MACRO- AND MICRO-MORPHOLOGICAL CHARACTERISTICS OF KICKXIA DUMORT. AND SOME RELATED TAXA IN SAUDI ARABIA

The morphological and anatomical aspects of 13 species belonging to genera Kickxia, Scrophularia , and Plantago of Saudi Arabia reveal that the species of Kickxia and Scrophularia are distinct from Plantago major . The most notable morphological differences are the absence of adventitious roots and the presence of acaulescent stem in the species of Kickxia and Scrophularia and the existence of adventitious roots and the absence of acaulescent stem in P. major . The species of Plantago possessed rose-shaped radical leaves, whereas the rest of the species studied had cauline leaves or both. The species of Kickxia and Scrophularia have pentamerous flowers with bilabiate corollas, whereas, P. major has tetramerous flowers with tubular corollas. The separation of P. major from the rest of the studied species is the most obvious result obtained from the dendrogram, and this result is consistent with some traditional taxonomic studies that placed Kickxia in the Scrophulariaceae species while separating P. major into an independent family Plantaginaceae. The anatomical findings revealed that the upper and lower epidermal cells of the leaves were irregular with undulate walls in all analyzed species, except in P. major , where they were polygonal with straight walls. Only P. major had amphianisocytic paracytic stomata, but eight Kickxia species had anomocytic stomata and K. hastata and K. abhaica , and the two Scrophularia species had anisocytic stomata. Therefore, this study suggests the retention of the Kickxia in Scrophulariaceae s.l. and maintaining Plantago in Plantaginaceae s.s.


Introduction
Plantaginaceae along with other species that were belonging to Scrophulariaceae, due to the molecular characteristics. In studying the relationship between plants and animals, (Knerl and Bowers, 2013) found that the species Kickxia spuria is of the preferred families for the Buckeye Butterfly. (Jeddi and Chaieb, 2010;Tarhouni et al., 2010) considered the species Kickxia aegyptiaca as pastoral plants. Kickxia elatine was also considered a pastoral plant by (Parlak et al., 2011).
The study of (Eraud et al., 2015) included the species Kickxia elatine in the plants of importance in bird feeding. (Adeux et al., 2017) considered Kickxia as an agricultural crop and can be used as an alternative to corn.
This research aims mainly to study the Kickxia species in Saudi Arabia due to the lack of taxonomic studies on this genus and compare these with the two species of the genus Scrophularia L. of Scrophulariaceae viz.: Scrophularia deserti, Scrophularia peyronii, and the species Plantago major as the typical representative of Plantaginaceae family, by using the morphological and anatomical taxonomic evidence and then analyzing these results statistically to obtain evidence to either endorse or reject the transferring of the species of Kickxia from Plantaginaceae to Scrophulariaceae.

Materials and Methods
In the present study, 13 species were collected from different localities in Saudi Arabia ( Table  1). The studied taxa include 10 species belonging to Kickxia, two Scrophularia species in addition to one species from Plantaginaceae (Plantago major).
For recording the morphological characteristics of vegetative and floral organs, the phenotype of both stem and leaves in each species was examined in terms of shape, margin, base, and apex of the blade, the dimensions of the lamina, the distribution of leaves on the stem and leaf veining were also measured, and the floral characteristics were examined using different optical microscopes. Photos were taken by camera Hawaii mate 20 pro. All results, observations, and measurements have been recorded.
For recording the micro-morphological (anatomical) characteristics, the method of Al-Duaiji et al. (1997) was used to prepare skinned leaves from plant species studied as follows: (i) Numbers were given to the samples to be stripped, then the dry leaves were cut and washed with running water to remove the dirt stuck, and then they were washed with distilled water. (ii) The dry leaves taken from the herbarium samples were soaked in distilled water for 24 hours; until the leaves became soft and easy to make a thin strip. (iii) A simple portion was made from the surface of the paper with a sharp mousse, and then, using pointed forceps and very carefully, a thin skin was prepared from the leaf skin. (iv) The stripes were placed on a glass slide making sure that the top was on the outside. (v) the strip was covered with drops of ethyl alcohol for 3-5 minutes to thicken the stripes and make them as transparent as possible. (vi) the stripes were stained with light green or saffron for several seconds, then the stripes were loaded with a drop of glycerin and covered with the slide cover, then placed into a 40 ° C oven for several hours. (vii) The slides were examined by light microscopy and imaged at force (40 ×) by the camera device installed with a light microscope.
The relevant terms of Metcalfe and Chalk (1950) were used to define the cell shapes, while stomatal types were determined from Stace (1989). The types of hairs (Trichomes) were also determined following Prabhakar (2004).

Numerical analysis
A dendrogram was constructed based on a data matrix using the NTSYS-pc 2.2 software package (Rohlf, 2009) by using both morphological and anatomical characteristics of the studied species.

