PHYCOLOGICAL STUDIES II. Some Algae From Arid Soils SRISUMON CHANTANACHAT A D HAROLD C. BOLD Phycological Studies II. Some Algae From Arid Soils by 1 2 SmsuMoN CHANTANACHAT AND HAROLD C. BoLD 1 Department of Botany, Chulalongkom University, Bangkok, Thailand. 2 Department of Botany, University of Texas, Austin, Texas. PUBLISHED BY THE UNIVERSITY TWICE A MONTH. SECOND CLASS POSTAGE PAID AT AUSTIN, TEXAS Acknowledgments The writers wish to express their appreciation to Professor Richard C. Starr for providing for their study certain cultures from the Culture Collection of Algae at Indiana University. They are grateful also to Professor S. P. Ellison, Jr. of the University of Texas, Dr. I. Friedmann of The Hebrew University, Jerusalem, Israel, Mrs. Marshall Johnston of the University of Texas, and Dr. and Mrs. R. Malcolm Brown of Pampa, Texas, for samples of arid soils used in this investiga­tion. Special appreciation is expressed herewith to the Arabian-American Oil Company (ARAMCO), especially to Tom Barger, President, and to 0. C. Seager, Manager of Exploration, both of Dahrhan, Saudia Arabia, for facilitating collec­tion of soil samples in that country. Mr. Dale Le Stourgeon and Mr. Douglass Hoese have contributed valuable technical assistance. Dr. Hannah Croasdale of Dartmouth College, Hanover, N. H., prepared the Latin descriptions. The editorial assistance of Mrs. Frances Denny is acknowledged with gratitude. Finally, the investigations herein reported have been supported in part by NSF Grant G 18746 and NIH grants RG 8211 and GM 08211-02, and publication has been financed by the Publications Fund of the Graduate School, University of Texas. Table of Contents Acknowledgments Introduction Materials and Methods Observations A. General aspects ~f the arid-soil algal flora B. Organisms new to science or otherwise of interest Neochloris oleoabundans sp. nov. Supplementary attributes in the genus Neochloris Key to the species of Neochloris Starr C hlorococcum diplobionticoideum sp. nov. Spongiochloris minor sp. nov. Spongiochloris incrassata sp. nov. Key to the species of Spongiochloris Starr Radiosphaera dissecta (Korschikoff) Starr Chlorosarcinopsis gelatinosa sp. nov. Chlorosarcinopsis eremi sp. nov. Key to the species of Chlorosarcinopsis Herndon C hlorosarcina Gemeck C hlorosarcina brevispinosa sp. nov. Chlorosarcina longispinosa sp. nov. Fr :edmannia gen. nov. F. israeliensis sp. nov. C. Facultative heterotrophy Summary Literature Cited Illustrations PAGE . 3 7 9 14 14 15 15 18 23 23 26 27 32 32 34 36 40 40 40 42 45 45 48 50 51 54 Introduction Although there have been a number of studies of algae of various types of soil, few have dealt especially with arid or desert soils. Killian and Fe'her ( 1939) made a survey of algae in soil of the Sahara Desert, North Africa, and L. Moewus ( 1953) reported on some algae isolated from semi-desert soils around Broken Hill, New South Wales, Australia. Because preliminary studies of the soil algal flora of certain sand samples from Saudi Arabia indicated that algae were present even in soils from a region of such low rainfall, it occurred to the writers that further investiga­tion of the algae in other arid soils might be of interest. Such an investigation, on a limited scale, has been made possible by the cooperation of a number of people to whom grateful appreciation is herewith expressed. Materials and Methods The algae described in this paper were isolated from samples of desert soils col­lected from various places, including Saudi Arabia, Arizona, Utah, Israel, and Mexico. For studying the soil algae present in each sample, 15-g portions were placed in sterile 125-ml Erlenmeyer flasks in quadruplicate, each containing 50 ml of: ( 1 ) modified Bristol's solution (Bold, 1949) ; (2) Kratz and Myer's ( 1955 ) medium D; (3) Knop's solution (Bold, 1942 ) ; and (4) sea water, respectively. The last was used because of evidence that some of the soils were from marine deposits containing high concentration of salts. Modified Bristol's solution was used with and without agar for routine cultiva­tion of the algae described in this paper. The media were prepared as follows: Modified Bristol's solution (Bold, 1949 ) Six stock solutions in 400 ml of distilled water were employed. Each contained one of the following salts in the amounts listed : NaNO:i 10.0 g K 2HPO, 3.0g CaCl,-2H20 1.0 g KH2PO, 7.0g MgS0,·7H20 3.0 g NaCl 1.0 g 10 ml of each stock solution were added to 936 ml of distilled water and then 1.0 ml of each of the 4 stock trace-element solutions per liter of macro-element solution was introduced. The stock trace-element solutions used were the following: ( 1) EDTA stock solution: 50 g EDTA plus 31 g KOH per liter. (2) Fe stock solution: 4.98 g FeS0.-7H20 per liter of acidified H 20. (Make acidified H 20 by adding 1.0 ml cone H 2SO, to 999 ml distilled water.) (3) Boron stock solution: 11.42 g H:iBO:i per liter. (4) H ,, stock solution": ZnS0.-7H20 8.82 g MnCl24H20 1.44 g Mo03 0.71 g CuS04·5H20 1.57 g Co (N03) 2·6H20 0.49 g all per liter of acidified• H20 (acidified as described under Fe stock) Kratz and Myer's ( 1955) Medium D NaN03 1.0 g per liter K2HPO. 1.0 g per liter MgS04·7H20 0.15 g per liter Ca(N03 ) 2·4H20 0.010 g per liter EDTA 0.050 g per liter Fe(SO.) 3·6H20 0.004 g per liter H5 micro-elements3 1.0 ml of stock solution, see ( 4) above This medium is used for the cultivation of some blue-green algae and has a pH of about 8.0. This high pH inhibits development of many bacteria. Knop's solution (Bold, 1942 ) Part A PartB Ca(NOa)2 4g KNO:i 1.0 g Distilled water 500ml KH2PO, 1.0 g MgS0,·7H20 1.0 g Distilled water 500ml Parts A and B were mixed just before using. Micro-elements were added as they were to the other solutions. The flasks with culture medium, having been inoculated with soil samples, were placed in the culture room at a temperature of 22°C under fluorescent illumina­tion controlled by an automatic timing device ( 12 hr light, 12 hr darkness). These conditions of cultivation are hereinafter referred to as "standard conditions." A phototactic ring of green, zoospore-producing algae appeared within 2 or 3 weeks in many of the flasks, and additional algae grew on the surface of liquid and on the submerged soil. lsolatiom of organisms which developed were made by several methods, includ­ing direct removal of cells with capillary pipettes and plating-out dilute suspensions in 2% Bristol's agar. Algal colonies appeared in the agar within 1 week. Single colonies of different kinds of algae were isolated by using finely drawn-out "Dis­p::rable Pasteur Capillary Pipettes" or platinum needles. Aliquots of each colony selected were inoculated into a small tube of Bristol's solution and also into a soil­water tube. The latter were prepared by placing a pinch of CaCOa in the bottom of a Pyrex test tu be ( 13 X 100 mm) , then adding 114-1 / 2 inch of soil5 and filling the tube approximately 2/3 full with distilled water. The tubes were steamed for 1 hr on each of the 3 successive days and inoculated after they had cooled and cleared. These unialgal cultures showed macroscopically visible growth under standard conditions about 2 weeks after inoculation. :; From Kratz and Myers, 1955. ·• Myers, personal communication. 5 Soil from a garden in Nashville, Tennessee. Chantanachat and Bold In order to study the morphology and life cycle of the organisms, and especially the distinctive physiological attributes, it was necessary to obtain bacteria-free cul­tures. The algae described in this paper were purified with the aid of 4% Tween-80 (Atlas Powder Co., Wilmington, Del.). The procedures employed were as follows: ( 1) a heavy suspension of the organisms (actively growing cultures) was placed in a 12-ml centrifuge tube and centrifuged for 1 or 2 min at 2,000 RPM until the organisms had sunk to the bottom of the tube. The supernatant was then decanted. (2) 8 ml of 4 % Tween-80 (prepared by adding 4 ml Tween-80 to 96 ml sterile distilled water ) was then poured into the tube; the latter was then placed in a Disontegrator" for 1 min in order to separate the clumps. The organisms then were left in the Tween-80 for 2-3 hr. It was found that frequently shaking the tube at this stage was helpful in attaining pure cultures. ( 3) The organisms were then centri­fuged again and the Tween-80 supernatant discarded. ( 4 ) After washing 5 times with sterile distilled water, with alternating centrifugations, the organisms appar­ently were rendered largely bacteria-free. (5) Isolations were made from these purified suspensions by streaking, plating, and single-cell isolation. (Bold, 194 2; Pringsheim, 1946, 1950). Bacteria-free cultures on Bristol's agar slants were maintained in triplicate Pyrex tubes ( 18 X 150 mm ), plugged with cotton or in Bakelite-capped tubes. These are hereinafter referred to as "stock cultures." Macroscopic observations were made on these stock cultures at 2-week, 1-month, and 2-month intervals to record color changes associated with the age of the cultures, and microscopic studies of cell morphology were recorded in drawings and photomicrographs. The morphology and life cycle of the organisms were studied in fresh mounts from 2-week-old cultures grown under standard conditions both in Bristol's solution and on Bristol's agar. Observations on fresh mounts were supplemented by using India ink and Methylene-blue to determine the extent of wall layers. Sudan IV was used to demonstrate the presence of lipid, and aqueous iodine ( L-KI) to de­termine the nuclear condition, characteristics of flagella, and presence or absence of starch and pyrenoids. The acetocarmine technique was used to supplement obser­vation of the nuclear condition in living cells. For this purpose, the procedure em­ployed was as follows: cells were affixed to clean glass slides by means of egg albumin and immersed in the fixative for 12-24 hr (or fixed overnight). The freshly prepared fixative contained the following: 3 parts of absolute alcohol; 1 part of glacial acetic acid; F eCL · 6H"O then was added until mixture became light brown. The excess fixativ~ was drained off and several drops of acetocarmine, prepared by the method of Cave and Pocock ( 1951 ) , were placed on the fixed material. After the addition of the coverslip, the slide was heated over a low flame until vapor arose from the stain. Observation of the preparation was made im­mediately. 