HETEROKONTOPHYTA, PHAEOPHYCEAE	439

the outer ones undergo divisions to form the cortex. The gametophytes are heterothallic, with the sex organs developing in clusters on the surface of the thallus. A superficial epidermal cell may develop directly into a male gametangium, or it may develop into a branched hair that bears several gametangia. The male gametangium consists of a stalk cell on which there are 20 or more tiers of cells, each tier composed of eight cells. The protoplast of each cell forms a biflagellate male gamete, which escapes through a pore in the gametangial wall to the outside. Female gametangia develop similarly to the male, but with a smaller number of larger cells. Female gametangia are four to seven tiers high, with only four cells in each tier. Free-swimming male gametes are pyriform, with a single reddish chloroplast at the place of flagella insertion. Free-swimming female gametes are also pyriform, but are much larger and have a dozen or so chloroplasts. The female gametes release a highly volatile, low- molecular-weight compound, multifidene (Fig. 21.8) (Pohnert and Boland, 2002) that attracts the male gametes (Müller, 1974). When the gametes fuse, the male gametes are actively swimming while the female are sluggish or immobile. Fusion of the two nuclei follows within a few hours, and the zygote begins to develop into the sporophyte within a day. Unfertilized female gametes develop parthenogenetically into gametophytes.

The zygote germinates to produce the sporophyte, which was first described as a separate genus, Aglaozonia. At first, growth is trichothallic and vertically upward into a columnar structure. Upward growth ceases when the plant is about 10 days old, and all further growth is laterally outward from the base of the column. Repeated cell division at the base of the column forms a flat, disc-like tissue that expands laterally as a result of division and redivision of the marginal cells. The sporophyte is homologous to a minute erect thallus subtended by an enlarged fertile holdfast. The disc-like portion of the thallus is several cells thick, and the outer cells are differentiated into an epidermis-like layer. The holdfast is attached to the substratum by numerous multicellular rhizoids growing from the ventral epidermal cells. The unilocular sporangia are formed in sori on the dorsal surface of the sporpohyte. A single epi-

dermal cell divides into one to six stalk cells and a single terminal unilocular sporangium. In the unilocular sporangium 8, 16, or 32 large, pyriform, haploid zoospores are formed, each with several chloroplasts. The zoospores escape through a large apical pore in the sporangial wall, swarm for 10 to 90 minutes, then settle down, round up, and secrete a wall. The zoospores then divide to form the gametophyte.

Although germlings from zygotes and from zoospores of the Aglaozonia sporophytes have not been grown to maturity in culture, they have been grown to a sufficiently advanced stage to show that the two stages are alternate generations of each other. In Europe, the sporophyte is perennial and fruits in winter or spring, whereas the gametophyte is a spring annual that disappears during the summer.

Fossils of a plant similar in structure to

Cutleria, called Limnophycus paradoxa, have been described from the Miocene (25 million years old) deposits in Germany.

Desmarestiales

In this order there is an alternation of a large macroscopic sporophyte with a small filamentous gametophyte (Fig. 21.15). The gametophyte forms oogonia and sperm, with the result that reproduction is oogamous. Growth of the sporophyte is trichothallic, and the main axis is corticated by downward-growing cells. In this treatment, the organisms sometimes considered in the Sporochnales are placed in the Desmarestiales, as suggested by Russell and Fletcher (1975).

Sporophytes of Desmarestia may reach a length of 2 to 3 m and occur primarily in the sublittoral region in colder waters of both the Northern and the Southern Hemisphere. The plants (Figs. 21.15, 21.16) have trichothallic growth from an intercalary meristem composed of flattened cells that cut off cells to the terminal hair above, and cells to the thallus below. The cells of the terminal hair continually wear away, and each of these cells usually bears one or two unbranched laterals. The cells produced below the meristem cut off two opposite laterals in one plane, which lengthen by means of a basal meristem. Cortication of the thallus begins by the basal cells of the lateral cutting off a number of cells that gradually form a

440 CHLOROPLAST E.R.: EVOLUTION OF TWO MEMBRANES

Fig. 21.15 The life cycle of Desmarestia. (Adapted from

Schreiber, 1932; Chapman and Burrows, 1971.)

