HISTO-ANATOMICAL OBSERVATIONS REGARDING VIOLA L. SPECIES IN THE GÂRBOAVELE RESERVE (COUNTY OF GALAŢI) Introduction

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1 Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi s. II a. Biologie vegetală, 2014, 60, 1: ISSN: , E-ISSN: HISTO-ANATOMICAL OBSERVATIONS REGARDING VIOLA L. SPECIES IN THE GÂRBOAVELE RESERVE (COUNTY OF GALAŢI) Anca MEREACRE 1 *, Angela TONIUC 1, Constantin TOMA 1 Abstract: The authors research the structure of vegetative organs of five different vernal species of Viola L. which grow in the Gârboavele - Galaţi nature reserve. The histological features, most of them quantitative ones, which differentiate the five species of Viola L., are pointed out: the thickness of the root and rhizomes peridermis, the number of palisadic layers of the leaf lamina, the presence or absence of tector hairs and of oxaliferous cells, their frequency and position. Key words: Viola, histo-anatomy, vegetative organs, Gârboavele-Galaţi reserve. Introduction There are 28 species of Viola L. in the Romanian flora (Grințescu et al., 1955; Sârbu et al., 2013), four of which known to grow in the Gârboavele - Galaţi nature reserve: Viola arvensis Murray, V. elatior Fr., V. hirta L. and V. odorata L. (Mititelu et al., 1968); to the above mentioned ones we bring a 5-th one, recently found by us: V. kitaibeliana Schult. Two of the species are annual ones (V. arvensis and V. kitaibeliana), while three of them are perennial plants (V. elatior, V. hirta and V. odorata), and have a thick rhizome, with or without aerial runners (V. hirta). The anatomic structure of Viola species has been little investigated until now, as can be seen from the two synthesis treaties, the one about the anatomy of dicotyledonous plants (Metcalfe and Chalk, 1972) and the one about the leaves of angiosperms in general (Napp- Zinn, 1973, 1974). When presentations of tissue structures of various vegetative organs are made, the authors mention, among others the Viola genre, too; Viola odorata being most frequently quoted. Burduja and Moţiu (1982) achieve a detailed histo-anatomical study of the aerial parts of Viola hymettia Boiss., a species closely related (often mistaken for ) to V. arvensis, V. tricolor and V. kitaibeliana. The aerial parts of V. arvensis are used (besides V. tricolor) in traditional medicine for their anti-inflammatory, expectorant and diuretic and other properties. The need to know more about the morpho-anatomic features of both species resulted in research work dedicated to these aspects (Meyer, 1916; Toiu et al., 2009, 2010). Reference to structure features of Viola species can be found in works of comparative (Petit, 1887; Howard, 1962; Uphof and Hummel, 1962) or ecological anatomy (Constantin, 1885), but also in some with a special focus on vernal species (Keller, 1934; Ubaidulaev, 1959; Ivanskaja, 1962, 1963; Goryşina, 1965). 1 Faculty of Biology, Al. I. Cuza University, Bd. Carol I, no.11, Iaşi Romania; anca_mereacre@yahoo.com (corresponding author*)

