The chloroplast pigments of the algal classes Eustigmatophyceae and Xanthophyceae. I. Eustigmatophyceae

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1 British Phycological Journal ISSN: (Print) (Online) Journal homepage: The chloroplast pigments of the algal classes Eustigmatophyceae and Xanthophyceae. I. Eustigmatophyceae S.J. Whittle & P.J. Casselton To cite this article: S.J. Whittle & P.J. Casselton (1975) The chloroplast pigments of the algal classes Eustigmatophyceae and Xanthophyceae. I. Eustigmatophyceae, British Phycological Journal, 10:2, , DOI: / To link to this article: Published online: 17 Feb Submit your article to this journal Article views: 448 View related articles Citing articles: 27 View citing articles Full Terms & Conditions of access and use can be found at

2 Br. phycolj, lo: June 1975 THE CHLOROPLAST PIGMENTS OF THE ALGAL CLASSES EUSTIGMATOPHYCEAE AND XANTHOPHYCEAE. I. EUSTIGMATOPHYCEAE By S. J. WHITTLE and P. J. CASSELTON Department of Botany, Birkbeck College, London WC1E 7HX Analysis of the chloroplast pigments of Pleurochloris commutata Pascher, Pleurochloris magna Boye Petersen, Polyedriella helvetica Vischer et Pascher and Vischeria stellata (Poulton) Pascher by several chromatographic methods has confirmed that they all contain violaxanthin as their major xanthophyll pigment. All had previously been assigned to the new algal class, the Eustigmatophyceae. Monodus subterraneus Boye Petersen may also belong in this class. Comparative studies have shown that the eustigmatophycean algae have different pigments from those found in Pleurochloris meiringensis Vischer, Mischococcus sphaerocephalus Vischer and Tribonema aequale Pascher which had previously been placed in the Xanthophyceae sensu stricto. The xanthophycean algae contain diadinoxanthin as their major xanthophyll and do not contain violaxanthin. It appears that it is possible to correctly assign an alga to the Chlorophyceae, Eustigmatophyceae or Xanthophyceae sensu stricto simply by examination of the absorption spectrum of the unseparated pigment extract. While considerable progress has been made in the study of algal pigmentation, the identification of those pigments characteristic of the Xanthophyceae has proved to be unexpectedly difficult. Two independent reports (Whittle & Casselton, 1969; Stransky & Hager, 1970a) that some but not all yellow-green algae contain violaxanthin as a major component provided an indication that variations in results obtained by different workers perhaps arose partly from the traditional Xanthophyceae comprising more than one major group of algae. This possibility was confirmed when Hibberd & Leedale (1970), as a result of detailed fine structural studies (Massalski & Leedale, 1969; Hibberd & Leedale, 1971a, 1972), proposed division of the Xanthophyceae into two classes, Xanthophyceae sensu stricto and Eustigmatophyceae. The chloroplast pigments characteristic of the Eustigmatophyceae are discussed in this paper. MATERIALS AND METHODS TABLE I. Algal species used and strain number Species Strain No. Mischococcus sphaerocephalus Vischer 847/1 Monodus subterraneus Boye Petersen 848/1 Pleurochloris commutata Pascher 860/1 Pleurochloris magna Boye Petersen 860/2 Pleurochloris meiringensis Vischer 860/3 Polyedriella helvetica Vischer et Pascher 861/1 Tribonema aequale Pascher 880/1 Vischeria stellata (Poulton) Pascher 887/2 The algae (Table I) were obtained in pure culture from The N.E.R.C. Culture Centre of Algae and Protozoa, 36 Storey's Way, Cambridge CB30DT. Methods for growth and harvesting of cultures, preparation of saponified and unsaponified pigment extracts and separation and 179 Published online 17 Feb 2007

