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. The

Chlorococcum species were streaked on to the surface ox a 0.1% starch agar plate, grown for two weeks under standard conditions and the agar then ilooded with aqueous 10

'^^Le ? resence of a clear halo surrounding the algal colony in contrast to a deep blue stained background was interpreted to represent a positive test for extracellular amylase activity. The absence of a halo was interpreted to indicate no amylase synthesis had occurred.

A sensitivity test was carried out on the different species with eight antibiotics (Difco multidisks), whose names and concentrations are enumerated in Table 1.

Table 1. Antibiotics, their abbreviations and concentrations in multidisks

Concentration Antibiotic (micrograms)

Penicillin (PEN) 10 Erythromycin (ERY) 15 Tetracycline (TET) 30 Novobiocin (NOV) 30

Neomycin (NEO) 50 Chloromycetin (CHL) 30

Streptomycin (STR) 30

The agents will hereafter be referred to by their abbre­

viations. To perform this sensitivity test, heavy suspensions

of each algal species were mixed with sterile, cooied- 11 bun-liquid Bristol's agar (1.5%) in separate Petri dishes. The multidisks were placed on the agar surface of each respective plate. After two-weeks ' growth under standard conditions, the results were observed. The presence of a clear zone around any disk was regarded as a positive test, indicating a sensitivity to the specific antibiotic

under examination. The differential susceptibility to UV light exhibited by each species was also tested. The algae were inocu­ lated on Bristol's agar and placed under a constant supply of UV emitted from a 30-watt germicidal lamp (General Electric, G$0T8) for a range from six minutes to eighteen minutes. This rang^e was established by preliminary tests.

Since the accurate strength of the UV source was not available, an estimation was set up by determining the

time required to kill the bacterium, ascner~cmm i under experimental conditions. it was found uo be four minutes. To avoid difficulty of UV penetration of dense cell concentration due to shading, precautions were taken to use a fairly dilute liquid inoculum which was evenly

spread on the agar surface. The growth of the algae was also compared under

different colored light sources obtained by using Bos. 3661S and 3661T Glass Filters (W. M. Welch Scientific Company). The filter was placed on the top of the plastic 12

Petri dish and the rest of the dish was covered by tin foil to prevent the penetration of white light. Again, each Petri dish of Bristol's agar was streaked with the six different species and placed under standard conditions. Results were observed after two weeks, four weeks, and

eight weeks. Sodium chloride agar with Bristol's solution as a base was prepared with added sodium chloride concentra­ tions ranging from zero to 1~>%, The aigae were streaked on the agar surface and placed under standard conditions. 'Tests were observed at the end of a two-week incubation

period. The effect of pH on the growth of the Chlorococcura

species were compared under diifereni; acid (Hoi) and

alkaline (NaOH) concentrations in Bristol's solution. The concentrations were measured and adjusted by a Beckman

Zeromatic pH Meter. HC1 or NaOH was added to Bristol s

solution until the desired pH was reached. The pH of the untreated Bristol's solution was found to be 6.3 and was used as a control. The growth was recorded after two weeks and after four weeks of incubation under stand­

ard conditions on the Gyrotory Shaker. The linal pH of the media was also measured with the same instrument This test was primarily designed to investigate the toler­

ance of the algae to various pH. 1 15

For the ultrastructural study, the six soecies of

U---Q- ocoeci-'i- were cultured in 250 ml Erlenmeyer flasks with 50 ml of Bristol's solution. These were placed on a Gyrotory Shaker under standard conditions.

Cells from two—month-old cultures were harvested by centrifugation. They were fixed for two hours in buffered

