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Though its unique features were recognized only recently, Prochloron—the only marine prokaryote that photosynthesizes by using a chlorophyll a+b system like that of land plants—is not a rare alga on many tropical seashores. Like the zooxanthellae of corals, it exists as a symbiont in zones where other algae are sparse or absent. We have found it especially abundant in low-littoral to sublittoral zones around Palau, in the West Caroline Islands, where at least six species of didemnids (colonial ascidians), occupying dissimilar and distinct ecological niches, harbor this prochlorophyte symbiont. Massed colonies of one of these hosts, Trididemnum cyclops, cover a large proportion of the exposed surfaces 1 or 2 m below mean sea level off Kamori island, in a swath several meters wide and several hundred meters long; its biomass maybe conservatively estimated at 0.1 tons (wet weight), of which at least 10 kg is represented by Prochloron. Per unit area, the chlorophyll a and b contents of such areas of symbiotic didemnids, and presumably therefore the productivity of the Prochloron, are comparable to those of, say, a field of grass or the phytoplankton in a column of reasonably fertile water.

Prochloron didemni, a photosynthetic symbiont of ascidians, first attracted attention because of its unusual pigment composition (Lewin and Withers, 1975; Newcomb and Pugh, 1975). It contained chlorophylls a and b but lacked phycobilins (Lewin and Withers, 1975; Thorne et al., 1977), and its ultrastructure revealed a prokaryotic cellular organization (see Swift, this volume). The discovery of Prochloron as a prokaryote containing chlorophyll b came at a time of accumulating evidence in support of endosymbiotic origins of mitochondria and chloroplasts (e.g., Margulis, 1970; Whatley et al., 1979). Although a number of extant models for the prokaryotic ancestor of the red algal chloroplast, namely, the unicellular cyanobacteria, were already recognized, a potential model ancestor for the green algal chloroplast was missing until the discovery of Prochloron. Consequently, many biochemical examinations have been directed toward comparisons between Prochloron and cyanobacteria and between Prochloron and chlorophyte chloroplasts in attempts to evaluate possible evolutionary relationships.

Prochloron is a prokaryotic oxygenic organism whose structure shows affinities with the green algae and higher plants. In particular it posseses Chl b and stacked thylakoids. However recent studies on its r-RNA (Seewaldt, Stackebrandt 1982, Van Valen 1982) do not rule out an acquisition of Chl b independent of that which gave rise to the green algae.

High-performance liquid chromatography (HPLC) was used to separate and quantify chlorophylls a and b as well as major carotenoid pigments present in freeze-dried preparations of diverse Prochloron- didemnid associations and in Prochloron cells separated from host colonies. Both chlorophyll a and b were consistently observed in each association. Chlorophyll a : b ratios ranged from 4.14 to 19.71. Ratio differences reflected species differences among didemnid hosts, which proved consistent over time and space. Generally good agreement was found between ratios determined in isolated cell preparations and in symbiotic colonies (in hospite). These values are 1.5 to 5-fold higher than ratios determined in a variety of eukaryotic green plants. The carotenoids in Prochloron are quantitatively and qualitatively similar to those found in various freshwater and marine cyanophytes from high-light environments. However, Prochloron differs from most cyanophytes in the absence of myxoxanthophyll and related glycosidic carotenoids. The consistent presence of chlorophyll b and individuality in carotenoid pigmentation render Prochloron biochemically distinct from cyanophytes, despite the fact that cells of both prokaryotic groups are often found in light-saturated environments.

Cyanobacteria (blue-green algae, blue-greens) are prokaryotic organisms that contain a photosynthetic apparatus similar in structure and function to that present in the chloroplast of the phototrophic eukaryotes (Stanier, 1977). They differ from Prochloron, a prokaryote that contains chlorophyll b instead of phycobiliproteins as light-harvesting pigments. They differ from the purple and green bacteria, particularly because they carry out oxygenic photosynthesis, but also because of differences in the ultrastructure, in the chemical composition of the photosynthetic apparatus, and in nutritional requirements and growth physiology. The mechanism of cyanobacterial photosynthesis is identical to that of photosynthetic eukaryotes. Accordingly, in this discussion of oxygenic prokaryotic photosynthesis, only those areas where cyanobacteria played a role in the understanding of the process or only those details which are characteristic of blue-greens will be reviewed.

Freeze-fracture replicas of the photosynthetic prokaryote Prochloron sp., collected at Coconut Island, Hawaii, show that the thylakoids are differentiated into stacked and unstacked regions much like the thylakoids of green algae and higher plants. On the exoplasmic (E) fracture face, the particle density is greater in stacked regions (≈3100 particles/μm 2 ) than in unstacked regions (≈925 particles/μm 2 ). On the complementary protoplasmic (P) fracture face, the particle density is lower in stacked regions (≈2265 particles/μm 2 than in unstacked regions (≈4980 particles/μm 2 ). The histogram of the diameters of E face particles in unstacked regions shows a single broad peak centered at 80 Å. In stacked regions, four distinct peaks, at 75, 105, 130, and 160 Å, are observed. These size classes are virtually identical to those found on E faces of thylakoids of the green alga Chlamydomonas and of greening pea chloroplasts. In the latter systems, the different size classes of E face particles are believed to represent photosystem II units surrounded by variable amounts of chlorophyll a/b light-harvesting complex. We propose that the same interpretation applies to the thylakoids of Prochloron , which contain a similar chlorophyll a/b complex. Our results add to the evidence supporting the view of the chlorophyll a/b complex as both a light-harvesting complex and a membrane adhesion factor. The similarity of the architecture of the thylakoids of Prochloron to that of green algal and plant chloroplasts also provides additional evidence of an evolutionary relationship between Prochloron and the chloroplasts of green plants.