6 Disontegrator System 80. Ultrasonic Industries, Inc., Albertson, L.I., N.Y. For providing more information on the taxonomy of soil algae, especially for the delimitation of species, several techniques and supplementary attributes were employed, as suggested, in part, by Deason and Bold ( 1960) and by Bold and Parker ( 1962 ) . These include a study of colony characteristics (macroscopic and under low magnification ) ; color changes on Bristol's agar upon aging; growth in different agar media, i.e., Proteose-peptone agar, soil-extract agar, 0.1 7o yeast extract agar, Bacto Nutrient agar, Bacto Nutrient Broth, Bacto AC Broth, Bacto Thioglycollate medium and 0.04% sulfathiazole in Bristol's solution; sensitivity to antibiotics, sulfonamides, and other agents, and, finally, growth in media con­taining various carbon sources. In order to study the differences in gross colony characteristics, a loopful of cells from 2-week-old stock cultures was streaked in circular fashion on the surface of Bristol's agar in small Petri dishes. After 2 weeks' growth under standard condi­tions, the colony characteristics, including color and topography, were observed and recorded. Observations were made both macroscopically and microscopically with low power ( 14 X ) magnification of a stereoscopic binocular microscope using reflected light. Photomicrographs of colony characteristics were made for the record. For comparative studies of growth in different solutions mentioned above, media were prepared as follows: Proteose-peptone agar was made by adding 1.0 g of proteose-peptone and 15.0 g agar to 1.0 liter of Bristol's solution. Soil-extract agar was prepared by adding 40 ml of soil-water supernatant (prepared by autoclaving a kilogram of soil in a liter of distilled water) and 15.0 g of Difeo agar to 960 ml of Bristol's solution. Yeast-extract agar was made by adding 1.0 g of yeast extract and 15.0 g of Difeo agar to 1 liter of Bristol's solution. Inoculations from 2-week­old stock cultures were streaked into small tubes ( 13 X 100 mm) of proteose­peptone agar, soil-extract agar, yeast-extract agar, and Bacto-Nutrient Agar slants. After 2 weeks under standard conditions, e5timation of the amount of growth was made and recorded. This was described by the adjectives "excellent," "good," "fair," "trace," or "none," based on standards used by Bold and Parker ( 1962 ). In maintaining the pure cultures, inoculations from stock cultures were made routinely into Bacto Nutrient-Broth tubes, AC broth and Thioglycollate media to insure that the cultures remained bacteria-free. Observations to determine bacterial contamination were made after 48 hr and subsequently. For comparative observa­tions and to test the ability of each alga to grow in these media, the observations were made after 2 weeks' growth under standard conditions. In the light of the work of Parker, Bold, and Deason ( 1961 ) and Bold and Parker ( 1962 ), study was made of the effects on the algae herein described, in light and in darkness, of adding carbon sources to Bristol's solution. Each organism tested was grown on Bristol's agar slants for 2 weeks. In order to get as uniform-as­possible an inoculum, the organisms to be tested were carefully washed off the agar slants into screw-cap tubes with sterile Bristol's solution. Uniform amounts of the Chantanachat and Bold suspensions were used to inoculate the several algae into other media. Test media included the following: Bristol's solution (control); 0.75 'fr arabinose, fructose, glucose, ribose, xylose, and sodium acetate, each in Bristol's solution. The bacteria-free cultures were inoculated into the tubes ( 18 X 150 mm) of sterile medium in duplicate by means of sterile disposable Pasteur pipettes. One of these sets was placed under standard condition<>, while the other was placed in darkness at approximately the same temperature. After 2 weeks, and periodically thereafter, estimates were made of the amount of growth in these tubes. These were described by the adjectives "excellent," "good," "fair," "trace" or "none," as stated previously. To determine the sensitivity of the organisms to antibiotics, sulfonamides, and other agents, the procedure employed was as follows: High-concentration "Mul­tidisks" were placed aseptically on the surface of Bristol's agar (prepared by using deionized water and Ion-agar instead of distilled water and Difeo agar) "pour·' plates previously "seeded" with a heavy suspension of 2-week-old cultures of the organisms to be tested. After 2 weeks' growth under standard conditions, obser\'a­tion was made of the occurrence of zones of inhibition. The "Multidisks" had been impregnated before use with the substances and at the concentrations listed in Table 1. TABLE l. Certain inhibitory agents, manufacturer's• abbreviations and concentrations used in "Multidisks." Code number Antihiotic agents Concentration 11-102A Chlortetracycline (A+) 30mcg Amphotericin B (AB+ ) 100 mcg Bacitracin (B+ ) 10 units Chloramphenicol (C+) 30mcg Carbomycin (CA+) 15mcg Colistin sulfate (CS+) lOmcg 11-102B Demethylchlortetracycline (D+) 30mcg Erythromycin (E+) 15mcg Kanamycin (K+) 30mcg Neomycin (N+) 50mcg Novobiocin (NV+) 30mcg Nystatin (NS+ ) 100 units ll-102C Oleandomycin (OL+ ) 15 mcg Penicillin (P+ J 10 units Polymyxin-B (PB+) 300 units Paromomycin (PM+ ) 300 mcg Ristocetin (R+ ) 30mcg Syncillin (SY+) lOmcg Some Algae From Arid Soils TABLE 1. (continued) 11-102D Cycloserine (CY+) 30mcg Dihydrostreptomycin (S+) lOmcg Staphcillin (SC+) 30mcg Oxytetracycline (T+) 30mcg Triacetyloleandomycin {TAO+) 15 mcg Tetracycline (TE+) 30mcg 1l-102E Sulfisomidine (EL+) 300 mcg Sulfisoxazole (G+) 300 mcg Sulfamethoxypyridazine (KY+) 300 mcg Triclobisonium chloride (TR+) 1 mg Viomycin (V+) lOmcg Vancomycin (VA+) 30mcg 1 l-102F Sulfadimethoxine (MA+) 300 mcg Sulfadiazine (SD+) 300 mcg Sulfamerazine (SM+) 300 mcg Triple-sulfa (SSS+) 300 mcg Sulfathiazole (ST+) 300 mcg Thiosulfil {TH+) 300mcg ll-102G Furaltadone (AL+) 100 mcg Nitrofurantoin (F+) 100 mcg Nitrofurazone (FC+) 100 mcg Furazolidone (FR+) 100 mcg Iso Nicotinic acid hydrazide (IH+) 25 mcg Methenamine mandelate (M+) 2.5mg Para amino salicylic acid (PS+) 100 mcg a Consolidated Laboratories. Chicago Heights. Illinois. In addition to the agents tested with "Multidisks," 0.04% sulfathiazole in Bris­tol's solution also was employed, as it had been by Bold and Parker ( 1962) . The drawings were made with the aid of Spencer Camera Lucida and the photo­micrographs were taken with a Bau~ch and Lomb microscope with apochromatic objectives and a Zeiss-Winkel camera attachment. Observations A. GENERAL ASPECTS OF THE ARID-SOIL ALGAL FLORA Isolations into unialgal cultures were made from 21 samples of arid soils from different places, including Saudi Arabia, Utah, Arizona, Israel, and Mexico. Itwas Chantanachat and Bold found that among the algae which developed were genera of the Chlorococcaceae, Chlorosphaeraceae, Xanthophyceae, and Cyanophyceae. The origins of the samples and organisms isolated are listed in Table 2. From Table 2 it is clear that the most prevalent genus of algae in arid soils is Chlorosarcinopsis, a chlorosphaeralean genus. Certain of the taxa listed in the table have been isolated into pure culture and have been investigated intensively; they are described in the following account. Others await further study. B. ORGANISMS NEW TO SCIENCE OR OTHERWISE OF INTEREST NEoCHLORIS oleoabundans sp. nov. (Fig. 1-8; 73-75) Cellulae sphaericae, 6-25µ. diam., sine matrice gelatinosa. Choroplastus parietalis poculiformis quasi dimidium tantummodo superficiei cellulae obducens; duo ( raro unum vel tria) pyrenoidea in quasi omni cellula matura. Membranae cellularum in culturis vel aliquot mensium aetate semper tenues, interdum, autem, incras­sationem unipolarem bulliformem praebentes. Cellulae uninucleatae. Reproductio per zoosporas ( 4-8) (raro 16) ex una cellula effectas, bipartitione iterata enascentes. Zoosporae ovatae 3.6-2. 7 µ., duo flagella aequa, stigma anterius, duas vacuolas contractiles anteriores, nucleum anteriorem et pyrenoideum posterius habentes, quiescentes sphaericae factae. Aplanosporae ( 4-8-16) quoque effectae. Reproductio sexualis non observata. Origo: E tumulis arenae Lat. 21°15 'E, Long. 55°15 'E, in loco Rub al Kali, Saudi Arabia dicto. Cult. num. Ch-1. Starr ( 1955) erected the genus Neochloris to include chlorococcalean algae with the following attributes: ( 1) vegetative cells with a hollow, cup-like chloroplast; ( 2) having at least a single pyrenoid in the chloroplast of the vegetative cells; and ( 3) producing zoospores without a wall and with 2 equal flagella. A species of this genus, apparently hitherto undescribed, was isolated into bac­ teria-free culture during this investigation from a soil sample collected from the top of sand dunes Lat. 21°15'N, Yong. 55°15'E in Rub al Khali, Saudi Arabia. The vegetative cells, which cohere in irregular masses, are spherical throughout their development (Fig. I ) . Cell size is variable (Fig. 73) and a maximum diameter of 25µ. has been observed. In actively growing cultures, the cell walls are character­ istically thin and smooth; they do not thicken with age, but in cultures 2 or 3 months old, some of the cells may have a bubble-like thickening at one pole (Fig. 3). The chloroplast is parietal and cup-like, and the single nucleus lies in a small area of colorless cytopla5m. Young vegetative cell5 recently derived from zoospores have a single pyrenoid, but, as the cells increase in size, 2 elongate pyrenoids are always present, these resulting from division of the original one (Fig. 2, 73). This Some Algae From Arid Soils TABLE 2. Samples of arid soils, their pH, and algae observed and/ or isolated therefrom Origin of soil samples pH Organisms Sand from top of sand dunes Lat. 8.0 N eochloris oleoabundans sp. nov. 21 ° 15'N, Long. 55° 15'E. Saudi Scytonema sp. Arabia Chlamydomonas sp. Rippled sand near base of sigmoidal 8.3 Scenedesmus sp. dunes Lat. 20 ° 40'N, Long. 54° Chlorosarcinopsis sp. 40'E. Saudi Arabia Chlamydomonas sp. ( palmelloid stage) Scytonema sp. As-Sanam, S.E. of Batnah Lat. 21 ° 8.2 Chlorosarcinopsis gelatinosa sp. nov. 46'N, Long. 53 ° lO'E. Saudi Arabia Anabaena sp. Nostoc sp. As-Sanam, surface of Zibar, Lat. 8.1 Chlorosarcinopsis sp. 21° 46'N, Long. 53° lO'E. Saudi Spongiochloris incrassata sp. nov. Arabia Bracteacoccus sp. Chlamydomonas sp. Oscillatoria sp. Large sigmoidal dunes near Lat. 8.1 Chlorosarcinopsis sp. 20° 40'N, Long. 54° 40'E. 20 miles Chlamydomonas sp. above Sabkhah. Saudi Arabia Anabaena sp. Top of Sigmoidal ridge Lat. 20° 8.0 Chlamydomonas sp. 25'N, Long. 53° 40'E. Saudi Arabia Chlorosarcinopsis sp. Oscillatoria sp. Six miles north of St. John, Arizona, 7.8 Bracteacoccussp. elevation 5800 ft. Chlorococcum sp. Chlorosarcinopsis sp. Spongiochloris minor sp. nov. Near Apache Junction, Arizona, ele­ 7.8 Chlorosarcinopsis sp. vation 1900 ft. C hlorococcum diplobionticoideum sp. nov. Chlamydomonas sp. Phoenix, Arizona 7.2 Protosiphon sp. Chlorosarcinopsis eremi sp. nov. Bracteacoccus sp. Chlamydomonas sp. Chlorococcum sp. Nostoc. sp. North of Moab, Grand County, 7.6 C hlorosarcino psis sp. Utah Chlorosarcina brevispinosa sp. nov. Chlamydomonas sp. Chantanachat and Bold TABLE 2. (continued) Samples of arid soils, their pH, and algae isolated therefrom Origin of soil samples pll Organis111s Near Superior, Pinal County, Ari­zona Four miles north of Glade, Arizona North of Samalayuca, Chihuahua, Mexico. (The west side of the high­way) North of Samalayuca, East side of the highway, Chihuahua, Mexico South of Samalayuca, East side of the highway, Chihuahua, Mexico. Loess soil, Sdeh Boku, Israel Coarse Sandy soil near Sodom (en­vironment of Dead Sea), Israel Hawmada soil near Sodom, Israel Sandy soil, Gevuloth, Israel Marly soil (environment of Dead Sea), Israel Rich organic soil, Ein Arus, Israel 8.1 7.1 8.0 7.8 7.2 8.3 7.5 7.4 8.0 8.1 7.4 Bracteacoccus sp. Chlorosarcina longispinosa sp. nov. Chlorococcum sp. Chlamydomonas sp. Chlorosarcinopsis sp. C hlorosarcino psis sp. Chlamydomonas sp. T etraspora sp. Oscillatoria sp. Radiosphaera sp. Bracteacoccus sp. Chlorococcum sp. C hlorosarcino psis sp. Scytonema sp. Chlorosarcinopsis sp. Chlorococcum sp. Chlamydomonas sp. Diatoms. Chlorosarcinopsis sp. Chlorococcum sp. Chlamydomonas sp. Nostoc sp. Anabaena sp. Diatoms. Chlamydomonas sp. Chlorococcum sp. Oscillatoria sp. Chlorosarcinopsis