oxidation method while cation exchange capacity (CEC) was by distillation method, following the standard method of the Association of Official Analytical Chemists (AOAC, 1985) at the Central Laboratory of National Institute for Oil Palm Research (NIFOR), Nigeria. The soil was a sandy soil with physico-chemical parameters of 5.48 pH, 20.42 ppm N, 3.56 ppm P, 3.56 (meg/100g) K, 2.32 (meg/100g) Ca, 2.60 (meg/100g) Mg, 8.2 (meg/100g) CEC, 3.67% C, 80.68% sand, 12.06% silt and 8.36% clay (Kekere et al., 2016).

2.3 Preparation of saline solution

Concentrations of 70 mmol/L, 140 mmol/L, 280 mmol/L and 560 mmol/L NaCl saline solutions were prepared in plastic kegs just before each treatment by dissolving weighed amount of available salt in tap water to make the desired concentrations. One molar solution was prepared by dissolving 58.5 g (molar mass of NaCl) in water to make 1 litre. This quantity (58.5 g) was divided by 1000 to make 1 mmol/L solution, and was subsequently multiplied by the desired value of mmol/L and number of litres to be prepared, to account for the amount of salt to be dissolved in water.

Plants were irrigated 3 times/week with saline water at concentrations of 0 (control), 70 mmol/L, 140 mmol/L, 280 mmol/L and 560 mmol/L mmol/L NaCl. Saline water treatment was initiated by gradual 70 mmol/L increments at 2-day intervals to reach the maximum salinity level of 560 mmol/L NaCl at 14 days, to prevent osmotic chock. Irrigation was done at the root zone of the plant to ensure that the relative level of soil salinity would be the primary cause of any observed effect rather than combined effect of soil and air-borne salinity. Each pot was treated with 150 ml (volume enough to keep the soil moist) per treatment; thus each pot received 450 ml of NaCl solution per week. The treatments lasted for 12 weeks; thus, each pot received a total of 4.5 litres of saline solution before the experiment was terminated. Meanwhile, pots were saturated with water and allowed to drain once per week to flush out excess salt and prevent salt build-up in the soil above the treatment levels. Besides, salt concentration in the pots was monitored by conductivity measurement in the leaked excess solution after application. The experiment was carried out from August to November, 2015. It was completely randomized with 5 single-plant replicates per treatment.

2.4 Anatomical studies

After 12 weeks of saline water irrigation, stomata number was estimated on the leaves sectioned for anatomical studies following the method earlier described by Hwang and Chen (1995). The stomata number per leaf was estimated as the product of stomata density and leaf area, measured with leaf area meter (LI-COR 300 model). Following the conventional method of free hand sections (Longstreth and Nobel, 1979), samples were taken from the centrally located region at each side of the mid-vein of the youngest fully expanded leaf. Stem sections were made at 5 cm from the tip of the apical bud based on the standard freehand section method (Ruzin, 1989). Segments from the root absorption zone (1.5 cm from the tip) were sampled. Unstained fresh free hand sections were cut in series, infiltrated with distilled water and examined. Measurements and micrographs were made using a digital camera (Nikon FDX-35) equipped with a Nikon stereo-microscope (Nikon 104, Japan). In leaf, the palisade cell length and diameter, spongy cell diameter, as well as the proportions of palisade and spongy cells expressed as a percentage of leaf thickness were measured. In stem and root, their diameter, cortex, pith and stele zone thickness expressed as a percentage of the cross section were determined.

2.5 Statistical analysis

Data were subjected to single factor analysis of variance and means were separated with Turkey Honest Significant Difference (HSD) test using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) at 95% level of significance.

3 Results

Diodia maritima leaf stomatal density decreased significantly with increasing salt concentration (Table 1). It reduced by 33.01%, 58.62% and 81.90% at 70 mmol/L 140 mmol/L and 280 mmol/L NaCl respectively relative to the control. In Kyllinga peruviana however, leaf stomatal density increased over the control by 24.35% and 21.61% at 70 mmol/L and 140 mmol/L respectively, but sharply decreased by 15.96% and 39.17% at 280 mmol/L and 560 mmol/L concentrations respectively (Table 1). The values of the number of stomata per leaf declined significantly in both plant species with increasing concentration of salt (Table 1, Plates E and F).

