Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LV, fasc. 2, s.ii a. Biologie vegetală, 2009 CONTRIBUTIONS TO THE STUDY OF ANDRYALA LEVITOMENTOSA (E. I. NYÁRÁDY) P. D. SELL REPRODUCTIVE STRUCTURES ANATOMY B. MINEA *, B. M. NEGREA **, IRINA GOSTIN * Abstract: The paper treats A. levitomentosa reproductive structures from an anatomical point of view. The species exhibited traits that are common in the Asteraceae family but also a few peculiarities such as a possibly five-carpel ovary, free non connate anthers and filaments fused to the corolla at different levels. Early stage embryos but no seeds were observed. Crystals have a presence limited to only certain areas: the integument, the ovary plateau, the anther walls and the connective. Key words: Andryala levitomentosa, anatomy, embryo, crystal Introduction Andryala levitomentosa is one of the rarest endemic species in Romania being strictly protected [10] which makes it difficult to harvest individuals for anatomical research. The recent discovery of three new locations where this plant grows [9] has made it possible for us to obtain some plant material to study. Being so rare this plant species is, anatomically speaking, one of the least studied in Romania. Literature about the anatomy of Andryala genus in general is very scarce [7] studied the taxonomic implications of trichomes in the Lactuceae tribe referring to A. levitomentosa too. Aiftimie-Păunescu and Vântu (2002) presented the stages of micropropagation of this species. Literature concerning the anatomy of the reproductive organs is scarcer still. Sáenz Laín and Gutiérrez Bustillo (1982) briefly describe the pollen grains of Andryala sp. as spheroidal, echinate and lophate. Regarding A. levitomentosa, only morphological descriptions have been done, in taxonomic studies [3, 11, 13]. This paper presents our research of the reproductive structures anatomy in A. levitomentosa. Another aim of this study is to add anatomical data to our knowledge of the species sexual reproduction which has been proving problematic since as early as it was discovered in the 1960's [13]. Mature seeds have never been recorded to germinate [9] hence the species has been reported to only reproduce vegetatively. Material and methods The plant material consisting of inflorescences in different stages of development and infructescences was collected in June July 2008 from the Pietrosul Bistriţei mountain. For the anatomical analysis the paraffin embedding [5] and the scanning electron microscopy [2] methods were used. For paraffin embedding the material was fixed in the Bouin fixative and then, using the Leica TP1020 tissue processor and the Leica EG1150 modular tissue embedding center, dehydrated with growing ethanol series, passed through * Al. I. Cuza University of Iaşi, Faculty of Biology, B-dul Carol I nr.11, 700506 Iaşi, Romania bogdanminea83@yahoo.com, irinagostin@yahoo.com * * Ştefan cel Mare University, Faculty of Forestry, Str. Universităţii nr. 13, 720229 Suceava, Romania bogdannm@yahoo.com 87
gradated ethanol/xylene mixtures (100% ethanol, 3:1, 1:1, 1:3 and 100% xylene) and embedded in paraplast x-tra (Sigma). Longitudinal and cross sections (15 µm thick) were made with a rotary microtome (Euromex - Holland). The tissues were stained with ruthenium red and methylene blue then mounted in Canada balsam. The slides were analysed and photographed using an Olympus BX50 microscope and an Olympus E330 camera. For scanning electron microscopy (SEM) investigations the plant material was fixed in FEA (formol: ethylic alcohol 70%: acetic acid 5:90:5) for 48 hours, washed with distilled water and stored in 70% ethanol [2]. After dehydration in a graded ethanol series (80%, 90% and 100%) and acetone, the material was critical point dried with CO 2 (using a EMS 850 Critical Point Dryer), sputter-coated with a thin layer of gold (30 nm) (using a EMS 550X Sputter Coater) and, finally, examined in a scanning electron microscopy (Tescan Vega II SBH) at an acceleration voltage of 27.88 kv. Results and discussions A. levitomentosa inflorescence is a calathidium containing only ray florets that are, at least anatomically, hermaphrodite. The receptacle is flat or slightly concave with cavities where the florets are placed. It becomes slightly convex after fructification. It consists of a cellulose thin-walled parenchyma and vascular bundles which supply the florets. Air cavities are frequent and relatively large. The perianth is double consisting of calyx and corolla. The calyx is modified into a pappus (Kotilainen [6] brought molecular evidence that pappus bristles are real sepals highly modified for seed dispersal) of approximately 30 bristles. The bristles are multiseriate, composed of elongated cells with projecting apices (Pl. I, fig. 1, 2). The florets (Pl. II, fig. 5) have five petals (exceptionally six) fused at the base to form a corolla tube. They are zygomorphic having a ligule, which represents more than half of the total corolla length, with the tip divided into five teeth. In cross sections the corolla appears formed of an upper (internal) and a lower (external) epidermis and a thin homogeneous mesophyll with small collateral type vascular bundles which are the mid veins of