sp. Oscillatoria sp. Lyngbya sp. Chlorosarcinopsis sp. S pongioc hloris sp. Scytonema sp. xanthophycean alga Chlorosarcinopsis sp. Chlamydomonas sp. Anabaena sp. Chlorosarcinopsis sp. Chlamydomonas sp. Oscillatoria sp. Some Algae From Arid Soils feature is very characteristic of the species. The pyrenoids, of course, are obscured by starch and oil in cells from cultures from the stationary phase of growth. In cells of cultures 3 weeks old and older, it has been possible, by use of Sudan IV, to demonstrate a considerable amount of oil. As the cultures age, these oil droplets increase in size and finally coalesce, thus crowding the chloroplast to one pole of the cell (Fig. 4-6, 7 4) . Asexual reproduction is accomplished by means of zoospores (Fig. 7) and aplanospores formed by successive bipartition. Zoospore size is between 2 and 3.5µ. in width and 3.6 and 4.5µ. in length. The individual ovoid zoospore possesses a single . nucleus, a parietal chloroplast with a posterior pyrenoid, an anterior stigma, 2 anterior contractile vacuoles, and 2 flagella of equal length (Fig. 8). The zoospores at quiescence become spherical ( Protosiphon-type; Starr, 1955) (Fig. 1 ) . The flagella, stigmata, and contractile vacuoles disappear, and the young vegetative cells then begin to grow. Reproduction is also effected by aplanospore formation. The aplanospores re­ semble zoospores soon after quiescence. They are liberated by rupture of the parent wall. Sexual reproduction has not been observed. Two-week-old colonies on Bristol's agar are dull-shiny and homogeneous except for lines made at inoculation (Fig. 75). Cultures remain green for as long as 3 months. Additional supplementary attributes will be compared with those of other known species in Tables 4-7. Up to date, 8 species of N eoc hloris have been described as follows : N . aquatic a (type species) Starr, 1955 N . gelatinosa Herndon, 1958b N. terrestris Herndon, 1958b N . fusispora Arce and Bold, 1958 N . pyrenoidosa Arce and Bold, 1958 N. minuta Arce and Bold, 1958 N. alveolaris Bold, 1958 N . pseudoalveolaris Deason and Bold, 1960 Cells from pure cultures of N. oleoabundans were compared carefully, with re­ spect to both morphological and physiological attributes, with the previously described species of N eochloris. The morphological comparisons (Table 3) were made of material grown under standard conditions. From these comparisons, it · became clear that the organism isolated from Arabian sand is unlike all of those described before the present investigation was undertaken. Supplementary attributes in the genus Neochloris Supplementary attributes investigated in most recent studies of soil algae in our laboratory (Deason and Bold, 1960; Bold and Parker, 1962) were not studied in TABLE 3. Morphological attributes of species of N eochloris Organism N. aquatica N. alveolaris N. fusispora N. gelatinosa N. minuta N. oleoabundans N. pseudoal­veolaris N. pyrenoidosa N . terrestris Vegetative cell si1.e 13.5µ. 25µ. 22µ. 17µ.-37 µ. 18µ. 6µ.-25µ. 25µ. 13µ.-25µ. up to 45µ. 35µ.--45µ. up to 75µ. .l.ell wall {al VBTIOUS phases of growth) outer wall layer not thickening markedlyh( o.w.n.t) (o.w.n.t) (o.w.n.t) (o.w.n.t) prominent, com­mon gelatinous matrix present outer wall layer thickening mark­edly with age (o.w.n.t) (o.w.n.t ) outer wall layer thickening mark­edly with age (o.w.n.t) No. of nuclei multi­nucleatea um-nucleate multi-nucleate multi-nucleate multi-nucleate multi-nucleate um-nucleate multi-a nucleate um-nucleate J'OSlt.Jon ana no. of pyrcnoids 1, excentric 1, excentric 1, excentric 1, or more, excentric 1, excentric 1 (young cells), 2, or rarely 3 (mature cells) , excentric 1, excentric ·­ 1-5, excentric 1, excentric Nature of 1.oospores 5.5µ. long, 2.5µ. wide, elongate 8-10µ. long 4-5µ. wide, fusiform 12-22.5µ. long, 2-3µ. wide, elongate, pointed both ends 5-9µ.long 2.5--4µ. wide, elongate 3-5µ.long 2-3µ. wide, almost spherical 3.6µ. long 2.7µ. wide, ovoid 8-10µ. long 4-5µ. wide, pointed anterior; rounded posterior 7-10µ. long 3-5µ. wide, compressed 5-12µ.long 2-6µ. wide, elongate • Nuclear number inrreases gradually as the cell ages. b In stationary-phase cultures Some Algae From Arid Soils previously described species of Neochloris. Accordingly, the writers have included in this investigation a comparative study of the known species of Neochloris with respect to supplementary attributes. This involved, as a necessary prerequisite, purification of the cultures of N. gelatinosa, N. pyrenoidosa, and N. terrestris, not previously available in this condition. The results of comparative studies of certain supplementary attributes are summarized in Tables 4-7. It is clear from these data that the supplementary attributes are useful in distinguishing among the several species of Neochloris. As indicated in Table 5, ribose and xylose markedly inhibited growth of most species of Neochloris. TABLE 4. Colonial attributes of 2-week-old cultures of N eochloris grown under standard conditions on Bristol's agar; color of aging (stationary-phase cells) on Bristol's agar slants Species N. aquatica N. alveolaris N. fusispora N. gelatinosa N. minuta N. oleoabundans N. pseudoalveolaris N. pyrenoidosa N. terrestris Macroscopic dry dry dry shiny shiny dull-shiny dull-shiny dull-shiny dry 14X. transmitted light rough (delicate, reticulate, vermiform) homogeneous (slightly granular) delicate, reticulate, rugose ; adherent to agar surface homogeneous, with dark lines, uniform translucent dots homogeneous homogeneous homogeneous homogeneous homogeneous yellow-green green green-orange orange at the edge of slant light green green green green with orange tinge orange at the edge of slants With respect to the Multidisk test (Table 7), the following conclusions are drawn: ( 1) all 9 species were inhibited by Paromomycin, Triclobisonium choride and Methenamine mandelate. ( 2) Dihydrostreptomycin slightly inhibited only 2 species. ( 3) Oxytetracycline, Sulfadimethoxine, and sulfadiazine each slightly · inhibited only 1 species. ( 4) None of the 9 species is inhibited by chlortetracycline, Bacitracin, Chloramphenicol, Novobiocin, Oleandomycin, Penicillin, Ristocetin, Syncillin, Cycloserine, Staphcillin, Triacetyloleandomycin, Tetracycline, Sulfisomi­ C hantanachat and Bold TABLE 5. Efjects of certain carbon compounds8 on growth of Neochloris species in light Bristot·s Sodium Species solution Arabinose Glucose Fructose acetate Ribose Xylose N. aquatica good+ fair good fair none good fair N. alveolaris trace+ trace good good fair trace trace N . fusispora fair trace+ fair+ trace ­ none trace none N. gelatinosa fair+ fair fair good fair fair trace N. minuta fair none fair+ good none good fair N . oleoabundans fair none fair none none trace- none N. pseudoalveolaris trace trace good good fair trace trace N. pyrenoidosa fair trace trace+ trace ­ trace ­ trace trace N. terrestris fair trace+ fair+ fair fair+ trace+ trace­ •Added at a nmcentration of O.i5% to Bristol's solution. TABLE 6. Comparative growth species of Neochloris on/in certain complex media Yeast-Proteuse· Barto Bacto thio-Bae to Baeto Species extrart agar peptone agar nutrient agar glycollate med. AC broth nutrient broth N. aquatica good excellent fair trace fair fair N . alveolaris excellent excellent fair fair trace ­ trace N. fusispora fair good fair trace trace + fair N. gelatinosa fair fair fair fair- none fair+ N. minuta good excellent good trace trace good N. o[eoabundans trace good trace good fair+ trace ­ N. pseudoalveolaris excellent fair fair trace ­ none trace N. pyrenoidosa fair good fair trace none trace N. terrestris good excellent excellent good+ fair fair TABLE 7. Sensitivity of species of Neochloris to certain agentsa 11-lO~A 11 -1028 IA 1.-\B rn re ( C:,\ .cs if) •E (K (:\ ,:-;y r:-;s +1 +1 +J +J +1 +. +, +1 +:1 + .1 +. +. N . aquatica + N. alveolaris N. fusispora + + + + N. gelatinosa + + + + N. minuta + + + + + + N. oleoabundans + + + _j_ N. pseudoalveolaris + + + N. pyrenoidosa + + + + N. terrestris + + + + Some Algae From Arid Soils TABLE 7.-(continued) lt-102C 11-102D (OL (P (PB (PM (R (SY (CY (S (SC (T (TPO (TE +) +) +l +) +) +) +) +) +) +) +) +) N. aquatica + N . alveolaris + N . fusispora + + N. gelatinosa + + + ( sl) N. minuta + + + N. oleoabundans + + N. pseudoalveolaris + + ( sl) N. pyrenoidosa + + N. terrestris + + 11-102E 11-102F (EL CG (KY insequentes saepe subeuntes aut magnitudine graditim auctae et per rup­turam membranae parentalis directe liberatae. Reproductio per coniunctionem isogametarum bipartitione successiva cellularum vegetativarum diploidearum atque aplanosporarum factarum. Gametae 14-18µ. long., 7-9µ. lat., ovatae et typi Chlamydomonadis (membranam habentes) chloro­plastum parietalem, unicum pyrenoideum posteriorem, nucleum anteriorem, duas vacuolas contractiles, stigma et duo flagella longitudine aequa quasi aeque longa ac corpus cellulae habentes. Gametae per parthenogenesim ad cellulas haploideas Chlamydamonadoideas binas quatemasque efficiendas saepe evolutae, hae aggre­ gatae aequae magnitudine ac cellulae vegetativae diploideae ( dissimiles hucusque cellulis haploideis in C. diplob.iontico). Origo: Ex exemplo soli prope locum Apache Junction, Arizona dictum. Cult. num. Ch-9. The genus C hlorococcum, according to Starr ( 1955 ) pos~esses the following 3 distinctive attributes: ( 1) a hollow, parietal chloroplast with or without a surface opening; ( 2) 1 or more pyrenoids; ( 3) biflagellate zoos pores which do not become spherical at quiescence. The organism here described as a new species, Chloro­coccum diplobionticoideum, was isolated into bacteria-free culture from a soil sample collected near Apache Junction, Arizona. This alga seems to be strikingly similar in life cycle to C. diplobionticum Herndon ( 1958b) but differs from the latter sufficiently to be considered a separate taxon. In the life cycle of these organisms, both haploid and diploid phases seem to occur simultaneously. The diploid vegeta­tive cells, which originate as zygotes, may develop thickened walls and become spherical as they enlarge (up to 36,u ), a typical C hlorococcum-like attribute (Fig. 76, 81, 85). These presumably diploid vegetative cells have a hollow, cup-like chloroplast with a single pyrenoid and nucleus (Fig. 10). At maturity, in actively growing cultures, they undergo successive bipartitions to form a large number (ca. 32) of biflagellate gametes (Fig. 12, 76 ), which are liberated by rupture of the parent cell wall. The individual gametes (Fig. 13, 77) are ovoidal, 14-18,u long and 7-9,u wide. Each has 2 anterior flagella, about body length, an anterior nucleus, stigma, 2 contractile vacuoles, and a parietal chloroplast with posterior pyrenoid. These motile cells swim for only an hour or so, and many of them unite isogamously (Fig. 16-20; 80, 81) . Inasmuch as the cultures studied were clonal, the organisms must be considered homothallic. Soon after plasmogamy, the walls of the gametes are shed posteriorly, and a new wall is secreted by the zygote (Fig. 20, 81). Flagella, stigmata, and contractile vacuoles disappear shortly. One of the 2 pyrenoids con­tributed by gametes begins to decrease in size, the latter a possible manifestation that one gamete plastid is disintegrating. The zygotes, as noted above, are actively photosynthetic; they enlarge, and function as vegetative cells. The fate of gametes which fail to unite in sexual