The relationship of Prochloron sp. isolated from four different didemnid ascidian hosts, namely Lissoclinum patella, Lissoclinum voeltzkowi, Diplosoma virensand Trididemnum cyclops was elucidated by comparative analysis of their 16S ribosomal ribonucleic acid (RNA). The oligonucleotide catalogues of the 16S rRNA obtained are almost identical, indicating a very close relationship among the prochlorophytes investigated. Phylogenetically Prochloron is a member of Cyanobacteriales.

Laboratory cultures have been established of the didemnid symbiont Prochloron, a unique prokaryotic alga that synthesizes chlorophylls a and b but no phycobilin pigments. Cell division in Prochloron cultures occurs under acidic conditions (pH 5.5) in the presence of tryptophan. The alga is a naturally occurring tryptophan auxotroph that survives in nature by close association with the host, Diplosoma similis. The metabolic dysfunction that renders Prochloron auxotrophic may involve only the initial step of the tryptophan biosynthetic pathway.

IN the rich biota of reef communities, one of the best known symbiotic relationships is that of dinoflagellates known as “zooxanthellae” with corals and giant clams1–3. In contrast, the presence of algae in ascidians (sea squirts: Phylum Chordata, Subphylum Tunicata), although known for many years, has been studied very little. It is known that the association is confined to tropical ascidians in the family Didemnidae, but even the phylum to which the algae belong, or indeed whether the green cells in question are algae at all, has remained uncertain. During a recent expedition of the RV Alpha Helix to the Great Barrier Reef, we encountered several species of colonial asdidians containing large numbers of bright green, spherical cells. We have established by optical and electron microscopy that these green cells are blue-green algae; their association with primitive chordates represents a considerable extension of the known host range of these prokaryotes.

UNICELLULAR algae associated with ascidians from tropical Pacific shores have been reported by various biologists1–5. They are bright green, generally spherical and about 10–20 µm in diameter, and they seem to have no clearly delimited nucleus or plastids. Such cells (identified as Synechocystis didemni), found associated with surfaces of Didemnum colonies on the Pacific coast of Mexico, have been shown by electron microscopy to be prokaryotic6,7, which suggests that they are cyanophytes, that is, blue-green algae. Although all known blue-green algae (other than a few apochlorotic types) contain phycoerythrin, phycocyanin, or both, however, these ascidian symbionts are apple green and contain no detectable bilin pigments. Furthermore, like the eukaryotic algae in the divisions Chlorophyta and Euglenophyta, they contain two chlorophyll components, separable by chromatography and provisionally identifiable as chlorophylls a and b (ref. 8), whereas no cyanophytes are known to contain chlorophyll b. The assignment of S. didemni to any of the established algal divisions, therefore, presents a major taxonomic problem.

Since the prokaryotic, green marine alga Prochloron has not, as yet, been cultured, lyophilized cells were used in a microadaptation of polyacrylamide gel electrophoresis (PAGE) in order to isolate the glucosyltransferase isozymes. The pattern obtained with these capillary gels was identical with those of cyanophytes. Besides two phosphorylase and synthase isozymes, three branching isozymes of the b.e. type were found to be present.

Genotypic relationships between seven Prochloron samples isolated from different didemnid ascidian hosts collected at the Palau archipelago and Munda (Solomon Islands) and one cyanobacterial (Synechocystis) strain were determined by DNA-DNA reassociations. Thermal stability values of DNA-DNA hybrids indicate that all Prochloron samples involved are mutually very closely related and only slightly related with the Synechocystis strain. It is concluded that the Prochloron samples tested are representatives of one and the same species.

Prochloron: A Microbial Enigma. Edited by Ralph A. Lewin and Lanna Cheng. Chapman and Hall: 1989. Pp. 129. £42, $53.

In the symbiotic association between the prokaryotic green alga Prochloron and three didemnid host species (Diplosoma similis, Lissoclinum bistratum, Trididemnum cyclops), between 6 and 51 % of the total carbon fixed during exposure for 1 h to H14CO3- in the light (150 �E m-2 s-1) becomes associated with the host tissue. Dark fixation of 14CO2 in these ascidian species and in Lissoclinum punctatum never exceeds 6% of photosynthetic fixation at saturating light intensity. The corresponding values for dark fixation of 14CO2 in isolated Prochloron cells fall within the same range. There is very little excretion of photosynthate from whole colonies of the above ascidian species nor from Didemnum molle, Lissoclinum voeltzkowi and Trididemnum miniatum (usually less than 1 % of total photosynthate at saturation light intensity), suggesting an efficient transfer mechanism from Prochloron to host. Evidence from pulse-chase experiments suggests that transfer probably involves the early products of photosynthesis. The extent of transfer of photosynthate between Prochloron and T. cyclops varies with the rate of photosynthetic 14CO2 fixation into the whole colony but there is some transfer even at low light intensities, which strongly limit photosynthesis.

The microscopic algae contained within the cloacal cavity of the ascidian Diplosoma virens have a high photosynthetic capacity [up to 5.3 �gC (�g chl a)-1 h-1], giving thecolony a high photosynthesis: respiration ratio (maximum 8.0-8 5). Photosynthesis is saturated at a quantum flux density of 120 × 1015 quanta cm-2 s-1 (400-700 nm) and occurs at its maximum at temperatures between 30 and 35� C. The photosynthetic capacity is inhibited by chloramphenicol but not by cycloheximide. This is interpreted as indicating an affinity of these algae with the prokaryotes rather than the Chlorophyta, in spite of their possession of chlorophyll b (chlorophyll a : b ratio = 6.0) as an accessory pigment. In this and other respects the algae resemble those (Prochloron) described from other didemnid ascidians. Use of a buffered extraction medium has allowed separation from the animal tissue of photosynthetically active algal cells.