every petal (Pl. III, fig. 14, 15; Pl. IV, fig. 16-19). At the base every mid vein has two bundles, a bigger external one that supplies the petal and smaller internal one that enters the stamen. Both the ligule and its teeth are formed by the split-up of the mid veins. At the base of the corolla tube there is a round nectary that surrounds the base of the style (Pl. II, fig. 6). The androecium is normally pentamerous. Exceptionally a sixth stamen appears. The filaments are fused to the corolla in the mid vein areas but not at the same level (Pl. III, fig. 15). The cross sections through the filament show a simple structure consisting of an epidermis, little parenchyma and a small vascular bundle in the middle (Pl. IV, fig. 16). The anther is tetrasporangiate (Pl. IV, fig. 17). The connective consists of some parenchyma and a small central vascular bundle. The two abaxial pollen sacs are distanced from each other being sometimes almost collinear to the adaxial ones. Longitudinally the adaxial sacs are shorter. The anthers do form a tube around the style but their walls are not fused (the anthers are not connate). In cross sections the anther wall consisted of three layers: an epidermis with tangentially lengthened cells, an endothecium with large cells and 88
an invasive tapetum. We suspect the middle layer to be not absent but rather ephemeral, present only in the early stages of development, but that remains to be proven by further research. The microspore tetrads are mainly tetrahedral and sporadically decussate (Pl. II, fig.8). The mature pollen grains are spheroidal and echinolophate which is consistent with other reports for Andryala sp. [12] In polarized light the connective and the anther walls exhibited prismatic and styloid crystals (Pl. II, fig. 9, 10; Pl. IV, fig. 19). We haven't tested their nature but we suspect them to be calcium oxalate crystals. The ovary is unilocular and inferior thus the floret is epigynous (Pl. III, fig. 11,12). Longitudinally the ovary appears narrowed at the base and having a wide plateau at the top where the corolla tube, the style and, on the rim, the pappus are inserted (Pl. II, fig. 6). The cross section is polygonal with five, exceptionally six, sides (Pl. IV, fig. 21; Pl. V, fig. 22). The ovary has five (exceptionally six) big ribs (the main angles of the polygon) and five (exceptionally six) small ribs. The small ribs have small vascular bundles in the center (Pl. IV, fig. 20, Pl. V, fig. 24). If we consider these bundles the mid veins of the carpels then A. levitomentosa has a pentacarpelar syncarpous ovary which is an important deviation from the general bicarpelar pattern in Asteraceae. A cause for this deviation could be the age of the species. It may have formed before the complete establishment of the bicarpelar pattern in Asteraceae. Further research of the ovary development in this species remains to confirm or deny our theory. Interestingly the exceptions are correlated, that is, a floret with a (presumed) sixcarpel ovary also has s six-petal corolla and a six-stamen androecium and can be therefore considered as trimerous. These can be considered as a manifestation of recessive genetic material or the effect of a possible mutation. The carpels have an external epidermis with radially elongated cells. The internal epidermis is fused to the integument of the ovule and has small flattened cells. The mesophyll has two areas: a narrower parenchymatous area towards the external epidermis and a wider area (more than half) towards the internal epidermis where the cells are degenerating leaving large air cavities (Pl. V, fig. 23). The plateau at the top is circular in cross section having an opening in the center corresponding to the style's transmitting tissue. In polarized light the same prismatic and styloid crystals can be observed (Pl. V, fig. 25). The single ovule is anatropous, tenuinucellate, unitegmic with central (basal) placentation. Crystals, visible in polarized light, are present and abundant in the integument (Pl. V, fig. 24). We have observed early stage embryos in some of the ovules (Pl. III, fig. 13). The style is cylindrical with two stigmatic lobes which contradicts the general rule of thumb that the number of stigmatic lobes equals the number of carpels. The style is semi-closed, cross sections showing transmitting tissue in its center. The internal epidermis of the stigmatic lobes is papillose (Pl. II, fig. 7; Pl. IV, fig. 18,19). After fructification the receptacle cells appear dehydrated and degenerate, air cavities being larger. Some vascular bundles are completely lignified. The receptacle becomes slightly convex. The achenes have thin and lignified walls; the cells are indistinguishable (Pl. V, fig. 26). At the top the plateau is mainly lignified, with a cellulosic central area, bearing 89
