NEW TAXONOMIC STUDIES on SIX CHLOROCOCCUM SPECIES By
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NEW TAXONOMIC STUDIES ON SIX CHLOROCOCCUM SPECIES 1 by Kwok Wah Lee 4 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Arts in the Department of Biology Fresno State College June1970 ACKNOWLEDGMENTS The writer wishes to express his sincere apprecia tion and gratitude to Professor Gina Arce for suggesting the problem and for her patient guidance during the research and completion of the manuscript. He is also particularly grateful to Professors Joseph Hsu and Ronald Meyer for giving fully of their time reading this manu script and for their helpful criticisms. He is deeply indebted to Professor Patricia Buckley, who taught the writer ultrastructure techniques and who also critically evaluated the section in electron-microscopic aspects of this investigation. Finally, special thanks are due to Miss Margie Wong, who spent much of her valuable time in typing the manuscript. TABLE OP CONTENTS INTRODUCTION i MATERIALS AND METHODS 5 OBSERVATIONS AND RESULTS 14 DISCUSSION 2? SUMMARY 56 LITERATURE CITED - 57 INTRODUCTION The generic name Chlorococcum appears frequently in reports of soil flora investigations, but there is a marked lack of agreement regarding the circumscription of organisms to which this genus name applies (Starr, 1955). Furthermore, there is confusion as to the limits of the soecies comprising this group. This disagreement may partly be explained by the fact that the greater number of the species of Chlorococcum were described before 1900 by investigators who studied algae only from mixed collections in nature. The intensive study of Starr (1955) on Chlorococcum and other spherical, zoospore—producing genera of o^e Chlorococcales laid a firm foundation for the taxonomy of this venus. The three most consistent cnaracoexiotios which are in occurrence and tend to group the species in putative natural alliance are: the type of zoospore, the type of chromatophore in the vegetative cell and one presence or absence of a pyrenoid in the vegetative ceil. The genus Chlorococcum is drfferentiated from other genera in the Chlorococcaceae by its possession of tnree constant attributes: (l) asexual reproduction by means of Ohlamydomonas type zoospores, (2) vegetative cells with 2 a parietal, hollow spherical chromatophore and (3) vege tative cells with at least one pyrenoid (Starr, 1955)• The importance of each as a taxonomic criterion at the generic level is fully discussed by Starr (1955)» Lhe zoospores of all species of this genus constantly retain their shape when becoming quiescent; all possess two flagella of equal length. Recently in classifying the taxonomically difficult unicellular algae, phycologists have found it necessary to resort to morphological descriptions together with certain physiological charactei-istics, such as hydrogenase activity, gelatin liquefaction, secondary carotenoid synthesis and the growth responses to various carbon and nitrogen sources (McLean, 1968)* Bold and co-workers (Brown and Bold, 1964); Chantanachat and Bold, 1962; Mattox and Bold, 1962; Smith and Bold, 1966) have employed nutritional requirements, utilization of various caroon and nitrogen sources, growth in light and dark, aoixiuy to reduce nitrite, antibiotic reactions and immunochemistry to supplement the attributes of species in several Chlorococcacean genera. McLean (1968) measured the total chlorophyll and carotenoid concentrations in relation to total pigment content for eighteen Gnlorococcum isolates which were grouped according to the color of the culture incubated under defined conditions for 6-7 weeks. He 3 suggested that pigmentation of old cultures be used as the first criterion for distinguishing species of Chloro- coccum. Comparative ultrastructure studies have also been utilized to supplement the microscopic morphological attributes. Gibbs (1962) employed the ultrastructure of the pyrenoids in the taxonomy of green algae above the generic level. Brown and Bold (1964) were the first to separate species of Tetracystis by comparing the ultra- structure of chloroplast, pyrenoid, mitochondria, Golgi apparatus and cell wall. Recently, Brown and McLean (1969) on the basis of studying the fine structure of pyrenoids in eighteen Chlorococcum species proposed three differentiating categories: (l) pyrenoids with unfragmented perforate starch plates, (2) pyrenoids with many separated fragmented starch plates, and (3) pyrenoids with hemi spherical unfragmented starch plates. Pyrenoids were further distinguishable on the basis oi the number Ox thylakoid disks or tubules which penetrate the ground substance. The objectives attempted in this study were tihree- fold: to compare the morphology of six diixereno species of the genus Chlorococcum growing on six different media under defined culture conditions, to determine any diag nostic physiological characteristics helpful in the sep 4 aration of these species, and to examine and study