Ultrastructure of post-fertilization development in the red alga Scinaia articulata (Galaxauraceae, Nemaliales, Rhodophyta)

Biology of the Cell 95 (2003) 27–38 www.elsevier.com/locate/bicell

Ultrastructure of post-fertilization development in the red alga Scinaia articulata (Galaxauraceae, Nemaliales, Rhodophyta) Stylianos G. Delivopoulos * Department of Botany, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece Received 2 July 2002; accepted 6 January 2003

Abstract The ultrastructure of the carposporophyte and carposporogenesis is described for the red alga Scinaia articulata Setch. After fertilization, the trichogyne disappears, and the pericarp develops to form a thick protective tissue that surrounds the carposporophyte. The hypogynous cell cuts off both one-celled and two-celled sterile branches. Patches of chromatin are frequently observed in evaginations of the nuclear envelope, which appear to produce vesicles in the cytoplasm of the cell of the sterile branch. Large gonimoblast lobes extend from the carpogonium and cleave to form gonimoblast initials. Subsequently, a fusion cell is formed from fusions of the carpogonium, the hypogynous cell and the basal cell of the carpogonial branch. The mature carposporophyte comprises the fusion cell that is connected to the sterile branch cells, gonimoblast cells and carpospores and is surrounded by extensive mucilage. Young carpospores possess a large nucleus and proplastids with a peripheral thylakoid, but they have few dictyosomes and starch granules and are indistinguishable from gonimoblast cells. Subsequently, dictyosomes are formed, which produce vesicles with an electron-dense granule, which indicates an initiation of wall deposition. Thylakoid formation coincides with incipient starch granule deposition. The nuclear envelope produces fibrous vacuoles and concentric membrane bodies. Carpospores are interconnected by pit connections with two cap layers. Dictyosome activity increases, resulting in the production of vesicles, which either continue to deposit wall material or coalesce to form fibrous vacuoles. The final stage of carposporogenesis is characterized by the massive production of cored vesicles from curved dictyosomes. Mature carpospores are uninucleate and contain fully developed chloroplasts, numerous cored vesicles, numerous starch granules and fibrous vacuoles. The mature carpospore is surrounded by a wall layer and a separating layer, but a carposporangial wall is lacking. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Scinaia articulata; Rhodophyta; Carposporophyte ultrastructure

1. Introduction The stages of reproductive development, particularly post-fertilization events, are important in the classification of red algae (Kylin, 1930, 1956; Dixon, 1973; Abbott and Hollenberg, 1976; Bold and Wynne, 1985). The majority of ultrastructural studies on overall carposporophyte development and carposporogenesis has been conducted on members of the Ceramiales, which are generally considered a more advanced order of the Florideophyceae (Tripodi, 1971, 1974; Chamberlain and Evans, 1973; Kugrens and West, 1973, 1974; Wetherbee and Wynne, 1973; Konrad Hawkins, 1974; Triemer and Vasconcelos, 1977; Wetherbee and West, 1977; Wetherbee, 1978, 1980; Broadwater and Scott, 1982; * Corresponding author. Tel.: +30-2310-998343; fax: +30-2310-998389. E-mail address: [email protected] (S.G. Delivopoulos). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 2 4 8 - 4 9 0 0 ( 0 3 ) 0 0 0 0 3 - 0

Delivopoulos and Diannelidis, 1991a, b, c). Several studies dealt with members of the Gigartinales (Tsekos, 1981, 1982, 1983; Tsekos and Schnepf, 1983; Delivopoulos and Diannelidis, 1990a, b; Kugrens and Delivopoulos, 1986; Delivopoulos and Tsekos, 1986); there were two studies on members of the Rhodymeniales (Gori, 1980; Delivopoulos and Kugrens, 1984) and one study on Cryptonemiales (Delivopoulos and Kugrens, 1985). Three studies dealt with Nemalion helminthoides (Velley) Batters (Duckett and Peel, 1978; RammAnderson, 1980; Ramm-Anderson and Wetherbee, 1982), a member of the Nemaliales, which is considered a phylogenetically lower Florideophyceae (Duckett and Peel, 1978). Probably no other order of the red algae had as many changes made in the understanding of its life history as the Nemaliales (Bold and Wynne, 1985). The family Galaxauraceae includes about seven genera (Bold and Wynne, 1985). The genus Scinaia was established

