Habit of Selaginella growing in xerophytic

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1 Text Systematic Position Division: Lycopodiophyta Class: Isoetopsida Order: Selaginellales Family: Selaginellaceae Genus: Selaginella Habit and Habitat Selaginella, with about 700 species, is cosmopolitan in distribution (Banks, 2009). The species are commonly known as spike moss or small club moss. Most of the species inhabit damp and shaded forests of tropics, but some (e.g., S. densa, S. rupestris, S. lepidophylla) grow in xerophytic habitats, such as exposed rock surfaces. S. oregano is an epiphyte that Fig 1: conditions Habit of Selaginella growing in xerophytic

2 grows on tree trunks in tropical rain forests. Several species of Selaginella are grown in gardens as ornamentals (Rashid, 1999). Some xerophytic species of Selaginella (e.g., S. lepidophylla, S. pilifefra) show caespitose habit; they curl and become ball like during dry season and again become green and fresh when moisture is available. These are called resurrection plants (Singh et al, 2000). Fig 2: Selaginella showing caespitose habit Selaginella is particularly interesting from comparative evolutionary perspective because it has retained the independent but water-dependent gametophytic generation that is typical of all non-seed plants. Because its gametophyte is not buried within maternal tissues of the sporophyte, Selaginella is also a useful experiment system for investigating how the alternation of generations (the switch between haploid gametophyte and diploid sporophyte) is regulated (Banks, 2009)

3 The genus is represented in India by more than 70 species. Among these species, Selaginella kraussiana, S. monospora, S. biformes, S. rupestris, S. megaphylla, S. bryopteris, S. ciliaris, S. chrysorhizos and S. pentagona are common. Morphology The sporophytic plant body of Selaginella is differentiated into root, stem and leaves. Besides some species also have rhizophores. 1. Roots. The primary roots are ephemeral and the adult plant has adventitious roots. The adventitious roots usually have specific locations in relation to stem dichotomies. In most of the creeping species with dorsiventral stems (e.g., S. kraussiana, S. laevigata), roots arise at or close to the point of dichotomy; in species like S. rupestris and S. wallichii they arise at the point of dichotomy as well as at other positions; and in S. selaginoides and S. spinulosa they arise from knot-like swellings present at the basal portion of the stem. The roots arise endogenously and are dichotomously branched. The dichotomies are at right angles to each other. The main function of root is to anchor the plant in the soil and absorb water and

4 mineral salts from the soil. Besides it form a passage way for water and dissolved substances from the root into the stem and also for foods from the stem down into the root. Fig 3: Adventitious roots in Selaginella 2. Rhizophore. In some species of Selaginella, many long, cylindrical, unbranched and leafless structures arise from the lower side of the stem at the point of dichotomy. These grow vertically downward and bear tuft of adventitious roots at their distal end. They are known as rhizophores (Goebel 1905; Bower, 1935). The rhizophore may develop into a typical leafy shoot under certain conditions. Like a typical root it grows downwards to the soil and absorbs water through its tissues in a direction reverse of that in which it has grown. It produces lateral endogenous

5 roots and helps in anchoring the plant to the substratum. Fig 4: Showing various organs like rhizophore, cone and leaves in Selaginella 3. Stem. The stem is erect and dichotomously branched in the sub-genus Homoeophyllum, and prostrate or sub-erect with lateral branching in the sub-genus Heterophyllum. The stem apex usually has a single well-defined apical cell, but in S. oregano a group of meristematic cells has been observed. 4. Leaves. The leaves of Selaginella are microphyllus, sessile and simple. Their shape varies from ovate to lanceolate. The leaf has a single midvein that remains unbranched throughout its course. Most of the species have thin and soft leaves, but in xerophytic species they are thick. The

6 vegetative leaf as well as sporophyll, has a small membranous projection on its adaxial (upper) surface, close to the base. The projection is known as the ligule. The basal part of the ligule has a distinct hemispherical foot-like structure, called glossopodium. It is composed of highly vacuolated thin-walled tubular cells. The ligule is embedded at the base of the leaf in a pit like structure, known as ligular pit. The projected part of the ligule is only one cell in thickness and is tongue-like (e.g., S. svogelli, S. martensii). It develops precociously and matures long before its associated leaf. Although the definite function of ligule is not known, it has been suggested that in some way they are associated with water absorption and secretion, and thus prevent desiccation of the shoot. Some consider that the ligules in Selaginella are concerned with upward movement of inorganic solutes, and thus compensate for smaller and less effective leaf primordia. In the sub-genus Homoeophyllum, all leaves are alike and spirally arranged. But the species belonging to the sub-genus Heterophyllum, have two types of leaves- two dorsal rows of small leaves (microphylls), and two ventral rows of large leaves (megaphylls).

