J. Phycol. 1042-49 (197) MORPHOLOGY AND NUTRITION OF PANDORINA UNICOCGA SP. NOV: William R. Rayburn Department of Hotany, Washington State University, Pullman, Washington 90163 and Richard ©. Starr Department of Plant Science, Indiana University, Bloomington, Indiana 47401 SUMMARY even strains of Pandorina unicocea sp. nov. were studied for morphological characterization. The spe- cies was delimited by having cells which contain a single basal pyrenoid and by colonies which have cells separate from cach other in the colonial enve- lope. A defined medium was developed for 4 strains of P. unicocca and the nutritional requirements of these strains examined. All 4 strains were capable of completely autotrophic growth and they could utilize nitrate, ammonium, or urea as nitrogen Optimum growth was attained at pH 8.0. ‘The strains of Pandorina used in this investigation were originally isolated because of their Eudorina- like appearance. After their mating behavior re vealed that they were indeed strains of Pandorina, it became obvious that their morphology did not match any published species description. For this reason and because several of the strains proved to be useful for experiments dealing with control of sextial reproduction, the present study was initiated. MATERIALS AND METHODS Cultures. The st ns used in this investigation were isslated smples (Fable 1), Culture methods, AML cultures were grown at 20 C. provided by standard 40.6 cool-white fluorescent | lated 10 give 16 hy of light and 8 hy of darkness. from re-wet 50 Light was nps regu Liquid ccaltures were exposed to 400 f-c of illumination. Bacterized ceultunes were grown in soil water medium (13). Axenic cule tures were obtained by repetitive sedimentation, decantation, id resuspension in sterile distilled water followed by plating, fon an agar medium made up of 4 parts Hvistol’s solution to | part Euglena medium (13). Axcnic liquid cultures were grown in 150% 18 mm culture tubes fitted with stainless” steel closures and containing 10 ml of M3 medium (Fable 2) Stock cultures were maintained in dim light of about 50 tec fon M3 medium solidified with 19% agar, Glassware and media were sterilized by autocaying at 121 G at 15 pst for 20 min. Glassdistilled water and analytical grade reagents were "This sa portion of the dissertation submitted by WRR to {uate School of Indiana University in partial fulfillment of the requirements for the Ph.D. degree. WRR was supported by a U.S, Public Health Service Training Grant ficrobiology aslministered by Indiana University = Received July 30, 1973; revised November 20, 1973. used in all media preparations sitional. studies glassware was heated in a dry oven at 200 G for 3 hr. Mating methods. Compatible colonies were mixed together in Pyrex triplespot plates enclosed in 100% 20. mim petsi dishes. Colonies were mixed by pipetting about 03 ml of each mating type into a spot, A small amount of distilled water was added to the bottom of the petri dish to retard evaporation he containers were exposed 10 400 fee from the mixtures of illumination from 40w coo-white Muorescent lamps at « Zygote germination. Following a mating reaction, the zygotes were permitted to remain in the spot plates for 48 hr. The rygotes were collected with pipettes and placed on 1% water aa jor was evaporated and the agar plates with the zygotes adhering to the surface were inverted over chloro: form for 30 sec to kill remaining, vegetative cells. "The zygotes ‘darkness by enclosing the petri dishes in paper at room temperature for 8-10 ah the zygotes were picked from the agar surface with pipettes, placed into soil water supematant, and illuminated. Growth measurement. Growth was measured twrbidimetrically at 750 nm in a Bausch and Lomb Spectronie 20 speetrophoto: meter, Meastrrement at 750 nm eliminated absorption by pig. ments. Optically matched culture tubes containing 10.0 mt ‘of medium were inocukated with 0.1 ml of colony suspension taken from cultures which measured 0.1 OD. Measurements were taken every 2 days during the second halt of the light period after