HETEROKONTOPHYTA, PHAEOPHYCEAE	441

Fig. 21.16 Desmarestia. (a)–(d) Cortication of an axial cell

(A) by corticating hyphae (CH). (e) Partial section of a mature thallus. (B,C) Laterals; (M) meristoderm.

complete one-layered envelope around the elongating axial cell that produced the lateral. The cells of this one-layered envelope soon undergo periclinal division, with the outer layer that is produced behaving as a meristem. This meristem then produces the cells of the cortex to the inside, with the result that the axial cells and laterals are progressively buried. In addition to this primary growth, the cells of the inner cortex are capable of secondary growth, enlarging to form hyphae that push their way downward between the cells of the cortex. Trumpet hyphal cells occur in the medulla. These cells have perforate end walls with callose and probably function in conduction of nutrients as do the sieve filaments in the Laminariales (Moe and Silva, 1981).

D. aculeata (Fig. 21.15) produces unilocular sporangia on the sporophytes in the winter by the tangential division of a surface cell of the cortex. Meiosis apparently occurs in the production of a few biflagellate zoospores formed in each small

unilocular sporangium. The zoospores have an eyespot and a single chloroplast. Released zoospores settle, lose their flagella, and round up within 24 hours. The cell contents move out into a germ tube immediately after settling. The sporelings produce female and male gametophytes in roughly similar numbers, the male gametophytes having small cells and being less densely pigmented than the female gametophytes. Gametophytes grow vegetatively only in red light, with differentiation of oogonia and antheridia occurring under blue or white light (Müller and Lüthe, 1981). About 11 days to 3 weeks after germination of spores, conical antheridia and lateral oogonia appear on the gametophytes. The tubular oogonia dehisce apically to liberate the egg, which usually adheres to the gelatinized aperture. A single spermatozoid is released from each antheridium through a narrow apical aperture. The freshly released eggs secrete three volatile chemicals that cause the antheridia to burst and attract free spermatozoids to the eggs. The three sexual hormones are desmarestene, ectocarpene, and viridene (Fig. 21.8). Desmarestene is the most potent of the three sexual hormones (Müller et al., 1982). Sporophyte development begins with the production of a tube from one side of the zygote and a lightly pigmented rhizoid from the opposite pole. The initial tube goes on to produce an oppositely branched uniseriate filament, which forms a trichothallic meristem below most of the lateral branches. Cortication of the thallus begins, as has already been described, to produce a mature sporophyte and complete the life cycle. Gametophytes of Desmarestia (Andersen, 1982) and the Laminariales have several features in common, including clusters of unicellular antheridia, vertically elongated intercalary oogonia, attachment of the extruded egg to the oogonial apex, and growth of the young sporophyte upon the oogonial apex.

Some of the species of Desmarestia accumulate large amounts of malic acid, lowering the pH of the vacuolar sap to as low as 2. In collecting seaweeds, the species of Desmarestia should be kept separate because some of their cells will rupture, releasing acid and killing the other seaweeds.

In the Antarctic waters, members of the Desmarestiales provide the bulk of the biomass of benthic seaweeds. They are perennial, covering

442 CHLOROPLAST E.R.: EVOLUTION OF TWO MEMBRANES

large areas of water to depths of about 40 m. The largest and most abundant species (D. anceps and D. menziesii) form thickets, but not the protective canopy characteristic of many kelps. The Antarctic possesses the only cold-water flora without Laminariales, although in sub-Antarctic waters there are vast stands of kelps (Macrocystis and Lessonia) (Moe and Silva, 1977).

The Desmarestiales and the Laminariales have a number of similar development characteristics (Tan and Druehl, 1996) that include (1) vegetative development of the gametophyte in red light;

(2) requirement of white or blue light for development of antheridia and oogonia; (3) existence of spermatozoid-releasing and -attracting factors secreted by eggs; (4) unusually long and flexible hind flagella; (5) lack of eyespots in spermatozoids; and (6) formation of sexual organs by the gametophyte, representing an exhaustive and almost lethal effort for the gametophytes.