3 180 S. J. WHITTLE AND P. J. CASSELTON characterisation of pigments have been described previously (Whittle & Casselton, 1969; Whittle, 1973). Although pigments were routinely separated by thin-layer chromatography (TLC), column chromatographic separations were also performed because the results of some previous workers using column chromatography appeared to contradict those obtained using TLC. Individual pigments were quantitatively estimated by a spectrophotometric method following the procedures of Hager & Meyer-Bertenrath (1966). The values of the extinction coefficients tt:lcrn~ zrl %-~ used in the calculations are shown (Table II). TABLE II. The extinction coefficient, E~m, for some pigments Pigments Solvent ~1% ~max References ~lcm (nm) B-Carotene Chloroform Gillam, 1935 Lutein Ethanol Zscheile et al., 1942 Neoxanthin Ethanol Davies, 1965 Violaxanthin Ethanol Goodwin, 1955 Other carotenoids Ethanol 2500* Amax Goodwin, 1955 Chlorophyll a Acetone MacKinney, 1940 diethyl ether Comar & Zscheile, 1942 Chlorophyllide a Acetone ") Calculated from data of Brown, 1968; and Phaeophytin a Acetone ) MacKinney, 1940 Chlorophyll b Acetone MacKinney, 1940 diethyl ether Comar & Zscheile, 1942 ~1% value was used when the actual extinction coefficient was not * This approximate ~lem available. The total amount of carotenoids in extracts from which the chlorophyll had been removed by saponification were calculated approximately from the E value (absorbance) at A max; the value ~ vl t~l% ~lcm for the carotenoid mixture being taken as 2500 (Goodwin, 1955). The concentration of chlorophyll a and total carotenoids were easily estimated in total pigment extracts of eustigmatophycean algae. Chlorophyll a was estimated from the E value at the ~ max in the red region (663 nm) where carotenoid absorption is negligible, and total carotenoid was calculated from the E value at 450 nm (in acetone) where carotenoids strongly absorb but chlorophyll a absorption is negligible. RESULTS IDENTIFICATION OF EUSTIGMATOPHYCEAN YELLOW-GREEN ALGAE When the absorption spectra of the total pigment extracts of seven yellowgreen algae were examined, four species were found to have a carotenoid peak at 469 nm (Fig. 1). These included species previously shown to contain violaxanthin (Whittle & Casselton, 1969). The remainder had their carotenoid peak at a higher wavelength, 472 nm, and these included two species previously shown not to contain violaxanthin (Fig. 1). This consistently observed property of the total absorption spectra reflects the difference in xanthophyll composition of the Eustigmatophyceae and Xanthophyceae and serves as a useful and easy means of classifying previously unexamined algae. PIGMENTS OF PLEUROCHLORIS COMMUTA TA Separation of pigments The total pigment extracts contained about 19 mg chlorophyll a and 7 mg

4 Pigments of the Eustigmatophyceae 181 I 1 I I I I A Z-O ~ ~,. O.C I I i i i I i i B I I I Wavelength (nm) FIG. 1. Comparison of the total absorption spectra (in diethyl ether) of the pigments of algae in the Eustigmatophyceae (A) and Xanthophyceae (B). 1, Monodus subterraneus; 2, Pleurochloris magna ; 3, Fischeria stellata ; 4, Pleurochloris commutata ; 5, Pleurochloris meiringensis ; 6, Mischococcus sphaerocephalus ; 7, Tribonema aequale. carotenoids g-t dry wt of alga. Separation of pigments was achieved by preparative adsorption thin-layer chromatography and by stepwise elution column chromatography. The results obtained using the TLC method of Riley & Wilson (1965) are shown in Table III for extracts chromatographed before and after saponification. Essentially similar results were obtained when Kieselgel G +