Morphological Characteristics of Vegetative and Floral Organs
The morphological analysis of vegetative, floral, and anatomical aspects of 13 species belonging to genera Kickxia, Scrophularia, and Plantago L. of Saudi Arabia was carried out. The morphological characteristics of vegetative and floral organs of these species are described below.
Lifeform: Through field trips, it was found that Kickxia (Table 2) and Scrophularia are spread in their environments as distant individuals while Plantago major in groups. The plant species differ in their lifeform. There are ephemeral annuals represented by K. elatine and K. hastata, as they are characterized by being small and their roots are shallow and spread horizontally to exploit rainwater and dew drops on the soil surface, and this observation is consistent with the study of Wood (1997) and Chaudhary (2001). There are Sclerophytes, which are shrubs or perennial herbs in most of the species under study, which have adaptations to withstand or avoid the dry season, and among these adaptations observed in some species are the reduction of their vegetative total and the epiphysis surface, rapid leaf fall, and the presence of dense hairs on the plant and leaves which reflect part of the sun's rays and form a moist medium around the leaves so, reduce the process of transpiration. The results of this study agree with Fischer (2004) and Hamed et al. (2014).
Habit: On the Habit side, the subshrub was found in the two species of Scrophularia, which corresponded to the result of Issa and Al-Ali (2018), while we found woody herb in five species of Kickxia (K. acerbiana, K. aegyptiaca, K. elatine, K. pseudoscoparia and K. spartioides). The herb appeared in Plantago major, as well as in the other five species of Kickxia, and this result was consistent with the results of Ghebrehiwet et al. (2000).
Stem: Some species were distinguished by being hairy as in Kickxia abhaica, and three species were sparsely hairy, which are: K. elatine, K. acerbiana, K. aegyptiaca, and the species Plantago major, while in other species the appearance of the plant was glabrous, and this result was consistent with Hamed et al. (2014). All the studied species were distinguished by the caulescent market except for Plantago major, the stem was a caulescent (stemless -dwarf), this result agreed with Bukari (2009), Zubair (2010), and Hamed et al. (2014). The stem erects also appeared in four species of Kickxia: K. acerbiana, K. aegyptiaca, K. pseudoscoparia, K. spartioides and two species of Scrophularia, while a week was extended or climbing in other species of Kickxia, and this is consistent with what was stated in the study of Chaudhary (2001). All the studied species were branched except for the Kickxia hastata, which was unbranched this matches what was stated in the study of Chaudhary (2001) and Hamed et al. (2014). The branching was monopodial for all species except for Scrophularia peyronii which was sympodial (limited growth), and this is consistent with a study by Issa and Al-Ali (2018).
Leaves: The leaves of Kickxia species were distinguished by the heterophylly, while this variation was not recorded in P. major and the two Scrophularia species which were characterized by identical leaves. This result is consistent with Chaudhary (2001) and Hamed et al. (2014). Also, the leaves of all Kickxia species were simple, entire, with reticulate veins, whereas in the two species of Scrophularia it was pinnately lobed with reticulate veins. The leaves of the studied species were organized alternate except for the leaves of the Scrophularia deserti, which were opposite (Issa and Al-Ali, 2018), while Plantago major leaves are broad, clustered at the base of the plant in the form of the rosette and prominent parallel veins, and this result is consistent with Weryszko-Chmielewska (2012) an Haddadian et al. (2014).
Flowers: In all Kickxia species, flowers were solitary, while in Plantago major they were regular on one axis in the form of spike inflorescence, and flowers of the two Scrophularia species were regular in racemose inflorescence. All the flowers of the studied species were pentamerous and zygomorphic, except for Plantago major, which was tetramerous and Actinomorphic. The results are consistent with the study of Bukari (2009), Ianovici et al. (2010) and Hamed et al. (2014). Hairy flowers appeared in all Kickxia species, while they had a glabrous appearance in Plantago major and Scrophularia. All the flowers of the Kickxia species were yellow, while Scrophularia species were characterized by a dark red colour, and Plantago major flowers were membrane brownish as in Bukari (2009), Ahmad et al. (2009) andHamed et al. (2014).
All the studied samples were distinguished by a continuous calyx with the fruit and free sepals, as well as by transparent membranous edges, which varied in their shapes between filiform and lanceolate. All species of Kickxia and Scrophularia were distinguished by a corolla with fused petals of a five-bilabiate shape and differed by the presence of a spur in the petals of the species belonging to Kickxia, which resulted from the mutation of the front petal and it differs in length according to the species. This result agreed with a study by Chaudhary (2001) and Hamed et al. (2014).
All the studied species contained four fertile stamens, and all the stamens were epipetalous and didynamous except for Plantago major, the stamens were equal. This result was consistent with the study of Hamed et al. (2014). Pollen in the two species of Scrophularia was distinguished by the presence of a sterile stamen, and this result agrees with the study of Uzunhisarcikli et al. (2015), Ranjbar and Rahchamani (2018). The stamens are found in all Kickxia species inside the corolla tube while they are exerted in the two Scrophularia species and Plantago major. This result agreed with the study of Bukari (2009) and Hamed et al. (2014).
Fruits: All fruits of the studied species were globose, except for Kickxia acerbiana and Plantago major the fruits were elongated. All the fruits of the studied species were dehiscence, multi-seeded capsule, but they differed in the method of blooming as the fruits of the Kickxia species opened with two apical pores, while in Plantago major they opened with a cover and with two valves in Scrophularia species as in Hamed et al. (2014).
The results obtained from the dendrogram (Fig. 1) and (Table 2) separated Plantago major in an independent series (SI), while the rest of the other species are combined in (SII) due to their similarity in many of the phenotypic characteristics and this result is consistent with many traditional taxonomic systems that include Kickxia within the Scrophulariaceae (Bentham and Hooker, 1876;Cronquist, 1981;Engler and Prantl, 1895). This result also matches the opinion of some studies in maintain Kickxia and keeping it within Scrophulariaceae s.l. (Hamed et al., 2014).

