178 EVOLUTION OF THE CHLOROPLAST

Fig. 5.41 The life cycle of Acetabularia mediterranea.

(Adapted from Egerod, 1952; Smith, 1955.)

leading to maternal inheritance of chloroplast genes (Kuroiwa, 1985).

Caulerpales

This order contains the coenocytic or siphonaceous Chlorophyta. The non-septate thallus thus resembles a garden hose without any cross walls

separating the usually large thallus, except during reproduction. The cells have numerous lensshaped or fusiform-shaped chloroplasts and, in some cases, amyloplasts. Two carotenoids, siphonoxanthin (Fig. 5.1) and siphonein, not normally found in the Chlorophyta, occur in this order (with the exception of Dichotomosiphon, which has only siphonein; Kleinig, 1969). Cellulose is usually not a wall component and is replaced by a -1,3 linked xylan or a -1,4 linked mannan (Parker, 1970). The

CHLOROPHYTA 179

Caulerpales are marine algae and occur as seaweeds in the warmer oceans.

The most important families in the Caulerpales (or Siphonales or Codiales) are as follows:

Family 1 Derbesiaceae: stephanokontic zoospores; no amyloplasts; no oogamous reproduction.

Family 2 Codiaceae: only biflagellate swarmers; thallus basically filamentous; amyloplasts may be present; no oogamous reproduction.

Family 3 Caulerpaceae: only biflagellate swarmers; thallus composed of a stem bearing blades; amyloplasts present; no oogamous reproduction.

Family 4 Dichotomosiphonaceae: oogamous reproduction.

Derbesiaceae

Derbesia (Fig. 5.42) is a filamentous alga found in tropical and temperate waters growing on stones near the low-tide line or on larger algae. The life cycle of Derbesia (Feldmann, 1950) involves the alternation of this filamentous sporophyte with a bulbous vesicular gametophyte, the Halicystis stage, which was originally described as an independent plant. The Halicystis stage is found in deep water, usually epiphytic on a coralline alga. The filamentous D. tenuissima sporophyte (Page and Kingsbury, 1968) has an interwoven basal portion that supports erect branched filaments, which are occasionally divided by plug-like septa.

This filamentous sporophyte forms ellipsoidal sporangia as short lateral branches cut off by a septum from the main axis. The sporangia form zoospores meiotically (Neumann, 1969) with a whorl of flagella at one end. The zoospores swim for a while, settle, and germinate to produce a filament that forms the vesicular Halicystis gametophyte (Fig. 5.43). The Halicystis plants have nuclei arranged in an outer layer under the cell wall, with the chloroplasts in an inner layer next to the large central vacuole. The first visible stage of gamete formation is the migration of protoplasm to the plant apex; this protoplasm becomes separated by a membrane and is now the gametangium. The nuclei in the gametangium undergo synchronous divisions, with the

protoplasm cleaving to form the uninucleate gametes.

Plants are heterothallic, producing only one type of gamete. The male plants have olive-green gametangia, whereas those of the female are blackish. Release of the gametes is induced by light, which causes an instantaneous increase in turgor pressure, rupturing the weakened pore area of the wall, and causing a forcible expulsion of the gametes. Following release, the pore is sealed by the gametangial membrane (Wheeler and Page, 1974). Within 12 hours the cell is uniformly green, and it is capable of forming a second gametangium after 24 hours. The biflagellate gametes have one chloroplast in the male and 8 to 12 in the larger female (Roberts et al., 1981). The female gametes are less active than the male. Immediately after mixing, the male gametes surround the female. One of the males begins to fuse with the female, and the whole group of gametes sinks. The zygote germinates to form the filamentous sporophyte. There is a debate as to whether the nuclei from the male and female gametes fuse after plasmogamy of the male and female gametes, or whether karyogamy occurs in the sporangia of the filamentous Derbesia phase (Lee et al., 2000; Schnetter and Eckhardt, 2000).

An endogenous rhythm controls gamete formation in the Halicystis stage of D. tenuissima (Page and Kingsbury, 1968). Gametogenesis has a basic period of 4 to 5 days in the laboratory, but in nature usually occurs in multiples of this figure, and is evidently timed by the tides. The rhythm is unaffected by changes in temperature or light intensity, indicating that it is an endogenous rhythm not directly linked to metabolic processes, such as photosynthesis. After induction of gametogenesis, about 7 hours of dark is necessary for maturation of the gametes, after which light causes their immediate release.

Bryopsis (Fig. 5.44(d)) is a common alga in quiet water of tide pools and other sheltered locations. The genus has a main axis that supports lateral upright branches.