daughter colony formation was completed. Experi in uiplieate and the results averaged. Every experiment was repeated at least twice. Differences of growth Among identical tubes rarely exceeded 0.01 OD. Inocula were Taken directly from cultures in log phase of growth except 4 nin Bro: In these experiments, ments were don for exper ng with vit inocula were starved for 48 hr in yitamin-free medium, RESULTS Cell morphology and colony organization. Kive heterothallic pairs and 1 other heterothallic strain of Pandorina were begun by single-colony isolations and represented populations from Indiana, Mass: chusetts, and Oregon. Colonies from actively grow- 1g cultures in soil water medium were examined over a period of 3 years. ‘The strains were tentatively identified as Pando- rina charkowiensis because the cells of older colonies were not compactly arranged in the colonial enve- lope but were separated from each other distinctly hollow colonies, However, the st fered from the colonies of P. charkowiensiy described by Korschikow (7) in having cells with a single basalPANDORINA UNICOCCA SP. NOV. 13 Source of soil samples from which Pandorina strains were isolated. Bern CoMlestor Tol & 102 E. Goldste 103 & 104 105 & 106 BE, Lippert 107 & 108 Cook log & 110 Goldstein Mm E. Goldste Isolator ‘State/County M. F, Golds - M. E, Goldsu ML E, Golds Indiana, Monroe Indiana, Monroe ook, Oregon, Tith usetis, Barnstable Indiana, Brown Indiana, Brown Massae M. E, Goldst M, FE. Goldst pyrenoid throughout the entire vegetative phase, except occasionally in older cells when the pyrenoid divided imo two. In the original description of P. charkowiensis, Korschikow reported the presence of up to 7 pyrenoids in older cells. This multipyrenoid condition was observed by Smith (12) collected in California, but Thompson (16) described P. charkowiensis collected in Kansas as having cells with single pyreni species, P. ininodi Chodat, was described from Switzerland as having multipyrenoid cells loosely arranged in the colonial matrix (6) Goldstein (4), Pocock (9), and Smith (12) considered the number of pyr an important char- acter for the separation of species in the volvocacean genera Goninin and Eudorina. ‘Therefore, since the strains of Pandorina examined in this study are cor sistently unipyrenoid, they are recognized as repre- senting a distinet species. PANDORINA UNICOC Coloniae cylindricae, ellipsoideac, ad ovato-ellip- soideas, ant subsphericac, involucro coloniae levi Coloniae ex 8, 16 vel 32 cellulis constantes, cellulac discretae, in stratis distinctis intra involucro gelati- noso confluente duplici ordinatae. Gellulae maturae magnitudine fere aut omnino aequae, forma ovoideae ad late ovoideas, extremitate lata satis applanata, ASP. NOV ‘Tame 2 M3 Medium. Flaal Goncentration 10x 108 50x 10m 20% 10m 10x Ws Gamponent KNO, Naxglycerophosphate on] com 5.6 10" a 21x 10% x08 a8 7X10 Na.MoO, ‘oC le GHLO nit Bre SAX 108M 1.0% 10 p/m v PV Tree following Cin g/liter): Na,etA, 4,0, (1081; ZoCl, 0.005; Na, Moo, extrorsus aspiciente, Omnis cellula singulum chloro- plastum poculiformem, longitudinaliter striatum, singula pyrenoide basali pracditum continens; singula pyrenoides in cellulis adultioribus in duas interdum se dividens. Stigmata in strato cellularum anteriore maxima, in stratisporterioribus subsequentibus progredienter minora, in strato postremo saepe nulla Omnes cellulae in colonia ad colonian-fitiam efficiendam se dividere potentes. Reproductio sexualis isogama, cellula gamcto potente. Coloniae libere natantes 26-88 p. long. ad 18-68 p lat: cellulae 6.5 ad 16.5 diam. Colonies cylindrical, ellipsoidal to ovate-ellipsoidal, or subspherical in shape, with the colonial envelope smooth in outline. Colonies of 8, 16, or 32 cells, ‘we from each other and arranged in distinct tiers within a confluent double-layered gelatinous envelope. Mature cells equal in size or ne: ovoid to broadly ovoid in shape with the broad end often somewhat flattened and facing the exterior of the colony. Each cell containing a single longi tudinally striated cup-shaped chloroplast with single basal pyrenoid; in older cells the single pyre noid occasionally dividing into wo, Stigmata largest in the amtrior tier of cells and progressively smaller in the subsequent posterior tiers often completely Jacking in the posterior tier, - Every cell in a colony capable of dividing to pro- duce a colony daughter exuual reproduction isogamous, with all cells of a colony as potential gamet Freeswimming colonies 26-88 yp long t0 18-68 broad; cells 65-165 in diameter. ' Source. Isolated by M stein from a reawet mple collected by B. F, Lippert at Lee's C Tillamook County, Oregon (strain numbers 10! 