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by Bivona-Bernardi (1822) for the individual species Scinaia forcellata Bivona-Bernardi. The genus Gloiophlea, which is similar to Scinaia in both habit and reproduction, was erected by J. Agardh (1872). A new genus, Pseudogloiophlea, was proposed by Levring (1953, 1955) to incorporate all the incorrectly placed species that conformed to Gloiophlea sensu Setchell. Pseudoscinaia, a fourth genus of Galaxauraceae, which is vegetatively identical to Scinaia, was proposed by Setchell (1914), mainly because the gonimoblast filaments radiate from several focal points, which is unlike Scinaia, where there is a single central focus for gonimoblast initiation. Of the genera in Galaxauraceae, only Scinaia and Gloiophlea (sensu J. Agardh 1872 and Levring 1953) have remained unchanged. The genus Scinaia, as has been redescribed by Huisman (1985, 1986), including Pseudoscinaia Setchell and Pseudogloiophlea Levring, contains 35 species. The family Galaxauraceae is recognized by the more complicated post-fertilization stages (Bold and Wynne, 1985). Due to the varied interpretations regarding Scinaia, it is evident that any critical examination of post-fertilization development in the type genus Scinaia could add new data to the reproductive and vegetative diversity of this otherwise conservative genus. This work was undertaken in order to present, for the first time, a complete ultrastructural study of carposporophyte development of the undescribed species Scinaia articulata Setch., in hopes of providing useful information to help clarify the phylogenetic relationships within this genus. 2. Results Pyriform cystocarps are scattered throughout the thallus. A prominent ostiole is present in the mature cystocarps that are immersed in the thallus. Before fertilization, the first cell of the carpogonial branch, known as the basal cell, produces several cells, which eventually form the pericarp. The second cell, which is the hypogynous cell, cuts off a one-celled and a two-celled sterile branch, while the third cell, which is the carpogonium, elongates and forms a trichogyne. After fertilization, the trichogyne disappears and the pericarp develops further, eventually forming a thick protective structure that surrounds the carposporophyte (Fig. 1). The fertilized carpogonium contains the zygote nucleus, and elongate proplastids in a lightly stained cytoplasm, and is surrounded by a thick cell wall (Fig. 2). The hypogynous cell is connected with one cell of the sterile branch cells (Fig. 3). The hypogynous cell and the cell of the sterile branch possess many mitochondria, some proplastids, some cisternae of endoplasmic reticulum and concentric membrane structures, but they are devoid of starch granules and have low dictyosome activity (Figs. 4, 5 and 9). In the cell of the sterile branch, there is a large central nucleus with evaginations, surrounded by numerous mitochondria (Fig. 4). Chromatin is mostly dispersed, and some patches are frequently observed in the evaginations of the nuclear envelope as well as in the interior

(Fig. 4). Often, chromatin is also isolated by sac-like structures in the nucleus of the hypogynous cell (Fig. 5). Nonmembrane associated chromatin masses are often observed in the cytoplasm of cells of the sterile branch (Fig. 9). Two nuclei can frequently be observed. There are also bands of ER and membranous structures (Fig. 4). The nuclear envelope is blebbing and appears to produce vesicles in the cytoplasm (Fig. 6). Meanwhile, neighboring pericarp cells are fusing with the cells of the sterile branches (Figs. 7 and 8). In the beginning of carposporophyte development, the fertilized carpogonium produces two multinucleate gonimoblast lobes (Fig. 9). These lobes enlarge significantly and cleave continuously to form gonimoblast initials (Fig. 10). Two gonimoblast initials arise directly from the carpogonium and initially produce two gonimoblast filaments (Fig. 9). With successive divisions and branching of these two filaments, more gonimoblast filaments are produced (Fig. 10). The developing carposporophyte is surrounded by extensive mucilage and elongate pericarp cells (Fig. 10). Gonimoblast initials are large cells with a large nucleus, proplastids and mitochondria occurring in the peripheral cytoplasm and in the central cytoplasmic area (Fig. 10). The gonimoblast initials divide to form gonimoblast cells, which in turn differentiate to form young carpospores (Fig. 10). Oblong carpospores are produced in chains at the ends of the gonimoblast filaments and are 5 × 15 µm (Fig. 10). A fusion cell is always formed from fusion of the carpogonium, the hypogynous cell and the basal cell of the carpogonial branch (Figs. 10 and 11). The nucleus of one cell of the sterile branch is transferred through the pit connection to the hypogynous cell (Fig. 12). As the fusion cell matures, it enlarges, and the sterile one- and two-celled branches on the hypogynous cell are connected to it (Fig. 13). Subsequently, these cells transport their nuclei and organelles to the fusion cell through cytoplasmic channels (Fig. 13). The cells of the sterile branches have a large nucleus, mitochondria and many vacuoles, but no starch granules (Fig. 13). Young gonimoblast cells (Fig. 14) appear indistinguishable from young carpospores (Fig. 15). Cytoplasmically, they are rather simple, similar in structure and have a large nucleus with a small prominent nucleolus, numerous proplastids with a peripheral thylakoid and a few starch granules, but they lack dictyosomes (Fig. 14). Young carpospores are embedded within mucilage produced by the gonimoblast cells. Starch granules are generally absent in most carpospores during the earliest stages of differentiation, but starch polymerization is often concomitant with the formation of thylakoids in the plastids and concentric membrane formation in the cytoplasm. Carposporogenesis begins with the production of vesicles that have an electron-dense granule within straight-profiled dictyosomes (Fig. 15). These dictyosome vesicles are responsible for initial wall deposition inside the compressed fibrils

