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For Fucus vesiculosus the microscopic analysis displays a cladomial parenchymal thallus, ramified dichotomically, differentiated clearly in rhizoidal, cauloidal ...

Annals of RSCB

Vol. XV, Issue 1

CYTOLOGICAL FEATURES OF SOME MACROPHYTIC ALGAE FROM MEDITERRANEAN SEA Elena Doroftei1, Maria Mihaela Antofie2, Daciana Sava1, Mariana Arcuş3 1

FACULTATEA DE ŞTIINŢE ALE NATURII ŞI STIINŢE AGRICOLE, UNIV.“OVIDIUS” CONSTANŢA; 2FACULTY OF AGRICULTURAL SCIENCES, FOOD INDUSTRY AND NATURE PROTECTION, UNIVERSITY “LUCIAN BLAGA”, SIBIU; 3FACULTATEA DE FARMACIE, UNIV. “OVIDIUS” CONSTANŢA

Summary The current paper comprises the comparative cytological study of algae in the Mediterranean Sea during the cold season in the Expedition Atlas 2007- the Kingdom of Morocco, organized by the Oceanic Club Institute for Oceanographic Explorations and the Protection of the Marine Environment. Five species of macrophytic algae from different phyla were collected and identified at the African shore. The studied species are macrophytic algae; among them some of the most developed plants existing now in the aquatic environment. Apart from the theoretical importance of the study of their ontogenesis, phylogenesis and cytology, many of these species can be used in human alimentation or are valorized for animal feed, while others are used as raw material in different branches of industry. For Cystoseira barbata the algal material is analyzed macroscopically and the thallus is observed to be cylindrical, ramified and differentiated in rhizoid, cauloid and phylloid. Along the phylloids, numerous floaters are observed, with role in supporting the plant in a floating state. The terminations of the phylloids have certain formations similar to the floaters, inside of which there are conceptacles. The conceptacle is a cavity in which there are oogonia and antheridia, organs for sexual reproduction. Microscopically, by means of an objective 20, the aerocysts are observed disposed among the cortical cells, while in the central part of the section, large cells are observed, different from those in the basal area. For Dictyota dichotoma the algal material is analyzed macroscopically and the lamellate thallus is observed ramified dichotomically in the basal part, with a fixing disc. A cortical layer made up of intensely colored cells is observed at the exterior of the thallus (under optical microscope, objective 20) in transversal section. In the apical part of the thallus, one can observe non-ramified colorless hairs resulted from the division of the superior part of the cortical cells. In the median section of the thallus (objective 20), intensely colored cortical cells can be seen while the central cells, colorless and large, are disposed in a single layer. For Fucus vesiculosus the microscopic analysis displays a cladomial parenchymal thallus, ramified dichotomically, differentiated clearly in rhizoidal, cauloidal and phylloidal portions, the latter having aerocysts. It is an alga with a relatively big thallus. Under microscope (objective 20), in transversal section through the apical zone of the thallus, the following can be observed: compact, solid tissue with an evolved parenchymal structure. Among the cortical cells, air vesicles (aerocysts) of different shapes and sizes can be found. The walls of the air vesicle are lined with small cells that contain photosynthetic pigment, arranged on several rows. For Laminaria saccharina the macroscopic thallus consists mostly of three distinctive portions. At the basis, there is the fixing mechanism made up of verticilated hapters forming a widened cone, ramified and ended in adhesive discs. Above, there is the longer or shorter stipule, conic or cylindrical. The main part of the thallus is made up of the phyllidium shaped like a simple blade. In transversal section through the median area of the thallus (under microscope, objective 20), three layers can be distinguished: a medullar area made up of ramified filaments disposed in all directions, colorless, with thin walls, surrounded by the main layer of the thallus made up of almost colorless cells, with thick walls, elongated in a longitudinal way. For Codium elongatum the macroscopic thallus appears lamellate, ramified dichotomically, erect, fixed to the substrate by a disc-like formation or similar to rhizoids. The thallus is dark-green, the branches have spongy consistency and can reach a diameter of 3-10 mm and a height of 15-20 cm. In a transversal section through the thallus (objective 10), the cells with thick wall can be seen, with numerous chloroplasts placed peripherally. On the exterior walls, numerous hairs grouped excessively in the median area can be observed. In conclusion in the cold season, the samples revealed a predominance of algae from the group Pheophyta in the Mediterranean Sea. The algal thallus is differentiated in terms of pigments, type and dimensions. The cytological differences observed under the optical microscope among the species analyzed can also constitute criteria for taxonomic identification. Key words: Macrophytic algae, Cystoseira barbata, Dictyota dichotoma, Fucus vesiculosus, Laminaria saccharina, Codium elongatum.