2 Mention must be made that three of the species studied by us (Viola arvensis, V. kitaibeliana and V. odorata) have been analyzed from an anatomical point of view besides other plants growing in Northern Iran (Yousefi et al., 2012). From the above we can conclude that there are few histo-anatomical data referring to Viola L. species, reason why, in our work, we are attempting a comparative analysis of the vegetative organs of the five vernal Viola species mentioned above, which grow in the Gârboavele - Galaţi nature reserve. Materials and methods The material under study was collected in the Gârboavele - Galaţi nature reserve in May 2012, at the anthesis stage. This forest-park is at 90 m altitude, and are predominantly: Quercus pubescens, Q. pedunculiflora, Ulmus foliacea, Tilia tomentosa, to which are added Acer campestre, A. tataricum, Pyrus pyraster, Cotinus coggygria and another species (Mititelu et al., 1968). The plants were fixed and preserved in 70% ethyl alcohol. The vegetative organs (the root, rhizome, aerial runner, aerial stem, and leaf) were sectioned and coloured (in iodine green and ruthenium red) according to the usual histo-anatomical technique in plant research. Cross-sections and the tangential/longitudinal ones have been analyzed under the Olympus CX31 optic microscope and photographed with an Olympus C 5060 camera (Andrei and Predan, 2003). Results and discussions THE ROOT (Plate I, Figs. 1-3) The rhizodermis of V. odorata and V. hirta occasionally presents long absorbent hairs; the rhizodermis, as well as the external layer of the bark of the other three species is exfoliates early. The bark of V. odorata and V. hirta is differentiated in the exodermis, cortical parenchyma and endodermis; the other species keep only the internal layers of the cortical parenchyma (at V. hirta with aerial gaps) and the casparian type of endodermis; some of the parenchymatous cells of V. hirta and V. odorata contain calcium oxalate ursines. The central cylinder is the anatomical area with a secondary structure, due to the activity of the vascular cambium which has produced a thin external phloem ring and a thick central xylem body. The phloem ring consists of sieve-tubes, companiong cells and numerous cells of amiliferous parenchyma (collenchymatous in V. arvensis, arranged in strictly radial rows in V. elatior). Only Viola odorata has an incipient secondary structure, the phloem has few parenchyma cells, and the xylem has very few libriform elements. The central xylem body comprises vessels of varied diameters, irregularly dispersed in the fundamental lignified mass. The axial part of the stem of V. arvensis, V. hirta and V. kitaibeliana is less lignited, wih fewer and smaller vessels which are separated only by cellulosic parenchyma. The species were determined by Ion Sârbu, Ph.D, whom we warmly thank in this paper, too. 14

3 Thus, the early passage from the primary to the secondary structure can be noticed both in perennial and in annual species, but only at the level of the central cylinder, on account of the vascular cambium. THE RHIZOME (Plate I, Fig. 5) Only Viola hirta and V. odorata present rhizomes, both are perennial species, but only one of them has aerial runners with adventitious roots from the nodes (V. odorata). The contour of its cross section is very irregular, with ribs of various size in V. hirta and a symmetrical structure in V. odorata. The structure of both species is typically secondary, resulting from the activity of both lateral meristems, but especially of the vascular cambium. The epidermis and the largest part of the cortical parenchyma have exfoliated. In the deep persistent internal parenchymatic layers of V. odorata some cells contain, like the ones in the pith, calcium oxalate ursines. The phellogen (or cork cambium), differentiated at the level of the internal cortical parenchyma or of the pericycle (V. hirta), has produced one or two areas of suber and the phellem. The vascular cambium has produced the secondary conducting tissues, of a fascicular type in V. odorata and ring type in V. hirta. The secondary phloem ring is relatively thick and contains conducting elements (sieve-tubes, companion cells) in the close vicinity of the cambium and numerous cells of amiliferous parenchyma towards the exterior, disposed in radial rows, some containing calcium oxalate ursines. The secondary xylem ring is much thicker, strongly sclerified and intensely lignified, made up of irregularly dispersed vessels of varied diameters, many libriform fibers and few parenchyma cells. The xylem ring is crossed by some parenchymaticcellulosic medullar rays In the thickness of the V. hirta ring we can distinguish several (4-5) rings, each of them having more numerous early vessels and libriform fibers with thinner walls, and fewer later vessels and libriform fibers with thicker walls. In the early xylem areas we can see some islands of cellulosic parenchyma. The pith is parenchymatic-cellulosic, and many cells contain calcium oxalate ursines. THE AERIAL RUNNER (Plate I, Fig. 4) The aerial runner, which we find only in V. odorata has a flat-elliptical contour in cross-section, with a slightly concave face (the inferior one), which corresponds to the place where the endogenously formed adventitious roots will come out; here the phloem is reduced and interrupted by the cord which forms the adventitious root. When dealing with thinner and younger material, the contour of the cross-section is circular, the cortical parenchyma is exfoliating, the suber zone has 3-4 layers of suberified cells, the phellodermis area is visibly collenchymatized, and the phloem ring has a different thickness on the circumference of the organ. The structure is typically secondary and asymmetrical for thicker and older material. The aerial runner surface is covered by a relatively thick peridermis, with 4-6 layers of suber (exfoliating) and an equal number of phellodermis layers, which are tangentially elongated and are disposed in radial series, some of them containing calcium oxalate 15