7 The leaves occur in pairs and the two leaves of a pair are always unequal. Anatomy 1. Root. A cross section of a root shows a simple structure. The epidermis is made up of tangentially elongated cells. In exposed roots, the outer wall of epidermal cells is cutinized, but in roots that penetrate the substratum, the epidermal cells are delicate and have root hairs. The cortex is usually homogenous, consisting of many layers of thin parenchymatous cells. But in some species the outer layers of the cortex become thickwalled and form hypodermis. In S. selaginoides, the parenchymatous cells of the cortex show mycorrhizal association. The innermost layer of the cortex forms endodermis. In species like S. densa and S. rubella endodermis is fairly distinct. The central part of the root is occupied by a protostele, surrounded by 1-3 layers of parenchymatous pericycle. The xylem, which forms the central solid core of the stele, is monarch to tetrarch and exarch. The phloem occurs in the form of a ring around the xylem. 2. Rhizophore.

8 The anatomy of the rhizophore resembles that of the root. Some variations in the internal organization are due to the fact that the rhizophore is an aerial structure, whereas the root is a subterranean organ. The epidermis is single layered and the outer wall of the epidermal cells is covered with a thick layer of cuticle. Root hairs, characteristic of roots, are absent on rhizophores. The cortex is differentiated into an outer sclerenchymatous and an inner relatively wide parenchymatous zone. The innermost layer of the cortex forms endodermis. The protostele of the rhizophore is surrounded by a parenchymatous pericycle. Usually the stele is monarch and exarch, but shows some variations. For example, in S. atro-viridis, the metaxylem is crescent-shaped with many protoxylem strands on its concave side, and in S. kraussiana the xylem is centrifugal. Fig 5: Cross section of rhizophore 3. Stem.

9 Internally, the stem is more complex than the root. The anatomy of the stem shows variations not only in different species, but also within the same species depending on stem diameter. A transverse section of the stem shows epidermis, cortex and central cylinder. Fig 6: Internal structure of Selaginella stem The epidermis is the outermost unistratose layer. The outer walls of the epidermal cells are highly cutinized. The epidermis is devoid of stomata and hairs. The cortex is usually composed of compactly arranged parenchymatous cells without intercellular spaces. But in mature stems of many species outer layers of cortex become partially sclerenchymatous, forming a tough hypodermis. In xerophytic species (e.g., S.

10 rupestris, S. lepidophylla), most part of the cortex is composed of thick-walled cells. A distinctive feature of Selaginella stem is the presence of radially elongated endodermal cells, called trabeculae. They have characteristic casparian bands on their lateral walls. Due to the presence of trabeculae, the central stele is separated from the cortex by large air spaces. In fact, in a transverse section the stele appears suspended in an axial air column with the help of trabeculae. The air spaces develop due to more rapid expansion of the cortical region than the stele. This differential growth also results in radial stretching of some endodermal cells. Xerophytic species of Selaginella, however, do not have trabeculae. Fig 7: Showing trabeculae in Selaginella stem The number of steles in the stem shows considerable variation in different species of Selaginella. For example, the stem is monostelic in S. spinulosa and S.

11 flabellata, distelic in S. kraussiana, and polystelic (with steles) in S. laevigata. Besides, the number of steles may also vary within different parts of the same plant. For example, the creeping branches of S. braunii are distelic, whereas the erect branches are monostelic; and in S. lyalli, the creeping branches are distelic and the erect branches are polystelic. The stele is surrounded by a single-layered pericycle. The shape and structure of the stele is also variable. It is circular in S. kraussiana and flat or ribbon-like in S. viridangula and S. vagelii. Most of the species have a protostele with a solid xylem core surrounded by phloem, but S. laevigata var. lyalii has a siphonostele. The xylem is usually monarch (S. kraussiana), or diarch (S. selaginoides). It usually consists of only tracheids; the protoxylem tracheids have annular or helical thickenings; whereas the metaxylem tracheids show scalariform thickenings. In S. oregana, S. densa and S. rupestris, however, the xylem has true vessels with transverse perforation plates. Although secondary growth is absent, some secondary xylem elements have been found in the basal part of the stem of S. selaginoides. 4 Leaf.