1% osmium tetroxide , made by mixing 2% aqueous osmium tetroxide with an equal volume of 0.2 M phosphate buffer,

pH 6.8. After washing four times with cold distilled

water, the material was placed in 0.5% uranyl acetate and left overnight at 4-5 C. The cells were dehydrated

in a standard series of ethyl alcohols. Acetone was used

as the final dehydrating agent and as a solvent for the

plastic mixture. The embedding medium was a mixture of 15 ml Araldite

6005, 25 ml Epon 812, and 55 ml DDSA (University of Texas

Mixture I, with dibutyl phthalate omitted). One drop of the catalyst DMP-30 was added for each ml of the .final

plastic mixture. The plastic was polymerized in a vacuum

oven at 60 C. Sections were made on a Porter-Blum MT-1

Ultramicrotome with glass knives, and poststained with Reynolds' (1965) lead citrate. Micrographs were taken

on a Hitachi HS-8-1 electron microscope. OBSERVATIONS AND RESULTS

The observations and results may conveniently be grouped into three sections. Some selected colonial and individual characteristics of the algae on six dif­ ferent media are compared in the first section. The second section deals with the growth responses of the different species when subjected to the physiological I tests. The last section deals with the comparative cell wall ultrastructure.

Suedes Separation Based on Comparative Morphology

The comparative size of individual cells of each of six Chiorococcuin species on different media are pre­

sented in Table 19 of the Appendix. The average cell size of each respective species

was rather constant regardless of growth on media, except for C. hvpnosporum, which showed variations. The six species grown under controlled conditions could be grouped

conveniently according to cell size, as summarized in

Table 2. 15

Table 2. Average cell size on all growth agars assayed

Size range Species

19 to 20 microns C. scabellum

20 to 22 microns C. minutum r*. multinucleatum

21 to 30 microns C. hynnosporum

32 to 35 microns C. polymorphum C, aolanosporum

The shapes of the individual cells of each respective suedes after two weeks' growth on different growth agars can be examined in the Appendix (Table 20). These results

permit a species separation into one of two categories,

as seen in Table 3*

Table 3. Cell shapes of Chlorococcum species

Characteristic shape Species

Spherical on all C* aplanosnorum ^ med ia tested 5. multinucleatum G. sca'oellum

Variable according to h.• hypnosporum C. minutum growth medium C. polymorphum 16

The thickness of the cell wall of the species was examined after a two-week growth period on various agars and found to some degree diagnostic in species separation. The results can be seen in the Appendix (Table 21). According to these results, the species can be grouped into two categories on the basis of algal wall thickness. These categories are presented in summary form in Table 4.

Table 4. Separation of Chlorococcum species according to wall thickness

Average wall thickness Species

Less than 0.50 microns C. aplanosrorum C. fayonosporum C. minutum

0.75 to 2.00 microns C. multinucleatum C. polymorphum 0. scabellum

The color of colonies growing on agars of various

composition were somewhat varxable but species xixed. The color produced in response to the agar type is pre­

sented in Table 22 of the Appendix. Tne six species

can be separated into three main categories (Table 5). 17

Table 5» Contrasting culture color of Chlorococcum species on different media

Range o.f color Species

Invariably green C. hyonosporum 0. minutum oi Green or yellow green aplanosporum oi polymorphum

Orange or orange green C. multinucleatum C. scabellum

The edge characteristics of the colonies of the different species are shown on Table 23 of the Appendix. The six species may roughly be categorized into three groups (Table 6).

Table 6. Species separation based on> colonial morpholog;

Edge Species

n Smooth and unlobed hypnosporum c. minutum oi multinucleatum Smooth and lobed oi scabellum O

I aplanosporum Rough and uneven or OI with outgrowths polymorphum

I 18 opecies Separation Based on Physiological Reactions

1. Sodium Acetate and Sugar Assays

The ooservations and results of the investigations concerning the use of physiological tests in the taxonomy of the algae are summarized in Tables 24-33 in the Appendix.

During the first part of this physiological investi­ gation, media with the organic sources (Table 24-) were used in an attempt to differentiate the different taxa. The results of this test can be analyzed in the form of a dichotomous key for the six species (Table 7)•

Table 7. A Key for species separation based on sodium acetate and sugar assays

1. Inhibited or slightly inhibited01 by sodium acetate ...... 2 2. Slightly inhibited by arabinose C. hypnosporum 2. Not inhibited by arabinose . . 3 3. Inhibited by fructose and xylose 4 4. Slightly inhibited by glucose and ribose . . C. multinucleatum 4. Not inhibited by glucose and ribose ...... C. scabeilum 3. Not inhibited by fructose and xylose C. aplanosoorum 1. Not inhibited by sodium acetate . . 5 I 5. Slightly inhibited by ribose, inhibited by xylose ...... C. minuturn 5. Not inhibited by ribose or G. uolymorphum xylose

a"Inhibited" is indicated as trace growth and slightly inhibited" as fair growth in Table 24. 2. Growth in the Presence of Crystal Violet

'The grow th responses of the species on different concentrations of crystal violet agar are recorded in Table 25 01 tne Appendix. According to the degree of inhibition of crystal violet on the growth, the species can be grouped into three categories (Table 8).