Ectocarpales

These algae consist of filaments or of filaments compacted together. In the order it is possible to see the gradual morphological evolution from a filamentous structure to pseudoparenchymatous (haplostichous) complex structures of compacted filaments (from the Ectocarpaceae to the Ralfsiaceae, and Splachnidiaceae). Along another line, the filamentous thallus has evolved by the division of the filament into true parenchymatous (polystichous) thalli (from the Ectocarpaceae to the Scytosiphonaceae). Most of the algae in the order are heterotrichous, with the thallus consisting of two different parts: (1) the prostrate creeping disc that functions as a holdfast, and (2) the erect filamentous, bulbous, or foliose stage. In some of the algae, both systems are evident (Scytosiphon, Fig. 21.19), whereas in others the erect stage is reduced to filaments of a few cells and the thallus is crustose (Ralfsia, Fig. 21.18), and in yet others the erect stage is predominant with the prostrate system reduced to a small holdfast (Petalonia, Fig. 21.20). Even within the same alga, there can be a stage that consists of only a thin crust, whereas the alternate stage has a welldeveloped erect stage.

Nucleic-acid sequencing studies have shown a strong relationship between the Ectocarpales,

Desmarestiales and Laminariales (Draisma et al., 2001).

Four of the families in the Ectocarpales will be considered here.

Family 1 Ectocarpaceae: plants with freefilamentous construction with no adherence of filaments to each other.

Family 2 Ralfsiaceae: algae with a basal layer supporting erect filaments that are compacted together to form a tissue.

Family 3 Scytosiphonaceae: parenchymatous thalli with mostly diffuse growth.

Family 4 Splachnidiaceae: plants with trichothallic growth and unilocular sporangia formed in conceptacles.

Ectocarpaceae

These organisms have free-filamentous construction with no adherence of the filaments to each other. Ectocarpus is the prevalent genus, and is composed of freely branched, uniseriate filaments differentiated into prostrate and erect systems. The prostrate parts are rhizoid-like and often penetrate the substrate. Growth can be diffuse or more or less clearly trichothallic, with intercalary cell divisions confined to certain areas of the filaments. Some workers divide up the family into different genera on the basis of cytology and morphology, whereas others consider that the family contains the single genus Ectocarpus (Russell and Garbary, 1978).

The life cycle of E. siliculosus (Fig. 21.17) can be taken as representative of the family (Papenfuss, 1935). The haploid and diploid phases are both filamentous, but the diploid filaments have longer cells than the haploid filaments. The diploid plants produce unilocular and plurilocular sporangia either on the same plant or on separate plants. These sporangia discharge their zoospores between 0600 and 1200 hours. The mother cell of a unilocular sporangium can be distinguished from a branch initial by the spherical shape and large nucleus of the mother cell. The cell is initially vacuolate, but the physodes and vacuoles are soon extruded from the cell and become lodged in the wall (Loiseaux, 1973). The chloroplasts and nuclei of the unilocular sporangium divide in regular sequence, with the

HETEROKONTOPHYTA, PHAEOPHYCEAE	443

chloroplasts next to the wall and the nuclei in the center of the cell (Knight, 1929). The nuclei divide meiotically. A chloroplast then becomes associated with a nucleus, and a zoospore is delimited around it. A small perforation occurs at the apex of the unilocular sporangium, and up to 32 haploid zoospores ooze out of the sporangium in a gelatinous matrix. The perforation is small, and zoospores are relatively large, being twice the size of gametes and zoospores from plurilocular sporangia. The zoospores initially swim in a straight pattern, then display circling movements as they explore appropriate surfaces for settling (Iken et al., 2001). The zoospores prefer to settle on a hydrophobic surface, preferably one with a microbial film. The zoospores germinate within 2 to 3 hours to produce haploid filaments.

The plurilocular organs (Fig. 21.17) are modified lateral branches that are divided into as many as 660 cubical cells, each containing a motile cell. The plurilocular sporangia on the diploid filaments produce zoospores that remain motile for 3 to 5 hours, settle, and within 2 to 5 hours germinate to produce diploid filaments like

the parent. The germ tube of the sporeling arises from the narrow, anterior flagellated end of the zoospore, which is always oriented toward the light. The plurilocular organs on the haploid filaments are smaller than those on the diploid filaments, and produce either zoospores or gametes. The motile gametes are all of the same size but differ physiologically. The female gametes settle down about 5 minutes after liberation and secrete a sexual hormone called ectocarpene [all- cis-1-(cycloheptadien-2 ,5 -yl)-1-butene] (Fig. 21.8) (Müller et al., 1971). Male gametes (Fig. 21.17) (Maier, 1997a,b) move very rapidly (269 m per second) in a straight line in open seawater when no female gametes are around (Müller, 1978). The motile male gametes (which can remain motile for up to 8 hours) swim in circular paths on encountering ectocarpene, the diameter of the circular path decreasing in response to increasing ectocarpene concentration (Müller, 1982). As soon as the female gamete is reached, a firm contact is established between the apical part of the front flagellum of the male gamete and the plasma membrane of the female gamete. The posterior