106). ‘Type cultures have been deposited in the Cul ture Collection of Algac at Indiana University, Colonies with 32 cells are ellipsoidal to ovate- ellipsoidal or infrequently subspherical in. shape and have cells arranged in 5 distinct tiers (Fig. 1). The anterior and posterior tiers each contain 4 cells, and the middle 3 tiers each contain 8 cells. Sixteen celled colonies are cylindrical to ellipsoidal in shape with cells arranged in 4 tiers of 4 cells each (Fig. 3). Colonies with 8 cells have the cells arranged in 2 tiers. In all of the colo omni coloniae soil P. ies, each tier of cellsWILLIAM R. RAYBURN AND RICHARD C. STARRPANDORINA UNICOCCA SP. NOV 45 js rotated with respect to the adjacent tier or tiers so that they interlock, Colonies with 16 and 32 cells are commonly pro- duced by all of the stains. Colonies with 8 cells are seldom produced in actively growing populations but in increasing numbers in declining popula- ns, Colonies with 64 cells have never been observed. All cells in a colony are equal or nearly equal in size. Cells vary in shape {rom avoid to broadly ovoid. The broad end of the cell is often somewhat flattened and faces the exterior of the colony. Bach cell con- tains a massive cupshaped chloroplast. In young, cells the chloroplast ns longitudinally striated, A prominent pyrenoid surrounded by starch grains is ed in the base of the chloroplast toward the ior of colony. The le _pyrenoid occasionally divides into two in older cells. Two whiplash flagella project from the broad end of each ppe: ha spinning motion, A definite polarity exists whil ming: this polarity can be recognized even in non. swimming colonies since the anterior cells of a colony have larger stigmata than the posterior cells. Fach stigma is located at the chloroplast surface in line with and posterior to 2 contractile vacuoles which are at the base of the flagella, “The nucleus is located in the cytoplasm surrounded by the cup- shaped chloroplast Asexual reproduction. Every cell in a colony is capable of dividing to produce a daughter colony 4). Colonies which are about to begin daughter jon can be recognized by their ex- The cells appear to separate by an in. Grease or loosening in the gelatinous matrix. Under the culture conditions used in this study, cell vision begins shortly after the beginning of the light period and can be delayed by keeping the cultures in darkness, ‘The first division takes place in a plane extending from the point of flagellar attach ment through the posterior end of the cell. ‘The second division is always perpendicular to the first and results in 4 equal-sized cells. ‘The third division appears more or less oblique, giving rise to the cruciform arrangement of the 8-celled plaka. In version may occur at this time to give an S-celled colony with 2 tiers of 4 cells. A fourth division results in the IGcelled plakea and inversion at this time gives a cylindrical 16-celled colony, The 4 ca before in- the colony is swim- panded size. cells which lie in the center of the pl version become the anterior 4 cells of the colony. A fifth division results in a 82-celled plakea. The 16- and S2-celled stages are no longer flat plites but are curved like a wind-blown sail. During s of the plakea simply fold back to give a cylindrical or ellipsoidal colony Vhe fates of the various cellular organelles have wersion the corn been followed thro The flagella usu cleava igh daughter colony format ly disappear prior to the first hence colonies in which