In Diodia maritima, the decline became significant at concentrations of 140 mmol/L and 280 mmol/L with 73.88% and 92.98 % reduction respectively. On the other hand, K. peruviana had significantly reduced the number of stomata per leaf at all levels of salt treatment relative to the control, with percentages of 25.81, 31.92, 57.33 and 76.42 at 70 mmol/L, 140 mmol/L 280 mmol/L and 560 mmol/L concentrations of NaCl respectively. Plants grown under salt application had thicker leaves than those irrigated with salt-free water (Table 2).

However, increase in leaf thickness became significant at 140 mmol/L and 280 mmol/L in Diodia maritima whereas it was significantly higher at all levels of salinity over the control in Kyllinga peruviana. The proportion of spongy cell per leaf also increased in both plants but there was no significant difference between the control and those exposed to salt stress in Diodia maritima while significantly higher values were obtained in Kyllinga peruviana subjected to saline water irrigation at all levels than in the control. In addition, one-way ANOVA showed that the palisade cell length was not affected by salt application. Furthermore, the palisade and spongy cell diameters had similar trends in both plant species. The parameters were improved by salt treatments in both plants. While Diodia maritima showed a significant difference at 140 mmol/L and 280 mmol/L only, Kyllinga peruviana gave significantly higher values at all salinity levels when compared to the control. Salinity increased stem diameter, percentage stele, pith and cross sectional area significantly at concentrations of 70 mmol/L and 140 mmol/L NaCl but without statistical difference at 280 mmol/L in Diodia maritima (Table 3, Plate G).

Table 1 Effect of salinity on leaf stomatal density and number of stomata per leaf on the abaxial leaf surface of Diodia maritime and Kyllinga peruviana

Plate E  Effect of salinity on the leaf stomata number of Diodia maritim Note: S = stomata, Magnification= 10 x 10

The percentage cortex was also not significantly affected at 280 mmol/L by salt treatment, but it was significantly reduced at 70 mmol/L and 140 mmol/L NaCl. In the root, the dimension of diameter, percentage cortex and cross section area declined significantly under salinity stress compared to the control whereas the proportion of stele was not affected significantly. In K. peruviana stem, the diameter, cross sectional area and proportions of stele and pith had higher values in plants subjected to salinity treatment than in those irrigated with salt-free water (Table 4, Plate H).

4 Discussion

Stomatal density and number of stomata reduction under salinity in D. maritima and at 560 mmol/L NaCl in K. peruviana conforms to the earlier findings in Alternanthera maritima and erecta subsp. maritima where salinity reduced stomata density and number of stomata per leaf (Kekere et al., 2016). Salinity has also caused changes in stomatal density, shape, and size in Populus euphratica (Chen et al., 2015) and Kandelia candel (Hwang and Chen, 1995). Salinity resulted in increased stomatal density at 50 mmol/L, 100 mmol/L, and 150 mmol/L NaCl but decreased at 200 mmol/L salt concentration in Populus euphratica (Alam et al., 2014), similar to what was obtained in K. peruviana in this study. Reduction in stomata is an adaptation to survive salt-stress-induced water deficit by minimizing loss of water through transpiration (Flowers et al., 1986). Salinity led to changes in the leaf cellular structure of the plants. The increase in leaf thickness in this study has similarity with the report on Leucaena leucocephala with increased length and diameter of leaf palisade cells and reduction in intercellular spaces (El-Lamey, 2015). The enlargement of palisade and spongy cells in the plants confirms a high ratio of mesophyll area/leaf area, which has been linked to increment in photosynthetic rate in plants (El-Lamey, 2015). In thick leaves, more mesophyll cells increase internal leaf surface area for gaseous exchange, which improves carbondioxide uptake, thus enhancing photosynthetic rates (Hwang and Chen, 1995).