12 Both, the upper and the lower epidermis of the leaf are unistratose. The epidermal cells have chloroplasts. The leaves are mostly amphistomatic, but sometimes they are hypostomatic, as in S. martensii. Stomata are distributed mostly in the midrib region. The mesophyll consists of loosely arranged thinwalled cells, with many small or large intercellular spaces. It is usually made up of only spongy parenchyma, but occasionally a distinct palisade layer may be present towards the morphological upper side. A mesophyll cell has 1-8 cup shaped chloroplasts, which have many spindle shaped pyrenoid-like bodies. The leaf has a median vascular bundle surrounded by a distinct bundle sheath. The xylem, which occupies the central part of the bundle, consists of only tracheids with annular or spiral thickenings. It is surrounded by phloem. Reproduction The sporophyte of Selaginella reproduces vegetatively and by spores. Vegetative reproduction Vegetative propagation in Selaginella takes place by tubers, bulbils, dormant buds and by fragmentation.

13 In S. rupestris, prostrate branches produce roots during favorable conditions. These root bearing prostrate branches separate from the parent plant and grow into new sporophytes. Species like S. chrysorhizos and S. chrysocaulos propagate with the help of tubers and bulbils. The tubers may be aerial, developing at the apices of aerial branches (e.g., S. chrysocaulos) or subterranean (e.g., S. chrysorhizos). During favorable conditions the tuber germinates into a new sporophyte. Aerial branches of S. chrysocaulos also bear some dormant (resting) buds which grow into new plants during favorable conditions. Fig 8: Tubers in Selaginella Reproduction by spores Selaginella is a heterosporous pteridophyte; it produces two types of spores megaspores and microspores. The megaspores form female gametophytes on germination and the microspores

14 give rise to male gametophytes. The sporangia are strictly dimorphic, i.e., micro and megaspores are formed in separate sporangia. The sporangia bearing microspores are called microsporangia, and those bearing megaspores as megasporangia. There are many microspores in a microsporangium, while each megasporangium usually has 1-4 (or rarely more) megaspores. The megaspores are much larger than the microspores. The sporangia are borne singly in the axils of sporophylls. The sporophyll-bearing micro-sporangium is called microsporophyll, and the one with megasporangium is known as megasporophyll. The sporophylls are spirally arranged around a central axis to form a strobilus. Fig 9: Parts of strobilus showing megaspores and microspores in Selaginella

15 Strobilus or cone. In most of the species of Selaginella, sporophylls are aggregated at the apex of the main stem and its branches in definite loose or compact cones, called strobili (singular = strobilus). The size of the strobilus varies from 5mm to 6-7 cm. It is often inconspicuous due to its small size, and similarity between sporophylls and vegetative leaves. Usually a branch terminates in strobilus, but in species like S. cuspidata and S. patula, vegetative growth of the branch may continue beyond the strobilus. In S. erythropus, a second strobilus is produced on the fertile branch after an intervening vegetative region. Thus, in this species sporophylls and vegetative leaves occur in alternate segments. Distribution of micro and megasporangia in strobilus. In most of the species of Selaginella, both micro and megasporangia are found within the same strobilus. Their distribution is specific. For example, in S. selaginoides, S. rupestris and S. helvetica, megasporangia are present in the basal part and microsporangia in the upper part of the strobilus; in S. kraussiana there is only a single megasporangium at the base of the strobilus, and the rest are microsporangia; and in S. inaequalifolia one side of the strobilus bears only megasporangia, and the other

16 microsporangia. In S. martensii and S. caulescens, mega-and microsporangia do not show any definite arrangement. In S. selaginoides, a series of basal sporangia are non- functional. The two types of spores are never present within the same sporangium. In S. gracilis and S. atroviridis, strobili are monosporangiate, i.e. micro and megasporangia are borne in separate strobili. Development of sporangium. The initial stages of the development of micro and megasporangium are similar. Both develop from the transverse row of initial cells, i.e. the development is of eusporangiate type. The sporangial initials divide periclinally, establishing outer jacket initials and inner archesporial initials. The archesporial initials undergo repeated anticlinal and periclinal divisions forming a mass of sporogenous cells. Simultaneous divisions also occur in the jacket initials and the derivatives eventually form a twolayered sporangial jacket. The cells of the outermost layer of the sporogenous tissue (adjacent to the inner wall layer) form a nutritive layer, known as tapetum. The tapetal layer disintegrates as spores mature. The last generation of sporongenous cells functions as spore mother cells. The micro and megasporangium differ in subsequent development.