5 Major peaks are shown in italics. * = inflection. 1. Pigments identified by co-chromatography with authentic samples. 2. & 3. Maxima measured in chloroform and acetone respectively. 4. Pigments not always separated from pigment extracts presumably because of their relatively low concentration. 5. Pigments would be masked by chlorophyll a on chromatograms of unsaponified extracts. 6. & 7. Tentative identifications based on absorption maxima and sorbability. 8. Cis isomers of violaxanthin are normally present in trace amounts (Stransky & Hager, 1970a): the saponification process increases their concentration. 9. Stransky & Hager (1970a) have identified this pigment as neoxanthin. 10. Furanoids sometimes formed from the parent pigments during the application of the pigment extract to the silica gel layer prior to chromatography. TAaLE III. Adsorption thin-layer chromatography of the pigments of Pleurochloris commutata. Absorption maxima and RF values of pigments separated, from both saponified and unsaponified methanolic extracts on 1 mm layers of Kieselgel G using as solvent a mixture (12:30:58 by vol.) of diethylamine, ethyl acetate and petroleum ether (b.p C) Pigment Mean Adsorption maxima (nm) Separated from saponified (S) R~ 100 in ethanol in ethanol/ha and/or unsaponified (U) extracts Pigments regularly found 1 D-Carotene Vaucheriaxanthin-ester Free Vaucheriaxanthin Violaxanthin Chlorophyll a Minor pigments sometimes found 4 Cryptoxanthin epoxide 6? Unknown carotenoid Zeaxanthin 7? Cis-violaxanthin Unknown monoepoxy xanthophyll 9 Furanoid derivatives 10 sometimes found Furanoid oxide of vaucheriaxanthin-ester Furanoid oxide of free vaucheriaxanthin Luteoxanthin (furanised violaxanthin) Auroxanthin (furanised luteoxanthin) ", 447, 472* (431", 458, 485) 2 S & U , 442, *, 421, 449 U 7 418, 441, , 421, 450 S , 439, , 400, 424 S & U 74 (377, 411,428, 531,579, 616, 664) 3 U , 447, 475* 403", 424, 444 S~ , 446, 479* Not measured S , 450, 477 No change S & U , 412, 435, , 399, 423 S , 439, , 420, 448 S & U , 421, 449 No change U , 422, 448 No change S , 424, , 401, 424 S & U , 401, 425 No change S & U

6 Pigments of the Eustigmatophyceae 183 MgO (1 -k 1 by wt) or Aluminium Oxide G (Merck) replaced the usual Kieselgel G stationary phase and when diethyl ether:petroleum ether (b.p C): acetone (3:2:1 by vol.) was used as developing solvent. Confirmation of the presence of the major carotenoids was obtained by separation of a saponified extract on a column of ZnCO3 -k Celite (Table IV). TABLE IV. Stepwise elution column chromatography of the carotenoids of Pleurochloris commutata. The pigment extract (after saponification) was chromatographed on ZnCO3 + Celite (3 + 1, by wt) using petroleum ether (b.p C) as the initial eluent followed by aliquots of this solvent containing an increasing amount of acetone (0"5, 1, 2, 4, 8, 16, 32, 64, 100% by vol.). Finally, aliquots of 5 % and 10% methanol in acetone were used. The fractions are listed in order of increasing adsorptive power Absorption maxima (nm) Amount of pigment in ethanol and recovered Fraction (in ethanol/hcl) Identification (~tg/g dry wt) 1 423*, 449, 474 ~-carotene (no change) 2 424, 448, 475 cryptoxanthin 30 (400", 425, 448) epoxide? z 3 425, 449, 475 zeaxanthin?3 158 (no change) 4 404", 427, 450, 474 partly furanised 998 (378, 399, 423) violaxanthin , 442, 470 vaucheriaxanthin 5 > 294 (397, 420, 447) Major peaks are shown in italics. * = inflection. 1. Fraction 1 was identified by co-chromatography with authentic l~-carotene from the Sigma Chemical Co. 2. Fraction 2 contained a very small amount of an epoxy xanthophyll which could be cryptoxanthin epoxide. 3. Fraction 3 could be zeaxanthin (Stransky & Hager, 1970a). 4. Fraction 4, which consisted of violaxanthin and a small amount of luteoxanthin derived from it by furanisation, was identified by co-chromatography with authentic violaxanthin separated from Urtica dioica. 5. Fraction 5 was identified by co-chromatography with vaucheriaxanthin separated from Tribonema aequale. Examination of separated pigments The major pigments found in Pleurochloris commutata were B-carotene, chlorophyll a and the xanthophylls vaucheriaxanthin ester and violaxanthin, their identity being confirmed by co-chromatography with authentic pigments. In particular, using layers of either Kieselgel G or Aluminium Oxide G and eight different developing solvents, it was impossible to separate authentic violaxanthin, obtained from several sources including both green algae and angiosperm leaves, from the diepoxy pigment with an identical absorption spectrum found in P. commutata. Thus there is strong evidence that the P. commutata pigment is violaxanthin. In addition, using the method of Kleinig & Egger (1967), the three vaucheriaxanthin-esters of P. commutata were found to be identical with those of Tribonema aequale. Several minor xanthophyus, which only ever occurred in low concentrations, were also isolated from P. commutata. These included an epoxide which might correspond to neoxanthin, an unidentified weakly sorbed carotenoid, and pigments which could be cryptoxanthin monoepoxide and zeaxanthin. Isomerisation of violaxanthin during chromatography sometimes produced detectable amounts of its cis isomer.

