MORPHOLOGY OF MARSILEA VESTITA. II. MORPHOLOGY OF THE ADULT LAND AND SUBMERGED LEAVES

Two distinct adult leaf forms, i.e., land and submerged leaves, of the aquatic fern Marsilea vestita were collected from nature and were obtained from plants in sterile culture. The venation patterns and anatomy of these 2 adult leaf forms are presented in detail. Pinna development and the morphology of a third leaf form, i.e., floating leaves, are briefly considered. The differences in leaf form are ascribed to differences in the activity of the marginal meristem of the leaf. THREE ADULT leaf forms occur in Marsilea, i.e., land, floating, and submerged. Of all these forms, the adult land leaf is the best known from a morphological point of view (Tournay, 1951). The ontogeny of this land leaf was described by Hanstein (1865), Johnson (1898) and Schneider (1913). These workers concluded that the leaf lamina develops from 4 protuberances at the distal portion of the leaf primordium. According to Williams (1921) and Tournay (1951), these 4 protuberances, while undergoing expansion and differentiation, come to lie together as a "sandwich" of 4 pinnae. The adult submerged leaf has been regarded by earlier workers as an adult land leaf which was inhibited at an early stage of its development (Goebel, 1928). Allsopp (1954) obtained adult land and submerged leaves from sterile cultures of M. drurnmondii and showed for the first time that both leaves are anatomically distinct. It can be said that much is now known about Marsilea, but details of the morphology of the leaf forms of the adult plant are lacking. It is hoped that this present study of the morphology of the adult leaves of Marsilea vestita will be of help to future workers interested in the experimental induction of different leaf forms. MATERIAL AND METHODS-Leaves and apical portions of rhizomes obtained from plants growing in sterile culture were supplemented with similar material collected in 2 different streams. The first stream was 4.5 miles north of Lakeport on State Highway #29 in Lake County, California. The second stream was 4.6 miles west of the Calaveras County line on State Highway # 8 in San Joaquin County, California. Collections were made in the first stream in July, 1962, and in the second in June, 1963. Plants were grown in sterile culture in liquid nutrient or on top of glass cloth which 1 Received for publication October 14, 1963. This study represents a portion of a thesis submitted in partial satisfaction of the requirements for the Ph.D. at the University of California, Berkeley, and was supported in part by NSF summer fellowships for teaching assistants. The help of Drs. A. S. Foster and R. Cleland and the services of the Scientific Photographic Laboratory, University of California, Berkeley, are gratefully acknowledged. was saturated with nutrient solution (see Gaudet, 1963, 1964 for techniques). Figure 1 shows plants from both situations. All plant material was killed and fixed in formalin-acetic acid-alcohol. Entire leaves were cleared in NaOH using the general procedure of Foster (1955). Perforated polyethylenie ice-cube trays (Horner and Arnott, 1961) were used to carry the leaves through the solution. All leaf material was thoroughly washed and transferred to chloral hydrate (saturated aqueous solution) for 4-5 days, after which the material was again washed and then dehydrated in alcohol and stained in alcohol-xylene-safranin. This technique made the leaf venation strikingly visible and also stained the stomata and cell walls of the upper and lower epidermis. Portions of leaves and whole apices were dehydrated using tertiary butyl alcohol and embedded in Tissuemat, and serial transections and longisections were made at 8-10 u and stained, using the tannic acid-iron chloride-safranin technique (Foster, 1934). RESULTS-Pinna development-The primordium of a quadrifid leaf originates from a single, large surface cell which occurs on the flank of the shoot apex (Fig. 2). This single cell functions as an apical cell during the early stages of ontogeny, and it cuts off cells which form a leaf primordium as seen in Fig. 3. Two lateral protuberances arise on the sides of this primordium, and a terminal meristematic "flattening" occurs. This terminal flattening, in turn, forms 2 additional protuberances. Expansion of these 4 protuberances occurs by periclinal and anticlinal cell divisions of a marginal meristem and results in the development of 4 pinnae closely appressed to one another. The cellular details of stages between early growth of the pinnae and the adult leaf were not investigated. In order to determine if a marginal meristem functions in the same way in the adult leaf as it does in juvenile leaves, some paradermal sections of the young leaf margin were made (Fig. 4). These sections show that a marginal meristem has produced cells which are differentiating into