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Figs. 1–5. Scinaia articulata. Electron micrographs of early post-fertilization development. Scale bars = 1 µm except Fig. 1. Fig. 1. The carpogonium, hypogynous cell and sterile branch cell. The hypogynous cell and sterile branch cell are surrounded by pericarp cells. The hypogynous cell is not visible. Fig. 2. Higher magnification of the carpogonium. Fig. 3. Cytoplasmic bridge connecting the hypogynous cell with one cell of the sterile branch. Fig. 4. Cell of the sterile branch. Chromatin is observed to the evagination of the nuclear envelope. Fig. 5. Chromatin separated by sac-like structures (arrows) in the nucleus of the sterile branch. Scale bars = 1 µm except Fig. 7. Abbreviations used in all figures: BC = basal cell, Ch = chromatin, CM = concentric membranes, Cpg = carpogonium, Cs = carpospores, CV = cored vesicle, CW = cell wall, D = dictyosome, ER = endoplasmic reticulum, FC = fusion cell, FS = floridean starch, FV = fibrous vacuole, GC = gonimoblast cell, GI = gonimoblast initial, GL = gonimoblast lobe, HC = hypogynous cell, M = mitochondrion, Mu = mucilage, N = nucleus, NE = nuclear envelope, P = plastid, PC = pericarp cell, SBC = sterile branch cell, SL = separating layer, V = vacuole.

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Figs. 6–8. Scinaia articulata. Electron micrographs of the cells of the sterile branch. Fig. 6. blebbing nuclear envelope appearing to produce vesicles (arrows) into the cytoplasm (double arrow). Fig. 7. Fused cells (arrows) in the sterile branch. Fig. 8. Higher magnification of the fused cell in the sterile branch of Fig. 7.

of the carposporangial mucilage (Fig. 15). Formation of starch granules also begins at this time (Fig. 15). The most notable feature of the intermediate stage of carpospores is the nuclear envelope, which exhibits an unusual activity by producing concentric membranes and formations like fibrous vacuoles (Fig. 16). Carpospores continue to increase in size, probably due to the increase of cytoplasmic components, and are interconnected by pit connections that have two cap layers (Fig. 17). The intermediate stage is characterized by considerable wall thickening (Fig. 18). Dictyosome activity is intense during this stage, resulting in the production of vesicles, which continue to deposit wall material (Fig. 18). Another feature of the intermediate stage is the continued thylakoid formation. At the end of this stage, large fibrous vacuoles have been formed (Fig. 19). The final stage of carpospore formation is characterized by the massive production of the cored vesicles from curved dictyosomes (Fig. 20). Some of these cored vesicles may become incorporated in the fibrous vacuoles, but the majority remains stored in the cytoplasm, and they are never released into the wall while the carpospores remain enclosed within the cystocarp. The mature carpospore consists of a separating layer, a carpospore wall, a nucleus, well-developed chloro-

plasts, numerous cored vesicles, floridean starch granules and fibrous vacuoles (Fig. 21). 3. Discussion This report represents the first ultrastructural study of carposporophyte development in a species of the genus Scinaia of the family Galaxauraceae, which includes all the post-fertilization stages from gonimoblast initial formation to complete carpospore maturation. Post-fertilization development in Scinaia articulata differs significantly from the descriptions provided by Setchell (1914) and Dawson (1949). After fertilization, the trichogyne disappears and the pericarp develops further. Two gonimoblast initials arise directly from the carpogonium of Scinaia articulata, instead of the three to four initials that are formed in other species of Scinaia that have been studied (Huisman, 1985, 1986; Bhatia and Vijayaraghavan, 1995). These initials divide repeatedly to produce numerous radiating gonimoblast filaments, from which chains of carposporangia are produced. Since the critical stages of the gonimoblast initial formation are often difficult to observe and easy to misinterpret, Magruder (1984) suggested that it would eventually be

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Fig. 9. Scinaia articulata. Electron micrograph of a young carposporophyte. The hypogynous cell is connected through a cytoplasmic bridge to the carpogonium, which produces two gonimoblast lobes extending to the left and to the right. Scale bar = 5 µm.