daughter colony formation is occurring are found on the botom of the container in which they are growing. If the Hlagella remain attached through the first’ division, they are passed along together. No flagella have been observed remaining attached beyond the 8-celled age of daughter colony formation. Before the first division, a cell may contain 1 or 2 pyrenoids. Follow: ing the first division a pyrenoid can be seen in each of the daughter cells, Atempts 10 follow pyrenoids through later divisions have not been successful. The stigma remains intact through several successive di visions and is often found in 1 of the posterior cells of the young colony after which it subsequently dis- appears. The chloroplast material is passed on to the daughter cells throu; portion of the parent plastid going to each of the daughter cells after every division. The process of daughter colony formation requires several hours. Following the last division, the plakea inverts in a fashion typical of volvocaccan algae to form an 8, 16:, or 32celled colony. One flagellum appears before the other on each cell. As the flagella increase in length, they begin to beat, moving the young colonies within the confines of the mucl. expanded. and watery parental mawix. Stigmata become visible in the anterior cells of the colonies and pyrenoids can be seen in all of the cells, Both 16- and 32celled daughter colonies may be formed within the same parent colony. Further development involves only increase in the size of the cells and colonies. Sexual reproduction this study are heteroth: mating. process is ess All of the strains used in lic. The morphology of the ntially the same in all of the strains. The first sign of sexual reproduction ir tures of comple 'y mating types is attraction of colonies into small groups or clumps (Fig. 5). ‘The colonies contact by flagellar tips. In strains 105-106, the colony clumps may eventually include 20. ot more colonies, Among the other strains, the clumps are composed of {ewer colonies. After the formation of colony clumps, the gametes begin to emerge from the colonies by squeezing out of their cell walls through a tiny opening (Fig, 6). ‘The anterior end of the gamete begins to slip through the opening, caus ing the protopla the constriction, The morphology of the gametes is that of the to become porarily bilobed by vegetative cells inasmuch as the gametes are vege- tative cells that have escaped from the colonial matrix. However, the flagella are no longer re stricted by the colonial envelope and move from the point of insertion in the protoplast. Once free, the naked gametes round up, swim actively, and sur round colonies of the opposite mating type whose416 105 6 ATION OF ts cove Fic, 7. ‘The effect of KNOs concentration on the growth of strains 103, 104, 105 and 106 as measured by optical density at 750. nm after 14 days of growth, Coneentrations given in molarity WCENTATION OF MS Fic, 9 ‘The effect of MgSO, concentration on the growth of strains 103, WH, 105 and 106 as measured by optical density it 730 nm after H days of growth. Concentrations given in molarity WILLIAM R, RAYBURN AND RICHARD CG. STARR os io as lull 5 ws 106 as Sous CEROPHOSTANTE CONCENTRATION OF taf Fic, 8 ‘The effect of Nayglyeerophosphate concentration on the growth of strains 108, 101, 105 and 106 as measured by optical density at 750 nm after 14 days of growth. Concen tations given in-mokaity MENTATION OF Cat Fic. 10. ‘The effect of CaCl, concentration on the growth oof strains 103, 10, 105 andl 106 as measured by optical density at 750-nm after 1M days of growth, Concentrations given in molarityPANDORINA UNICOCCA SP, NOV. 17 loo 9.0} pH 80 7.0) Time Elopsedin Days Daily change of pH in unbuffered M3-medivm owth of strain 105. Lowest daily valwe me Highest daily Fic. 1. during the g beginning of the light cycle end of the light cycle gametes have not as yet escaped, All gametes escape in the same manner and finally they become associ ated in clumps by the agglutination of their flagell: tips. An initial fusion bridge is formed between the anterior ends