reproduction is illustrated in (Fig. 14, 82). Soon after cessation of motility, these gametes undergo bipartition (s) to form 2 or 4 Chlamydomonas-like daughter cells (Fig. 14, 82-84), as in Chloro­coccum diplobionticum. These daughter cells remain within the parental wall. After some growth, each daughter cell may undergo further division to form either haploid gametes or aplanospores. Those which fail to produce gametes or aplano­spores may be liberated directly from the parental wall. C hantanachat and Bold In addition to the formation of gametes by the diploid vegetative cells, under certain conditions, aplanospores may develop (Fig. 78). The latter may undergo gametogenesis while still enclosed within the original parental cell wall (Fig. I5). In the stationary phase of growth, many of the cells develop excentrically thick­ened walls (Fig. I I), which may reach 7p. in thickness. Cells from such cultures are bright orange and filled with oil droplets. Two-week-old Bristol's agar (Fig. 86) are dry and coarse-granular; 3-month-old cultures remain green, except the thick-walled cells which become bright orange. Additional supplementary attributes which serve to distinguish C. diplobiontic­oideum from C. diplobionticum are shown in Tables 8, 9. With respect to colony TABLE 8. Colonial attributes of 2-week-old cultures of Chlorococcum diplobionti­cum and C. diplobionticoideum grown under standard conditions on Bristol's agar; color of aging (stationary-phase) cultures on Bristol's-agar slant Species 1\1arros<·opic 20 X, transmitted light Color at 3-months old C. diplobionticum dull, dry rough, slightly vermiform green• C. diplobionticoideum dry coarse granular green" • Diploid cells orange. b Thirk-walled cells orange. TABLE 9. Efjects of certain carbon compounds• on growth of C. diplobionticum and C. diplobionticoideum in light. nristol"s Sodium Species solution Ara hi nose FructflSC" acetate Ribo~e Xylose C. diplobionticum good good trace trace fair trace C. diplobionticoideum trace trace-none none trace-noneh •Added at a concentration of O.i5% to Bristol's solution. b ProhRhly requires growth fartor(s ) . characters, there are only minor differences; but with respect to growth in organic carbon sources, several differences are evident. With respect to the Multidisk tests, the following conclusions are drawn: ( 1) Chlortetracycline, Carbomycin, Demethylchlortetracycline, Erythromycin, Di­hydrostreptomycin, each inhibited only growth of C. diplobionticoideum. (2) Nitrofurazone and Iso-nicotinic acid hydrazide each inhibited only the growth of C. diplobionticum. ( 3) Amphotericin B, Colistin sulfate, Kanamycin, Neomycin, Nystatin, Polymyxin­B, Paromomycin, Furaltadone, Nitrafurantoin, Methenamine mandelate and Para­amino-salicylic acid each inhibited growth of both species. As noted above, C hlorococcum diplobionticoideum is very similar in its mor­phology and life cycle to C. diplobionticum Herndon ( 1958b). With respect to the latter, both seem to have in their population diploid and haploid cells.7 However, there is a striking difference between the 2 organisms. In C. diplobionticum, the haploid cells, which originate from single gametes, increase very little in size, and thereupon release their 2 (typically) division products. In contrast, in C. diplo­bionticoideum the haploid cells, which also originate from single gametes, fail to release their 2 or 4 division products, but, instead, increase gradually in size, ulti­mately equaling the diploid cells in this respect. Further divisions to form either gametes or aplanospores occur, or each daughter cell may be liberated directly from the parental cell wall. One might well contrast the ephemeral and transitory nature of the haploid cells in C. diplobionticum with the persistent haploid phase of C. diplobionticoideum; and, furthermore, in light of this, one might consider the former to exhibit a heteromorphic and the latter an isomorphic diplobiontic life cycle. Cytological study, however difficult, is strongly indicated for these anomalous taxa of the genus Chlorococcum. SPONGIOCHLORIS Starr The generic attributes of Spongiochloris, as defined by Starr ( 1955), are: ( 1 ) chloroplast net-like; ( 2) with 1 or more pyrenoids; ( 3 ) zoospores becoming spherical at quiescence. Two new species of Spongiochloris have been studied dur­ing the present investigation. SPONGIOCHLORIS minor sp. nov. (Fig. 21-28; 87-92) Cellulae omnis aetatis sphaericae; chloroplastus cellulae iuvenis parietalis poculi­formis, gradatim spongiosus reticulatus transformatus; cellulae ad 40µ. diam., membranis aetate paululum incrassatis, non stratifactis; cellulae maturae multi­nucleatae. Reproductio asexualis per zoosporas aplanosporasque fissione progrediente ef­f ectas, membrana parentali rupta liberatas. Zoosporae ovate chloroplastum par­ietalem, stigma anterius, nucleum, duas vacuolas contractiles et flagella habentes, 8.85µ. long., 3--3.5µ. lat.; pyrenoideum posterius; zoosporae quiescentes sphaericae factae. Reproductio sexualis non observata. Origo. a loco distante 6 milia passuum ab oppido North St. John, Arizona dicto. Cult. num. Ch-8. The organism herein described as a new species, Spongiochloris minor, wai isolated into bacteria-free culture from a soil sample collected 6 miles from Nortl 7 The cytological evidence on this point is not yet adequate for either organism. St. John, Arizona. Its generic attributes are clearly those of the genus Spongiochloris as set forth by Starr ( 1955). The vegetative cells are spherical at all ages (Fig. 87). Cells with a maximum diameter of 4011 have been observed. The cell wall is thin, but it may thicken slightly in cultures 1 month old or older. The chloroplast of S. minor varies in appearance with age. Young vegetative cells have a parietal chloroplast (Fig. 21 ) , but as the cells increase in size, the chloroplast segments (Fig. 22), finally becoming a net-like structure. The chloro­plasts of mature cells are typically net-like (Fig. 23, 88). The net-like character of the chloroplast becomes less distinct as the cells age because of the presence of starch and oil in large quantities (Fig. 89). A single, excentric pyrenoid is always embedded in the chloroplast; occasionally 2 or more are present. In older cells of S. minor, there may be many pyrenoids resulting from fragmentation of the original one (Fig. 24). The vegetative cells of S. minor are uninucleate only when very young (Fig. 21 ) ; as the cells enlarge, they become multinucleate (Fig. 23). The nuclei can be demonstrated with aqueous iodine ( 12-KI) and acetocannine. Asexual reproduction is accomplished by means of aplanospores (Fig. 25-26, 90) and zoospores (Fig. 27), formed by progressive cleavage of the protoplast. The zoospores (Fig. 28) are ovoid, 8p. in average length and 3p. in average width. They are of the Protosiphon type (Starr, 1955) in that they become spherical immediately at quiescence (Fig. 91 ) . Individual zoospores possess an anterior stigma, 2 anterior contractile vacuoles, 2 flagella of equal length, a single anterior nucleus and a parietal chloroplast with posterior pyrenoid (Fig. 28). The aplanospores and zoospores vary in number, depending on the size of the cell which produced them; young aplanospores are 4-6p. in diameter. They are released by rupture of the parent wall. Sexual reproduction has not been observed. Two-week-old colonies on Bristol's agar are dry (macroscopically) and min­utely glomerulate (Fig. 92). Comparison of S. minor with other known species of S pongioc hloris has been made in Tables 10-14. SPONGIOCH LORIS incrassata sp. nov. (Fig. 29-35; 93-96) Cellulae omnis aetatis sphaericae, chloroplastus cellulae iuvenis parietalis poculi­fonnis, in plastidem spongiosam reticulatam, filia in superficie relative tenuia, prope centrum grossa habentem, gradatim transfonnatus; cellulae 54p. diam. attingentes; membranae in culturis duarum hebdomadum aetate manifeste in­crassatae stratifactaeque; cellulae maturae multinucleatae. Reproductio asexualis per zoosporas aplanosporasque fissione progrediente ef­fectas, membrana parentali rupta liberatas. Zoosporae ovatae, chloroplastum parietalem, pyrenoideum posterius, stigma anterius, duas vacuolas contractiles et flagella habentes, 7-8µ long., 2-4µ lat., quiescentes sphaericae factae. TABLE 10. Morphological attributes of known species of S pongiochloris Organisms Vegetative cell size Cell wall Nature of chloroplast l'osiuon and no. of pyrenoids Nature of zoospores S. excentrica 30µ.-60µ. not thickening markedly (bubble-like thickening may be present) net-like (coarse strands) 1, excentric 15µ. long X 2µ. wide, elongate S. incrassata 38µ.-54µ. outer wall layer thick and stratified (3µ. or more) net-like (fine strand at surface, coarse at the center) 1, central 7µ. long X 2µ. wide, ovoid S. lamellata 80µ. outer wall layer thick (25µ.), com­posed of several layers sponge-like rather than net-like several, excentric 9-13µ. long x 3-3µ. wide, elongate S. minor 36µ.-40µ. outer wall layer thickening . markedly with age (2.7µ.) net-like (coarse strands) several, excentric 8-8.5µ. long x 3-3.5µ. wide, ovoid S. spongiosa 30µ.-100µ. outer wall layer not thickening markedly net-like (very fine strands) 1, central 15µ. long X 2µ. wide, elongate Reproductio sexualis non observata. Origo: a loco As-Sanam, Lat. 21 °461 N, Long. 53 ° 101 E, Saudi Arabia dicto. Cult. num. Ch-4. The organism here described as a second new species of Spongiochloris, S. in­crassata, was isolated into bacteria-free culture from a soil sample collected from As-Sanam. Lat. 21°461 N, Long. 53°101 E Saudi Arabia. The vegetative cells are spherical and vary in size (Fig. 93). Cells with a maxi­mum diameter of 54µ. have been observed. The cell wall is markedly thickened and stratified even in 2-week-old cultures (Fig. 29, 94, 95) and may be as much as 2 or 3µ. thick. The chloroplast of S pongiochloris incrassata varies in appearance with age. Young vegetative cells, recently developed from zoospores, have a parietal chloro­ Chantanachat and Bold TABLE 11. Colonial attributes of 2-week-old cultures of S pongiochloris grown under standard conditions on Bristol's agar; color of aging (stationary-phase) cells on Bristol's agar slants Species 1\1acroscopic appearance 14 X. transmitted light Color at 3 months S. excentrica very shiny smooth, homogeneous yellowish-green S. incrassata dry scabellate, cracked into brick-red flecks irregular polygonal patches on green S. lamellata dry homogeneous, but granular orange at the edge of slant S. minor dry homogeneous, minutely entirely bright glomerulate orange S. spongiosa dry homogeneous, but granular green tinged with orange TABLE 12. Efjects of certain carbon compounds8 added to Bristol's solution on growth of S pongiochloris species in light Bristol 's Sodium Species solution Arabi nose Glucose Fructose acetate Ribose Xylose S. excentrica good trace fair+ fair fair fair trace S. incrassata tracec trace fair trace+ trace- trace- trace- S. lamellata fair trace ­ trace+ fair- trace- fair trace S. minor trace+c trace fair+h fair- fair- trace+ none S. spongiosa fair trace good trace none trace trace •Added at a concentration of 0.75% to Bristol's solution. b C.olor <·hanged to orange. « Probably has growth fanor requirement. TABLE 13. Comparative growth of Spongiochloris species on/in certain complex media Yeast-Proteose-Baeto Bacto thio-Bae to Bacto S1>et·ies extract