208 EVOLUTION OF THE CHLOROPLAST

After a resting period, the zygote germinates by splitting the outer zygote wall, with the middle layer of the wall (mesospore) protruding through the fissure in the outer layer. The protoplast has two flagella that beat weakly in the watery interior of the mesospore. After the zoospore is released from the outer zygote wall, the mesospore wall breaks down, leaving the protoplast inside the endospore wall. This protoplast then behaves similarly to a gonidial protoplast and divides to form a young colony of approximately 1000 somatic cells with four gonidia in a single tier.

Sexual type is inherited in a 1 : 1 ratio, and there is no parthenogenetic development of the eggs.

In Volvox carteri, the males make and accumulate a sexual inducer that is a 30-kilodalton glycoprotein (Starr and Jaenicke, 1974). The sexual inducer is released when the sperm are released from the sperm packets. The inducer is effective at 6 10 17 M. One sexual male releases enough inducer to convert all the related males and females in a volume of 1000 liters from asexual to sexual reproduction. Under the moderate growth conditions present in a large stable body of water, it is normally only the males that produce the sexual inducer. However, it is possible to force asexual males and females to make the inducer by subjugating the cells to a high temperature for a period of time. One hour at 42.5 °C is a sufficient heat shock to induce the formation of sexuality in V. carteri asexual males and females. Without the sexual inducer, the asexual males and females will produce asexual gonidia. In the presence of sexual inducer, the males and females produce sperm packets and eggs, respectively. The heat shock response is an adaptation to life in shallow temporary bodies of water where Volvox is often found. In the spring, in such bodies of water there is abundant water and the temperature is relatively low. Volvox grows asexually under these conditions. As summer progresses, the temperature in these bodies of water rises, and the organisms begin to dry out. The increase in temperature shocks the Volvox into producing the sexual inducer and initiating sexual reproduction. This results in the formation of drought-resistant zygospores, which sur-

Fig. 5.75 David Kirk Born 1934 in Clinton, Massachusetts. He obtained his B.Sc. from Northeastern University in 1956 and his Ph.D. from the University of Wisconsin in 1962. From 1962 to 1969 he was at the University of Chicago, eventually becoming Associate Professor. In 1969 he joined the faculty in the Department of Biology at Washington University in St. Louis where he is a tenured professor. Dr. Kirk has been a leading researcher in the cell biology of Volvox.

vive the dry conditions and serve as an overwintering spore. The above-described research by Kirk and Kirk (1986) (Fig. 5.75) has provided an explanation for Powers’s (1908) observation that he had great difficulty finding sexual Volvox in large bodies of water. Powers further noted that “in the full blaze of Nebraska sunlight, Volvox is able to appear, multiply and riot in sexual reproduction in pools of rainwater of scarcely a fortnight duration.” It took another 80 years for Kirk and Kirk to discover the heat-shock phenomenon and to explain Powers’s observations.

Tetrasporales

These algae have immobile vegetative cells that are capable of cell division, unlike those in the Chlorellales or Volvocales. The colonies are non-filamentous, and flagellated cells are formed by many genera. Asexual reproduction occurs via the formation of zoospores, aplanospores, or akinetes. Sexual reproduction is isogamous, by

CHLOROPHYTA 209

Fig. 5.76 The life cycle of Tetraspora gelatinosa. (Adapted

from Klyver, 1929.)

fusion of biflagellate gametes. Almost all the organisms are freshwater.

Conventional wisdom has stated that the Tetrasporales evolved from the Volvocales by loss of motility in the vegetative condition. However, data from small subunit ribosomal DNA indicate the line leading to the Tetrasporales is more primitive than the line leading to the Volvocales (Booton et al., 1998).

Two families will be considered here:

Family 1 Tetrasporaceae: cells with pseudocilia. Family 2 Palmellaceae: cells without

pseudocilia.

Tetrasporaceae

The elongated gelatinous thalli of the Tetrasporaceae have vegetative cells in groups of two to four, with each cell having two pseudocilia. Pseudocilia are longer than flagella but are evidently related to them because the pseudocilia have a normal basal body but an abnormal 9 0 configuration of microtubules near the base of the pseudocilia (Lembi and Herndon, 1966; Wujek, 1968). The number of microtubules lessens and becomes more irregular as the end of the pseudocilium is approached.