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Figs. 10,11. Scinaia articulata. Electron micrographs of a developing carposporophyte. Scale bars = 5 µm. Fig. 10. The two gonimoblast lobes cleave continuously (arrows) to form gonimoblast initials from which two gonimoblast filaments are produced, by the subsequent branching of which more filaments are produced. Fig. 11. Part of the fusion cell that originates from the carpogonium, the hypogynous cell and the basal cell.

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Figs. 12,13. Scinaia articulata. Electron micrographs of the fusion cell connected with the cells of the sterile branch. Scale bars : Fig. 12 = 5 µm, Fig. 13 = 1 µm. Fig. 12. Nuclear transfer from the cell of the sterile branch to the hypogynous cell through cytoplasmic channel. Fig. 13. Fusion cell connected to the two cells of the sterile branches through cytoplasmic channels. Nuclear transfer from the cell of the sterile branch to the fusion cell. Figs. 14,15. Scinaia articulata. Electron micrographs of young gonimoblast cell and young carpospore. Scale bars = 1 µm. Fig. 14. Young gonimoblast cell showing a large nucleus with a small nucleous, numerous proplastids and few starch granules. Fig. 15. Young carpospore with straight profiled dictyosomes producing vesicles with electron-dense granule (arrows).

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Figs. 16–21. Scinaia articulata. Electron micrographs of intermediate-aged and mature carpospores. Scale bars = 1 µm except Fig. 20. Fig. 16. Nuclear envelope producing concentric membrane bodies and formations like fibrous vacuoles in an intermediate-aged carpospore. Fig. 17. Pit connection with two cap layers between intermediate-aged carpospores. Fig. 18. Intermediate-aged carpospore forming fibrous vacuoles. Fig. 19. Nearly mature carpospore with fibrous vacuole. Fig. 20. Dictyosome producing cored vesicles in a nearly mature carpospore. Fig. 21. Typical mature carpospore with cored vesicles, fully developed chloroplasts and fibrous vacuoles.

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proven that the primary gonimoblast initials are produced directly from the carpogonium in all species of the Galaxauraceae. Huisman (1985), in his meticulous study, revealed that in the majority of the Galaxauraceae, the gonimoblast initials do arise directly from the carpogonium and not from the hypogynous or fusion cell as Svedelius (1915) asserted. Huisman’s (1985) observations are confirmed in the present ultrastructural study in Scinaia articulata. Interestingly, for some Scinaia species from Indian waters, the hypogynous cell origin of gonimoblast initials has been reported (Krishnamurthy and Sundararajan, 1987; Bhatia and Vijayaraghavan, 1995), whereas for other Scinaia species from the Sea of Japan, the carpogonial origin of gonimoblast initials has been described (Zablackis, 1987; Kajimura, 1988, 1995). Based on this contradiction, Bhatia and Vijayaraghavan (1995) suggested further work on the gonimoblast development of various species of Scinaia in order to clarify whether there are biogeographical patterns. The mature carposporophyte consists of three components: the fusion cell, gonimoblast cells and carpospores. The fusion cell is formed from fusions of the carpogonium, the hypogynous cell and the basal cell of the carpogonial branch similar to the majority of Scinaia species studied (Huisman, 1985, 1986; Zablackis, 1987; Krishnamurthy and Sundararajan, 1987; Kajimura, 1988) and unlike Scinaia pseudocrispa (Bhatia and Vijayaraghavan, 1995). As the fusion cell matures and enlarges, the sterile one- and two-celled branches of the carpogonial branch remain connected directly to it. Eventually, they form a part of the fusion cell and transport some of their organelles and their nuclei through cytoplasmic channels to the fusion cell. Consequently, this multinucleate fusion cell contains haploid and diploid nuclei. It is presumed that the haploid nuclei degenerate or become inactivated, and only diploid nuclei participate in subsequent carposporophyte development. Based on visual evidence, the patches of chromatin found in the evaginations of the nuclear envelope or isolated by sac-like structures, as well as the dark staining spherical masses of material observed in the cytoplasm of the cell of the sterile branch, may represent dehydrated chromatin similar to Faucheocolax attenuata (Kugrens and Delivopoulos, 1985). The haploid nuclei are possibly inactivated through dehydration and chromatin condensation and can remain in the cytoplasm throughout development. DNA inactivation by dehydration and condensation has been observed during animal spermatiogenesis (Fawcett, 1975; Dixon et al., 1977), resulting in dark staining chromatin and a dramatic decrease in nuclear size (Browder, 1984). By the time the carposporophyte is fully developed, the fusion cell is quite extensive. The organelles of the fused cells have a degenerate appearance, and starch granules are absent. Therefore, ascribing a nutritive function to the fusion cell in Scinaia articulata is speculative, as in Nemalion helminthoides (Ramm-Anderson and Wetherbee, 1982), unless the material from the degraded organelles can be utilized by the developing carposporophyte. Thus, an indirect nutritive role could be assigned to the fusion cell of Scinaia articulata similar to the