4 ursines. From place to place there are remains of the primary cortical parenchyma, adhering to the suber. The central cylinder is very thick, but irregular thickness along the circumference of the aerial runner, so that it gets the shape of a cordiform contour in cross-section, the xylem getting into direct contact with the suber along one of the sides. We see that the cambium produces an incomplete, but thick, phloem ring (sieve-tubes and companion cells) towards the interior and numerous parenchyma cells, disposed in radial rows and with moderately thickened walls, towards the exterior. Cambium also produces a thick, completely lignified, xylem ring (vessels, libriform fibers, parenchyma cells), pierced by 1-2 parenchymatic-cellulosic medullar rays. Many of medullar rays cells, as well as those of the liberian parenchyma, contain calcium oxalate ursines. At a closer analysis one can notice, inside the secondary xylem ring, 6-7 incomplete, concentric rings which can be recognized by the early vessels of a larger diameter and by the terminal libriform areas. THE AERIAL STEM (Plate II, Figs. 6-11) The structure of the two annual species (V. arvensis and V. kitaibeliana) is and remains only a primary one. The structure of the three perennial species (V. elatior, V. hirta and V. odorata) is mostly of secondary origin in the lower third of the stem, but only at the level of the central cylinder, due to the activity of the vascular cambium. The contour of the cross-section varies from circular to elliptically-circular (modified by two lateral wings (V. elatior), to trapezoid (V. arvensis) or square shaped at the tip and triangle shaped at the basis of the stem (V. kitaibeliana). The epidermis has isodiametric cells, with thicker internal and external walls than the other ones; the external wall is covered by a thin, lightly striped cuticle. From place to place, at least in the upper third of the stem, we can notice stomata and tector hairs. There are numerous unicellular relatively short hairs on the lateral wings in the lower third of the stem in V. arvensis, V. elatior and V. kitaibeliana. The bark is parenchymatic, of meatic type, but it also has aerial gaps (V. elatior, V. odorata), and it ends with an endodemoid (V. arvensis) or with a casparian endodermis (V. hirta, V. odorata). The hypodermic layer is collenchymatized in the ribs most frequently of an angular or tangential type (V. kitaibeliana).cells with calcium oxalate ursines have been notice in V. elatior, V. hirta and V. odorata. The central cylinder of annual species has conducting tissue of fascicular type (with a primary structure) while the perennial species have ring type tissue (towards the basis of the stem, with a secondary structure). The number of vascular bundles varies from the tip (4) towards the basis of the stem (8-12) in V. kitaibeliana, it is constant (4) in V. arvensis or it is higher (8-10) at the tip of the stem. In perennial species the vascular bundles unite giving birth to two concentric rings at the stem basis. In the primary structure the phloem has sieve-tubes and companion cells, while the xylem has vessels and cellulosic parenchyma type of cells. The secondary phloem ring has, in addition to this, parenchyma cells, and the secondary xylem ring has also libriform fibers. The medullar rays, sclerified and lignified, form, together with the secondary xylem, 16