I Table 8. Effect of crystal violet on the growth of Chlorococcum species

Degree of inhibition Species

No inhibition at any C. aplanosporum concentration C. minutum G. polymorphum

Complete inhibition at higher concentrations C. scabellum

Complete inhibition at 0. hypnosporum all concentrations C. multinucleaturn

3. Ability to Digest Starch Digestion of starch by Chlorococcum species was examined by testing for amylase activity. The results are tabulated in Table 26 of the Appendix. As a result of the starch test, two differentiating categories can

be established (Table 9)- 20

_ao_Le 9. Starch digestion by Chlorococcum species

Amylase activity Species

Present G. aplanosporum C. hypnosporum G. polymorphum Absent C. minutum C. multinucleatum G. scabellum

4. Antibiotics Reactions The differential sensitivity of the species to the antibiotics used is recorded in Table 27 of the Appendix. I It appears that no significant differentiating categoriza­

tion can be derived from the experimental results. Further consideration on the use of antibiotics as a

species differentiating agent will be given in the

Discussion section.

5. Tolerance to Ultraviolet Radiation The exposure time necessary to destroy all cells of the colony was examined. All the six species were eventually killed by UV at various time lengths of expo­ sure. The experimental data are recorded in Table 28

of the Appendix. The species tolerance permits the arrangement of 21 species into groups according to the time required by UV treatment to kill the algal cells (Table 10).

Table 10. Relative tolerance of species to ultraviolet radiation

Lethal dose Species

12 minutes C. aplanosnorum C. hypnosporum C. muitinucleatum

16 minutes C. minuturn C. scabel-Lum

18 minutes C. polymorphum

6. Response to Light No significant differences were observed after both

the two—week and four—week incubations, except for the fact that there was a general retardation of growth in all species with the black filter (no light). The results

observed after an eight-week incubation are recoroed in Table 29. Exaggerated retardation of growth of all species

in darkness is evident. Poor growth of all species in green light is also evident. However, the treatment with

different colored lights, in the writer's opinion, is

not a good species-differentiating attribute. 22

7. Sodium Chloride Tolerance

The inhioioory effect of different concentrations

01 sodium cnlonde v;as inv estigated; the experimental results are recorded in the Appendix in Table 30.

The Chlorococcum species selected showed differential tolerance to RaCl concentration and can thus be categorized

into three groxips as presented in Table 11,

Table 11. Relative tolerances to NaCl

NaCl concentration Species Completely inhibitory to growth

3c /o C. hypnosporum C. minutum C. scabellum

M-c/o C. anlanosporum C. mu11in uc 1eatum

yp C. polymorphum

8. Relative pH Tolerance The effects of pH were investigated, the results

of which are recorded in Tables 31-35-of the Appendix. The limits of pH tolerance for each species after

x- • - ^ tn Bristol's solution is recorded a two—week incubatron m ^ _. species fall into onree it Table 31 of the Appendix. me species acidic conditions required to com- classes according to aci 1 23 pletely repress ^urther algal growth, as shown in Table 12.

-able 12. Limits of tolerance to lower pH ranges

pH completely suppressing growth Species

3 C. aplanosporum 2 C. hypnosporum C. polymorphum , 1 G. minutum G. multinucleatum G. scabellum

According to their tolerance to alkaline conditions, however, they can be tentatively separated into two groups that bear little correspondence to the tolerance by any respective species to acid conditions. The two groups are presented in Table 13»

Table 13. Tolerance limits at_high pH after two weeks' incubation

pH completely suppressing growth Species

12.0 C. anlanosporum C. scabellum

12.3 G. hvpnosporum G. minutum C. multinucleatum C. nolymorphum 24

nfter a two—month incubation, the growth responses and linal pK values were recorded in the Appendix (Tables $2 and 33;. Accordingly, the six species can be separated into two definite groups (Table 14).