Table 2 Effect of salinity on the leaf thickness, ratio of spongy and palisade parenchyma to leaf thickness, and mesophyll cell dimensions in the leaves of Diodia maritime and Kyllinga peruviana

Table 3 Diameter, cross sectional area and proportion of cortex, stele and pith to cross section in the stem and root of Diodia maritima grown in soil with different concentrations of NaCl

Plate F Stem anatomy of Diodia maritima under different levels of salinity Note: E= Epidermis, C= Corte, S = stele, P = pith. Magnification = 10 x 40

Plate G Stem anatomy of Diodia maritima under different levels of salinity Note: E= Epidermis, C= Cortex, P= pith, S = stele. Magnification = 10 x 40

Plate H Root anatomy of Diodia maritima under different levels of salinity Note: E= Epidermis, C= Cortex, S = stele

The vascular bundle being an integral part of the stele, the changes in the dimension of the stele is an evidence that salinity induces structural changes in xylem and phloem vessels. The improvement in stele proportion due to salinity in this study agrees with the earlier results obtained on jatropha plants (Abou-Leila et al., 2012) and Alternanthera maritima and erecta subsp. maritima (Kekere et al., 2016).

Salinity increased stem cross-sectional area and diameter in D. maritima and K. peruviana by increasing the percentage stele as influenced by the pith. Salinity usually results in a biochemical loosening of the cell wall under turgor pressure, which initiates cell expansion followed by water and solute uptake, and an increased succulence (Chen et al., 2015). Any increase in cortical area under salt stress in the salt tolerant species may be vital under physiological drought for its better water storage, which is an adaptation for ion dilution to minimize the effect of Na⁺ and Cl^- in plant tissues (El-Lamey, 2015). A reduction in cell size under high level of salt has also been observed in Tagetes patula and Ageratum mexicanum (Zapryanova and Atanassova, 2009). Reduction in cell size was also attributed to the plant ability to reduce its size in order to minimize salt uptake (Zapryanova and Atanassova, 2009). The negative effect of salinity on stem and root cells was occasioned by reduction in the vascular area and cortical cells, which was in line with the results earlier obtained in Gazania harlequin (Younis et al., 2014). The reduction in D. maritima root size by salt stress and at high concentration in K. peruviana agrees with the results on Prosopis tamarugo in which root diameter, cortex and vascular system size declined at NaCl concentration up to 400 mmol/L (Serrato-Valenti et al., 1991). Salt stress usually results in water deficit, which causes cell collapse, thus a reduction in cell size (Junghans et al., 2006; Ficho et al., 2009; Escalante-Perez et al., 2009). The root anatomy of the plants were also affected by salinity. It has earlier been reported that plants roots are potentially the first sites of damage or line of defense due to the direct contact with soil solution and the first to encounter excess salinity (Rewald et al., 2013). Increased dimension in the root size at 70~140 mmol/L NaCl in K. peruviana agrees with the report that the root cortex thickness was promoted under 100~200 mmol/L NaCl in the xerohalophyte shrubs Nitraria retusa and Atriplex halimus (Boughalleb et al., 2009). Root cell decrease was obtained in Portulaca oleracea treated with saline water (Alam et al., 2014) similar to the results on D. maritima at all concentrations and at 560 mmol/L NaCl in K. peruviana. Likewise, the cortical parenchyma in Chloris gayana decreased under saline conditions (C´eccoli et al., 2011), and reductions of cortex thickness under high salinity were also reported in shrubs (Boughalleb et al., 2009) and tree roots, which could be as a result of ion toxicity caused by Na⁺ and Cl^- accumulation in the tissues (Reinoso et al., 2004).

Plate I Root anatomy of Diodia maritima under different levels of salinity Note: E= Epidermis, C= Cortes, S = stele. Magnification= 10 x 40