Micro-Morphological (Anatomical) Characteristics
Data Matrix was constructed from the obtained results (Table 3) of the anatomical features of the studied species so that the presence of the trait was expressed by the number (1) and in the absence of the trait by (0) to be used in the numerical analysis, and a dendrogram was created between the plant samples using NTSYS-pc 2.2 software package according to the method of (Rohlf, 2009), cluster analysis of a matrix of similarity and dissimilarity was implemented among the species under study. It is from the results obtained from the dendrogram (Fig. 2) the number of Series, Clusters and Groups between the studied species.
The results showed the upper and lower epidermal cells of the leaves are identical on both isodiametric surfaces. They appeared irregular with the undulate surface in all studied species except for Plantago major. The results of this study agree with the results of Bahadar et al. (2018).
The results also showed the presence of the amphistomatic leaves i.e., the stomata are located on both lower and upper surfaces of the leaves alike, and they were distinguished by three types: The first type: Anomocytic (Ranunculaceous) stomata observed in eight species of Kickxia, and this result is consistent with the results of Lahari and Rao (2018).  The second type: Anisocytic (Cruciferous) stoma, surrounded by three cells. It was observed in only two species: Kickxia abhaica and K. hastata. It was also observed in two species of Scrophularia. This result agreed with the results of Ranjbar and Rahchamani (2018).
The third type: Amphianisocytic-Paracytic. This type was seen only in Plantago major, and this result agrees with the results of Bahadar et al. (2018) but disagrees with Mesquita et al. (2017), where it was mentioned that the stomata in Plantago major are anomocytic.
Most of the studied species were characterised by the absence of hairs on the epidermal leaf surface while the others were distinguished by the presence of non-branching glandular hairs with two and multiple cells arranged in one row and differed in the shape of the terminal cell, so it took the glandular spherical shape in the species K. abhaica, K. acerbiana, K. aegyptiaca, K. elatine, acute while non-glandular in Plantago major.
Thus, it is evident from the anatomical study of the leaves of the studied species that there are some characteristics which distinguish Plantago major from the rest of the studied species in terms of the shapes of the polygonal epidermal cells from 4-5 while the rest of the species are irregular in shape, as well as the type of stomata in the Plantago major amphianisocyticparacytic, unlike other species. Also, by a special type of non-glandular, multicellular, unbranched acute hairs.
Thus, this study demonstrates that Plantago is unique in its anatomical characteristics from the rest of the species under study, which confirms that Plantago is kept in a separate family (Plantaginaceae) and not included in the Scrophulariaceae family, which includes Kickxia and Scrophularia.
This result agreed with the anatomical and morphological study (Hamed et al., 2014) on some genera of the Scrophulariaceae family with Plantago and emphasized the need to keep Plantago within a separate family (Plantaginaceae) and for more accuracy in separating the species, many studies must be conducted. The other is to create new taxonomic indications that support the separation or not.
Also, the results obtained from the dendrogram (Fig. 2) and (Table 3) separated Plantago major into an independent series, while the rest of the other species are grouped in another series due to their similarity in many anatomical features; the shape of epidermal cells, the type of stomata, the terminal cell shape of the hair, and this result is consistent with many traditional taxonomic systems that include Kickxia within Scrophulariaceae (Bentham and Hooker, 1876;Cronquist, 1981;Engler and Prantl, 1895). Also, agree with the opinion of some studies that suggest that Kickxia should not be separated and kept within the Scrophulariaceae s.l. (Hamed et al., 2014).
The Macro and Micro-Morphological characteristics of species under study are important in defining, separating, and studying the evolutionary relationships between taxa. This observation gives extra support to the taxonomic views that suggest the retention of the Kickxia in the family of Scrophulariaceae s.l. and maintaining Plantago in a separate monogenetic family of Plantaginaceae s.s.