of 2 adjacent compatible gametes Fusion is lateral and rapid, requiring only 8 to 4 n ‘The gametes are usually similar in size but Irequently they are unequal. ‘The size of the gametes depends on the size of the cells in the individual colonii from which they emerged; colonies of both mating types produce gametes of various sizes The quadriflagellate zygotes active or inactive “swimmers, depending upon the strains. Strains 109-110 produce zygotes which are motile for many hours, Alter the swimming period, the zygotes settle on the bottom of the container or float on the surface of the liquid. For this reason the zygotes are found singly. In contrast, the zygotes of strains 107 108 andl 105-106 are usually found in loosely arranged lumps, their boundaries becoming shaped. by the position of neighboring zygotes. In suains 101-102, 111-102, and 103-104, the zygotes become arranged in tight cohesive clumps. The flagella eventually disappe: are visible in the zygotes throughout the light p in which mating takes place and may be seen on the subsequent day. ‘The pyrenoids are soon difficult to identify because of the accumulation of starch in the 7ygotes, However, with IsKI solution the pyre: noids may be distinguished for a day or two. The zygote is naked during the first day, but by the beginning of the next light period a smooth zygote wall appears. "The zygote wall increases in thickness during the second day. ‘The zygote contents become less and less green and by the filth day the zygote are tan in color Zygotes of strains The stigmata fod 105-106, 107-108, and 109— 015 & 8 010 » 104 3 S008 105 65707580 «8S 80 Initial pH Fic. 12. Effect of initial pH on growth of strains 101 and los after 16 days as measused by optical density at 790 tin Media were buffered with glycylglyeine and. histidine sure 10-20 in diameter. Strains 101-1 111-102, and 103-104, which have compact zygote form zygotes which measure 8-H ye in clumy diameter. Zygole red Ww 105-106. G gote germination was hr degree of success with strains tion occurred within 24 hr. Just prior to germination a tiny bulge appears on’ the surface of the zygote wall. Soon alter, the protoplast rges through a small opening much in the manner gamete escapes trom its cell well. ‘This protoplast (gone) is biflagellated and surrounded by a vesicle. The flagella project outside of the vesicle. After tive period of swimming, which lasts for an ht period, the gone settles to the bottom of the container. At the beginning of the next light criod daughter colony formation takes place. ‘The ng gone colonies are tiny in comparison to nor mal vegetative colonies. Several days pass before daughter colony formation occurs again. Inheritance of mating type. Singlecelled gones were left together in the containers until daughter colony formation was completed and the resulting gone colonies had increased in size. ‘Then colonit were isolated and transferred to culture tubes con- taining soil water medium, Alter the cultures had grown sufficiently, the gone populations were back- crossed with the lonies from the 2 parental strains were placed into separate de- iple-spot plates and an approxi mately equal number of colonies from the gonal pop: ulation to be tested for mating type were mixed with cach of the parents. Colonies of the gonal strain were placed in the third depression as a control to insure uniformity of the clone. ‘The presence of zygotes in one of the spots indicated compatibility with that parental strain and therefore an opposi mating type. germination. ha hi pressions of Pyrex.48 WILLIAM R. RAYBURN AND RICHARD C. STARR From a cross of 105 x 106 the resulting zygotes were germinated and 160 gone colonies were iso lated.” OF these, 156 produced sufficiently large populations for backcrossing, The inheritance of ing type was 78:78. Two f; clones of opposite ing type were selected and crossed. The zygotes were germinated and 100 fz gone colonies were iso- lated. ‘The surviving 97 populations were crossed with the original parental strains, 105-106, and gave a mating type ratio of 50:47. uclear