the pappus (Pl. I, fig. 1). Inside no seeds were observed, only membranous spongy fusiform structures which seem to be the result of a failed embryogenesis (Pl. V, fig. 27). Conclusions Our investigation of this little known species anatomy shows the presence of typical characteristics for the Asteraceae family [4, 14] such as the calyx reduced to pappus, corolla composed of five fused petals which form a ligule, five stamen androecium, anthers which form a tube around the style, tetrahedral microspore tetrads and spheroidal echinolophate pollen, single anatropous tenuinucellate unitegmic ovule with central (basal) placentation and two stigmatic lobes. However A. levitomentosa has a few peculiarities that make it stand out. The most important is the possibly five-carpel ovary which would be a serious deviation from the normal bicarpelar pattern in the Asteraceae family. Other less common traits are the stamens fused to the corolla at different levels within the same floret and the anthers that are not connate but free. Trimerous florets only appear accidentally. Another goal of our investigation was to take a step further into the solving of this species reproduction mystery. We observed early stage embryos in some of the ovules but we didn't find seeds in any of the achenes. Further research remains to clarify the faith of the embryos. Crystals were reported in other Asteraceae species too [8]. In A. levitomentosa their presence is limited to certain areas of the reproductive organs namely the integument, the ovary plateau, the anther walls and the connective. Crystals were not observed in any of the vegetative organs (unpublished data). REFERENCES 1. AIFTIMIE-PĂUNESCU A., VÂNTU S., 2002 - Micropropagation of the endemic species for Romanian flora Andryala levitomentosa (E. Nyar.) Sell. Revue Roumaine de Biologie, 47: 9-11 2. BOZZOLA, J. J., RUSSELL, L. D., 1999 - Electron Microscopy: Principles and Techniques for Biologists. 2th Edition, Jones & Bartlett Publishers, 48-72 3. CIOCARLAN V., 2000 Flora ilustrată a Romaniei. Edit. Ceres, Bucureşti 4. IVĂNESCU L., TOMA I., 2003 Embriologie vegetală. Edit. Junimea, Iaşi 5. JOHANSEN D. A., 1940 - Plant microtechnique. McGraw-Hill Co., New York, NY. 6. KOTILAINEN M., 2000 (2001) - Flower development in Gerbera hybrida, Asteraceae. Academic dissertation. Institute of Biotechnology and Department of Biosciences, Division of Genetics, University of Helsinki, Finland 7. KRAK K., MRÁZ P., 2008 - Trichomes in the tribe Lactuceae (Asteraceae) taxonomic implications. Biologia, 63/5: 616 630, Section Botany 8. MERIC C., DANE F., 2004 Calcium oxalate crystals in floral organs of Helianthus annuus L. and H. tuberosus L. (Asteraceae). Acta Biologica Szegediensis, 48(1-4): 19-23 9. NEGREA B. M., PRICOP E., 2009 - Rediscovery of Pietrosia levitomentosa E. I. Nyárády ex Sennik., an endemic and endangered plant species from Pietrosul Bistritei Mountain, Romania. Rom. J. of Biol.- Pl.Biol., 54 (1): 101-114 10. NEGREA B. M., PRICOP E., 2009 - The endemic plant species Pietrosia levitomentosa, a real conservation challenge. AES Bioflux 1(1):1-11 11. NEGREAN G., 2004 Genul Pietrosia a fost reabilitat. Buletinul Grădinii Botanice Iaşi. 12. SÁENZ LAÍN C., GUTIÉRREZ BUSTILLO M., 1983 - El contenido polínico de la atmósfera de Madrid. Anales Jard. Bot. Madrid, 39 (2): 433-463 13. SĂVULESCU T. et al., 1964 Flora R. P. Române, Fam. Compositae (9: 210 214). Edit. Acad. Rom., Bucureşti 14. TOMA I., TOMA C., 2003 Citodiferenţiere şi morfogeneză vegetală. Edit. Corson, Iaşi 90
Explanation of plates Original images. Where not specified otherwise scale bar = 100 µm Fig. 1 Achene plateau (disc[13]) with pappus; Scale bar = 500 µm Fig. 2 Pappus (detail) Fig. 3 Pollen grains; Scale bar = 20 µm Fig. 4 Pollen grain; Scale bar = 10 µm Fig. 5 A. levitomentosa floret Fig. 6 Ovary plateau (longitudinal section) nectary visible at style base Fig. 7 Floret (longitudinal section): stigmatic lobes with papillose internal epidermis Fig. 8 Microspore tetrads Fig. 9 Floret in polarized light (longitudinal section) crystals visible in anther walls Fig. 10 Idem. fig. 5 (detail) Fig. 11 Ovary with ovule (longitudinal section) Fig. 12 Ovary with ovule in polarized light (longitudinal section) crystals visible in ovary plateau and ovule integument. Fig. 13 Ovule with early stage embryo (longitudinal section) Fig. 14 Floret at corolla tube level (cross section) Fig. 15 Floret at filament-corolla fusion area level (cross section) Fig. 16 Floret: corolla tube splits to form the ligule (cross section) Fig. 17 Floret: tetrasporangiate anthers (cross section) Fig. 18 - Floret: mature opened anthers (cross section) Fig. 19 - Floret at anther level in polarized light (cross section) Fig. 20 Receptacle in polarized light: (cross section) Fig. 21 - Normal five(?)-carpel ovary at mid level (cross section) Fig. 22 Abnormal six(?)-carpel ovary at mid level (cross section) Fig. 23 Idem. fig. 18 (detail) Fig. 24 Ovary at mid level in polarized light (cross section): xylem is fluorescent blue Fig. 25 - Ovary at plateau level in polarized light (cross section) Fig. 26 - Fruits (longitudinal section) Fig. 27 - Membranous structure inside the fruits (longitudinal section - detail) 91
B. MINEA and colabs. PLATE I Figure 1 Figure 2 Figure 3 Figure 4 92
B. MINEA and colabs. PLATE II Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 93
B. MINEA and colabs. PLATE III Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 94
B. MINEA and colabs. PLATE IV Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 95
B. MINEA and colabs. PLATE V Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 96