5 a thick and compact ring. We can observe thin sclerenchymatic fiber cords on the external face of the phloemic ring (V. elatior). The pith is parenchymatic-cellulosic and many of its cells contain calcium oxalate ursines (V. odorata, V. elatior, and V. hirta). Many pith cells of the annual species become disorganized thus creating a smaller or larger central aerial cavity, along the whole stem. Such small aerial cavities in the pith have been noticed in V. elatior, too. THE LEAF (Plates III, IV, V) The petiole (Plate III, Figs ) The contour of the cross-section varies from triangular (V. kitaibeliana) to semicircular modified by two lateral-adaxial wings (three in V. odorata). The epidermis has isodiametric cells, whose exterior walls are slight thicker than the others and they are covered by a very thin cuticle. From place to place one can notice stomata and rare unicellular tector hairs (more frequent on the lateral-adaxial wings), shorter (V. elatior) or longer ones (V. hirta). The fundamental parenchyma is slightly collenchymatised under the epidermis (especially in the lateral-adaxial wings), with aerial cavities on adaxial face (V. arvensis, and V. odorata); some of the cells contain calcium oxalate ursines (V. elatior and V. odorata). The conducting tissues form 3 vascular bundles: a large median one and two small lateral-adaxial ones, all of them with a primary structure. The lamina (Plate IV, Figs ; Plate V, Figs ) The epidermis, in front side view, presents cells with irregular contours, with slight waving lateral walls on the upper face of the lamina and strongly waving ones on its lower face; only V. kitaibeliana presents lateral walls with high amplitude waves on both faces of the limb. Anisocytic type stomata (cruciferous ones) can be noticed in the two types of epidermis, with a higher frequency on the surface unit of the lower epidermis, this indicating an amphystomatic leaf. In V. hirta the stomata are not present or are extremely rare in the upper epidermis, indicating a hypostomatic leaf. The mesophyll comprises two types of assimilating tissues: palisadic and spongy ones, that the limb has a bifacial heterofacial structure (dorsiventral leaf) in all five Viola species we have investigated. The differences between species refer to the number of palisadic layers (one in V. hirta and two in the other species), to the length of the composing cells (longer in V. kitaibeliana and shorter in V. elatior and V. hirta ), to the presence or absence (V. arvensis ) of unicellular tector hairs, to the presence (V. hirta and V. odorata) or absence of oxaliferous cells. Conclusions The general structure plan of all investigated vegetative organs is similar in all five Viola species. The passage from the primary to the secondary structure of the root takes place very early both in perennial and annual species, but only at the level of the central cylinder, that is on account of the vascular cambium. 17