activity during zygote formation and germination was not followed. In Pandorina morum, meiosis occurs during zygote germination (3). In the closely related Gonium pectorale nuclear fusion occurs shortly after syngamy and meiosis occurs dur- ing zygote germination (I4). Cultures of Pandorina unicocea begun by isolation of gone colonies showed a 1:1 segregation for inheritance of mating. type. This genetic evidence indicated that meiosis occurred in the zygote, in agreement with observations on Pandorina morum and Gonium pectorale. Nutrition. The nutrition of strains 103, 104, 105, and 106 was studied to provide a basis for mating experiments. Bacteria-free colonies were grown orig inally in autoclaved supernatant from soil water medium, Colonies were transferred to a defined medium consisting of 5 x 10-4 at Ga(NOg)s, M K,HPO,, 1.5 x 10-1 M MgSO,, PIV tr mix (17), and 10 pg/ml vitamin Bys. ‘The macro- nutrients were those used by Wilbois (17) for the culture of Pandorina morum. Doubling the conce trations of the macronutrients increased growth. Although this defined medium was superior to soil water supernatant for growth, it had certain limita- tions, Attempts to increase the concentration of K.HPO, resulted in precipitation of calcium phos- phate during autoclaving. ‘Therefore K,HPO, was replaced by Nagglycerophosphate. Comparison of growth in the 2 phosphate sources revealed no detectable difference, Calcium was provided in lower concentrations as GaCls, and Ca(NOs)z was replaced by KNO,. ‘The new medium was named M3 (Table 2). It was used as the basis for further nutritional s. ‘The concentration of each of the macro- ie the optimal con- in. stu nutrients was v. centration for growth of each str Vitamin nutrition. The effect of vitamin Byz con- centration on growth was studied. Vitamin By, con- centration was varied from 10-* to 10° g/ml, Colo- nies were also n-free medium. No difference in growth was measured after 15 days of culture in any of the vitamin Bys concentrations. Colonies were transferred from each culture tube ‘nto a new one containing an identical vitamin By nd subcultured for 15 more days. At nt difference culated into vit concentration that time, there was still no signifi between growth in any of the vitamin Byz concen- trations or in the vitamin-free medium. Vitamin Bs was not needed in the mediun has been shown to be required by several volvocacean algae for growth, only vitamin By was studied since good growth occurred in M3 medium whieh lacked any other supplemented vitamin. Nitrogen nutrition. All of the strains utilized ni: trate as a nitrogen source (Fig. 7). The optimal con: centration of KNO, for all strains was 1x 10-8 Slightly higher concentrations reduced total growth in strains 105 and 106, Since nitrogen depletion of the medium played an importa reproduction of strains 103-104, the effects of other sources of nitrogen on growth were studied. In the absence of KNOg, KCL w: dium. Urea and NH,GI were supplied in concentrations that provided an amount of nitrogen equa 10M KNOs, Urea supported growth equiv nitrate, and) ammo supported slightly less growth, Two other nitrogen compounds were of interest. Histidine and glycylglycine were nontoxic when added to M3 medium and both could serve as buffers. When histidine and glycylglycine were i cluded in the medium as the only nitrogen sources, neither one sustained growth. Other macronutrients. Optimal concentrations of cach of the other macronutrients for growth were de termined (Fig. 8, 9, 10). For all 4 5x 10M Nagglycerophosphate supported growth, ‘This conc ion was one-half the KNOs concen: uation that supported the best total growth, “The al concentration of MgSO, was between 1 x Although thiamine opt 10-1 to 1.25 x 101m, ‘The optimal concentration of CaCl, varied among the strains over a range of 7.5 X 10 to 1.25 X 10-1 wt and appeared to be correlated with the total amount of growth characteristic of each strain under the culture conditions used for this study, Trace metals, ‘The concentration of P TV trace metals was increased 2- and 3fold without any in