Taole 14. Tolerance limits at higher pH after two-months' incubation pH completely suppressing growth Species

1 12.0 C. aplanosnorum C. hypnosDorum C. scabellum

12.3 C. minutum C. multinucleatum C. polymornhum

Final pH values were measured after 2-months' incuba­ tion (Table 33). All species show no difference in tolerance

and ability to recover in the lower pH limics; all were killed at oH 3 or less and the final pH was not signif­ icantly chapged. With an initial highly alivalj-ne oh,

the final pH was drastically changed.

Species Separation Based on Comparative Cell Wall Ulora- structure

The comparative cell wall ultrastructure of the

different species are presented in Figures 1-12 ana m

Table 34 of the Appendix. Ail the species used in this study have a double-

layered cell wall. According to the thickness of the 25

0U.L.8X* an^ inn©!* wallss the SDeci.es can be separated as follows (Table 15).

Table 15. Species separation based on outer and inner wall thickness

1 Comparison of thickness Species

Outer wall thicker C. nolymornhum Almost equal thickness C. 'multinucleatum Inner wall thicker C. aplanosporum C. hynnosporum C. minutum C. scabellum

On the basis of the comparative electron density

of the outer and inner walls, the species fall into three

categories (Table 16).

Table 16. Species separation based on outer and inner wall electron density

Comparison of electron density Species

Outer wall denser C. aplanosporum C. hypnosporum C. multinucleatum 0. scabellum

Both wall densities equal C. minutum C. pol.ymorphum Inner wall denser 26

v/ith reference to the inner wall texture the species can be separated as follows (Table 17).

•Table 17. Species separation based on inner cell wail.texture

exture Species

Finely striated C. scabellum Co'arselv granular C. aplanosporum C. minutum

Heterogeneously granular C. hypnosnorum

Finely granular C. multinucleatum C. polymorphum

The species can be separated in a similar manner

by making use of their outer wall texture (Table 18).

Table 18. Species separation based on outer cell wall texture

Texture Species

Very finely granular G. multinucleatum C. polymorphum Finely granular C. minutum Coarsely granular C. hvpnosporum Densely granular C. aplanosporum

C. scabellum Solid DISCUSSION

The need for prolonged study of unialgal cultures under standard conditions as the prerequisite for eluci­ dating their taxonomy has been emphasized by Bold (1950,

1953), Trainor and Bold (1955), Starr (1955), Herndon (1958a, 1958b), Arce and Bold (1958), Deason and Bold (I960), Bold and Parker (1962), Chantanachat and Bold (1962), Bischoff and Bold (1963), Brown and Bold (1964), and Smith and Bold (1966). The identification of species as compared to that of higher taxa is considerably more difficult and time consuming. It is necessary first to develop and standard­ ize techniques to facilitate the identification of large numbers of organisms. More specifically it has become increasingly apparent that other criteria in audition to strictly morphological ones must oe used, if possible, to delimit species (Deason and Bold, I960). As. an aid 10 taxonomic characterization special attention has been devoted to devising techniques which would provide addi­

tional differentiating criteria. Recently, in the classification of the difficult unicellular algal species, phycologists have employed physiological tests (Bold and Parker, 1962; Chantanachat 28 and i30_G, 1962; Bischoff ana Bold, 1963; Brown and Bold,

196A; Smith and Bold, 1966), comparative pigmentation

analysis (mcLean, x.968;, and ultrastructure characteristics (Gibbs, 1962; Brown and McLean, 1969) as taxonomic tools.

The resuxts indicate that the separation of the six species in this investigation was enhanced by comparing

their morphology when grown on differential media. Further means of distinguishing among the species were obtained by examining the growth responses of each species under different physiological conditions and its cell wall ultra- structure characteristics. Although the morphology of each species has been described in detail by its respective

isolators (Arce and Bold, 1936; Starr, 1955; Bischoff and Bold, 1963; Deason and Bold, I960), in this study, the characteristics of each species were compared by contrast­ ing the soecies—specific morphology when they were grown on six differential media. It was xound ohat cell size, cell share, wall thickness, color of culture and macros­ copic colonial characteristics are convenient criteria

for the separation of the species (Tables 2-6). The results obtained for the culture color in general

agree with the results worked out by McLean (1968), who grew the algal species for six weeks on one medium only.

