THE CYTOHISTOLOGICAL AND CYTOHISTOCHEMICAL ZONATION OF THE SHOOT APEX OF BOTRYCHIUM MULTIFIDUM

Cytohistologically, the shoot apex of Botrychium multifidum is composed of three zones-a zone of surface initials in which there is usually a centrally located apical cell, a zone of subsurface initials, and a cup-shaped zone that is subdivided into a pheripheral zone and a rib meristem. The results of cytohistochemical tests for total protein, RNA, total carbohydrate, histones, and DNA localization support this concept. Thus, the cytohistological zonation of the apical meristem of the Ophioglossales is essentially identical to that of the Filicales, and furthermore, is comparable to that of the seed plants. THE APICAL MERISTEM (shoot apex) of the three genera of the eusporangiate Ophioglossales has been previously described and illustrated as consisting of a single apical cell in Ophioglossum spp. (Holle, 1875; Rostowzew, 1891; Bruchmann, 1904; and Campbell, 1911), in Botrychium spp. (Bruchmann, 1906; Campbell, 1911; Baas-Becking, 1921; and Bierhorst, 1971), and in Helminthostachys zeylanica (Farmer and Freeman, 1899; and Campbell, 1911). In contrast, Gewirtz and Fahn (1960) concluded that the apical meristem of Ophioglolssum lusitanicum ssp. lusitanicum is composed of a group of initials, whereas McAlpin and White (1974) concluded that in both Ophioglossum and Botrychium the apical meristem of these eusporangiate taxa exhibits a zonation pattern comparable to that they described for numerous leptosporangiate taxa in the Filicales. Thus, McAlpin and White (1974) viewed the apical meristem of both eusporangiate and leptosporangiate ferns as consisting of two zones-a zone of surface initials and a zone of subsurface initials. Based on my observations on Dennstaedtia cicutaria (Stevenson, 1976) and the illustrations of numerous other taxa by Hagemann (1964), Michaux (1971), and McAlpin and White (1974), I came to the conclusion that the zonation of the apical meristem of the Filicales is best viewed as consisting of three zones-a zone of surface initials (in which a prominent apical cell may or may not be evident), a zone of subsurface initials, and a cup-shaped zone in which two regions, a peripheral zone and rib meristem, can be recognized. Thus, the present investigation of shoot apical zonation in Botrychium multifidum (Gmel.) Rupr. was undertaken 'Received for publication 1 July 1975. I thank Ernest M. Gifford, Jr., Rudolf Schmid, and Arthur R. Spurr for their critical reading of the manuscript and their invaluable suggestions and comments. 2Present address: Fairchild Tropical Garden, Miami, Florida 33156. to determine whether this concept is applicable to a representative of the Ophioglossales, and furthermore to ascertain whether it is supported by the results obtained when cytohistochemical methods are employed. MATERIALS AND METHODS-Shoot tips of Botrychium multifidum (Gmel.) Rupr. were field collected (for collection data see Stevenson, 1975) and fixed in FPA (formalin-propionic acid-50 % ethanol, 1:1:18 v/v). The material was dehydrated in an ethanol-toluene series, infiltrated and embedded in Paraplast (melting point 57 C), and serially sectioned at 8,am. For the analysis of cytohistological zonation, a staining schedule with Safranin 0, Fast Green FCF, and Orange G was employed. To determine the cytohistochemical zonation, the following stains or reactions were used-Aniline Blue-Black for total protein, Azure B for RNA, alkaline Fast Green for histones, Periodic Acid-Schiff reagent (PAS) for total carbohydrate, and the Feulgen reaction coupled with Schiff's reagent for localization of DNA. In all instances, the procedures (including the appropriate controls) outlined by Riding (1971) were followed. Voucher specimens are deposited in the Herbarium of the University of California, Davis. OBSERVATIONS-Cytohistological zonation-The following description of cytohistological zonation is based on the results of staining with Safranin 0, Fast Green FCF, and Orange G. In median longisection, the shoot apical meristem is composed of three zones (Fig. 1-3, 15)-a zone of surface initials, a zone of subsurface initials, and a cup-shaped zone. Although these zones are not usually sharply delimitable, it is nevertheless useful to describe them in a generalized fashion. The zone of surface initials, which usually but not always contains a prominent centrally located apical cell (cf., Fig. 1 with Fig. 2-3), contributes to and main-