17 Further development of microsporangium. In microsporangium about 80-90% spore mother cells are functional, and behave as microspore mother cells. The remaining spore mother cells degenerate and form a viscous nourishing fluid. The functional spore mother cells undergo meiosis and form haploid microspores, which are arranged in tetrahedral tetrads. Further development of megasporangium. In megasporangium, all spore mother cells but one, degenerate. The functional spore mother cell behaves as megaspore mother cell. It divides meiotically forming four tetrahedrally arranged haploid megaspores. All the four megaspores derived from a megaspore mother cell may not always be functional. For example, in S. sulcata only one, and in S. rupestris two megaspores are functional. Sometimes there are more than one megaspore mother cells in a megasporangium and in such cases the megasporangium has 8 or more megaspores. The megaspores are much larger than microspores. The expression of maleness or femaleness is not genetically determined; it appears to be influenced by the nutritional factor, and the specific environment in which the sporangium develops.

18 Mature sporangium. Mature sporangia are stalked structures, with a two-layered sporangial jacket. The cells of outer jacket layer are elongated and contain chloroplasts. The micro and mega sporangia differ in shape, size and colour. The microsporangia are slightly elongated, yellow, red or orange in colour. The megasporangia are larger and paler and assume the shape dictated by the enlarging megaspores within. The mature sporangium dehisces along the line of dehiscence present at its distal end and oriented transverse to the axis of the sporophyll. Structural modification of the surface cells along this line and at its flanks results in splitting of the distal part of the sporangium into two valves. The lower cup-shaped part of the sporangium shrinks on drying and throws out spores violently. Gametophyte The spore is the mother cell of the gametophytic generation. As mentioned earlier, Selaginella is heterosporous and produces two types of spores- the smaller microspore and the larger megaspores. This difference in the size of the spores is related to their fate and function; microspores develop into male

19 gametophyte and megaspores into female gametophytes. In Selaginella both microspores and megaspores begin to germinate while still inside the sporangium (i.e., they germinate in situ). Thus, spores are shed at multicellular stage. Microspores and development of male gametophyte Microspores: The microspores are small, spherical structures, ranging mm in diameter. A microspore is surrounded by a thick ornamented exine and a relatively thin intine. The ornamentations in the exine may be papillate, echinulate or granulate. The spore has a single haploid nucleus and granular cytoplasm, rich in fatty substances. The fats probably provide food to the developing male gametophytes as spores contain no chlorophyll. Development of male gametophyte: The microspores germinate inside the microsporangium and are shed at 13- celled stage. The first division of the microspore is asymmetrical and as a result a small lenticular prothallial cell and a large antheridial initial is established. The prothallial cell does not divide

20 further and the entire sporangium develops from the antheridial initial. The first division of the antheridial initial is nearly at right angles to the prothallial cell. It results in the formation of two antheridial cells of almost equal size. Both these cells divide by a vertical wall to produce a group of four cells. Thus, at this stage the gametophyte consists of five cells (four cells derived from the antheridial initial and a prothallial cell). The two basal cells, derived from the antheridial initial, do not divide further, whereas the upper two daughter cells divide repeatedly and form ten cells. At this stage the gametophyte has 13 cells (10 cells derived from the upper daughter cells of the antheridial initial,2 basal daughter cells and 1 prothallial cell).of these, four central cells function as primary androgonial cells and eight peripheral ones as jacket cells. The male gametophyte is shed from the microsporangium at 13-celled stage. It enters into partially opened megasporangium where further development of the male gametophyte takes place in close proximity of the developing female gametophyte. In some species, it is believed, that further developed of the male gametophyte takes place in the soil.