The cell walls of the gametophytic phases of

Derbesia tenuissima and Bryopsis plumosa contain large amounts of xylans, whereas the walls of the sporophytes contain large amounts of mannans (Huizing et al., 1979).

180 EVOLUTION OF THE CHLOROPLAST

Fig. 5.42 The life cycle of Derbesia. (Adapted from Smith,

1955; Zeigler and Kingsbury, 1964.)

Codiaceae

These algae differ from those in the Derbesiaceae in having biflagellate swarmers. The structure of the thallus is basically filamentous. The family can constitute a significant proportion of seaweed populations in tropical waters, including some attractive forms such as “mermaid’s fan”

(Udotea, Fig. 5.44(f )) and “Neptune’s shaving brush” (Penicillus, Fig. 5.44(e)).

Codium (Fig. 5.45) occurs from the low-tide mark up to 70 m depth in tropical and temperate marine waters. The genus was originally absent from much of the East Coast of North America. In 1957, C. fragile was found along the central Atlantic Coast (Bouck and Morgan, 1957), and it has subsequently spread as far north as Maine. It is probable that the alga was introduced on

CHLOROPHYTA 181

Fig. 5.43 Derbesia tenuissima. (a) Vegetative Halicystis gametophyte. (b) Mature male gametophyte (arrow points to gametangium). (c) Mature female gametophyte (arrow points to gametangium). (d) Sporangium with fully cleaved protoplasm. (e) Mature sporophyte. (From Lee et al., 2000.)

oysters transplanted from Europe. Since its introduction, Codium has become a pest in oyster beds, attaching to oysters and causing them to be cast adrift during heavy storms (Fig. 5.46).

The thallus has a crustose prostrate portion that bears several cylindrical dichotomously branched shoots. The shoots have a central medulla composed of interwoven colorless filaments that give rise to inflated branchlets, the utricles, which surround the medulla. The utricles have a thick peripheral layer of cytoplasm around a large central vacuole. The discoid chloroplasts are in the outer part of the cytoplasm and the small nuclei in the interior. The colorless filaments of the medulla are divided in places by walls, especially near the base of the utricles. Dark-green female and brown male gametangia are produced from the utricles of the diploid thallus. The gametes are formed meiotically and are released when the lid-like apical portion of a gametangium ruptures, extruding a

gelatinous mass with a central canal through which the gametes move. The gametes initially lack flagella and are carried passively. After flagella extrusion, the gametes swim away, with the male gamete fusing with the side of a larger female gamete. The flagella from the male gamete are lost, and the flagella of the female gamete propel the zygote. After settling and flagella retraction, the zygote germinates immediately into a new Codium thallus. The gamete thus constitutes the only haploid structure in the life cycle.

Whole plants of C. fragile are able to fix nitrogen, owing to an association between the alga and a nitrogen-fixing bacterium (Azotobacter) on the surface of the alga (Head and Carpenter, 1975). The alga secretes 0.7 to 1.3 mg glucose per gram of dry weight of the alga per hour, or 16% to 31% of the carbon assimilated to the outside of the thallus. The bacterium uses the secreted glucose and in turn fixes the nitrogen. The nitrogen fixation occurs only under conditions of nitrogen deficiency and is probably an important factor in the growth of Codium in shallow bays under oligotrophic conditions.

Codium fragile shows a number of adaptations to its habitat. During winter months when the

182 EVOLUTION OF THE CHLOROPLAST

Fig. 5.44 (a) Caulerpa prolifera. (b) Caulerpa floridana.

(c) Caulerpa microphysa. (d) Bryopsis plumosa. (e) Penicillus capitatus. (f) Udotea conglutinata. (After Taylor, 1960.)

availability of dissolved inorganic nitrogen is at its highest in the water, C. fragile accumulates reserves of nitrogen which are utilized in times of relative nitrogen deficiency (Hanisak, 1979). The

period of maximum carbon fixation, pigment content, and chloroplast size occurs during the early winter when competition from other algae is minimal and variation in tidal amplitude is decreased (Benson et al., 1983). In the summer, the physical environment is more extreme, owing to increased drying in the intertidal zone and

CHLOROPHYTA 183

Fig. 5.45 The life cycle of Codium sp.

increased competition from other algae. Codium fragile effectively retires from much of this competition by undergoing reproduction in the summer. This is accompanied by the development of frond hairs which may increase nutrient uptake.