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fusion cell of Faucheocolax attenuata Setch. and Gloiosiphonia verticillaris Farl. (Delivopoulos and Kugrens, 1984, 1985). Finally, free gonimoblast filaments were not observed in the cystocarpic cavity of Scinaia articulata, unlike in Scinaia forcellata (Huisman, 1985) and Scinaia cottonii (Kajimura, 1995). Chiang (1970) observed only one gonimoblast initial in Scinaia pseudojaponica Yamada et Tanaka. He suggested that the number of the gonimoblast initials produced from the carpogonium or from the hypogynous cell can be used as a criterion for interpreting phylogenetic relationships among the genera of the Galaxauraceae. According to his suggestion, the more primitive members of this family have more than one gonimoblast initial, which is a conspicuous feature of many genera of the lower Nemaliales, while the species having only one gonimoblast initial, like Scinaia pseudojaponica, are presumably advanced. However, the present study shows that Scinaia articulata produces more than one gonimoblast initial, similar to the majority of Scinaia species studied (Huisman, 1986). Therefore, Chiang’s (1970) suggestion may not be valid. Post-fertilization development in Scinaia articulata is not as complex or advanced as in some other red algae. Mature carpospores possess typical red algal chloroplasts, but young carpospores and the gonimoblast cells contain proplastids with a peripheral thylakoid only. Therefore, they may depend on the inner carposporophyte cells and the fusion cell for nutrition, unlike in Nemalion helminthoides (RammAnderson and Wetherbee, 1982), where carpospores contain fully functional chloroplasts throughout development and hence are nutritionally independent even at a young stage. Another difference between these two species is that the developing carposporophyte of Scinaia articulata is surrounded by a protective pericarp, unlike the Nemalion helminthoides naked carposporophyte (Womersley, 1965; RammAnderson and Wetherbee, 1982). In the cell of the sterile branch, the nuclear envelope is blebbing and appears to produce vesicles in the cytoplasm. Extensive blebbing of the nuclear envelope has also been observed in developing carpospores of Levringiella (Kugrens and West, 1973), Polysiphonia and Pterosiphonia (Tripodi, 1974), in differentiating tetraspores of Osmundea spectabilis var. spectabilis (Delivopoulos, 2002), in fern eggs (Bell and Duckett, 1976) and in meiotic tissues of angiosperms (Dickinson and Heslop-Harrison, 1977). Blebbing of the nuclear envelope is one of the events collectively termed nucleo-cytoplasmic interactions (Kessel, 1973; Peel et al., 1973; Franke, 1974; Franke et al., 1974; Spring et al., 1974; Bell and Duckett, 1976; Dickinson and Heslop-Harrison, 1977). These interactions are indicative of intense transcriptional activity at critical stages in the life history of red algae (Duckett and Peel, 1978). Moreover, in the intermediate stage carpospores, the nuclear envelope produces concentric membranes and formations like fibrous vacuoles. Formations similar to the fibrous vacuoles also produced by the nuclear envelope have previously been reported during carposporo-