Fig. 21.17 The life cycle of

Ectocarpus siliculosis.

444 CHLOROPLAST E.R.: EVOLUTION OF TWO MEMBRANES

ends of the two gametes fuse to form the zygote. The process of fusion takes about 20 seconds, and after fusion the zygote loses its attraction for male gametes as indicated by the dispersion of the male gametes near the zygote. The zygotes take 2 to 3 days to germinate, and the sporelings develop more slowly than those from diploid zoospores. Some of the unfused gametes have the ability to germinate parthenogenetically to give rise to haploid filaments again. This germination is slow, requiring 36 to 48 hours. Clonal populations of E. siliculosus from different parts of the world are interfertile, indicating that there has been little genetic isolation of the species (Müller, 1979). Ectocarpene isolated from E. siliculosus attracts male gametes of two other species of Ectocarpus, indicating that ectocarpene can be artificially synthesized and is effective in attracting male gametes.

Ectocarpus has a fairly wide tolerance to changes in temperatures and salinity. Several studies (Boalch, 1961; Edwards, 1969) have shown that E. siliculosus will grow and produce sporangia at temperatures between 10 and 29 °C. Müller (1962) found that at 13 °C this species produces unilocular sporangia, at 19 °C plurilocular sporangia, and at 16 °C both types of sporangia. It has the ability to grow in salinities from 0.5 to 1.5 times that of seawater at 20 °C, and 0.25 to 1.75 times that of seawater at 15 °C. The alga is an obligate photoautotroph and will not grow on any supplied carbon source in the dark. Although it will not grow in the dark, it has the ability to survive up to 150 days of darkness and still remain viable.

Ralfsiaceae

These algae have a basal layer of branched, radiating, laterally coalesced filaments attached to the substratum by the cell wall or by rhizoids. From this basal layer, chloroplast-bearing filaments arise that are compacted together to give a firm tissue. Ralfsia (Fig. 21.18) probably has an isomorphic alternation of generations. The brown crustlike diploid plants produce unilocular sporangia at the base of loosely associated multicellular paraphyses. Zoospores are most likely produced by meiosis in the unilocular sporangium, and the zoospores give rise to haploid crusts. These gametophytes form plurilocular sporangia that are ter-

minal on erect filaments and probably produce motile cells that can act as gametes or zoospores (Kylin, 1934; Edelstein et al., 1968; Hollenberg, 1969).

Some of the algae that have been placed in this family are the alternate phase of the life cycle of other higher algae and as such have been removed from the family.

Scytosiphonaceae

The Scytosiphonaceae, Dictyosiphonaceae, and Punctariaceae are sometimes grouped together in a separate order, the Dictyosiphonales. The members of these three families all have parenchymatous thalli resulting from diffuse or apical growth, and both gametes are motile. These algae, though, have many structural similarities to the more complex members of the Ectocarpales such as the Splachnidiaceae, and will be considered as members of the Ectocarpales, as suggested by Russell and Fletcher (1975).

In the Scytosiphonaceae, growth is diffuse although in some older plants growth can be suprabasal. The macroscopic phase of the plant produces plurilocular sporangia, but not unilocular sporangia. Scytosiphon lomentaria (Fig. 21.19) is a common intertidal rock-pool alga. It has a narrow, cylindrical thallus up to 50 cm long, gradually tapering from apex to base. The thallus has occasional constrictions, and the plants grow in tufts with smaller individuals showing no or few constrictions. Petalonia (Fig. 21.20) has flat leafy fronds consisting of larger medullary cells covered with smaller cortical cells. In the North Atlantic,

Petalonia fascia and Scytosiphon lomentaria occur in the same area. This area is limited on the north by the 0 °C summer isotherm and on the south by the 17 °C winter isotherm (Fig. 17.9) (van den Hoek, 1982).