Symbiotic associations between a number of molluscs and flatworms with chloroplasts of the Codiales are fairly common. Some molluscs (Elysia, Tridachia, Placobranchus) normally feed on siphonaceous Chlorophyceae such as Codium and Caulerpa by puncturing the cells and sucking out the contents. The chloroplasts are not always digested, and many chloroplasts lodge in the body

184 EVOLUTION OF THE CHLOROPLAST

Fig. 5.46 Codium sp. on a scallop.

of the animal and actively photosynthesize (Trench et al., 1969, 1973a,b). In E. viridis, the chloroplasts can remain functional for at least 3 months when the animals are starved in the light. The rates of photosynthesis of chloroplasts intact in Codium and of chloroplasts in Elysia are of the same order (Trench et al., 1973b). Chloroplasts isolated from Codium release only 2% of their fixed carbon into the medium, mainly as glycolic acid. Isolated chloroplasts in animal homogenate release up to 40% of the fixed carbon, mostly as glucose with some glycolic acid. The animal cells obviously cause the symbiotic chloroplasts to release a large amount of their photosynthate, calculated to be at least 36% of the fixed carbon (Trench et al., 1973b).

The plants of Halimeda (Fig. 5.47) consist of calcified segments separated by more or less flexible little calcified nodes. Coenocytic filaments make up the thallus. At the surface of each segment the

filaments are inflated to form utricles which are closely appressed to one another in mature plants and form an unbroken surface separating the intercellular spaces from contact with the outside. The cell walls of the utricles are calcified. Occasionally, Halimeda plants bear clusters of bead-like reproductive structures on branched stalks arising from the surface of the segments. These form biflagellater swarmers, but it is not known whether they are gametes or zoospores.

Two types of plastids exist in Halimeda – amyloplasts and chloroplasts. Both types develop from proplastids (Borowitzka and Larkum, 1974). Actively growing plants of Halimeda may add a segment a day, the newly formed segment being noncalcified and white, containing only amyloplasts (Wilbur et al., 1969). At this stage the utricles have not completely closed, leaving intercellular spaces continuous with the outside medium. After the segment is 36 to 48 hours old, the utricles have closed with one another, and aragonite CaCO3 crystals begin to appear on fibrous material outside the walls of the utricles. By the time that calcification has begun, the segment contains well-developed chloroplasts and is green. The older the segment becomes, the fewer amyloplasts and the more chloroplasts there are present.

Incorporation of radioactive 45Ca is stimulated by light, with calcium incorporation into the thallus showing a diurnal rhythm, being greater during the day than during the night (Stark et al., 1969), even under constant illumination. This difference in calcification rates is reflected in the movement of chloroplasts to the periphery of the segments during the day and away from the periphery during the night. The calcification process appears to be a two-step process: first the ions are bound to the wall, with the consequent increase in their concentration, and then the CaCO3 is precipitated. Halimeda and Penicillus (Fig. 5.44(e)) have lower rates of CaCO3 deposition than the calcified red algae (Goreau, 1963).

Halimeda is particularly successful at colonizing bottom habitats where ambient light intensities are from 10 to 20 times less than at the surface. The Halimedas are exceptional among the calcareous Chlorophyta in that they tend to be more heavily calcified in deep than in shallow

CHLOROPHYTA 185

Fig. 5.47 (a) Halimeda tuna. (b) Fertile segment of

Halimeda. (c) Nodal region of Halimeda opuntia. (d) Central filament of Halimeda discoidea with lateral branches forming outer utricles. ((a) after Taylor, 1960; (c),(d) after Egerod, 1952.)

water. Although this difference may be due to a decreased amount of organic matter rather than an increased calcification, the overall effect is opposite to that of other calcareous green algae such as Penicillus (Fig. 5.44(e)), Udotea (Fig. 5.44(d)), and Rhipocephalus, which are invariably less calcified in deep water. Halimeda often performs the major role in calcification in lagoons. Hoskin (1963) examined the sand of Alacran Reef, Mexico, and found that it consisted of 35% Halimeda, 29% coral, 8% other coralline algae, 8% mollusks, 6% foraminifera, 1% miscellaneous skeletal grains, 9% fecal pellets, and 4% aggregates by volume.

The family Codiaceae is one of the most important groups of rock-building algae, and, in the course of its long history, has been represented by a large number of genera (Johnson, 1961). In extant (living) plants the calcified genera usually have the outer portion calcified, whereas the inner portion is not. This type of calcification

exists in fossil forms, resulting in good preservation in the outer part of the fossil, but with the structural features gradually fading toward the center. A section of Paleocodium from the lower Carboniferous (Fig. 5.48(a)) shows a structure similar to Codium (Fig. 5.46). Restoration of a branch of Ovulites margaritula from the Eocene (Fig. 5.48(b)) results in a structure similar to Halimeda.

