MORPHOLOGY OF MARSILEA VESTITA. I. ONTOGENY AND MORPHOLOGY OF THE SUBMERGED AND LAND FORMS OF THE JUVENILE LEAVES

During their ontogeny, the primordia of the juvenile leaves of Marsilea plants in sterile culture develop 1, 2 or 4 marginal meristems, and these, in turn, contribute cells to the young leaf by antiand periclinal cell divisions. The final leaves are unifid, bifid, or quadrifid, depending on how many marginal meristems develop, and this is determined early in the ontogeny of the leaf. The mechanism which determines whether or not a marginal meristem develops may fluctuate, as shown by the existence of trifid leaves. Two forms of juvenile leaves are produced, those in a liquid medium, which in many respects resemble the adult quadrifid submerged leaves, and those on a solid medium, which in many respects resemble the adult land leaves. MANY studies have been made of the growth and development of the fern Marsilea, but little is known of the ontogeny and morphology of the leaves of the juvenile stage of this plant. Hanstein (1865) described the ontogeny of the subulate first leaf ("Keimblatt") produced by Marsilea salvatrix up to the point at which procambium appeared. He also described the appearance of a flat "swelling" of the distal portion of the second leaf primordium, which later developed into a spatulate leaf. In order to illustrate the general organography of Marsilea, Braun (1870, 1872) and Williams (1921) used sketches of the juvenile leaves. In general, the juvenile leaves of Marsilea appear in this sequence: a subulate first leaf, followed by spatulate leaves (both types are herein referred to as unifid leaves), then bifid, and finally the adult quadrifid leaves. There is evidence in the literature that the juvenile leaves of Marsilea may be of 2 anatomically different forms, i. e., they may be either land or submerged leaf forms. This difference in form was noted in nature by Sadebeck (1902) and was induced in sterile cultures of Marsilea by Allsopp (1963). This paper describes the ontogeny and morphology of the submerged and land forms of the juvenile leaves of Marsilea vestita. MATERIAL AND METHODS-Juvenile leaves and apices for this study were obtained from young plants ("sporelings") of Marsilea vestita growing in sterile culture (see Gaudet, 1963, for culture techniques). The sporelings were allowed to grow I 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. in culture tubes containing 20 mil of modified White's nutrient medium, under weak continuous fluorescent lighting, until they were exactly 15 days old. At this age the first and second leaves of the plant had been produced. The reason for allowing these 2 leaves to develop was to be able to start plants on glass cloth (to simulate land conditions) at the same time they were started in the liquid medium. If these first 2 leaves were not present, the plants did not become established on the glass cloth. Some sporelings were transferred to sterile tubes which contained pieces of glass cloth and sufficient nutrient medium almost to cover the glass cloth. Before use, this glass cloth was boiled for 3 hr in concentrated nitric acid, washed until acid-free with tap water, and rinsed thoroughly with glass-distilled water. Entire leaves were cleared, and apices were sectioned and stained using the same procedures as those outlined in a concurrent investigation of the adult leaf (Gaudet, 1964). RESULT,S-Ontogeny of the leawe8s-In the liquid medium, the third, fourth, fifth and, occasionally, a sixth leaf are spatulate (Fig. 1). These leaves arise on the shoot apex (Fig. 2} by means of an ntogenetic "flattening" of the distal portion of a leaf primordium. The leaf primordium per se develops from an initial cell on the flank of the shoot apex. This initial functions as an apical cell at the tip of the primordium (a small primordium is shown on the left in Fig. 3). At a certain point in the ontogeny of the leaf primordium, this apical cell ceases to function, and a group of initials takes its place. These initials divide periclinally and anticlinally mostly in 1 plane. This spatulate leaf develops further by means of a marginal meristem. It is evident in Fig. 3 that this marginal meristem is maintained by repeated divisions periclinal and anticlinal to the margin. The primordium begins to develop just behind