7 184 S. J. WHITTLE AND P. J. CASSELTON PIGMENTS OF MONODUS SUBTERRANEUS, PLEUROCHLORIS MAGNA, POL YE- DRIELLA HELVETICA AND VISCHERIA STELLATA Separation by column chromatography D-Carotene, vaucheriaxanthin and violaxanthin were the main carotenoid pigments obtained by stepwise elution column chromatography of saponified extracts of Pleurochloris magna and Vischeria stellata. Whilst violaxanthin was the most abundant carotenoid, some minor xanthophylls were also isolated but none of these was positively identified. Comparable results were obtained by zone chromatography on sucrose columns (Tables V and VI), and the pigments of Polyedriella helvetica appeared to be identical to those of the other algae. An unsaponified pigment extract of V. stellata was also separated by gradient elution column chromatography on ZnCOa because both Allen et al. (1964) and Thomas & Goodwin (1965), using this technique, did not find violaxanthin in a Vischeria sp. The results (Table VII) correlated with the separations on columns of MgO, sucrose and Ca(OH)2. Separations by thin-layer chromatography Separation of the total pigments ofpleurdchloris magna, Polyedriella helvetica, Monodus ~ubterraneus and Vischeria stellata by TLC showed that these four algae have identical major chloroplast pigments, namely chlorophyll a, ~- carotene, vaucheriaxanthin-ester and violaxanthin, the latter constituting approximately one half of the total carotenoids in both Monodus subterraneus and Vischeria stellata (Table VIII). Saponification, which removed chlorophyll, converted vaucheriaxanthin-ester into its strongly adsorbed free xanthophyll (RF = 0.07). Otherwise the separations of extracts after saponification correlated well with the separations of total pigments. Six different TLC systems, based on Aluminium Oxide G, Kieselgel G and mixtures of these two adsorbents, produced comparable separations with various developing solvents. Four minor xanthophylls were sometimes detected (possibly cryptoxanthin in V. stellata, cryptoxanthin epoxide in Pleurochloris magna and Polyedriella helvetica, zeaxanthin in V. stellata and Polyedriella helvetica, and an unknown, strongly adsorbed monoepoxide in all four algae). The separations obtained by thin-layer chromatography correlated well with the column chromatograms previously described. Further extensive use of cochromatography showed that the diepoxy major xanthophylls of Pleurochloris commutata, Pleurochloris magna, Monodus subterraneus and Vischeria stellata are identical to one another and to authentic violaxanthin. Co-chromatography of the strongly adsorbed monoepoxides of Monodus subterraneus and Vischeria stellata indicated that they are identical, but this neoxanthin-like xanthophyll was not identified, although chromatographic systems were found which could separate it from authentic samples of heteroxanthin, neoxanthin and free vaucheriaxanthin. Similarly, the other minor xanthophylls were not positively identified by co-chromatography. The use of comparative reversed phase partition chromatography indicated that the three vaucheriaxanthin-esters are identical in Pleurochloris commutata, Monodus subterraneus, Tribonema aequale and Vischeria stellata.

8 TABLE V. Stepwise elution column chromatography of the carotenoids ofpleurochloris magna. The pigment extract (after saponification) was chromatographed on a column of MgO -t- Celite (2 + 1, by wt) using petroleum ether (b.p C) as the initial eluting solvent and 4 ~ methanol in acetone as the final solvent. The fractions are listed in order of increasing adsorptive power of total Absorption maxima (nm) in: Fraction Pigment carotenoids Eluent recovered Eluent Ethanol Ethanol/HCl 1 ~-Carotene ~ 25.7 Pet. ether 424, 448, Cryptoxanthin monoepoxide? ~ acetone 418, 443, , 443, *, 425, Violaxanthin 1 trans- 40'1 32~ acetone 416, 439, , 401, 426 cis- 413, 437, , 437, , 400, Unknown monoepoxide ~ acetone 418", 440, , 441, , 425, Vaucheriaxanthin 1 trans Acetone 418, 443, , 441, , 423, 451 ClS- 418, 440, , 439, , 423, Unknown monofuranoid oxide 3 trace 4~ methanol 400, 423, , 423, , 423, 450 Major peaks are shown in italics. * = inflection. 1. Identity confirmed by co-chromatography. 2. Tentative identification based on absorption maxima and sorbability. 3. Possibly derived from vaucheriaxanthin.