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cytology, many of these species can be used in human alimentation or are valorized for animal feed, while others are used as raw material in different branches of industry. The biological role of the macrophytic algae is well known in all the aquatic basins, as they contribute to the biological balance and represent the basis of the primary productivity in these basins, they help to purify the water and offer the oxygen needed in the respiration process. Numerous researchers in the world have been attracted to the huge exploration possibilities in various domains (Bold and Wynne, 1978; Doroftei et al., 2000; Doroftei, 2007; Lobban and Harrison, 1997; Reviers, 2002; Van den Hoeck, 1997) and they have been searching for solutions not only for study but also for the exploitation of this precious marine flora and its use in different fields of activity to the use of humankind.

Introduction The current paper comprises the comparative cytological study of algae in the Mediterranean Sea during the cold season in the Expedition Atlas 2007- the Kingdom of Morocco, organized by the Oceanic Club Institute for Oceanographic Explorations and the Protection of the Marine Environment. The Mediterranean is an almost closed sea, with Europe in the north, Africa in the south and Asia in the east. In the west, it communicates to the Atlantic Ocean through the Gibraltar Strait, to the Marmara Sea through the Dardanelles Strait and to the Black Sea through the Bosphorus Strait. The Marmara Sea is considered part of the Mediterranean Sea. The term mediterranean comes from the Latin meditteraneus, which means “middle of the Earth”, this sea being the center of the Earth for the ancient Romans. The fact that it is surrounded by land affects the properties of the sea, the evaporation being excessive in the east causing the sea level to drop and salinity to rise. This is why the nutrient quantity is low. Numerous tributary rivers flow into the Mediterranean Sea and their flow is higher than the Danube (a tributary to the Black Sea), but they flow over the entire surface of the sea. The phytoplankton is poor because of the low amount of nutrients. The phytoplankton is not homogenous in the water mass and when winds blow, it can be concentrated or dispersed in one area or another. The waters of the Mediterranean Sea are very transparent due to the low amount of organic matter, which is why it is famous for its blue and deep waters. Five species of macrophytic algae from different phyla were collected and identified at the African shore. The studied species are macrophytic algae; among them some of the most developed plants existing now in the aquatic environment. Apart from the theoretical importance of the study of their ontogenesis, phylogenesis and

Material and methods In view of accomplishing this paper, samples were collected from the Mediterranean Sea (the shore of Morocco) in February when the water temperature was 15.2˚C and the air temperature was 19˚C, from the intertidial zone during reflux, from rocky substrate. The algae were collected from the substrate with the help of blades and then taken to the laboratory for the taxonomic identification. In the laboratory, the algae from the Mediterranean Sea were pressed on herbarium sheets for their determination and detailed analysis. The samples are taken out of the containers with fixing material, are washed of impurities and then introduced into a container with clean water. Most times, the samples contain algae from different phyla, which is why their sorting on systematic groups is necessary. At this stage, the algae are determined as much as possible because some features are difficult to identify after drying and pressing. A piece of polished glass or a tile is used to place the paper on which the alga 317