agar peptone agar nutrient agar glycollate med. AC broth nutrient broth S. excentrica excellent excellent good excellent trace fair S. incrassata fair fair fair good trace ­ trace+ S. lamellata fair fair trace trace- trace- trace S. minor fair good fair fair- trace fair- S. spongiosa fair good trace trace trace ­ trace­ plast (Fig. 30 ) ; later, as the cells increase in size, the chloroplast segments (Fig. 31 ) , finally becoming a net-like configuration which is relatively fine-stranded at the surface of the cell, becoming coarse at the center (Fig. 32). The nature of the plastid, of course, becomes obscured as the cells age because of the presence of starch and oil in large quantities. TABLE 14. Sensitivity of S pongiochloris species to certain agents"­ 11-IOZA 11-102B (A (AB (B (C (CA (CS (D (E (K (N (NV (NS +J +) +) +) +) +) +J +J +) +) +J +) S. excentrica + + + + S. incrassata + + + S. lamellata + + + + S. minor + + + + + + (sl) (sl) S. spongiosa + + + + + 11-102C 11-102D (OL (P (PB (PM (R (SY (CY (S (SC (T (TPO (TE +) +) +) +) +) +) +) +) +) +) +) +) S. excentrica + + S . incrassata + + S. lamellata + + S . minor + + + S. spongiosa + + + + ( sl) ( sl) 11-102E 11-102F (EL (G (KY (TR (V (VA (MA (SD (SM (SSS (ST (TH +J +) +) +) +) +) +J +> +) +) +) +l S. excentrica + S. incrassata + S . lamellata + S. minor + S. spongiosa + 11-102G (AL (FC (FR (IH (M (PS 0.04% sulfathiazole +) ?) +J +> +) +J +) S. excentrica + + + trace S. incrassata + + + + trace+ S. lamellata + + fair S. minor + + + + + + + trace- S. spongiosa + + + fair a The agents and the abbreviations for them are listed in Table 1; + indicates inhibition; .... indicates no inhibition; ( sl) means slight inhibition. The pyrenoid of the young vegetative cells is parietal when the plastid is entirely parietal. As the latter becomes more complex and as its net-like character becomes apparent, the pyrenoid becomes central. At least one pyrenoid always is embedded in the chloroplast (Fig. 29-32, 93) ; occasionally 2 or more are present. C hantanachat and Bold The cells are uninucleate only when very young (Fig. 93, 95) ; as they enlarge, they become multinucleate (Fig. 29). The nuclei are suspended within the hyaline cytoplasm of the living cells. Asexual reproduction is accomplished by means of aplanospores and zoospores (Fig. 33, 34) formed by progressive cleavage of the protoplast. The zoospores are ovoid, 7-8µ. in length and 2-4µ. in width. The individual zoospore possesses 2 flagella of equal length, 2 anterior contractile vacuoles, an anterior stigma, a single anterior nucleus and a parietal chloroplast with a posterior pyrenoid. The most prevalent, obvious, and characteristic method of multiplication of the organism is by aplanospore formation; zoosporogenesis is less frequently encounter­ed. The aplanospores and zoospores vary in number, depending on the size of the cell which produced them. Sexual reproduction has not been observed. Two-week-old colonies on Bristol's agar are dry, rugose and cracked (Fig. 94) . Additional supplementary attributes are listed in Tables 11-14. In appraising the morphological and supplementary attributes of Spongiochloris minor and S. incrassata, comparative study of these attributes in the other known species was included. Table 10 summarizes the morphological and supplementary attributes of all the known species of Spongiochloris which have been studied comparatively in this investigation. Appraisal of the morphological and supplementary attributes of S. minor and S. incrassata has impelled the writers to consider these 2 isolates of Spongiochloris from desert soils as new species. Morphologically, S. incrassata and S. minor are closest to S. spongiosa Starr. They differ from each other especially in cell size and thickening of the outer wall layers in stationary phase cultures. With respect to supplementary attributes, several points are noteworthy. For example, the color of S. minor becomes orange in Bristol's solution supplemented with glucose, while the other species remain green. Growth of S. excentrica is excellent on yeast-extract agar, Proteose-peptone agar and Bacto Thioglycollate media (Table 13), in contrast to that of the other species. With respect to the "Multidisk" tests (Table 14), the following conclusions can be drawn: ( 1) All 5 species are inhibited by Amphotericin B, Colistin sulfate, Neomycin, Polymyxin-B, Paromomycin, Triclobisonium chloride, Nitrofurantoin and Methena­mine mandelate. ( 2) Demethylchlortetracycline, Erythromycin, N ystatin, Cycloserine, Nitrofurazone and Iso-nicotinic acid hydrazide each inhibited only 1 species. ( 3) Dihydrostreptomycin, Furaltadone, and Para-amino-salicylic acid inhibited only 2 species. ( 4) No 2 species of the 5 subjected to the "Multidisk" test had identical patterns of sensitivity. The following key will aid in distinguishing all the currently known species of S pongiochloris. Key to the species of S pongiochloris Starr 1. Cell wall thickening markedly with age .. ..... ......... ... ......... ... .......... .... ...... 2 1. Cell wall not thickening markedly with age .. . .. ............................................ 4 2. Mature cells with 1 pyrenoid ......................................S. incrassata sp. nov. 2. Mature cells with more than 1 pyrenoid ...................... ............................ 3 3. Young cells coherent by their cell walls; 3-month-old cultures on Bristol's agar becoming orange at the margin of colony .................. ....................................S. lamellata Deason & Bold ( 1960) 3. Young cells isolated, separate; 3-month-old cultures on Bristol's agar becoming bright orange all over .................... S. minor sp. nov. 4. Pyrenoid central in mature cells . .......................... S. spongiosa Starr (1955) 4. Pyrenoid excentric in mature cells ..................... S. excentrica Starr ( 1955) RADIOSPHAERA dissecta (Korschikoff) Starr. (Fig. 97-104) Two organisms isolated from arid soils clearly possess all the generic attributes of Radiosphaera, namely: ( 1) vegetative cells with an asteroid chloroplast con­sisting of peripheral lobes joined with a central mass; ( 2) vegetative cells with at least 1 pyrenoid; (3) production of Chlamydomonas-type (walled) zoospores which do not immediately become spherical at quiescence. Two organisms were isolated into bacteria-free culture from soil samples from 2 different collections. One (Ch-14) was isolated from a sample collected from the west side of the highway, North of Samalayuca, Chihuahua, Mexico. The other (K-9) was isolated from a sample of algae from South Dakota dusts (cour­tesy of Mr. R. M. Brown, Jr., Department of Botany, The University of Texas). The 2 organisms were carefully studied and compared with R. dissecta (Korschi­koff) Starr ( 1955). The 3 taxa are morphologically alike. The vegetative cells are non-motile and spherical, except that young cells (those recently derived from walled zoospores) are ellipsoidal (Fig. 97). As the cells age, they gradually become spherical. Cell size is variable (Fig. 103) and, as the cultures age, cells up to 150µ. in diameter are not rare, although smaller cells are always present. The cell wall is slightly thickened with age. The chloroplast is asteroidal, consisting of peripheral lobes joined with a central mass (Fig. 98). In young cells, the chloroplast is parietal and the lobes are simple. As the cells increase in size, the periphery of the chloro­plast becomes more lobed, the lobes being joined with the central portion which contains a single pyrenoid. The pyrenoid appeared to be composed of many radiat­ing, crystal-like bodies. The nature of chloroplast and pyrenoid is obscured in old cultures because of the presence of oil and starch granules (Fig. 104). Young vegetative cells are uninucleate, but as the cells increase in size, they become multi­nucleate; the nuclei lie between the lobes of the chloroplast. Asexual reproduction is accomplished by means of zoospores (Fig. 99-102) Chantanachat and Bold and aplanospores formed by progressive cleavage of the protoplast. They are of the Chlamydomonas-type (Starr, 1955), possess a thin wall, parietal chloroplast with posterior pyrenoid, single anterior nucleus, anterior stigma, 2 flagella of equal length and 2 contractile vacuoles. The zoospores retain their shape upon qui­escence. Reproduction is also effected by aplanospore formation. The aplanospores resemble immature vegetative cells. They are liberated b~ rupture of the parental cell wall. Sexual reproduction has not been observed. At the time ( 1955) that Starr worked with Radiosphaera, physiological at­tributes had not been considered in the taxonomy of chlorococcalean algae. In the present investigation, the writers have compared certain supplementary attributes in Starr's strain and with those in the 2 newly isolated ones. The results indicated that although morphologically indistinguishable, the 3 isolates differ physiologi­cally. Tables 15-18 show that with respect to colony characters (dry and rough), the 3 organisms are similar, but with respect to sensitivity to antibiotics and growth in -organic media (in the light ) , some differences are evident. There are only minor differences in tests with organic carbon, but with yeast-extract agar and sensitivity to antibiotics, greater differences among the isolates are apparent. CHLOROSARCINOPSIS Herndon Two isolates of Chlorosarcinopsis have been studied intensively in the present investigation and described as new taxa. TABLE 15. Colonial attributes of 2-week-old cultures of Radiosphaera grown under standard conditions on Bristol's agar; color of aging (stationary-phase) on Bristol's agar slants Species ~facroscopic l.J X. tran~mitted light Color at ~ months R. dissecta dry rough, (coarse granular) light green R. dissecta (K-9) dry rough, (coarse granular) light green R . dissecta (Ch-14) dry rough, (coarse granular) light green TABLE 16. Effects of certain carbon compoundsa on the growth of Radiosphaera in light Bristol·s Sodium Spec-ies solution Arabinose Glucose Fructose acetate Rih"se Xylose R. dissecta fair fair trace + trace- trace ­ trace+ none R . dissecta (K-9) fair fair+ trace+ trace none fair+ fair ­ R. dissecta (Ch-14) trace trace- trace- none none trace ­ trace ­ • Added at a concentration of 0.75% to Bristol's solution. TABLE 17. Comparative growth of Radiosphaera on/in certain complex media Yeast-Proteose· Bae to Baeto Bacto thio-Bae to Species extract agar peptone agar nutrient agar AC broth glycollate med. nutrient broth R . dissecta good fair+ fair+ trace- fair trace R. dissecta (K-9 ) trace fair trace- none trace none R . dissecta (Ch-14) trace trace- trace- trace- trace none TABLE 18. Sensitivity of isolates of Radiosphaera to certain agents"' 11-102A 11-lOZB (A (AB (B (C (CA (CS (D (E (K (N (NV (NS+l +) +) +) +) +) +) +) +) +) +) +) R . dissecta + + + + + + + + R. dissecta (K-9) + + + + + + R . dissecta (Ch-4) + + + + 11-t02C 11-1020 (OL (P (PB (PM (R (SY (CY (S (SC (T (TAO (TE +) +) +) +) +l +) +) +) +) +) +) +) R . dissecta + + + + R . dissecta (K-9) + + + + R. dissecta (Ch-14) + + + 11-102E 11-102F (EL (G (KY (TR (V (VA (MA (SD (SM (SSS (ST (TH+l +) +) +) +) +) +) +) +) +) +) +) R. dissecta + R . dissecta (K-9 ) + R. dissecta (Ch-14 ) + 11-102G (AL (FC (FR (IH (M (PS 0.04% sulfathiazole +) r) +) +) +) +) +) R . dissecta + + + + + fair R. dissecta (K-9) + + + + fair R. dissecta (Ch-14) + + + + trace a The agents and the abbreviations for them are listed in Table 1; + indicates inhibition; .... indicates no inhibition; (sl) means slight inhibition. CHLOROSARCINOPSIS gelatinosa sp. nov. (Fig. 36-42; 105-108) Cellulae typice in fasciculis tridimensionalibus, in matrice communi inclusae; cellulae tantummodo iuvenes ( e zoosporis nuper oriundae) singulares, hae sphaeri­cae 6-10,u. diam. Cellulae uninucleatae chloroplastum parietalem poculiformen et pyrenoideum immersum typice excentricum habentes. Membrana cellulae semper teriuis. C hantanachat and Bold Reproductio asexualis per zoosporas typice 4-8 in unaquaque cellula bipar­titionibus succes.5ivis effectas; zoosporae ovatae 6-8µ. long., 2-5µ lat., duo flagella anteriora aequa, vacuolas contractiles nucleum stigmaque anterius et chloro­plastum parietalem, pyrenoideo posteriore praeditum, habentes; zoosporea quies­centes sphaericae factae. Reproductio sexualis non observata. Origo: a loco as-Sanam, air-strip surface of Zibar; Lat. 21°46' N, Long. 53 °1 O' E, Saudi Arabia dicto. Cult. num. : Ch-5. The organism herein described as a new species, Chlorosarcinopsis gelatinosa, was isolated into bacteria-free culture from a soil sample collected from As-Sanam, air-strip surface at Zibar, Lat. 21°46' N, Long. 51°1 O' E, Saudi Arabia. Vegetative cells are spherical when solitary, although when in groups they may be angular as the result of mutual compression (Fig. 107). Young solitary vege­tative cells attain a diameter of 6-1 Oµ. (Fig. 36). They are spherical, possess a parietal chloroplast and an excentric pyrenoid; the cytoplasm is granular and usually contains abundant starch granules. The cells are uninucleate (Fig. 36). As they approach 6-10µ. in diameter, they either undergo zoosporogenesis or divide vegetatively (Fig. 38-40; 106) . Vegetative cell division occurs in actively growing cultures in Bristol's liquid or agar media. Cytokinesis is preceded by di­vision of the pyrenoid and by nuclear division. A partition is laid down across the center of the parent cell, dividing it into 2 daughter cells (Fig. 37) which remain in close association with the parent cell wall. Vegetative growth is resumed by the daughter cell<; which, in tum, may undergo cell division. The direction of each vegetative division is, usually, perpendicular to the preceding, so that packets of cells are produced (Fig. 39, 105). Cells in these packets may partially dissociate, but the groups of cells are quite characteristic, especially in older cultures ( 2­month-old cultures) . The packets in this organism are composed typically of 2 or 4 cells usually lying in 1 plane, and they adhere in large clusters with a common gelatinous matrix (Fig. 105) . The cell wall is delicate. Aqueous mounts in India ink and Methylene blue of material from I-month-old cultures demonstrate the presence of an external, gelatinous layer, a common matrix (as defined by Deason and Bold, 1960). Cells of cultures 1-month-old become bright orange, and it has been possible, by the use of Sudan IV, to demonstrate a considerable amount of oil in such cells. The cells are uninucleate throughout their development (Fig. 36, 3 7, 40, 4 2). This attribute usually is evident from observations of living cells but has been con­firmed by preparations stained by aqueous I2-KI and acetocarmine. Asexual reproduction is accomplished by dis.5ociation of the cell packets and by zoospore formation. The zoospores arise by successive bipartitions of the protoplast (Fig. 40). Usually 4-8 zoospores are produced by each parent cell. The zoospores Some Algae From Arid Soils are liberated by rupture of the parent wall (Fig. 106). They are of the Proto­siphon-type (Starr, 1955). Individual zoospores (Fig. 41) are 6-8µ. in length and 3-5.u in width. They are elongate with the anterior pole slightly pointed. Each has 2 flagella of equal length, a single anterior nucleus, parietal chloroplast with pos­terior pyrenoid, an anterior stigma and 2 contractile vacuoles. At quiescence, they become spherical and then begin vegetative growth. Sexual reproduction has not been observed. Two-week-old colonies on Bristol's agar are dry, scabellate vermiform, and adhere to the agar surface (Fig. 108) . Three-month-old cultures become bright orange. Additional supplementary attributes are compared with those of other known species in Tables 20-23. CHLORSARCINOPSIS eremi sp. nov. (Fig. 43-46, 109-112). Cellulae in fasciculis tridimensionalibus ad structuram pseudofilamentosam formandam typice ordinatae; matrix communis nulla; cellulae tantummodo iuvenes ( e zoosporis nuper oriundae) singulares, hae sphaericae 6-8,u diam.; cellulae uninucleatae, chloroplastum parietalem poculiformem et pyrenoideum typice excentricum habentes; membrana cellulae semper tenuis. Reproductio asexualis per zoosporas typice 8-16.u in unaquaque cellula bipar­titionibus successivis effectas. Zoosporae ovatae 9-11 .u long., 4-6,u lat., duo flagella aequa duas vacuolas contractiles nucleum anteriorem stigma et chloroplastum parietalem, pyrenoideo posteriore praeditum, habentes; zoosporae quiescentes sphaericae factae. Reproductio sexualis non observata. Origo: ex exemplo soli a loco Phoenix, Arizona dicto. Cult. num. Ch-10. The second species of Chlorosarcinopsis investigated was isolated into bacteria­free culture from a soil sample collected from Phoenix, Arizona. Its vegetative cells are spherical when solitary, but when aggregated they may become angular as the result of mutual compression. Young, solitary vegetative cells attain a diameter of 6-8,u (Fig. 43). The cell wall is relatively thin at all ages. Aqueous mounts in India ink and Methylene blue have failed to demonstrate a gelatinous matrix. The chloroplast is parietal with a single excentric pyrenoid (Fig. 43-45, 110). The plastids of young vegetative cells possess a unipolar opening. This opening is not usually evident in mature cells. Each cell contains a single nucleus in the central cytoplasm. As the cultures age, pyrenoid and nucleus become obscured because of the formation of abundant starch granules and oil. Asexual reproduction is accomplished by zoospores formed by successive bipar­titions (Fig. 109 ). The zoospores vary in shape and average between 4 and 6.u in width and 9 and 11,u in length. Each zoospore possesses 2 flagella of equal length, 2 anterior contractile vacuoles, an anterior stigma and nucleus, and a parietal Chantanachat and Bold TABLE 19. Morphological attributes of known species of Chlorosarcinopsis Species Vegetative cell size Cell wall liab1t ol growth under standard conditions Nature 01 akinete-like cells Nature of 7.00spores C. aggregata 13.6µ. thin, common matrix absent packets of 2-4 cells none 8-22µ. long x 2-3.4µ. wide, elongate C. dissociata 15-25µ. thin, common matrix absent cells dissoci­ated, packets irregular thick wall size 25-30µ. 6-15µ. long X 3-6µ. wide, spherical to elongate C. eremi 6-8µ. thin, common matrix absent slightly dissociate, packets regu-Jar arranged to form pseu­dofilamen tous groups none 9-11µ. long X 4-6µ. wide, ovoid C. gelatinosa 6-10µ. thin, common matrix present packets regular none 6-8µ. long X 3-5µ. wide, elongate C. minor 12µ. thin, common matrix absent packets regular none 7-12µ. long X 3-51-' wide, fusiform TABLE 20. Colonial attributes of 2-week-old cultures of Chlorosarcinopsis grown under standard conditions on Bristol's agar; color of aging (stationary-phase cultures) on Bristol's-agar slants Species Macroscopic 14 X , transmitted light Color at 3 months C. aggregata dry minutely glomerulate; orange flecks plain margin on green C. dissociata dull-shiny nerved, venose green flecks on orange C. eremi dry rough; adhere entirely closely to agar surface orange C. gelatinosa dry rugose vermiform; bright closely to agar surface orange C. minor dry minutely glomerulate; orange flecks radiate margin on green TABLE 21. Effects of certain carbon compoundsa on growth of Chlorosarcinopsis species in light Bristol 's Sodium Species · solution Arabinose Glucose Fructose acetate Ribose Xylose C. aggregata good good goodb good fair+ good none C. dissociata excellent good excellentb goodb none good fair+ C. eremi good fair good fair fair fair trace+ C. gelatinosa fair+ trace+ good fair- fair trace trace- C. minor excellent fair good+ good trace excellent none 8 Added at a concentration of 0.75% to Bristol's solution. b With orange ring on top of the tube. TABLE 22. Comparative growth of Chlorosarcinopsis species on/in certain complex media Yeast-Protease· Bae to Bacto thio-Bacto Bacto Species extract agar peptone agar nutrient agar glycollate med. AC broth nutrient broth C. aggregata good excellent fair fair excellent" fair C. dissociata fair good fair fair+ excellent" fair C. eremi trace trace trace+ excellent excellent fair C. gelatinosa trace+ trace trace excellentb fair trace+ C. minor good good fair excellentc trace+ fair • Excellent growth only at the bottom of the tube. · b Excellent growth at the top. c Excellent growth at junction of aerobic and anaerobic layers; fair at top. chloroplast with posterior pyrenoid. At quiescence, the zoospores become spherical and the flagella, contractile vacuoles and stigmata disappear. The zoospores then begin vegetative growth. Reproduction is also effected by vegetative cell division, which results in the formation of many-celled packets, usually in 1 plane (Fig. 45, 110). It is quite typical in this organism, even in older cultures ( 1 month old ), that the cells remain in packets which are arranged in pseudofilamentous configurations (Fig. 111 ) . In stationary-phase cultures, however, the cells become somewhat dissociated, but most remain together. Sexual reproduction has not been observed. Two-week-old cultures on Bristol's agar are dry and depress the agar surface (Fig. 112) . Three-month-old cultures become orange. Additional supplementary attributes are compared with those of other known species in Tables 20-23. In addition to Chlorosarcinopsis gelatinosa and C. eremi, 3 other species of Chlorosarcinopsis were available for comparative study in culture. They are C. minor (Herndon, 1958a), C . dissociata (Herndon, 1958a) and C . aggregata (Arce and Bold, 1958). The writers have included a comparative study of all the known species of Chlorosarcinopsis with respect to the supplementary attributes. This Chantanachat and Bold TABLE 23. Sensitivity of Chlorosarcinopsis species to certain agents8 11-102A 1l-I02B (A (AB (B (C (CA (CS (D (E (K (N (NV (NS +) +) +) +) +) +1 +J +J +J +J +i +) C. aggregata + + + + + C. dissociata + + + + + C. eremi + + + + C. gelatinosa + + + + C. minor + I1-I02C 11-1020 (OL (P (PB (P:\I (R (SY (CY (S (SC (T (TPO (TE +J +J +1 +i +) +J +J +) +.1 +i +1 +1 C. aggregata + + C. dissociata + + + + ( sl) ( sl) C. eremi + + C. gelatinosa + + C. minor + + I 1-I02E 1l-102F (EL (G (KY (TR (V (VA (MA (SD (S:'-1 (SSS IST (TH +) +) +J +! +) +J +J +J +i +i +! +1 C. aggregata + C. dissociata + C. eremi + C. gelatinosa + C. minor + 11-IO~G (AL (F (FC (FR (IH (:\! (PS 0.04% sulfathiazole +) +) +) +i +1 +i +i in Bristol"s solution C. aggregata + + + + + excellent ( sl) C. dissociata + + + trace C. eremi + + + + trace C. gelatinosa + + + trace C. minor + + + trace • The agents and the abbreviations for them are listed in Table 1; + indicates inhibition; .... indicates no inhjbition; (sl) means slight inhibition. involved, as a neces.5ary prerequisite, purification of the cultures of C. minor and C. dissociata not previously available in the axenic condition. On the basis of both morphological (Table 19) and physiological attributes (Tables 20-23), it is clear that C. gelatinosa and C. eremi differ from each other and from the species previ­ously described. With respect to colonial attributes, the color change is noticeable. With respect to defined media, several points are noteworthy. For example, arabinose and ribose inhibited growth only in 1 species, C. gelatinosa. Xylose inhibited growth of 4 of the 5 species of Chlorosarcinopsis. The color of 2 species changed in Bristol's solu­tion supplemented with glucose and fructose. Furthermore, the 2 new species did not grow well on yeast-extract agar, Proteose-peptone agar and Bacto Nutrient Agar. With respect to the "Multidisk" tests, the following conclusions are drawn: ( 1) All 5 species were inhibited by Colistin sulfate, Paromomycin, Triclobisonium chloride, Nitrofurantoin, Furazolidone and Methenamine mandelate. (2) Nitrofurazone, Cycloserine each inhibited only 1 species. (3) Nystatin, Dihydrostreptomycin, Furaltadone each inhibited only 2 species. Key to the species of Chlorosarcinopsis Herndon 1. Cells not remaining aggregated in Sarcina-like packets ........................................ C. dissociata Herndon ( 1958) 1. At least some cells remaining aggregated in obvious Sarcina-like packets even in old cultures ........................................................ 2 2. Cells remaining aggregated in packets of more than 4, usually arranged in 2 or 3 planes ........ C. minor. Herndon ( 1958) 2. Cells remaining aggregated in packets of 4 or more cells, usually in 1 plane .... .. ............ . .. . . . . . . . ......... .... . .. ........ ... 3 3. Common matrix present in cultures in stationary phase of growth .......................................... C. gelatinosa sp. nov. 3. Common matrix absent in cultures in stationary phase of growth . .......... .. .. ...... . .... ....... . . .. .... ........ .... .... .... .. ......... 4 4. Packets of cells not arranged to form pseudo-filamentous configuration ..........C. aggregata Arce and Bold ( 1958) 4. Packets of cells arranged to form pseudo-filamentous configuration ...... ...............................C. eremi sp. nov. CHLOROSARCINA Gemeck, The genus C hlorosarcina, according to Vischer ( 1933) and Herndon ( 1958a) includes green algae with the following attributes: ( 1) a cup-like parietal chloro­plast; ( 2) without a pyrenoid; and (3) zoospores (wall-less) which become spheri­cal immediately upon quiescence. Two new species of this genus are described below and subsequently compared with the only other species available in culture, namely, C hlorosarcina stigmatic a Deason ( 1959) (Tables 24-27). CHLOROSARCINA brevispinosa sp. nov. (Fig. 4 7-53; 113-121) Cellulae singulares, aut in aggregationibus pseudofilamentosis, tractatae multo frigore ( e.g.,-8°C per unam habdomadem, deinde ad solutionem Bristolii novam translatae) fasciculas tridimensionales Sarcinoideas formentes. Cellulae singulares Chantanachat and Bold mque ad 25µ. diam., membranis etiam in culturis vestustioribus non perspicue incrassatis; matrix communis semper nulla. Chloroplasti cellularum vegetativarum typice parietales poculiformes bipartiti sine pyrenoideis; cellulae uninucleatae; cultura in incrementi periodo immobili sublutea facta; akineta, spinis brevibus ( usque ad 4µ. long.) praedita, culturis senescentibus abunde facta, ad aplanosporas zoosporasque (?) formandas germinantia, membrana akineti per fissuram aequa­toriam aperta. Reproductio quoque per zoosporas aplanosporasque bipartitionibus successivis effecta5. Zoosporae ovatae, 6-11 µ. long., 3-5µ. lat., chloroplastum parietalem duas vacuolas contractiles anteriores et nucleum anteriorem habentes, stigmata nulla; zoosporae quiescentes sphaericae factae. Reproductio sexualis non observata. Origo: e solo deserti versus septentriones a loco Moab, Grand County, Utah dicta. Cult. num. Ch-11. The organism herein described as a new species, C. brevispinosa, was isolated into bacteria-free culture from a soil sample collected from the desert north of Moab, Grand County, Utah. The vegetative cells are spherical when solitary (Fig. 47--49, 113-115) although pseudofilamentous configurations may be formed (Fig. 11 7). If cultures of this organi5m are stored in the freezing compartment of an electric refrigerator at 8°C for 1 week and then transferred to Bristol's solu­tion, they respond by undergoing intensified vegetative cell divisions to form Sarcina-like packet<;. These are absent in cultures grown at 22-25°C. Young, solitary, vegetative cells (those recently derived from zoospores) have diameters of 4-7µ., while mature cells may reach 25µ. in diameter. The cell wall remains rela­tively thin at all ages. Some of the cells, however, rapidly increaŁe in size, attaining a diameter of 30--35µ.. The cell walls of these are slightly thickened ( 3--4µ.), and their outer wall layers begin to form spiny protuberances (Fig. 49-50, 113, 114, 116, 11 7). These cells are properly termed akinetes. Cell size is variable in any population, as is typical of all zoosporiferous chforococcalean algae, because of differences in the ages of the component cells. The chloroplast of vegetative cells is parietal and cup-or bowl-like and is typi­cally bipartite (Fig. 48). Occasionally, as during zoosporogenesis, more lobes are present. It has been possible by use of aqueous 12-KI to demonstrate abundant starch granules in these cells, but pyrenoids are absent. The chloroplasts of the akinetes are different. They are more or less massive and a considerab'e amount of oil can be demonstrated within them (Fig. 50) by u~e of Sudan IV. A single nucleus is present within the hyaline cytopla5m of the living cells (Fig. 4 7--49). Asexual reproduction occurs by means of aplanospores and zoospores (Fig. 51­53, 118-120) formed by successive bi partitions of vegetative cells. The morphology of zoospores is similar to that of Protosiphon zoospores (Starr, 1955). They range from 6 to l lµ. in length and from 3 to 5µ. in width. Each zoospore (Fig. 52) is biflagellate with the flagella of body length; 2 anterior contractile vacuoles, an anterior nucleus and a parietal chloroplast are present. Stigma and pyrenoid are absent. The number of zoospores in each cell is less than 16, usually 4 or 8. The zoospores are liberated by rupture of the parent wall (Fig. 51 ) . The motile period of the zoospores is apparently short (less than 1 hr). Soon after quiescence, zoo­spores rapidly become spherical to form tiny vegetative cells (Fig. 4 7) ; the flagella and contractile vacuoles soon disappear. Sometimes, some of the spiny akinetes produce aplanospores which resemble zoospores at quiescence. These aplanospores are released by rupture of the spiny wall (Fig. 53, 118). The spiny akinetes apparently represent the resistant phase of this alga. At 2 weeks, cultures of Chlorosarcina brevispinosa consist largely of single cells (Fig. 115), although pseudofilamentous branches may be formed (Fig. 117). As the cultures age, these cells dissociate and the number of spiny akinetes increases; 2-week-old cultures in darkne:s tend to form abundant spiny akinetes. Sexual reproduction has not been observed. Two-week-old colonies on Bristol's agar are dry and rough (Fig. 121 ) . Three­month-old cultures become tinged with orange. Additional supplementary at­tributes are considered comparatively with those of known species in Tables 25-28. CHLOROSARCINA longispinosa sp. nov. (Fig. 54-61; 122-128) Cellulae singulares aut in incrementi periodo immobili fasciculas cubicas effici­entes; periodus pseudofilamentosa tantummodo tempore frigoris gravis (e.g., -8°C per unam hebdomadem) evocata, cellulae deinde ad solutionem Bristolii novam translatae. Cellulae singulares usque ad 12µ. diam. Membranes senescentes non perspicue incrassatae, matrice communi nulla; chloroplastus cellularum vegetativarum typice parietalis poculiformis multipartitus (plerumque quadri­partitus) sine pyrenoideis; cellulae uninucleatae. Culturae in periodo incrementi immobili subluteae factae; akineta membranas crassas necnon spinas longas ( usque ad 9µ.) habentia, culturis senescentibus abunde producta, sine luce per duas heb­domades saltem ad aplanosporas aut fasciculum quatuuor cellularum aut zoosporas ( ? ) formandas germinantia. Reproductio quoque per zoosporas, bipartitione successiva cellularum vegetati­varum plerumque 4 vel 8 in unaquaque cellula effectas; zoosporae ovatae, 8-lOp. long., 3-5µ. lat., duo flagella aequa duas vacuolas contractiles nucleum anteriorem et chloroplastum parietalem habentes; stigmata pyrenoideaque nulla; zoosporae quiescentes sphaericae factae. Reproductio sexualis non observata. Origo: e solo deserti, e regione loci Superior, Pinal County, Arizona dicti collecta. Cult. num. Ch-12. The second species of C hlorosarcina here described as a new taxon was isolated Chantanachat and Bold into bacteria-free culture from a soil sample collected from near Superior, Pinal County, Arizona. Vegetative cells of C. longispinosa are spherical when solitary (Fig. 54, 122), or pseudofilamentous (Fig: 123), or both types may be present in a given culture. The cells occur typi.cally in Sarcina-like configurations or packets formed by vegetative cell division (Fig. 55, 61; 124, 126). The cell wall of vegetative cells is thin and does not thicken with age. The cell walls of the spiny akinetes, present also in this species, are slightly thickened, and the outer layers develop prominent, elongate spines (Fig. 59, 60, 125). The latter may be as long as 5-9,u.. This characteristic alone (although there are others) can be used to distinguish C. brevispinosa from C. longispinosa. The chloroplast of vegetative cells is parietal and divided into several (usually 4) large segments (Fig. 55). Abundant starch granules may be present. As the cultures age, the chloroplasts become less distinct because of the production of oil and starch in large quantities. This is true, especially for the spiny cells ( akinetes) at all ages. The alga is uninucleate (Fig. 54, 55, 59, 61) throughout development. Asexual reproduction is accomplished by means of zoospores formed by suc­cessive bipartitions (Fig. 56, 58) ; the zoospores (Fig. 58) are ovoid or slightly pointed at both ends, 8-10,u. in length and 3-5,u. in width. Each zoospore possesses 2 flagella of equal length, 2 anterior contractile vacuoles, an anterior nucleus and parietal chloroplast. The zoospores are liberated by the rupture of the parent wall (Fig. 61). At quiescence, they become spherical ( Protosiphon-type [Starr, 1955]) and begin vegetative growth (Fig. 54, 122). Sometimes some of the spiny akinetes produced 4-cell packets.These packets are released by rupture of the spiny wall (Fig. 61, 127). This evidently usually occurs in old and dry cultures (more than 6 months old). Sexual reproduction has not been observed. Two-week-old colonies on Bristol's agar are dry and rough (Fig. 128). Three­month-old cultures become tinged with orange. Additional supplementary at­tributes are compared with those of other known species in Tables 24-28. Three species of the genus C hlorosarcina, as delimited by Vischer ( 1933) and Herndon ( 1958a), are now available for comparative study in bacteria-free cul­tures. These are: Chlorosarcina