21 The four central primary androgonial cells of the male gametophyte divide repeatedly forming a mass of antherozoid mother cells or androcytes. Each androcyte metamorphoses into a spindle-shaped biflagellate antherozoid. The antherozoids of Selaginella are perhaps the smallest amongst the vascular plant. With the formation of antherozoids, the jacket cells decompose and form a mucilaginous substance. The antherozoids float in this substance. Until this stage the male gametophyte is completely enclosed within the wall of the microspore. Thus it is entirely endosporic and extremely reduced structure. Unlike other pteridophytes, vegetative prothalli are not formed in Selaginella. The gametophyte is not set free and is dependent on the parent sporophyte for nutrition. Megaspore and development of female gametophyte Megaspores: Megaspores are much larger than the microspores. Their diameter varies from 0.15 to 0.5 mm. Usually all megaspores in a megasporangium are approximately of the same size, but in S. molliceps one megaspore is larger than the other three, and in S. stenophylla there are two large and

22 two small megaspores. The megaspores are also arranged in tetrahedral tetrads. The wall of the megaspore is differentiated into an outer massive exine and an inner thin intine, but in S. rupestris and S. apus it is differentiated into three distinct layersthe outer exospore, the middle mesospore and the inner endospore. The megaspore has a single haploid nucleus, surrounded by granular cytoplasm, rich in fatty substance. Development of female gametophyte: Like male gametophyte, the development of the female gametophyte of Selaginella also begins while it is still within the megasporangium. In S. kraussiana, the gametophyte is liberated from the megasporangium after the first archegonium is differentiated, whereas in S. rupestris and S. apus it is retained in the megasporangium even after the development of embryo has started. However, in S. spinulosa and S. helvitica the development of female gametophyte starts only after the megaspore is shed from the sporangium. Immediately after the development of female gametophyte initiates, a large vacuole appears in the centre of the megaspore and as a result the cytoplasm is pushed along the spore wall in the form of thin

23 membrane. There is considerable enlargement of the megaspore. The outer spore wall (exospores) grows more rapidly than the mesospore and endospore, consequently a large gap is formed in between the exospore and mesospore. At this stage, the exospore is attached to the mesospore only at one point. The space between the exospore and the mesospore is filled with a homogenous liquid. The haploid nucleus of the megaspore divides repeatedly without any wall formation. The free nuclei are unequally distributed in the peripheral cytoplasm; they are clustered beneath the triradiate ridge of the spore and sparsely distributed elsewhere. Now, wall formation begins in the apical region and a lensshaped pad of small cells is formed at the apical end. It is separated from the rest of the female gametophyte by a distinct diaphragm. The cytoplasmic layer becomes thicker gradually and pushes the mesospore outward. As a result the mesospore again comes in contact with the exospores. With the increase in the amount of cytoplasm, the central vacuole diminishes and eventually disappears. The part of the gametophyte below the diaphragm is multinucleate in early stages but becomes multicellular as wall formation proceeds inward. At

24 this stage, the spore wall ruptures along the triradiate ridge exposing the apical cellular pad. The exposed part of the female gametophyte may develop chloroplasts but the photosynthetic ability of this part is of limited importance as food for the developing embryo is stored in the lower multicellular part of the gametophyte. Many rhizoids develop from the exposed part of the gametophyte. They attach the gametophyte to the substratum and also help in absorption of water. Development of archegonia: Archegonia develop from the apical tissue of the gametophyte. All superficial cells of this tissue have the potential of forming archegonia. The archegonial initial divides periclinally into a primary cover cell and a central cell. The primary cover cell divides by two vertical divisions at right angles to each other and forms four neck initials. The neck initials divide transversely so as to form eight neck cells, arranged in two tiers of four each, in the meantime, the central cell divides by a periclinal wall and an outer primary neck cell and an inner primary venter cell is established. The former does not divide further and directly functions as neck canal cell, whereas the latter divides transversely into a venter canal cell and an egg.