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sections were accomplished, the blade cutting through the vegetal material perpendicularly on its axis. The piece of vegetal preparation is placed on a slide, it is covered with cover slip and then it is analyzed under the microscope. If the preparation is too thick and does not offer a clear image of the algal cells, the squash technique is employed, meaning that the slide with the preparation is pounded with a small stick in order to separate the cells.

will be fixed. The following procedure is used: the piece of glass or tile is places in the water container with the alga; with the help of a spatulated needle or a pair of tweezers, the alga shape is adjusted and its ramifications are arranged as close as possible to their natural orientation; the plate is inclined softly in order to remove the water excess. The paper on which the alga is fixed is detached from the glass plate and stuck to a vertical support in order to drain the water. The drying and pressing stage is accomplished placing the paper with algae between sheets of newspaper or blotting paper. In order to prevent the algae from sticking to the newspaper, we protected the thalli with pieces of gauze. In order to press the algae, we placed a few kilo weights on top of the pile of sheets. The drying time interval is not fixed as it depends on the algae consistency and the rhythm of changing the gauze and newspapers between the herbarium sheets. Thus, among the Mediterranean Sea algae, we managed to identify four species of brown algae and a single green one: Cystoseira barbata, of Class Cyclosporeae, Order Fucales, Family Cystoseiraceae; Dictyota dichotoma, of Class Pheophyceae, Order Dictyotales, Family Dictyotaceae; Fucus vesiculosus , of Class Phaeophyceae, Order Fucales, Family Fucaceae; Laminaria saccharina, of Class Phaeophyceae, Order Laminariales, Family Laminariaceae and Codium elongatum, of Class Chlorophyceae, Order Bryopsidales, Family Codiaceae. Transversal sections were made through the thallus of the identified algae in order to realize the microscopic preparations. The vegetal material from the Mediterranean Sea was preserved, the advantage of the latter being that it can be kept indefinitely. The sections were realized with an unused shaving blade, an instrument frequently used in the laboratory. The advantage is that a considerable number of sections are done very quickly with a minimum of instruments. Transversal

Results and discussions Cystoseira barbata: They are algae characterized by a particular polymorphism. The thallus can have a length of 1.5-2 meters. It attaches itself to a substrate by means of a disc from which several stalks emerge. The caulid is cylindrical and has a large number of branches which make the thallus look like a tree. At the ends of different types of branches, there are cylindrical, conical-cylindrical, siliquous or tubercular receptacles (fig. 1). In certain biological seasons, air vesicles can form on the braches, isolated or in a chain.

Fig. 1. Macroscopic aspect of Cystoseira barbata thallus

Most species are grouped in the northern hemisphere (especially in the Mediterranean basin) and some in the southern hemisphere. The Cystoseira barbata tissues have reached the differentiation into epidermoid, cortex and medulla. In the apical area of the thallus, there is a single meristem cell 318

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because of the changes in the environment of these algae from one season to the next. In section through the attachment disc, the cortical cells are observed with very thick walls and with no particular organization. Dictyota dichotoma is an alga with lamellate thallus, ramified dichotomically, erect and attached to the substrate by means of a formation similar to a disc or rhizoids (Hwang et al, 2005).The thallus growth is realized by a biconvex apical cell, present at the end of each branch (fig. 4).

which determines the growth in length and the thallus ramification. On the edge, there is the meristoderm, which is permanently dividing, determining thus the enhancement of the thallus diameter. The cells are placed in pallisadic shape and are full of photosynthetic pigments which reveals thus the passing from the epidermoid area to the cortex area, which are not arranged specifically and are less colored as well (fig. 2). In the center of the section there is a group of different cells which constitute the medulla. These cells can participate in transporting the photosynthesis products from the apical area to the base of the thallus (fig. 3).

Fig. 4. Macroscopic aspect of Dictyota dichotoma thallus Fig. 2. Section of apical region of the Cystoseira barbata thallus (800x)

On the outside of the thallus, in transversal section, a cortical layer is observed made up of intensely colored cells. In the apical part of the thallus there are colorless, non-ramified hairs resulted from the division of the superior part of the cortical cells (fig. 5).

