THE MORPHOLOGY OF RIELLA AFFINIS. I. GERMINATION OF THE SPORE AND DEVELOPMENT OF THE THALLUS

THE PRESENT investigation resulted from the discovery in California of a species of Riella previously known only from the original description (Thompson, 1940). This species, R. a,flnis, was described by Howe and Underwood in 1903 from material collected five years earlier near Tafira, Grand Canary, by Mrs. 0. F. Cook (Howe and Underwood, 1903). It is the only homothallic species of Riella in which the archegonial involucre is ornamented with eight longitudinal flange-like wings. The California specimens of R. afflnis were found growing in an artificial lake, Lagunita, on the Stanford University campus. The bed of the lake is completely dry for six or seven months each year. With the advent of the rainy season the lake fills rapidly, and the water remains at a uniform level from early in February to early in May. When full the greatest depth of the water is about 35 feet. Between the first of May and the middle of June the water is completely withdrawn from the lake for irrigation purposes and the lake bed is entirely dry from the first of July until it begins to fill late in the fall. At the time when the lake is being emptied, isolated plants of Riella become exposed when the water level has dropped about five feet. As the water level drops further, more and more plants are exposed. A survey of the lake bed after withdrawal of the water reveals that R. affinis grows most abundantly at a depth of 15 to 20 feet. The plants grow in thick clusters and on becoming dry, form very distinctive sods on the exposed lake bed. There are seventeen species of Riella (Wigglesworth, 1937), four of which are South African and eleven Mediterranean. Of the remaining two species, one, R. americana Howe and Und., is known only from the United States and at present only from Texas and possibly South Dakota. R. affinis is now known from two widely separated stations, the Canary Islands and California. METHODS.-Cultures for a study of development in the adult thallus were made by breaking dry Riella sod into an ordinary finger-bowl culture dish and covering with pond water to a depth of an inch or more. The spores germinated within two to three weeks and the resultant thalli developed sex organs about two months after the culture was started. This material was used for study of the living plant and also fixed for embedding and sectioning. 1 Received for publication June 10, 1941. The writer is indebted to Dr. Gilbert Morgan Smith of Stanford University under whose direction this investigation was carried out. He is also indebted to Mrs. E. Bodenstein and Mrs. E. Varneck for aid in the translation of Italian and Jugoslavian papers. Cultures were also started with spore masses, intact within the capsule wall, which had been dissected from the dry plants. These were kept intact and placed in Syracuse watch glasses. Pond water was used for some of these cultures; for others a mixture of equal parts of pond water and filtered soil extract was used. The latter was made by boiling 500 grams of soil from the lake bed in one liter of pond water after the procedure given by Bold (1936). Such extracts have been used with much success in the culture of algae. The spores germinated more rapidly in the soil extract medium than in ordinary pond water and also germinated sooner when kept in masses than when the individual spores were dispersed. It was also found that, while spores from plants of the current season would germinate soon after drying, a much higher percentage of germination was obtained if they had been kept dry for a year or more before a culture was started. It was soon found that the food content, particularly the oil globules, of the germ tube and young sporeling so obscured the nuclei and division walls that an accurate knowledge of early growth could not be obtained from a study of living material. Hence, clearing and staining were necessarv. All material was fixed in chromic anhydride-formalin. That to be sectioned was carried through a tertiary-butyl alcohol dehydrating series and embedded in paraffin. All material was stained in safranin or eosin at some stage in the dehydrating series so that it would be visible in the paraffin. Most material was sectioned at eight microns. The stains used were Heidenhain's iron-alum haematoxylin and gold orange, or safranin and fast green. The materials selected for whole mounts were first stained and then passed from absolute alcohol into terpinol. Material in the stages from germination to young juvenile thalli were taken graduallv into terpinol and from that gradually into pure balsam. Keeping a mass of germinated spores intact facilitated the handling of it during staining and balsam infiltration. The adult thalli were passed from alcohol directly into terpinol and from that directly into pure balsam. It was found most useful to mount the adult thalli between two cover slips so that the entire mount could be turned over and examined on either side. The necessity for mounting germination and earlv growth stages whole is obvious. The whole mounts of all stages of adult thallus growth are of considerable value. They are like thick sections and are exceptionally useful in furnishing a three dimensional picture of structures and aiding in the interpretation of serial sections.