Caulerpaceae

The coenocytic plants of this family have two types of plastids – chloroplasts and amyloplasts. Caulerpa (Fig. 5.49) is the only genus in the family and is a common inhabitant of intertidal and infratidal tropical and semitropical marine waters. The plants have a creeping green rhizome with root-like colorless rhizoids and frond-like erect shoots. The erect shoots exhibit a considerable variation in morphology, many times resembling the blades of higher plants, after which some of the species are named. The thallus derives support from turgor pressure and from wall ingrowths, the trabeculae. The walls have a -1,3 linked xylan as the main structural component.

Chloroplasts are prominent in the leaves and rhizome but totally lacking in the rhizoids and the extreme apex of the growing rhizome tip

186 EVOLUTION OF THE CHLOROPLAST

Fig. 5.48 (a) Diagrammatic sketch of a section of the thallus of Paleocodium from the Lower Carboniferous, showing arrangement of branching filaments. (b) Restoration of a branch of Ovulities margaritula from the Eocene. ((a) after Johnson, 1961; (b) after Munier-Chalmas in Johnson, 1961.)

Fig. 5.49 Caulerpa taxifolia, non-

fertile (a) and fertile (b) thalli. (From

Zuljevic and Antollic, 2000.)

and growing blade. Amyloplast distribution is the reverse of chloroplast distribution, large numbers of amyloplasts being present in the rhizoids and blade tip with few amyloplasts in the rhizome and blades. There is a large central vacuole except at the growing tips.

Mature regions of Caulerpa have two systems of protoplasmic streaming: large longitudinal streams in the vacuole and smaller streams oriented 45° to the blade axis in the peripheral cytoplasm. Bundles of microtubules are associated with the cytoplasmic streaming (Sabnis and Jacobs, 1967). The rate of streaming is relatively slow, 3–5 m s 1, as compared to 60 m s 1 for

Nitella.

In sexual reproduction, male and female gametangia are formed on the same frond by migration of the cytoplasm from the rhizomes into the fronds and subsequent cleavage to yield gametes (Fig. 5.49(b)) (Goldstein and Morral, 1970). The large female gametes and smaller males are released in a greenish viscous fluid. After a few minutes the gametes agglutinate in groups of up to 50, followed by separation of pairs and the formation of zygotes. The development of Caulerpa has not been followed beyond zygote formation.

The development of aquaculture, aquariums and international shipping has led to worldwide exposure of marine environments to

CHLOROPHYTA 187

Fig. 5.50 Wounding the coenocytic thallus of Caulerpa taxifolia results in conversion of cytoplasmic caulerpin into oxytoxin 2 by an esterase followed by a dehydration. The reactive sites on oxytoxin 2 immediately cross link cytoplasmic proteins, resulting in the formation of a plug that excludes water from the thallus. The whole process is complete within seconds.

non-indigenous species of algae. More than 60 macroalgal species have been introduced into the Mediterranean Sea (Piazzi et al., 2001; Philips and Price, 2002; Verlaque et al., 2003). Species of Caulerpa are the more aggressive of these introduced algae, quickly overgrowing native algae by the rapid elongation of its stolons (2 cm d 1). The spread of Caulerpa is also due to a unique method of asexual reproduction. No zoospores are formed; instead breakage of the thallus results in new viable plants. On breakage of the coenocytic thallus, the thallus fragments are sealed within seconds by a gelatinous external wound plug that prevents the cytoplasm from coming into contact with seawater (Adolph et al., 2005). The cytoplasm of Caulerpa contains caulerpenyne, which on wounding of the thallus is converted by an esterase into oxytoxin 2 (Fig. 5.50). Oxytoxin 2 is a very reactive 1,4 dialdehyde which

cross links cytoplasmic proteins, producing the wound plug.

These Caulerpa spp. in the Mediterranean Sea may be Red Sea migrants through the Suez Canal or Lessepsian species. The term refers to Ferdinand de Lesseps who designed and led the team that built the Suez Canal. Curiously, there is no similar term for the Panama Canal (which de Lesseps also initially designed). The Panama Canal has a series of higher freshwater lakes which effectively prevent marine species from traveling from one side to the other.

Dichotomosiphonaceae

Dichotomosiphon differs from the rest of the Caulerpales in having oogamous sexual reproduction. The thallus is a dichotomously branched tubular coenocyte bearing colorless rhizoids. Both lensshaped chloroplasts and amyloplasts are present