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genesis (Wetherbee and Wynne, 1973; Broadwater and Scott, 1982; Delivopoulos and Tsekos, 1985) and spermatogenesis (Cole and Sheath, 1980). It has been suggested that in those cases, the nucleus is directly involved in the active synthesis of storage material (Wetherbee and Wynne, 1973). Cytological differentiation of carpospores in Scinaia articulata is relatively simple and involves three developmental stages. The first step is the formation of the carpospore wall; during the second stage, the fibrous vacuoles are formed; and the third stage is characterized by the production of cored vesicles that remain stored in the cytoplasm until carpospore release. Carpospore differentiation in Scinaia articulata is signaled by distinct changes in the morphology of the dictyosomes and the dictyosome-derived vesicles, and by accumulation of starch granules as in Nemalion helminthoides (Duckett and Peel, 1978; Ramm-Anderson, 1980; RammAnderson and Wetherbee, 1982). However, in Scinaia articulata, there are no granular vacuoles (Delivopoulos and Kugrens, 1985), ephemeral or unusual vesicles, such as striated vesicles (Tripodi, 1971; Kugrens and West, 1973, 1974; Wetherbee and Wynne, 1973; Wetherbee and West, 1977; Wetherbee, 1978) and striped vesicles (Kugrens and West, 1974). In addition, carpospores in Scinaia articulata do not exhibit any organelle polarity, unlike those of Nemalion helminthoides, where there is a distal plastid and proximal nucleus (Ramm-Anderson and Wetherbee, 1982). Moreover, in Scinaia articulata, there is no proliferation of multiple carposporangia in several directions, as that reported in other species of Nemaliales (Svedelius, 1911, 1942; Brown, 1969; Ramm-Anderson, 1980; Ramm-Anderson and Wetherbee, 1982). In Scinaia articulata, the mature carpospore wall consists of two layers, while confluent mucilage still surrounds the carpospores. The inner layer would be the carpospore wall layer, which was formed during carpospore maturation, while the outer one represents a region arising from the compression of fibrils due to carpospore enlargement. From a structural point of view, a carposporangial wall in Scinaia articulata does not exist, which is similar to the situation in Faucheocolax attenuata and Gloiosiphonia verticillaris (Delivopoulos and Kugrens, 1984, 1985) and unlike Nemalion helminthoides (Ramm-Anderson and Wetherbee, 1982). The only wall that could be considered a carposporangial wall is the outer layer of mucilage that surrounds the young carpospores. This layer is later compressed by additional wall material to form the separating layer of mature carpospores, similar to Faucheocolax attenuata and Gloiosiphonia verticillaris (Delivopoulos and Kugrens, 1984, 1985). In conclusion, the present study, coupled with previous ones on Batrachospermum moniliforme Roth (Brown, 1969) and Nemalion helminthoides (Ramm-Anderson and Wetherbee, 1982), reinforces the idea that the phylogenetically lower Florideophyceae, such as the Batrachospermales and Nemaliales, exhibit a simple developmental pattern of carposporogenesis, while the phylogenetically higher red algae

(Tripodi, 1971, 1974; Chamberlain and Evans, 1973; Konrad Hawkins, 1974; Triemer and Vasconcelos, 1977; Wetherbee and West, 1977; Wetherbee, 1978; Tsekos, 1981, 1983; Delivopoulos and Kugrens, 1984; Delivopoulos and Tsekos, 1986; Kugrens and Delivopoulos, 1986) have a remarkably more complex developmental sequence during carpospore formation.

4. Materials and methods Thalli of Scinaia articulata Setch. bearing cystocarps of varying sizes were collected during low tide from rocks of Campus Point on the Santa Barbara campus of the University of California, Santa Barbara. Small tissue samples were fixed in situ immediately for 5 h in 5% glutaraldehyde buffered by a mixture of equal amounts of 0.2 M Na-cacodylate buffer and seawater, which was adjusted to pH 7.0 (Kugrens, 1974). The fixed material was rinsed in decreasing concentrations of seawater and buffer, and the final rinse was in 0.1 M Nacacodylate buffer. Post-fixation of the samples for 5 h in 0.1 M Na-cacodylate buffered 2% osmium tetroxide was followed by dehydration with a graded (30%, 50%, 70%, 90%, 100%) ethanol series. The samples were then embedded in graded series of propyleneoxide-Spurr resin mixtures over a period of 2 days, followed by several changes in pure Spurr resin, and polymerized at 70 °C for 8 h. Thin sections were obtained with a Diatome diamond knife on a Reichert-Jung Ultracut E ultramicrotome and were post-stained for 45 min with 1% aqueous uranyl acetate and 15 min with lead citrate. Sections were examined with a Zeiss 9S-2 electron microscope.

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