6 The rhizome, present only in perennial species (V. hirta and V. odorata) has a secondary structure, as a result of the activity of both lateral meristems; the vascular cambium and the cork cambium (phellogen). In the very thick secondary xylem ring we can distinguish several areas, each with early and late wooden vessels (V. hirta). Both species present many oxaliferous cells in the primary and medullar cortical parenchyma. The aerial runner, present only in V. odorata, has, like the rhizome, a secondary structure, with a thick peridermis, very thick xylem ring, oxaliferous cells in the medullar rays, phelloderm and liberian parenchyma. The five species we have investigated are differentiated only by the presence or absence of tector hairs, of oxaliferous cells and central aerial cavity. The leaf petiole has a different cross-section contour, with two lateral-adaxial wings always visible. The species under study differ by the presence or absence of tector hairs, oxaliferous cells and aerial cavities in the fundamental axial parenchyma. All five species present 3 vascular bundles. The foliar lamina has a homogenous, bifacial, heterofacial (dorsiventral) structure, the difference between the species being mostly quantitative ones (the number of palisadic layers, the length of their cells), more rarely qualitative ones: the presence or absence of tector hairs and oxaliferous cells. REFERENCES Andrei, M., Predan, G.M.I., Practicum de morfología şi anatomía plantelor. Edit. Ştiinţelor Agricole, Bucureşti. Burduja, C., Moţiu, T., Cercetări morfologice şi histo-anatomice ale organelor vegetative de la specia Viola hymettia Boiss. et Heldr. Culeg. de studii şi artic. de Biol., Grăd. Bot. Iaşi. 2: Costantin, J., Recherches sur l influence qu exerce le milieu sur la structure des racines. Ann. des Sci. Nat., Bot., ser. 7. 1: Goryşina, T.K., Anatomiceskoe stroenie list ev rannevesennyh efemeroidov dubovogo lesa. Vestnik Leningradskogo Universiteta, Ser. Biol., 20, vyp 1(3): Grinţescu, G., Guşuleac, M., Nyárády, E., Viola L. In Flora R.P.R., 3. Edit. Acad. Rom., Bucureşti: Howard, R.A., The vascular structure of the petiole as a taxonomic character. Proc. 15-th Internat. Hort. Congress, Nice, 1958: Ivanskaja, E.N., Sur quelques particularités de la structure anatomique de la feuille de plantes en rosettes et en coussinet, d altitude. Dokl. AN SSSR. 146, 2: Ivanskaja E.N., O stroenie mezofila list ev nekotoryh vysokogornyh ratenij ţentral nogo Kavkaza. Zapiski Ţentral. Kavkazkogo otd. Vsesoiuz-go botanici-go obscestva, Ordjonikidze. 1: Keller E.F., Osobennosti anatomiceskogo stroenia list ev u vesennih efemero-odnoletnikov. Sovremennaia botanika. 2, 4: Metcalfe, C.R., Chalk, L., Anatomy of the Dicotyledons, 1. Clarendon Press, Oxford: Meyer, F.J., Bau und Ontogenie des Wasserleitungssystems der vegetativen Organe von Viola tricolor var. arvensis. Thesis, Marburg (cf. Bot. Zbl, 141, 1919: ). Mititelu, D., Gociu, Z., Patraşcu, A., Gheorghiu, V., Flora şi vegetaţia pădurii-parc Gîrboavele-Galaţi. Analele Științ. Univ. Al. I. Cuza Iaşi, s. II a. Biol. 14, 1: Napp-Zinn, Kl., 1973, Anatomie des Blattes. II. Blattanatomie der Angiospermen. Handbuch der Pflanzenanatomie, Bd. VIII, 2 A 1-2, Gebrüder Borntraeger, Berlin. Petit, L., Le petiol des Dicotyledones au point de vue de l anatomie comparée et de la taxinomie. These, Bordeaux. Sârbu, I., Ştefan, N., Oprea, A., Plante vasculare din România. Edit. VictorBVictor, Bucureşti: Toiu, A., Oniga, I., Tămaş, M., 2009 Cercetări morfologice şi anatomice asupra speciei Viola tricolor L. (Violaceae). Rev. Med. Chir. Soc. Med. Nat. Iaşi. 113 (2, suppl. 4):