9 TABLE VI. Zone chromatography of the carotenoids of Vischeria stellata. Zone chromatograms of unsaponifiable pigments of two different cultures of Vischeria stellata produced on columns of sucrose using 0-5 % r~-propanol in petroleum ether (b.p C) as the developing solvent Zone t Carotenoid V. stellata Culture 1 Absorption maxima (nm) in: Ethanol Ethanol/Ha % of total 1I. stellata Culture 2 Absorption maxima (nm) in: % of total carotenoids Ethanol Ethanol/HCl carotenoids recovered ~ recovered 1 13-Carotene (421,* 448, 474) (422*, 445, 472) Cryptoxanthin? 443, , ", 447, Violaxanthin 418, 439, , 399, , 440, , 399, Vaucheriaxanthin 418, 440, , 421, , 440, ,420, Unknown monoepoxide 417, 439, , 420; not detected Major peaks are shown in italics. * = inflection. The two cultures yielded slightly different absorption maxima for both the /3-carotene and cryptoxanthin zones. This was probably due to variable contamination of the zones. 1. Zones are listed in order of increasing adsorptive power. 2 & 3. % recovery of carotenoids was 87 and 58 % respectively. 4. Absorption maxima in diethyl ether. 5. A small amount of a pigment, possibly zeaxanthin (maxima in ethanol and ethanol/hcl = 422, 488, 473), was separated from this violaxanthin by TLC. When the carotenoids were separated on a column of Ca(OH)2 with 4% acetone in petroleum ether (b.p C)a discrete "zeaxanthin" band appeared between cryptoxanthin and violaxanthin.

10 TASLE VII. Gradient elution chromatography of the carotenoids of Vischeria stellata. The pigment extract (after saponification) was separated on a column of ZnCO8 + Hyflo Supercel (3 + 2, by wt), using as solvent petroleum ether (b.p C) containing progressively increasing amounts of acetone Fraction t Carotenoid Time of Mean conc. Absorption maxima (nm) in ethanol, and Vo of total carotenoids elution (min) of eluent (in ethanol/hcl) recovered 1 13-Carotene 6-10 Pet. ether 420*, 445, Cryptoxanthin? % acetone 425, 448, (no change) 3 Violaxanthin a % acetone 416, 439, (378, 400, 424) 4 Vaucheriaxanthin % acetone 419, 441, (399, 421, 447) 5 Unknown monoepoxide % acetone 418, 441, 468 1"9 (398, 420, 447) Major peaks are shown in italics. * = inflection. 1. Fractions are listed in order of increasing adsorptive power. 2. Measured in petroleum ether (b.p C). 3. The last portion of the violaxanthin fraction had absorption maxima of 410, 434, 464, and was probably the cis isomer. 4. The last portion of the vaucheriaxanthin fraction had absorption maxima of 417, 439, 469 and was probably the cis isomer. 5. Pigment can be separated from neoxanthin by co-chromatography.