One exception was that C. hypnosnorum was found to be yellow green in McLean's (1968) investigation. The 29 difference is perhaps due to the difference in age of the cultures reported herein and those of McLean at the time that color was recorded.

The agreement of these and other observations in this investigation deoend on controlled standard conditions in which the algae are grown. This and previously mentioned investigations stress the importance of standardized artificial conditions in which the algae are cultured and their differential species characteristics identified and compared. Certain supplementary carbon sources were inhibitory to the growth of some Chlorococcum species as compared with the inorganic standard Bristol's solution alone; for example, xylose and sodium acetate were found oo inhibit these species more than the ouher sources. This test might possibly be improved by using a wider range of concentrations of the respective carbon sources. Mattox and Bold (1962) employed tolerance to crystal

violet as a criterion in the classification of Ulotricha- cean algae. Crystal violet was found to be useful in distinguishing the genera Ul.othrix, Kormidium, and Sticho-

coccus, but the same substance had no usefulness m differentiating the species within the genus ulororix and only limited usefulness in differentiating the species

within the genera Hormidium and Stichococcus. Brown and 30 o0i.d (19o4) utilized it successfully to compare the species

"*"9 .-.2 °racysuis. The use of crystal violet as per^aihs to brie separation of the species of Chlorococcum in one present investigation indicates that it is a desirable and useful taxonomic tool.

Extracellular amylase activity of green algae has been explored as a possible taxonomic criterion by Mattox and Bold (1962), Bischoff and Bold (1963), and Brown and Bold (1964). According to the results in this study, only three Chlorococcum species show amylase activity. This may be an indication of a fundamental difference in metabolism or in induction of alternative biochemical pathways between the different species under investigation. Although such investigations are considered beneficial in algal taxonomy, as suggested by Mattox and Bold (1962), more work is required to perfect this technique. Antibiotics have been employee! to differentiate algal taxa by Deason and Bold (i960), Chantanachat and Bold (1962), Mattox and Bold (1962), Bold and Parker (1962;, and Bischoff and Bold (1963). Mattox and Bold (1962) were skeptical about the taxonomic significance of the differential sensitivities of the taxa studied because isolates of the same species from different locations showed variations and the results were not entirely repro­ ducible. In this study the effect of antibiotics was found 31 oo oe .unsatisfactory as a species-differentiating criterion -for omorococcum. It is believed that with the standard­ ization ox the antibiotic discs by the manufacturers, the multidisk rest may become a valuable taxonomic tool. The lethal and mutagenic effects of UV on micro- organisras through its action on DNA are well documented.

The genetic effect of UY irradiation is lucidly discussed by Thimann (1963) and Brock (1970).

On applying UV radiation, inconsistent results might arise with simple streaking method or with too heavy an inoculum. Such inconsistency could possibly be caused by the inability of UV to penetrate into the thickly packed ceils. Algal cells being shaded by others might thus survive the UV treatment. The effect of light rays of different wavelengths on the growth of the different Chiorococcum species does not seem to be a good differentiating criterion. All the species are inhibited by the absence of light. They also are generally retarded in growth when they are supplied with only green light. This is due to ohe fact that chlorophyll a and b utilize optimally those light rays in the red and blue regions of the visible light spectrum in the nrocess of photosynthesis (Brock, 1^70). /ery little green light is absorbed, but most of it is either transmitted or reflected by the green algae; therefore, 32 the role of green light in photosynthesis is minimal.