25 The mature archegonium of Selaginella has two cell long neck (consisting of eight cells in two tiers of four each), a neck canal cell, a venter canal cell and an egg. The four terminal cells of the neck project beyond the surface of the gametophyte as asymmetric nipples. Rest of the archegonium remains embedded in the tissue of the gametophyte Fig 10: Spores and their fate Fertilization Fertilization usually takes place after the megasporangium has fallen on the soil, but in some species it may occur while the female gametophyte is still within the sporangium. Just before fertilization, the neck cells of the archegonium separate from each other and form a passage for the entry of

26 antherozoids. After liberation from the male gametophyte, antherozoids swim in rain or dew water and reach the archegonia. Usually only one antherozoid enters into an archegonium and fuses with the egg to form a diploid zygote. Some species of Selaginella (e.g., S. rupestris, S. apoda) show seed habit. In these species, the sporangium has only a single megaspore and at maturity of the archegonium the spore wall ruptures, but the developing female gametophyte does not come out of the spore wall. The developing male gametophyte, when shed from the microsporangium (present in the distal part of the strobilus) lands on the partially open megasporangium. Thus, at this stage, both the male and the female gametophytes lie within the megasporangium. As such fertilization and embryo development take place inside the megasporangium. The sporangium is shed after the development of root and primary shoot of the new sporophyte. This feature is of considerable importance from the point of view of seed habit because when the megaspore with young sporophyte is shed, it has all typical characters of a seed. Development of embryo

27 The diploid zygote is the mother cell of the sporophytic generation. It divides transversely, establishing an epibasal (upper) suspensor cell and a hypobasal (lower) embryonic cell. As development proceeds, the suspensor cell repeatedly divides to form a suspensor, which pushes the developing embryo deep into the female gametophyte. The rest of the embryo develops from the embryonic cell. It divides by two vertical walls at right angles to each other, and thus a four-celled embryo is formed. One of the four cells of the embryo divides by an oblique vertical wall, and thus an apical cell with three cutting faces is established. This eventually functions as the apical cell of the embryonic shoot.the remaining three cells of the 4-celled embryo and the sister cell of the apical cell (i.e., total four cells) divide transversely to form two tiers of four cells each. The cells of both the tiers divide irregularly forming a multicellular embryo. Usually the cells of lower tier divide more rapidly than the upper tier and due to this differential growth the entire embryo apex rotates at and emerges through the apical part of the gametophyte. The derivatives of the lower tier form the foot. At first the foot grows on one side but eventually comes to lie opposite the suspensor. The foot acts as a haustorial

28 organ; its main function is to absorb nutrition for the developing sporophyte from the female gametophyte. At this stage, a superficial cell in each of the two diagonally opposed quadrants of the upper tier differentiates as the apical cell of a foliar appendage, which eventually forms a cotyledon. In the axil of each cotyledon a ligule develops. The part of the embryo immediately posterior to cotyledons develops into hypocotyledonary part of the stem. The stem grows with the help of the apical cell of the embryo. After the formation of cotyledons and stem, the apical cell of the root differentiates on the lateral surface of the foot. The derivatives of this cell develop into a root-like structure, called rhizophore. Roots, in fact, develop at the apex of the rhizophore. In early stages of development the young sporophyte is attached to the megaspore and derives its food from the female gametophyte with the help of its foot. But after the establishment of root and stem, the sporophyte becomes independent.

29 Fig 11: General life-cycle of Selaginella Medicinal uses Many species of Selaginella have been used as traditional medicines. In India, S. bryopteris is referred to as Sanjeevani one that infuses life for its medicinal properties (Sah et al. 2005). In Columbia, S. articulata is used to treat snakebites and neutralize Bothrops atrox venom. Throughout southern China, Selaginella is used as a popular herb for the treatment of various ailments (Lin and Kan, 1990; Pan et al 2001 and Maa et al 2003). Although most reports of the medicinal uses of Selaginella are anecdotal, researchers have begun to identify and characterize the active compounds in Selaginella extracts (Kang et

30 al. 2004; Chen et al and Yin et al. 2005). Among the best characterized are uncinoside A and uncinoside B, biflavonoids that have potent antiviral activities against respiratory syncytial virus (Ma et al 2003). Other biflavonoids from S. tamariscina inhibit the induction of nitric oxide (NO) and prostaglandins (Pokharel et al 2006; Woo et al 2006; Yang et al, 2006), which are involved in the pathogenesis of some cancers (Lala and Chakraborty, 2001; Zha et al 2004). The biflavone ginkgetin from S. moellendorffii selectively inhibits the growth of some cancer cells by inducing apoptosis (Sun et al 1997; Su et al 2000).