11 d TABLE VIII. Adsorption TLC of the total pigments of Pleurochloris magna, Polyedriella helvetica, Monodus subterraneus and Vischeria stellata. Absorption maxima (nm), RF values and quantitative estimates of the pigments separated from methanolic extracts of Pleurochloris magna, 9 Polyedriella helvetica, 9 Monodus subterraneus and Vischeria stellata, by TLC on 1 mm layers of Kieselgel G using as solvent petroleum ether (b.p C): ethyl acetate: diethylamine (58: 30:12 by vol.) Pleurochloris magna Polyedriella helvetica Monodus subterraneus Vischeria stellata Mean Abs. max. Mean Abs. max. Mean Abs. max. ~ of total Mean Abs. max. ~ of total RF in ethanol RF in ethanol RF in ethanol carotenoids RF in ethanol carotenoids Pigment ( (in ethanol/hcl) ( (in ethanol/hc1) ( (in ethanol/hc1) recovered 5 ( (in ethanol/hc1) recovered s 1oo) loo) loo) loo) B-Carotene ", 449, 474 (No change) Vaucheriaxanthinester , 442, 471 (400, 422, 450) 1 ~ Violaxanthin , 439, 469 (379, 400, 425) Chlorophyll a ", 414, , 618, 6664 Cryptoxanthin epoxide? or 76? 2 z cryptoxanthin -- (402, 426, 451) Unknown epoxide 8?? 469 (401, 423, 450) Furanoid of , 424, 450 vaucheriaxanthin- (No change) 33 ester Auroxanthin , 400, 425 (No change) Luteoxanthin , 423, 449 (379, 400, 425) 92 Not measured , 442, 471 (Not measured) , 439, 469 (Not measured) ", 416, , , 439, 469 (Not measured) , 422, 446 (Not measured) , 449, 474* 7 (No change) , 442, , 421, , 439, (379, 400, 425) 74 Not measured , 440, (399, 419, 446) *, 400, 425 (No change) , 421,447 (379, 400, 425) *, 449, 474 e , 442, (400, 422, 449) , 439, (379, 400, 424) , 411, , 616, , 44L (No change) , 43~ (399, 418,446) , 422, 450 (No change) , 425, 449 (379, 400, 424) Major peaks are shown in italics. * = inflection. 1. Pigments regularly found. 2. Traces of pigments detected on some chromatograms. 3. Pigments sometimes found as a result of furanisation of vaucheriaxanthin-ester and violaxanthin. 4. Measured in methanol ~ of the carotenoids recovered , 485, in chloroform. 7. Measured in acetone % of the carotenoids recovered. 9. Quantitative data not available.

12 Pigments of the Eustigmatopbyceae 189 DISCUSSION All of the early investigations of the pigment composition of the yellow-green algae resulted in erroneous identifications of some or all of the xanthophylls (Kuhn & Brockman, 1932; Carter, Heilbron & Lythgoe, 1939; Seybold, Egle & Htilsbruch, 1941 ; Heilbron, 1942; Strain, Manning & Hardin, 1944; Jamikoru, 1954; Strain, 1958; ~est~ik, 1963; Allen et al., 1964; Thomas & Goodwin, 1965; Mattox & Williams, 1965; Chapman & Haxo, 1966). This is hardly surprising as recent work with modern techniques has also led to controversy. However, more curious is the fact that, in studies prior to 1966, all yellow-green algae, irrespective of whether they would now be classified as Xanthophyceae sensu stricto or Eustigmatophyceae, were reported as having identical pigments (Seybold et al., 1941; Strain, 1958; ~est~ik, 1963; Thomas & Goodwin, 1965; Mattox & Williams, 1965). The work of Whittle & Casselton (1969) and the present study clearly show that there are two pigment groups of yellow-green algae, those resembling Pleurochloris commutata in having violaxanthin as the major xanthophyll and those resembling Tribonema aequale in having diadinoxanthin as the major xanthophyll and no violaxanthin. Strong supporting evidence for two pigment groups within the yellow-green algae was provided by Stransky & Hager (1970a) and Guillard & Lorenzen (1972). More significantly, Hibberd & Leedale (1970, 1971b, 1972), on the basis of detailed cytological and ultrastructural studies, separated certain taxa from the Xanthophyceae as a new algal class the Eustigmatophyceae. It is interesting that the diadinoxanthin-types are left in the Xanthophyceae sensu stricto and the violaxanthin-types have been moved into the Eustigmatophyceae (Table IX). Three algae were examined in the present study, in addition to those studied by Whittle & Casselton (1969). Pleurochloris meiringensis and Vischeria stellata were both found to contain the carotenoid complement expected on the basis of Hibberd & Leedale's (1970, 1971b) classification, Pleurochloris meiringensis (Xanthophyceae sensu stricto) is a diadinoxanthin-type and Fischeria stellata (Eustigmatophyceae) is a violaxanthin-type. Monodus subterraneus was found to be a violaxanthin-type and, on the basis of a preliminary structural investigation, Dr D. J. Hibberd (personal communication) has suggested that this alga also probably belongs in the Eustigmatophyceae although it lacks some of the morphological features which are typical of the class. It seems probable that the Eustigmatophyceae and the Xanthophyceae will be found to differ in biochemical characteristics other than pigmentation. It has already been shown that the polysaccharides of Monodus subterraneus show considerable differences to those of Tribonema aequale (Beattie & Percival, 1962; Cleare & Percival, 1972) and Hibberd & Leedale (1972) have pointed out that vesicles with unusual lamellate contents both surround the pyrenoid and occur freely in the cytoplasm in the Eustigmatophyceae. In addition to chlorophyll a and [3-carotene, the xanthophylls vaucheriaxanthinester and violaxanthin have been found in large amounts in all species of the Eustimatophyceae studied in recent years (Table IX). Other xanthophylls have been found in some of the species but usually in small amounts. According to Stransky & Hager (1970a) these include vaucheriaxanthin, neoxanthin, antheraxanthin and zeaxanthin. In fact the presence of the latter two is to be expected