It was lound that the six Chlorococcum species fall conveniently into three distinct tolerance groups when gro«^n on various con.c0Tifci*Q."bioiis o f* NsiCl. TI10 p iriiricipy cause o^ s alt inhibition of growth is believed to be due to osmotic effects. Using the Van Hoff equation (Bartho­ lomew, 1967), a 5% NaCl solution is equivalent to approxi­ mately 4-0 atmospheres osmotic pressure. This seems to indicate that the growth response to sodium chloride might be due to adverse osmotic effects. Since the response is species variable, tolerance to NaCl may represent a useful test in conjunction with others in the classifica­ tion of Chlorococcum species. The limits of hydrogen ion concentration for growth of microorganisms are from around pH 4-.0 to 9-0 (Rose, 1968). In preliminary tests no significant, diiference in growth response was observed when the six species were grown in Bristol's solution supplemented with HC1 or NaOH adjusted at a pH range of 5 to 10. However, differential responses were observed when che algae were treated in the same growth medium adjusted at more extreme pH values. Since the effect of pH on microbial activity is believed to be the interaction between hydrogen ions and enzymes (Rose, 1968), the difference in growth response among the species might indicate a fundamental difference in 33 the physiological responses peculiar to each individual species.

Organisms can alder the pH values of the environments through their activities. Since most ionized metabolic products are acidic, organisms are more likely to lower than to raise the pH of their environments. When the pH is changed through microbial action it is usually because ammonia or other nitrogenous organic compounds are released (Brock, 1970). A pH increase was observed in the present investigation from the measurements of the final pH values of the growth medium after a two- month culture period (Table 21). Growth was completely inhibited with an initial pH of 1 through 35 an<^ sinal pH values were not significantly changed. A drop in final pH values was evident with cultures started with high pH. With further improvement on the technique, pH tests

might be considered to be a valuable taxonomic criterion

for the separation of the species of Chloroccccum. Ultrastructure characteristics have been utilized

in algal taxonomy at both generic (Gibbs, 1962) and species levels (Brown and. Bold, 1964; Brown and McLean, 1969). In both studies, young and rapidly growing ce^ls were used and different stable, separating criteria were

set up. Leason (1965) studied the fine structure of 34 vegetative and dividing cells of 0h1 o rococ cum echinozygotum cultures in Bristol's solution and in the same medium with strontium substituted for calcium.

Brown and Bold (1964) found the outer and inner wall thicknesses to De a useful criterion for separating actively growing ueuracystis species into four morphological categories.

In this study, cells of a two-month-old culture were studied and the cell wall ultrastructure compared , and

analyzed (Tables 15-18, 34). The fact that the cell wails of the six species are composed of an outer and an inner wall layer agrees with the reports of Brown and Bold (1984). The pyrenoid, which was used as a stable differentiating characteristic

for Chlorococcum species (Brown and McLean, 1969), is not clearly identifiable in the old cells due to the development and accumulation of starch grains and vacuoles.

Other organelles described by Brown and Bold are also not clearly recognizable in the electron micrographs

in this present investigation. According to the results obtained in this stuay, it appears that the relative thickness, comparative electron

density, and the texture of the inner and outer walls

are useful criteria for the separation of the six Chloro-

coccum species. 35

Owing uo the restricted facility of the newly installed electron microscope, and to the limited back­ ground o^ one writer in electron microscopy, extensive ulorastructare study was not possible. It is hoped that this initial study will lead to further ultrastructural investigations.

In this investigation, the need for comparative morphological, physiological, and ultrastructural studies for the separation of species of green algae is empha­ sized. Hopefully, the principles of working in this direction may be utilized in the classification of other

microalgae. SUMMARY

Six n_crococcum species were cultured under defined conditions in aonerapts to establish criteria for separating Deoween them on the bases of comparative cellular and colonial m orphologies, physiological responses to dif­ ferent environments, and characteristic cell wall ultra- structure.

The species can be grouped into definite taxonomic

categories according to average cell size, cell shape, algal wall thickness, and characteristic morphology and

color of the colony. The growth responses of the algae under a variety

of physiological tests resulted in a set of separation criteria based on sodium acetate and sugar assays, search digestion, and tolerance to various conditions, includ­ ing UV radiation, NaCl, crystal violet, and pH. Antibiotic tests and the response to the light spectrum of different

wavelengths were found to be unsatisfactory criteria

for the separation of Chlorococeum species. • ..op cel l wall ultrastructure Preliminary attempts oO us , +. +->,0+- such comparative studies characteristics suggest tnat i for separation between might lead to additional crimen

Chlorococcum species. I

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