Fig. 3. Section of apical region of the Cystoseira barbata thallus (400x)

The basal area is represented by what is left of the alga during the winter when the rest of the thallus detaches. This small Cystoseira can avoid the disaster caused by winter storms and the strength of the waves. This adaptation occurred only

Fig. 5. Section of apical region of the Dictyota dichotoma thallus (200x)

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emerge. The caulid is narrow at the base and widens gradually as the median nervure becomes more evident. The edge of the thallus is undulated and there are air vesicles between the edge and the median nervure (fig. 8). In transversal section through the apical area of the thallus, there is compact solid tissue with an evolved parenchymal structure. Among the cortical cells there are air vesicles (aerocysts) of different sizes and shapes (fig. 9). The growth is due to one small initial cell located apically in a mucilaginous crypt.

In section through the median area of the thallus, there are cortical cells, strongly colored while the central colorless cells are disposed in one layer (fig. 6,7). In this alga, the tetrasporocysts can be isolated or grouped in 2-3 or more, placed on the entire surface of the thallus or in suns. The gametocysts occur on the surface of the thallus as whitish spots. The antherozoids have one flagellum. The evolution cycle is diplophasic, digenetic and with isomorphic generations.

Fig. 6. Section of medial region of the Dictyota dichotoma thallus (400x) Fig. 8. Macroscopic aspect of Fucus veziculosus thallus

The walls of the air vesicles are lined with small cells which contain photosynthetic pigment, arranged in several layers (fig. 9,10).

Fig. 7. Section of bazal region of the Dictyota dichotoma thallus (400x)

Fucus vesiculosus has parenchymal cladomial thallus, ramified dichotomically, differentiated clearly in rhizoidal, cauloidal and phylloidal portions, the latter having aerocysts (Knight and Parke, 1950; Kalvas and Kautsky, 1993). It is an alga with a relatively big thallus. The attachment to substrate is done by means of a disc (rhizoid) from which one or more thalli

Fig. 9. Section of apical region of the Fucus veziculosus thallus (400x)

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The Fucaceae are dioic plants therefore they display heterothallism. On the male thallus, in conceptacles, certain cells become antheridia in which the male sexual elements will be formed. In the conceptacles on the female thallus, certain cells become oogonia in which eight oospheres will be formed by nucleus division. The loose antherozoids swarm around the oospheres fertilizing them and producing thus the zygote. Laminaria saccharina: The thallus is made up of three distinct parts. The attachment apparatus is located at the base and it is made up of verticilated hapters, which form a widened cone. They are ramified and ended in adhesive discs. Above, there is the stipule, shorter or longer, conic or cylindrical. The main part of the thallus is made up of the phyllidium shaped like a simple blade. The growth occurs in an area located between the phyllidium and the caulid. In a transversal section through the apical area of the phyllidium, layers of small cells can be distinguished, with many discoid chromatophores (fig. 13).

Fig. 10. Section of apical region of the Fucus veziculosus thallus (400x)

In the median part of the thallus, many crypts are formed like bags deepened in the thallus, called conceptacles. They communicate with the exterior through a small ostiole in which the reproductive organs are formed (fig. 11,12).

Fig. 11. Section of medial region of the Fucus veziculosus thallus (400x)

Fig. 13. Section of apical region of the Laminaria saccharina thallus (400x)

In a transversal section through the median area of the thallus there are three layers: a medullar zone made up of ramified filaments disposed in all directions, colorless, with thin walls, surrounded by the main layer of the thallus made up of

Fig. 12. Section of medial region of the Fucus veziculosus thallus (1600x)

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almost colorless cells, with thick walls and elongated longitudinally. There are growth areas similar to the annual rings of Phanerogamae (fig. 14,15). The thallus structure is evolved, with differentiations in “tissues”: cortical, assimilating, meristematic, mechanical, conducting. The alga is represented by the sporophyte, which is well developed, with dimensions of about 1 meter, while the gametophyte is microscopic, reduced to a few cells.

Fig. 16. Macroscopic aspect of Codium elongatum thallus

In a transversal section through the thallus, there are tubular filamentous cells with thick cell walls and with numerous small discoid chromatophores, without pyrenoids. The thallus is differentiated into green cortex and colorless medulla. On the outside walls, there are numerous hairs grouped excessively in the median area (fig.17,18,19).

Fig. 14. Section of medial region of the Laminaria saccharina thallus (800x)

Fig. 17. Section of medial region of the Codium elongatum thallus (800x)

Fig. 15. Section of bazal region of the Laminaria saccharina thallus (800x)

Codium elongatum: The thallus is lamellate, ramified dichotomically, erect, attached to the substrate by a formation similar to a disc or to rhizoids (Israel et al, 2010). The thallus is dark-green; the branches have spongy consistency and can reach a diameter of 3-10 mm and a height of 15-20 cm (fig. 16).

Fig. 18. Section of medial region of the Codium elongatum thallus (800x)

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Doroftei, E., 2007-Comparative ultrastructure of Rhodophyta and Phaeophyta from the Black Sea coast. In: Proceedings of the 6th International Conference of PhD students, Miskolc, Hungary, Natural Science volume, 191-198. Hwang, I.K., Kim, H.S., Lee, W.J., 2005Polymorphism in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae) from Korea. In: Marine Biology, Springer. Knight, M., Parke, M., 1950- A Biological Study of Fucus vesiculosus L. and F. serratus L.. Journal of the Marine Biological Association of the United Kingdom, 29, 439514. Kalvas, A., Kautsky, L., 1993-Geographical variation in Fucus vesiculosus morphology in the Baltic and North Seas, European Journal of Phycology, 28, 2 , 85 – 91. Israel, A., Einav, R., Silva, P.C., Paz, G., Chacana, M.E. and Douek, J., 2010-First report of the seaweed Codium parvulum (Chlorophyta) in Mediterranean waters: recent blooms on the northern shores of Israel. Phycologia 49, 2, 107–112. Lobban, C. S., Harrison, P., 1997-Seaweed ecology and Physiology, Cambridge University Press, United Kingdom, 366 pp. Reviers, B., 2002- Biologie et Phylogenie des Algues, Ed. Belin, Paris, Tome 1, 351 pp. Van den Hoeck, 1997-Algae: an introduction to phycology, 300-342; 419-436. Ed. Cambridge University Press, Cambridge.

Fig. 19. Section of medial region of the Codium elongatum thallus (1600x).

Conclusions In the cold season, the samples revealed a predominance of algae from the group Pheophyta in the Mediterranean Sea. The thallus is differentiated in terms of pigments, type and dimensions. The type of thallus can be a criterion for the taxonomic identification. For example, the thallus is lamellate in Dictyota, Codium and Laminaria or filamentous ramified in Fucus. The cytological differences observed under the optical microscope among the species analyzed can also constitute criteria for taxonomic identification:  The type of cells in the algal tissue and their organization within the tissue;  The shape and size of algal cells;  The size, aspect and position of the chromatophore in the cell;  The number and shape of pyrenoids;  The aspect of the reproductive organs on the algal thallus.

References Bold, H.C., Wynne, M.J., 1978-Introduction to the Algae. Structure and reproduction, 706. Ed. University Press, Cambridge. Doroftei, E., Bavaru, A., Brezeanu, A., Sava, D., 2000-Comparative ultrastructure of some Chlorophyta and Rhodophyta from the Romanian coast of the Black Sea. In: Current problems in cellular and molecular biology, V, 462 – 470. Edited by Crăciun C. şi Ardelean A. Ed. Risoprint, Cluj-Napoca.

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