Colonies of Tetraspora gelatinosa (Fig. 5.76) are green, amorphous masses with an outer layer of vegetative cells (Klyver, 1929). They are found in quiet freshwater and can be attached or

210 EVOLUTION OF THE CHLOROPLAST

Fig. 5.77 Scanning electron micrographs of Botryococcus braunii. (a) Colony from the wild. (b) Colony showing the cup-shaped mucilaginous bases. (c) Two cells in mucilaginous bases. (d) Mucilaginous base with no cells. (From Plain et al., 1993.)

free-floating. Each cell has a large cup-shaped chloroplast with a central pyrenoid and two pseudocilia. Growth of the thallus results from vegetative division of the cells. In the formation of isogametes, a vegetative cell divides two to three times, resulting in four or eight pyriform gametes, each with an eyespot and a cup-shaped chloroplast. The biflagellate gametes break free from the colonial mucilage and fuse with each other at their anterior ends. The quadriflagellate zygote swims for a while before settling, retracting its flagella, and forming a cell wall. The zygote germinates, forming four or eight aplanospores without pseudocilia. These aplanospores enlarge, and when they have reached the size of vegetative cells, they divide to form daughter cells that have pseudocilia. The aplanospores and their daughter cells are held together by mucilage, and the aggregation makes up the typical thallus of

Tetraspora.

Palmellaceae

Members of the Palmellaceae have their cells united in small gelatinous colonies that are generally amorphous but may be of definite shape. Palmella (Fig. 5.63) is a freshwater alga composed of cells united by a gelatinous matrix forming colonies of indefinite shape. Asexual division involves the formation of zoospores, and sexual reproduction occurs by formation of isogametes.

Botryococcus braunii (Fig. 5.77) is a free-floating colony of indefinite shape within a hyaline or orange envelope. The colonies are composed of radially arranged cells embedded in a tough mucous envelope. It forms water blooms that have been implicated in the death of fish (Chiang et al., 2004). The cells accumulate a large amount of oil in the autumn, which often obscures the cell contents. This alga has been postulated as the cause of the boghead coals (e.g., torbonite) and the oil shales of the Tertiary Period (Blackburn and Temperley, 1936; Cane, 1977; Wolf et al., 1985). If these deposits are examined under a microscope, it is possible to see plant remains that are similar to extant colonies of B. braunii. A hydrocarbon derivative exclusively attributable to Botryococcus comprises 1.4% of a Sumatran petroleum

CHLOROPHYTA 211

Fig. 5.78 The structure of hydrocarbons isolated from

Botryococcus braunii. (Modified from Metzger et al., 1990.)

(Moldowan and Seifert, 1980). The cultivation of the alga has been proposed as a renewable source of liquid hydrocarbon fuel (Wake and Hillen, 1980; Yamaguchi, 1997). The resting stage of living B. braunii contains up to 70% of its dry weight as alkadienes, botryococcenes or lycopadiene (Fig. 5.78) (Metzger et al., 1990). The hydrocarbons are produced primarily during the exponential and linear growth phases. The dense matrix surrounding the cells is impregnated with the hydrocarbons.

Prasiolales

The Prasiolales and the following two orders, the Chlorellales and the Trebouxiales, are closely related and sometimes placed in a separate class, the Trebouxiophyceae (Sherwood et al., 2000; Lewis and McCourt, 2004).

The members of the order are characterized by a stellate chloroplast with a central pyrenoid. These algae occur in a large variety of habitats, including freshwater, marine, and terrestrial habitats (such as concrete walls, rocks, and tree bark (Rindi and Guiry, 2003)). The principal genus, Prasiola, has a unique type of life history, with meiotic divisions in mature sexual thalli resulting in a haploid apex and diploid base.

Prasiola stipitata consists of small, thin, broadly ovate blades appearing as dirty green patches at or above high-tide level, commonly in the spray zone or areas fouled by bird excrement. The cells have

a central stellate chloroplast. The diploid thalli can form either diploid spores or haploid gametes (Fig. 5.79), the spore-forming plants growing higher on the shore than the sexual gameteforming plants (Friedmann, 1959; Friedmann and Manton, 1959).

In the formation of the diploid aplanospore, the vegetative cells in the upper part of the thallus divide, making the upper part of the thallus multilayered. Each cell in this area forms a nonflagellated spore that settles, germinates, and develops into a new diploid plant like the parent (Fig. 5.80).

In the production of gametes, the cells in the upper part of a diploid thallus undergo meiosis, with the subsequent division of the haploid cells resulting in a multilayered upper part of the thallus. The haploid tissue is divided into a patchwork of rectangular darker and lighter areas containing the male and female cells, respectively. The difference in shading is due to the difference in size of the chloroplasts, the larger female cells having the larger chloroplasts. The gametes are liberated after the thalli are wet by the incoming tide. More male gametes are released because of their smaller size and greater production per unit area. The anteriorly biflagellate male gametes swim around the non-motile egg, and one of the male flagella touches the egg. The flagellum fuses with the egg, a step followed by fusion of the bodies of the gametes. The pear-shaped zygote swims vigorously by means of the posteriorly directed remaining male flagellum. At 5 °C, the