the cell wall ultrastr uetur e of each. It was intended that such data might provide additional information for the separation of the species under investigation, and hope fully, the principles might be applied to the classification of other microalgae. MATERIALS AND METHODS For this study, six species of Ohlorococcum were selected. They are: G. aplanosnorum Arce G. nypnosporum Starr minutum Starr C. multinucleatum Starr C. polr/morohum Bischoff and Bold C. scabellum Deason and Bold All cultures were obtained from the Algae Collection at the Department of Botany, Indiana University, Bloom- ington, Indiana (Starr, 1964-). The following culture media (Arce, 1956) were used in the comparative morphology: Bristol's Solution Six stock solutions, 4-00 ml in volume, were prepared, each containing one of the following salts in the con- centration listed: 1 6 NaNO-, 8 „ 5 . 10.0 g CaCl2 . 1.0 g K2HPO4 . 3.0 g KHgPO^ . 7.0 g MgS0v7H20 . 3.0 g NaGl . .. 1.0 g 10 ml of each stock solution were added, to 9^-0 ml of distilled water. To these was added 1 ml of minor elements solution (McVeigh and Bell, 1951). Bristol's Solution with Ferric Citrate 5 g of sodium citrate and 0.1 g of ferric chloride were added to 100 ml of distilled water. 1.29 ml of this solution were added to 500 ml of Bristol's solution. Bristol's Solution with Yeast Extract Bristol's solution was enriched hy the addition oi 1 ml of l°/o aq ueous solution of yeast extract (Difco) to 99 ml of the former. Bristol's Solution with Proteose Peptone To 99 ml of Bristol's solution was added 1 ml of 1% proteose peptone (Difco) Bristol's Solution with Dextrose To 99 ml of Bristol's solution was added 1 g of dextrose. 7 Nutrient Agar To 100 ml of distilled water were added 3 g of "Nutrient Agar" (Difco). All the media mentioned were solidified with 1.5% purified agar. The six species of Chlorococcurs were maintained as stock cultures on Bristol's agar and transferred period ically into capped sterile 25 mm. test tubes of the same agar slants. B'or grow th studies each of the six species were evenly streaked on equally divided Petri cisnes 15 cm in diameter. The tests were done in triplicate. Prior to inoculation each dish was filled with 25 mx of the respective agar madia. Each species was streaked axenically and grown under identical environmental condx- tions to increase the probability that any ooservable differences would be characteristic of each species. Growth conditions were standardized by supplying approximately 300 foot candles of light emitted from flourescent lamps set at a 12-12 hr light-dark cycle and maintaining a constant temperature of 22±1 0. These will hereafter be referred to as standard conditions. The morphology of the individual cells was observed after various periods of growth by hanging-drop, wet mount and simple methylene blue stain methods. The cells were observed under oil immersion at 1000X magnification. 8 Measurements were made with a calibrated e.ye-piece which was standardized by a stage micrometer. ^uitural characteristics were observed after six weer^s grow on. Colonial characteristics were studied hoch macroscopically and under low—power (100X) magni fication, mainly for the edge conformation. Average values in the comparative morphology section were made from twenty-five randomly chosen cells. A number of physiological tests also were employed. Most of these techniques were modified from earlier work (Deason and Bold, I960; Bold and Parker, 1962; Mattox and Bold, 1962). The following list of materials was used in the preparation of media for physiological tests; the concentrations indicated were grams per liter, liquid media were used with items 1 through 7, and tests were made in Pyrex glass culture tubes (13 x 100 mm). Media prepared with items 8 and 9 were solidified with 15 g Difco agar per liter® 1. D(-) glucose 5.0 2. Sodium acetate 5.0 3. L(+) arabinose 5.0 4. D(—) ribose 5.0 5. D(-) fructose 5.0 6. D(+) xylose 5.0 7. Sodium chloride 10, 20, JO, 40, 50 8* Crystal violet 0.02, 0.04, 0.10 9. Starch 1.0 Bristol's solution was used as a base for all the media listed above. The algal inocula for all the tests were transferred from 2-week-old cultures on Bristol's agar slants under standard conditions. All the liquid tests were carried out under standard conditions in test tubes positioned on a Gyrotorv Shaker (New Brunswick Scientific Company) to enhance the growth of the algal cells. Results were observed two weeks after inoculation with the comparative growth being described as excellent, good, fair, trace or none. Crystal violet commonly employed as a bacteristatic agent because it exhibits selective inhibition on Gram positive bacteria has also been shown to be useful in differentiating Ulothnx, c.orraxdiurn and uuicnococcus (Mattox and Bold, 1962^. its usefulness as a possioxe taxonomic tool in species ox Gnxorococcurn was examined in this study. Starch agar was used to test for the activity ox extracellular amylases synthesized by each species.