stigmatica Deason (1959), C. brevispinosa and C. longispinosa. The last 2 species are in one respect intermediate between chloro­sphaeralean and chlorococcalean algae. For example, although vegetative cell division ( sensu Fritsch [ 1935] and Herndon [ 1958], non Smith [ 1950]) is charac­teristic of chlorosphaeralean algae, this attribute is not readily observable in C. brevispinosa except following cold treatment (-8°C for 1 week) . On the other hand, vegetative cell division occurs at ordinary temperatures in C. stigmatica and C. longispinosa, with the result that characteristic Sarcina-like packet5 are formed. Thus, depending on environmental conditions, the unicellular ( chlorococcalean) or packet phase ( chlorosphaeralean) may prevail. Of the 3 species here assigned to Chlorosarcina, both C. brevispi,nosa and C. longispinosa may form short filaments or pseudofilaments. This attribute might be considered adequate to exclude these 2 species from C hlorosarcina, but the writers have been conservative in this connection. Similarly, although cells of the genus Chlorosarcina clearly possess single, cup-like parietal plastids (according to Hern­don, 1958a), as exemplified by C. stigmatica, the plastids of C. brevispinosa and C. longispinosa are parietal and cup-like, but bipartite ( C. brevispi,nosa) or multi­partite (C. longispinosa ). In the last instance, this suggests the disc-like plastids of the genus Bracteacoccus; but, here again, the writers have been conservative in not erecting a new genus to include taxa with segmented plastids, preferring to treat these 2 organisms as species of Chlorosarcina. With respect to morphological and physiological attributes, the 3 species of Chlorosarcina now available in culture may be compared as shown in Tables 24-28. It is clear from the data in Table 24 that these 3 species differ consistently in a number of morphological attributes. TABLE 24. Morphological attributes in species of Chlorosarcina Vega tive cell Habit of size, diam. N ature of growth under Nature of Nature of Organisms (solitary) Cell wall chloroplast standard conditions akinetes zoospores C. brevispinosa 25µ common cuplike, stigma ab-matrix mostly 35µ in diam. bipartite solitary with short­ sent,6-11µ. absent long X filamentous or pseudo-spines(2-4µ.) 3-5µ. wide, packets of ovoid cells '? .•• .. -:. CD. _·,..­ ' ~ : ' •, .·-.-·. . . . . ·. . ..,,.....c::,.... . . ...... . . : .. ·.. ~~;.>/ W0 @. '\ . @ @ ® 6/ @ Some Algae From Arid Soils Figs. 29-53 Figs. 29-35. Spongiochloris incrossoto.-Fig. 29. Mature vegetative cell; note multinucleate.condition ~nd net-like chloroplast.-Fig. 30. Young vegetative cell; note single nucleus and parietal chloroplast.­.fig. 31 . Vegetative cell; note segmentation of chloroplast.-Fig. 32. Mature cell from cultur ein stationary phase of growth; note thickening and striated outer wall layer, net-like chloroplast with central pyrenoid .and abundant starch granules.-Fig. 33. Aplanosporangium.-Fig. 34. Zoosporangium.-Fig. 35. Single ~oospore. Figs. 36-42. Ch/orosorcinopsis ge/otinoso.-Fig. 36. Young vegetative cell.-Fig. 37. Vegetative cell ·division.-Figs. 38-39. Packets of cells.-Fig. 40. Zoosporogenesis.-Fig. 41. Individual zoospore.­Fig. 42. Packets of cells from culture in stationary phase of growth; note common matrix; abundant oil droplets. Figs. 43-46. Ch/orosorcinopis eremi.-Fig. 43. Young vegetative cell.-Figs. 44-45. Vegetative cell ·division to form packets of 2 and 4 cells.-Fig. 46. Individual zoospore. Figs. 47-53. Ch/orosorcino brevispinoso.-Figs. 47-48. Young vegetative cell; note parietal and bipartite chloroplast, single nucleus.-Figs. 49-50. Development of spiny akinete.-Fig. 51. Zoosporan­gium and empty cells.-Fig. 52. Individual zoospore.-Fig. 53. liberation of aplanspores; note equatorial ·rupture of spiny akinete wall. Magnifications: Figs. 29, 33, 34, 44-46, 50-51, X 1250; Figs. 30, 35, X 1170; Fig. 31, X 2500; Fig. 32, X 1562; Figs. 36-40, 43, 47-49, 52, X 1875; Fig. 41 , X 2084; Fig. 42, X 1042; Fig. 53, x 625. Chantanachat and Bold J ® @) ..__ ( lo @ Some Algae From Arid Soils Figs. 54-72 Figs. 54-61. Chlorosarcina /ongispinosa.-Fig. 54. Young vegetative cell recently derived from zoospore; note single nucleus and multipartite parietal chloroplast.-Fig. 55. Packets of cells.-Fig. 56. Zoospore formation; note dividing chloroplast.-Fig. 57. Zoospore within zoosporangium.-Fig. 58. Individual zoospore.-Fig. 59. Median optical section of spiny akinete.-Fig. 60. The same in surface view.-Fig. 61 . Rupture of spiny akinete wall; note packet of 4 cells. Figs. 62-72. Friedmannia israeliensis.-Fig. 62. Individual zoospore.-Fig. 63. Young vegetative cell recently derived from zoospore.-Fig. 64. Vegetative cell division.-Fig. 65. Tetrad of cells.-Fig. 66. Malure vegetative cell; note multinucleate condition resulting from nuclear division before zoospore formation.-Fig. 67. Mature cell; note dividing chloroplasts.-Fig. 68. The same in median optical section.-Fig. 69. Zoosporangium.-Fig. 70-71 . Liberation of zoospores within vesicle.-Fig. 72. Aplano· spores and their liberation. Magnifications: Figs. 54, 58, X 5000; Figs. 55, 61, 69-71, X 1150; Fig. 56, X 938; Fig. 57, X 625; Figs. 59, 60, 62, X 3125; Figs. 63, 64, X 1875; Figs. 65, 67, 68, 72, X 2084; Fig. 66, x 1562. C hantanachat and Bold ,' / ® Some Algae From Arid Soils Figs. 73-80 Figs. 73-75. Neochloris o/eoabundans.-Fig. 73. Mature vegetative cell; note 2 pyrenoids in each cell and variation in cell size.-Fig. 74. Cell from 6-week-old culture; note abundant oil droplets in protoplast. -Fig. 75. A portion of 2-week-old colony on Bristol's agar. Figs. 76-80. Ch/orococcum dip/obionlicoideum.-Fig. 76. General view; note the formation of biflagellate gametes and thickened outer wall layer of diploid cells.-Fig. 77. Young (haploidl vegetative cells recently derived from motile cells.-Fig. 78. Aplanospore formation.-Fig. 79. Early stage of zygote; note empty walls of gametes shed posteriorly.-Fig. 80. Mature zygote. Magnifications: Fig. 73, X 700; Fig. 74, X 1365; Fig. 75, X 30; Fig. 76, X 230; Fig. 77; X 800; Fig. 78, X 750; Fig. 79, X 440; Fig. 80, X 275. 61 Chantanachat and Bold Some Algae From Arid Soils Figs. 81-88 Fig. 81-86. Chlorococcum diplobionticoideum.-Fig. 81. Zygote; note 1 pyrenoid beginning to de­generate.-Fig. 82. Oblique division of nonmotile gametes (zoosporesl to form 2 daughter cells.­Fig. 83. Two daughter cells within parental wall.-Fig. 84. Four daughter cells increasing in size.­Fig. 85. General view from 2-week-old culture on Bristol's agar.-Fig. 86. A portion of 2-week-old colony on Bristol's agar. Figs. B7-88. Spongiochloris minor.-Fig. 87. Group of vege:ative cells showing variation in cell size.­Fig. 88. Mature vegetative cell; note net-like chloroplast. Magnifications: Fig. 81, X 400; Fig. 82, X 1336; Fig. 83, X 1100; Fig. 84, X 756; Fig. 85, X 810; Fig. 86, X 30; Fig. 87, X 530; Fig. 88, X 1100. Some Algae From Arid Soils Figs. 89-96 Figs. 89-92. Spongiochloris minor.-Fig. 89. Cell from culture in stationary phase of growth; note obscured chloroplast and abundant starch granules.-Fig. 90. Aplanosporangium.-Fig. 91. Young vegetative cells recently derived from zoospores; note cup-like chloroplast.-Fig. 92. A portion of 2-week­old colony on Bristol's agar. Figs. 93-96. Spongiochloris incrassata.-Fig. 93. Vegetative cells; note central pyrenoid and variation in cell size.-Fig. 94. Mature vegetative cells.-Fig. 95. Mature cell; note thickening and striated outer wall layer and abundant starch granules.-Fig. 96. A portion of 2-week-old colony on Bristol's agar. Magnifications:. Fig. 89, X 675; Fig. 90, X 825; Fig. 91, X 1050; Fig. 92, X 30; Fig. 93, X 675; Fig. 94, X 625; Fig. 95, X 780; Fig. 96, X 30. Some Algae From Arid Soils Figs. 97-104 Figs. 97-104. Radiosphaera dissecta Starr.-Fig. 97. Young vegetative cells recently derived from zoo­spores.-Fig. 98. Mature vegetative cells; note asteroid chloroplast with central pyrenoid.-Fig. 99. Vegetative cell, zoospores and empty cells.-Fig. 100. Zoospores within zoosporangium.-Fig. 101. En­larged view of zoosporangium.-Fig. 102. Liberation ofzoospores.-Fig. 103. General view from 2-week­old culture.-Fig. 104. Cells from culture in stationary phase of growth; note obscured chloroplast. Magnifications: Fig. 97, X 900; Fig. 98, X 405; Fig. 99, X 250; Fig. 100, X 580; Fig. 101, X 800; Fig. 102, X 930; Fig. 103, X 180; Fig. 104, X 447. ® Some Algae From Arid Soils Figs. 1 05-11 2 Figs. I 05-108. Chlorosarcinopsis ge/atinosa.-Fig. 105. Packet of cells mounted in India ink to show common matrix.-Fig. I 06. Enlarged view of zoosporangia.-Fig. 107. Young vegetative cells showing the result of mutual compression.-Fig. 108. A portion of 2-week-old colony on Bristol's agar. Figs. 109-112. Chlorosarcinopsis eremi.-Fig. 109. Zoospores.-Fig. 110. Vegetative cell division.­Fig. 111 . Packets of cells forming a pseudofilamentous configuration.-Fig. 112. A p.ortion of 2-week-old colony on Bristol's agar. Magnifications: Fig. 105, X 750; Fig. 106, X 4150; Fig. 107, X 1130; Fig. 108, X 30; Fig. 109, X 1365; Fig. 110, X 2500; Fig. 111, X 700; Fig. 112, X 30. Some Algae From Arid Soils Figs. 113-120 Figs. 113-120. Ch/orosorcino brevispinoso.-Fig. 113. General view of 2-week-old culture on Bristol's 1:1gar.-Fig. 114. Pseudofilamentous phase and spiny akinete.-Fig. 115. Young vegetative cells recently derived from zoospores.-Fig. 116. Enlarged view of akinete; note short-spiny wall, abundant starch granules.-Fig. 117. Surface view of spiny akinete and pseudofilamentous configuration.-Fig. 118. liberation of aplanospores; note equatorial rupture of spiny akinete wall.-Fig. 119. Spiny akinete which produced packets of cells (under low temperaturel-Fig. 120. Zoosporangium. Magnifications: Fig. 113, X 357; Fig. 114, X 647; Fig. 115, X 1000; Fig. 116, X 940; Fig. 117, X 600; Fig. 118, X 650; Fig. 119, X 505; Fig. 120, X 850. Some Al{?ae From Arid Soils Figs. 121-128 Fig. 121. Chlorosarcina brevispinosa. A portion of 2-week-old colony on Bristol's agar. Fig. 122-128. Chlorosarcina longispinoso.-Fig. 122. Young vegetative cells recently derived from zoospores.-Fig. 123. A portion of pseudofilamentous configuration formed under low temperature.­Fig. 124. General view of 2-week-old culture; note spiny akinetes and packets of cells.-Fig. 125. En· larged view of spiny akinetes; note long spines.-Fig. 126. Cells from culture in stationary phase of growth; note packets of cells dissociating.-Fig. 127. Rupture of spiny akinete; note packets of cells inside. -Fig. 128. A portion of 2-week old colony on Bristol's agar. Magnifications: Fig. 121, X 30; Fig. 122, X 1098; Fig. 123, X 530; Fig. 124, X 170; Fig. 125, X 720; Fig. 126, X 550; Fig. 127, X 600; Fig. 128, X 30. Some Algae From Arid Soils Figs. 129-136 Figs. 129-136. Friedmannia israeliens;s.-Fig. 129. Telrads of cells.-Fig. 130. Further division of letrads to form 8 daughter cells.-Fig. 131 . Dissociation of tetrads into individual cells.-Fig. 132. En­larged view of zoosporangium.-Fig. 133. Individual zoospore.-Fig. 134. Aplanospores and their libera­tion.-Fig. 135. Mature vegetative cells; note division of parietal plastid before zoospore formation.­Fi(). 136. A portion of 2-week-old colony on Bristol's agar. Magnifications: Fig. 129, X 111 O; Fig. 130, X 1020; Fig. 131, X 1365; Fig. 132, X 2140; Fig. 133, X 1875; Fig. 134, X 1850; Fig. 135, X 1560; Fig. 136, X 30.