The life cycle of Petalonia (Fig. 21.20) and Scytosiphon (Fig. 21.19) has precipitated some controversy. Workers are more or less divided into the North American School (Wynne, 1969; Edelstein et al., 1970; Loiseaux, 1970; Kapraun and Boone, 1987), who claim that meiosis and fusion of gametes do not occur, and the Japanese and Australian school (Nakamura, 1965, 1972; Tatewaki, 1966; Clayton, 1980), who believe that meiosis occurs in the unilocular sporangia and

HETEROKONTOPHYTA, PHAEOPHYCEAE	445

Fig. 21.18 The life cycle of Ralfsia confusa. (Adapted from

Hollenberg, 1969.)

446 CHLOROPLAST E.R.: EVOLUTION OF TWO MEMBRANES

Fig. 21.19 Scytosiphon lomentaria. (a) Whole plant. (b) Portion of a section of the hollow plant showing hairs (h), paraphyses (p), and plurilocular sporangia (ps). (After Taylor, 1957.)

that the motile cells from the plurilocular sporangia function as gametes. The difference in results may be due to different local strains of the same alga. Both groups agree, however, that there are two different morphological phases, a macroscopic phase that produces plurilocular organs and a crustose phase that forms unilocular sporangia.

According to Wynne (1969), P. fascia (Fig. 21.20) has the following life cycle. The foliose thalli are annual and usually have erect lanceolate blades on a small discoid holdfast. The blades produce plurilocular sporangia that release negatively phototactic zoospores, with a period of motility lasting up to 24 hours. These zoospores settle and form filamentous germlings with the protoplast not being evacuated from the spore cell. The filaments branch, with the branches spreading laterally to form discs, with each cell having a single parietal plastid and a large pyrenoid. These discs can develop along two different lines, depending on the environmental conditions. Under short days and low temperature, the discs produce erect, uniseriate processes, the blade initials; these then

become parenchymatous by subsequent longitudinal divisions, yielding the upright flattened blade. The surface cell of the blade undergoes numerous antiand periclinal divisions to form dense lateral files that are the plurilocular organs. The sori cover most of the thallus except for the holdfast and the margin of the blades. The zoospores of the plurilocular organs settle and germinate to form new discs like the original ones. Under long days and high temperature, the discs develop into polystromatic crusts that resemble species of Ralfsia. After 4 weeks in culture these crusts reach maturity and are composed of a basal layer of cuboidal cells seldom exceeding six to ten cells in thickness and a layer of paraphyses supported by the basal layer. The paraphyses consist of four to six cells with a conspicuous cuticle covering the paraphyses. This cuticle is evidently secreted by the terminal cells of the paraphyses. A unilocular sporangium is formed by the basal cell of a paraphysis undergoing an unequal division to produce a cell that protrudes from one side. This cell enlarges laterally and upward, a basal cell is laid down, and a unilocular sporangium is formed. The sporangium cleaves up into 128, 256, or more zoospores, which are released by dissolution of the apex of the sporangium. The zoospores free themselves from the enveloping mucilage, swim away, exhibiting negative phototaxis, and, after several hours, settle down. The germlings give rise to the original discs again. Therefore, in the above life cycle the motile cells produced by both the unilocular and plurilocular organs behave similarly, germinating to form discs.

From the above discussion it can be seen that the morphology of the plant is dependent on environmental conditions. In addition, Lüning and Dring (1973) have shown that the type of light will affect the morphology of Petalonia and Scytosiphon. Among other effects, in red light the prostrate system consists of sparsely branched, uniseriate filaments, whereas under blue or white light it consists of profusely branched filaments. Hsiao (1969, 1970) showed that there are certain minimal concentrations of iodine in the water necessary for the different forms of P. fascia. In order to have the formation of the crust-like Ralfsia stage, 4.0 10 5 M KI was necessary, and, for the formation of blades, 4.0 10 6 M KI. Filamentous stages

HETEROKONTOPHYTA, PHAEOPHYCEAE	447

Fig. 21.20 The life cycle of Petalonia fascia. (After Smith,

1969; Wynne, 1969.)