7 Toiu, A., Oniga, I., Tămaş, M., Morphological and anatomical researches on Viola arvensis Murray (Violaceae). Farmacia. 58, 5: Ubaidulaev, U., Anatomo-ekologhiceskoe issledovania list ev nekotoryh efemeroidov i efemerov zapadnogo Tian-Şan. Avtoreferat, Taşkent. Uphof,T.C.J., Hummel, K., Plant Hairs. Encyclopedia of Plant Anatomy, 4. Gebrüder Borntraeger, Berlin. Yousefi, N., Mehrvarz, S.S., Marcussen, T., Anatomical studies on selected species of Viola (Violaceae) in Iran. Nordic Journal of Botany. 30, 4: EXPLANATION OF THE PLATES PLATE I Fig. 1.Viola arvensis - cross-section through the root (Oc.10 x Ob.4) Fig. 2. Viola kitaibeliana - cross-section through the root (Oc.10 x Ob.4) Fig. 3. Viola hirta - cross-section through the root details (Oc.10 x Ob.20) Fig. 4. Viola odorata - cross-section through the aerial runner details (Oc.10 x Ob.20) Fig. 5. Viola hirta - cross-section through the rhizome (Oc.10 x Ob.4) PLATE II Fig. 6. Viola elatior - cross-section through the upper level of the stem (Oc.10 x Ob.10) Fig. 7.Viola elatior - cross-section through the upper level of the stem details (Oc.10 x Ob.20) Fig. 8. Viola kitaibeliana - cross-section through the middle level of the stem (Oc.10 x Ob.10) Fig. 9. Viola hirta - cross-section through the middle level of the stem (Oc.10 x Ob.10) Fig. 10. Viola arvensis - cross-section through the basal level of the stem (Oc.10 x Ob.40) Fig. 11. Viola elatior - cross-section through the basal level of the stem (Oc.10 x Ob.20) PLATE III Fig. 12. Viola.kitaibeliana - cross-section through the petiole (Oc.10 x Ob.10) Fig. 13. Viola elatior - cross-section through the petiole (Oc.10 x Ob.10) Fig. 14. Viola arvensis - cross-section through the petiole (Oc.10 x Ob.10) Fig. 15. Viola odorata - cross-section through the petiole (Oc.10 x Ob.10) Fig. 16. Viola elatior - cross-section through the petiole details (Oc.10 x Ob.20) Fig. 17. Viola hirta - cross-section through the petiole details (Oc.10 x Ob.20) PLATE IV Fig. 18. Viola arvensis the lower and upper lamina epidermis, in front view (Oc.10 x Ob.40) Fig. 19. Viola elatior - the lower and upper lamina epidermis, in front view (Oc.10 x Ob.40) Fig. 20. Viola hirta - the lower and upper lamina epidermis, in front view (Oc.10 x Ob.40) Fig. 21. Viola odorata - the lower and upper lamina epidermis, in front view (Oc.10 x Ob.40) PLATE V Fig. 22. Viola arvensis - cross-section through the lamina in the midrib part (Oc.10 x Ob.20) Fig. 23. Viola odorata - cross-section through the lamina in the midrib part (Oc.10 x Ob.20) Fig. 24. Viola elatior - cross-section through the lamina in the midrib part (Oc.10 x Ob.20) Fig. 25. Viola kitaibeliana - cross-section through the lamina in the midrib part (Oc.10 x Ob.10) Fig. 26. Viola.hirta - cross-section through the lamina between the veins - details (Oc.10 x Ob.40) 19

8 PLATE I Figure 1. Viola arvensis Figure 2. Viola kitaibeliana Figure 3. Viola hirta Figure 4. Viola odorata Figure 5. Viola hirta 20

9 PLATE II Figure 6. Viola elatior Figure 7. Viola elatior Figure 8. Viola kitaibeliana Figure 9. Viola hirta Figure 10. Viola arvensis 21 Figure 11. Viola elatior

10 PLATE III Figure 12. Viola kitaibeliana Figure 13. Viola elatior Figure 14. Viola arvensis Figure 15. Viola odorata Figure 16. Viola elatior 22 Figure 17. Viola hirta

11 Lower epidermis Upper epidermis PLATE IV Figure 18. Viola arvensis Figure 19. Viola elatior Figure 20. Viola hirta Figure 21. Viola odorata 23

12 PLATE V Figure 22. Viola arvensis Figure 23. Viola odorata Figure 24. Viola elatior Figure 25. Viola kitaibeliana Figure 26. Viola hirta 24

Irina Berciu *, Constantin Toma Department of Biology, Al. I. Cuza University, Iasi

HISTO-ANATOMICAL ASPECTS OF VEGETATIVE ORGANS OF THYMUS DACICUS BORB. AND THYMUS GLABBRESCENS WILLD. Irina Berciu *, Constantin Toma Department of Biology, Al. I. Cuza University, Iasi * Correspondence: