THE , FRUITS, AND SEEDS OF TESTUDINUM KOENIG'

PHILIP A. ORPURT AND LINDA L. BORAL2 Manchester College, North Manchester, Indiana

ABSTRACT The flowers of turtle-grass, Konig, are redescribed from specimens collected in Biscayne Bay, Florida. An account is given of fruit development and structure. Anatomy of the seed and germination of this are described for the first time.

INTRODUCTION The herbaceous known as turtle-grass, Thalassia testu- dinum Konig (Fig. 1), is found in extensive submarine beds throughout the Caribbean and the (Humm, 1956; Thorne, 1954). The plant occurs along both coasts of Florida and is particularly abundant in the vicinity of Biscayne Bay, where it is considered to be of ecological importance as a primary producer of carbohydrate in tropical waters (Voss & Voss, 1955). Its distribution in the United States and some aspects of its ecology have been discussed by D. R. Moore (1963). Our attention was attracted to Thalassia in the early spring of 1963 when a previously undescribed fungus was isolated from necrotic lesions on the leaves of the plant. While acquainting ourselves with T. testudinum, its life history, floral structure, fruit formation, and seed structure and germination, it became apparent that there was a considerable lack of infor- mation on these important aspects of the plant. Thalassia testudinum is one of only a few true marine angiosperms. It produces submerged flowers, undergoes pollination, forms fruit, and accomplishes seed germination while remaining totally submerged in saIt water. such as Thalassia, pollinated beneath the water, are termed "hydrophilous." This adaptation represents an advancement relative to an aquatic environment found in few hydrophytes (Arber, 1920). According to Rydberg (1909), the first description of T. testudinum by Konig as a flowering plant in Konig and Sims, "Annals of Botany" in 1906, was based on staminate flowers only. Grisebach (1864) placed Thalassia in the Naiajadaceae and noted that pistillate flowers were un- known. Small (1903) in his treatise, "Flora of the Southeastern United States," placed Thalassia in the , where it is now clas- sified, and like Grisebach was unable to give a description of the pistillate flowers of this common marine flowering plant. One is not surprised.

'Contribution No. 542 from The Marine Laboratory. Institute of Marine Science, University of Miami. Supported in part by project No. 103-305, Microbiology Branch, Office of Naval Research, and by US PHS Grant No. 5489. 2N.S.F. Undergraduate Research Participant. 1964] Orpurt & Boral: Thalassia Testudinum Konig 297 therefore, at the expressed elation of Rydberg (1909) when he writes, "One of the most interesting plants recently collected by Mr. Percy Wil- son ... on his trip to the Bahamas, was the turtle-grass, Thalassia testu- dinum, in flowers and young fruit." From this material given him by Wilson, Rydberg described the pistillate and a young fruit of the plant. The descriptions and the accompanying diagrams by Rydberg have since been quoted and copied (Lawrence, 1951; Muenscher, 1944; Small, 1933) as an accurate report on the floral and fruit structure of the plant. But our observations of these structures of T. testudinum conducted during the spring and summer of 1963 on material from Biscayne Bay, Florida, have indicated discrepancies in these published descriptions. In addition to these observations, an account is given of the adaptations of the fruits and germinating seeds to their role in the ecology of this plant.

DESCRIPTION OF FLOWERS The plants of Thalassia are dioecious, the unisexual flowers developing in early April and continuing through September in the Miami area. The earliest bloom is the largest although ordinarily fewer than 1 per cent of the total plants in a bed are in flower at a given time. The shorter plants in the shallower beds flower earliest followed by those in the deeper beds as the season progresses. Although most turtle-grass beds are composed of either male or female plants (Phillips, 1960), at Matheson Hammock, in Biscayne Bay, they occur intermixed in approximately equal numbers. The staminate flower of T. testudinum is essentially as described pre- viously (Rydberg, 1909). It consists of 9 nearly sessile, basifixed, 4-thecate anthers closely appresed and surrounded by a perianth of three elliptical to oval segments (Figs. 4, 5). The perianth may be colorless or in some specimens lightly marked with pink to violet spots or small streaks. The pedicel and bud is enclosed by the 2-cleft spathe, but the anthers, when mature, project beyond the spathe as the pollen is freed into the water. Each theca of the anther is occupied by a mass of pollen (Fig. 8). The thecae dehisce by means of a vertical slit (Fig. 6) through which the pollen escapes attached together in long mucilaginous strands (Fig. 9). Individual grains are spherical, 50-60 JL in diameter, appear granular internally, and are hyaline (Fig. 7). The exine is spinulose and there are no germ pores or furrows. The pistillate flower is nearly sessile within a 2-c1eft spathe (Figs. 2, 3). There is a simple, inferior ovary (Fig. 10) containing usually 1 to 3 (rarely more) ovules in individual locules (Figs. 11, 12). The style, projecting above the ovary, is a tube similar in structure to that described in Halophila ovalis by Balfour (1878). It is filled with mucilage (Fig. 298 Bulletin of Marine Science of the Gulf and Caribbean [14(2)

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FIGURES 1-20. Thalmsia testudinum Konig. 1, Habit: a, blade; b, decomposing blades; c, ; d, root.-2, 2 flower: a, bud; h, spathe; c, inferior ovary.-3, 2 flower: a, stigmas; h, perianth; c, ovary; d, spathe.-4, 0 flower: a, bud; b, spathe.-5, 0 flower: a, stamen; b, perianth; c, spathe.-6, Stamen.-7, Pollen grain.-8, Stamen, x section: a, microsporangium; h, theca.-9, Pollen strand: a, grain; b, mucilage.-lO, Sectioned 2 flower: a, stigma; b, style element; c, cycle of styles; d, segments of perianth; e, core of style tube; f, style tube; g, hypanthium; h, ovary.-11, Longitudinal section 1964] Orpurt & Baral: Thalassia Testudinum Konig 299 IOe) and is nearly equal to the ovary in length (Fig. lOt). There are three perianth segments spreading from the apex of the stylar tube (Fig. I Od). From the base on the inside of the perianth segments, 7 or 8 stylar elements arise. Each of the elements divides into two stigmas (Fig. I Oa,b). Thus, contrary to the reported 9 to 12 stigmas (Ryd- berg, 1909), there are usually 14 or 16 stigmas. The stylar elements and the perianth are quite fragile, breaking away easily, and it is likely that the specimens examined by Rydberg were so damaged in transit that an accurate description was not possible. In any case, it is apparent that the number of stigmas is greater than reported by Rydberg. The perianth segments of the pistillate flower, as in the staminate flower, are commonly spotted or streaked by pink to violet pigment. The first observed change in the female flower following pollination is the loss of the stylar elements, which break off at their bases. The perianth also soon drops off leaving an apical prolongation or beak at- tached to the ovary (Fig. 11a) .

DEVELOPMENT OF FRUIT

The mature fruit of T. testudinum is an elliptical to globose capsule, 20-25 mm wide by 15-20 mm high, with a beak of 3-7 mm (Fig. 13). As the fruit matures it completely fills and splits the enclosing spathe. The surface of the fruit is at first echinate, becoming tuberculate at maturity. As it ripens, the fruit softens and changes from a bright green to a yellow-green. Occasional mature fruits are red. The mature fruit may remain attached to the mother plant, where it dehisces, or it may break free during heavy wave action. Free fruits remain afloat, thus affording an excellent means of dispersal. Fruits from the earliest flowers mature in about 8 weeks at which time dehiscence results in the formation of 5 to 8 irregular valves (Fig. 14). Within the fruit there occur usually 3 seeds (Fig. 12), occasionally 1 or 2, and rarely up to 6. Rydberg (1909) states that the seeds are numerous. Our observations do not confirm his report for in no case have we found a fruit with numerous seeds. It is our opinion that this discrepancy stems from the fact that Rydberg of fruit: a, beak; b, pericarp; c, placenta; d, seed.-12, Cross section of fruit: a, tuberculate exocarp; b, seed coat; c, seed; d, pericarp.-13, Mature fruit: a, beak (remains of style tube); b, tuberculate fruit surface; c, spathe.-14, Dehiscent capsule: G, style; b, valve.-15, Cotyledon cell: G, wall; b, starch grain.-16, Seed: G, chalaza; h, plumule; c, cotyledon.-17, Seedling: G, leaf; h, scale; c, chalaza.-18, Seedling: G, leaf; h, scale; c, root hairs.-19, Seedling: a, chalaza; b, leaf; c, scale; d, epicotyl; e, radicle; j, vascular strand of cotyle- don.-20, Lower surface of embryo: G, cell with starch grains; b, cell with tannin: c, root hair. 300 Bulletin of Marine Science of the Gulf and Caribbean l14(2) had for study only one immature fruit, in very poor condition. To quote Rydberg, "The writer intended to make a more careful investigation of the morphological structure of the fruit, but the dry material, more or less crushed and distorted by pressing, could not be restored to its original condition and the sections were as a whole unsatisfactory" (italics ours). Since the seeds contain numerous starch granules, and since his material was crushed, it appears very likely that Rydberg mistook the hard starch granules for the numerous seeds he reports as characteristic of T. testu- dinum.

STRUCTURE AND GERMINATION OF SEEDS The inner layer of the pericarp becomes mucilaginous as the fruit ripens. The seed coat, loosely surrounding the embryo, also becomes viscid and slips off easily to release the embryo into the water. While the fruits of Thalassia float, the seeds (embryos) sink immediately upon release from the fruit, settling with the flat side down. The pyriform seeds average 10mm in width by 8mm in height (Fig. 16). With the flattened side down, the chalazal end of the embryo is pointed upward so that the emerging plumule (Figs. 16b; 17a) is also directed upward. The plumule, at first enclosed in three scale leaves (Figs. 17b; 18b), may actually begin to emerge from the seed coat prior to the opening of the valves of the fruit. After the seed is released from the fruit, and follow- ing anchorage by root hair formation and plumule emergence. the young rhizome emerges and gives rise at the nodes to secondary roots (Figs. lc; Id). The germinating embryo exhibits an interesting mechanism by which it readily becomes anchored in the bottom sand. A large number of root hairs develop from the lower, flattened (cotyledon) side (Figs. 18c; 20c). The root hairs develop within 3 days after the seeds settle to the bottom, and thus quickly anchor the young plant on the unstable substrat~. This manner of root hair formation and anchorage mechanism is similar to that reported for Elatine hexandra and Zannichellia polycarpa (Arber, 1920) . A longitudinal section through the axis of the embryo reveals the internal organization of the embryo. The vascular axis and the cotyledonary node forms what may be roughly described as an S (Figs. 19d,f). The radicle is directed laterally (Fig. 1ge) and is nearly surrounded by the single, flattened cotyledon. It is the flattened cotyledon consisting of large cells filled with heavy starch granules (Fig. 15) which forms the base of the embryo as it comes to rest on the bottom in the water. Some of the epidermal cells of the cotyledon are darkened with tannin deposits while others give rise to the root hairs. 1964] Orpurt & Boral: Thalassia Testudinum Konig 301

SUMMARY A redescription of the unisexual flowers of T. testudinum Konig, based upon material from Matheson Hammock, Biscayne Bay, Florida, is given. Observations on flowering and fruit development indicates that it takes approximately 8 weeks from the time of pollination for fruits to mature. Contrary to a statement by Rydberg that Thalassia fruits contain nu- merous seeds, we found that the usual number is 3. Fruits with more than 3 seeds were rare. The pyriform seeds and their germination are described for the first time. Thalassia fruits float and thereby serve as an excellent means of plant dispersal. The seeds sink upon their release from the mature capsule, with their flattened cotyledon side oriented downward. This particular orientation is due to the formation of numerous starch granules in the cotyledon cells. Such an orientation thus directs the plumule upward and at the same time numerous root hairs from the lower, flattened side of the cotyledon quickly anchor the young plant in the unstable sand Oll the bottom. ACKNOWLEDGEMENTS The authors express their gratitude to Dr. Leonard Greenfield of the Institute of Marine Science, University of Miami, for his helpful advic~ with this study and for his able assistance in the collection of field data and materials. Our gratitude is also extended to Dr. S. P. Meyers for his counsel and assistance in the preparation of the manscript.

SUMARIO LAS FLORES, FRUTAS Y SEMILLAS DE Thalassia testudinum KONIG En el area de Miami las f10res unisexuales de Thalassia testudinum se presentan mezcladas en lechos submarinos, desde Abril a Septiembre. Aproximadamente el 1 % de las plantas de un lecho florecen a la vez, cmpezando desde los Iechos mas bajos hacia los mas profundos, a me- dida que progresa la estaci6n. Aunque la flor estaminada es esencialmente como se ha descrito por autores anteriores, se encontr6 que en la flor pistilada hay usual mente 14 6 16 estigmas en vez de 9 6 12 que es 10 reportado anteriormente. Toma alrededor de 8 semanas desde el momenta de la polinizaci6n para que las frutas maduren. La dehiscencia resulta de la formaci6n de 5 a 8 valvas irregulares. Contrario a 10 dicho por Rydberg de que las frutas de Thalassia con- tienen numerosas semillas, nosotros encontramos que el numero usual es 3. Mas de 3 semillas es cosa muy rara. Se describen por primera vez, las semillas piriformes y su germinaci6n. Las frutas de Thalassia flotan, resultando un excelente medio de disper- si6n de la planta. Las semillas se hun den al liberarse de la capsula de la 302 Bulletin of Marine Science of the Gulf and Caribbean (J 4(2) fruta madura, con el lado lisa del cotiled6n orientado hacia abajo. Esta especial orientaci6n es debida a la presencia de numerosos granulos de almid6n en las celulas del cotiled6n. Tal orientaci6n dirige la plumula hacia arriba en la germinaci6n y al mismo tiempo numerosos pelos de raiz se forman en la parte inferior del lado del cotiled6n, para fijar la planta joven en la arena inestable del fondo.

LITERATURE CITED ARBER, A. 1920. Water plants-a study of aquatic angiosperms. Cambridge Univer- sity Press, 436 pp. ASCHERSON, P., AND M. GURKE 1889. Hydrocharitaceae. In Engler and Prantl. Natur. Pflanzenfam. 2(1): 238-258. BALFOUR, B. 1878. On the genus Halophila. Trans. bot. Soc. Edinb. 13: 290-343. BRITTON,· N. L. AND C. F. MILLSPAUGH 1920. The Bahama Flora. Pub\. by the authors, N. Y. GRISEBACH, A. H. R. 1864. Flora of the British West Indian Islands. Lowell Reeve and Co., London. HUMM, H. J. 1956. of the northern Gulf coast. Bull. Mar. Sci. Gulf & Carib. 6: 305-308. LAWRENCE, G. H. M. 1951. of vascular plants. MacMillan and Co., New York, xiii + 823 pp. MOORE, D. R. 1963. Distribution of the sea grass, Thalassia, in the United States. Bull. Mar. Sci. Gulf & Carib., 13: 329-342. MUENSCHER, W. C. 1944. Aquatic plants of the United States. Comstock Publ. Co., Ithaca, N. Y., 374 pp. PHILLIPS, R. C. 1960. Observations on the ecology and distribution of the Florida sea- grasses. Fla. State Bd. Conserv. Mar. Lab., Professional Pap. Ser., No.2: 1-72. RYDBERG, P. A. 1909. The flowers and fruits of the turtle-grass (Thalassia). J. N. Y. bot. Odn., 10: 261-265. SMALL, J. K. 1903. Flora of the southeastern United States, Pub\. by the author, N. Y., through New Era Printing Co., Lancaster, Penna. 1394 pp. 1933. Manual of southeastern flora. Publ. by the author, N. Y., through Sdence Press Printing Co., Lancaster, Penna. 1554 pp. THORNE, R. F. 1954. Flowering plants of the waters and shores of the Gulf of Mexico. Fish. Bull., U. S., No. 89, 55: 193-202. Voss, G. L. AND N. A. VOSS 1955. An ecological survey of Soldier Key, Biscayne Bay, Florida. Bull. Mar. Sci. Gulf & Carib., 5: 203-229.