13 TABLE IX. Sub-division of yellow-green algae into two groups having different xanthophyll compositions Major xanthophyll Algae Based on the work oft Hibberd and Leedale's classification* DIADINOXANTHIN (formerly identified as antheraxanthin; 1, 2,3) VIOLAXANTHIN Botrydiopsis alpina Vischer Botrydium granulatum (L.) Greville Bumilleria sicula Borzi Bumilleriopsis filiformis Vischer Heterococeus caespitosus Vischer Heterothrix debilis Vischer Mischococcus sphaerocephalus Vischer Pleurochloris meiringensis Vischer Tribonema aequale Pascher T. minus West T. viride Pascher Vaucheria sessilis (Vaucher) de Candolle V. terrestris Lyngbye Vaucheria spp. Monodus subterraneus Boye Petersen Pleurochloris commutata Pascher P. magna Boye Petersert Polyedriella helvetica Vischer et Pascher Vischeria stellata (Poulton) Pascher 6 X 1,4,6 X 6 X 6 X 6 X 6 X 3, 7 X 7 X 2, 3, 5, 6, 7 X 2 X 2 X 1,4 1,4 1,5 7 3,6,7 E 3, 7 E 3,7 E 7 E *X = Xanthophyceae sensu stricto, E = Eustigrnatophyceae. treferences: 1. Kleinig & Egger, 1967; 2. Falk& Kleinig, 1968; 3. Whittle & Casselton, 1969; 4. Egger et al., 1969; 5. Strain et al., 1968, 1970; 6. Stransky & Hager, 1970a; 7. Whittle & Casselton, this paper. Interpretation of the data of Guillard & Lorenzen (1972) suggests that they found diadinoxanthin in Botrydium becherianum Vischer, Ophiocytium majus Naegeli, Tribonema aequale and Vaucheria sessilis, and violaxanthin in Pleurochloris magna. Hibberd & Leedale (1970) also placed Chlorellidium tetrabotrys Vischer et Pascher, Chloridella neglecta Pascher, Ophiocytium majus and Sphaerosorus composita Moewus in the Xanthophyceae and Ellipsoidion acuminatum Pascher, Chlorobotrys regularis (West) Bohlin and Vischeriapunctata Vischer in the Eustigmatophyceae.

14 Pigments of the Eustigmatophyceae 191 as these are derived from violaxanthin by a reversible light-induced deepoxidation (Stransky & Hager, 1970a). In the present work, a neoxanthin-like pigment and either cryptoxanthin or cryptoxanthin monoepoxide were found in certain cases whilst zeaxanthin was confirmed. It was concluded from cytological and ultrastructural studies that the Eustigmatophyceae is fundamentally different from all other classes of algae and that it is less like the Xanthophyceae sensu stricto than the latter is like the Chrysophyceae, Phaeophyceae and Bacillariophyceae (Hibberd & Leedale, 1972; Hibberd, 1972). In view of the fact that, in general, pigment studies support the separation of the Xanthophyceae sensu stricto and the Eustigmatophyceae, it should be pointed out that they are the only algal classes which at present are known to contain the unique xanthophyu, vaucheriaxanthin-ester. The other pigments of the Xanthophyceae sensu stricto are discussed in the following paper. ACKNOWLEDGEMENTS We are indebted to Dr B. H. Davies, Dr D. J. Hibberd, Dr H. Kleinig and Dr L. R. G. Valadon for irfformation and to Mrs L. Lawes, Miss G. Morgan, Mr V. Morris and Mr P. Randall for technical assistance. An equipment grant from the Central Research Fund of the University of London allowed the purchase of the Unicam S.P. 800A spectrophotometer. REFERENCES References for papers I and II are given together at the end of paper II.