growth after I days. ‘Therefore, the 1 concentration was not limiting to growth. Hydrogen-ion concentration. During exponential yowth in unbuffered M3 medium a daily fuetuation Of pH was measured in which the lowest pH occurred at the start of the light period, and the highest pH occurred at the end of the light period (Fig. 11). To avoid possible undesirable effects of pH fluctua tion on growth, media were buffered. Glycylglycine and histidine were found to be nontoxic and suitable s buffers. Glycylglycine was tested over a concen: tration range of 5X 104 to 5 x 102 M. Histidine was tested at 5X 10-4 and 1 x 10m, Glycylglycine ut 1 10-8 Mt and histidine at 1 x 10-# a4 together, titrated with either NaOH or HCI, buffered media effectively between pH 5.0 and 9.0, ‘The growth response to initial pH by strains 108 and 104 was identical as was the response by strains 105 and 106. ins 103 and 104 occurred at Optimum growth in strPANDORINA UNICOCC PH 8.0. Optimum growth in strains 105 and 106 occurred at pH 8.0 to 8.5 (Fig. 12). DISCUSSION Nearly all nutritional investigations of colonial volvocalean genera have revealed vitamin require ments, Biotin, Bys, and thiamine, either singly or in combination, were required for growth of Astre- Phomene gubernaculifera (I), Volvulina steinii and V. pringsheimii (2), Platydorina caudata (3), Volvox globator and V. tertius (11), and Gonium pectorale (5). One comparative nutritional study of 23 spe- cies of colonial Volvocales, belonging to sphaeral- lacean, spondylmoracean, and volvocacean genera found that all strains examined required both Bi and thiamine (10). Only 2 reports indicate that strains of Goniwn pectorale (15) and Pandorina morum (8) were capable of completely autotrophic growth. ‘The nutritional requirements of 4 strains of Pandorina unicocca studied in this investigation were very similar to those of 3 Pandorina morum strail sl by Palmer & Starr (8). They utilized mmonitm, oF urea as nitrogen sources for growth, and in all strains growth occurred without €xogenous supplies of vitamins. The results of this study support the view of Palmer & Starr that dif ferent strains may represent different: physiological races of the same species and that completely auto- trophic strains of Pandorina and probably other volvocalean genera may be as common as vitamin- auxotrophic strains. ACKNOWLEDGMENT c for We gratefully acknowledge Dr. Hannah 'T. Croasth writing the Latin description of Pandorina unicoren sv. Nov 49 REFERE crs, Brooxs, A. E. 1972. ‘The physiology of Asrephomene gubernaculifera, J. Protozool, 19:195-, Canmroor, J. R. 1967, Nutrition of Volvulina Playfair, J. Protozool, Ve CouEMAN, AL W. 1950, Sexual isolation in Pandorina morum. J. Prat 1 GowstEs, M.E, 1961, Speciation and mating behavior in Eudorina. J. Protozool. W:317-44 Hawais, D. 0. 1969, Nutrition of Plarydovina caudate Kofoid. J. Phiyeol. 5205-10. Hewee-PesraLozzi, G. 1961, Das Phytoplankton des Suss wassers. Sy tind Biologie. Chlorophyceae. Volvo cles. Die Binnengewasser, Band XVI, 3 Teil, 17H. Kowemikow, A. 1923. Uber zwei neue Organismen aus der Gruppe der Volvocales. rch, Sor. Russ. Protistol. 2 170-8 (with German eésuine), Painter, EG. & Srane, RC. 1971. Nutrition of Pando- rina morn, J. Phycot. 7: Pocock, M.A. 1955. St dies in North American Volvo ales. 1. The gems Gonium, Madrone 18:19-64 Pancsnrint, E.G, & Peinesisint, O, 1959, Die Eenahrung. koloniebildender Volvocales. Biol. Zentral, 78:937-71 Provasous, La & PINENER, I. J. 1959, Avtificial media for freshwater algae: problems’ and suggestions. Jn ‘Tryon, A. Jr. & Hartman, R.T, [Rds The Ecology of Algae Spec. Publ. No. 2 Pymatuning Lab, Field Biol. Univ Pittsburgh, 81-96, Summ, G. M. 1910, Notes on the Volvocates. Bull Torrey Bot. Club 53:350-69, Sram, R. C. 1964. The culture collection of algae at Indiana University. aun. J. Bot. UNOS. sry, J. Re 1958. A morphologic and genetic study of Gonium pectorale, Am. J. Bat, AB0G1-72. 1865, Growth and mating of Gonium pectorale (Volvocales) in defined media. J. Phiyeol, 225-8 Thomson, R. H. 1954, Studies on the Volvocales. 1 Sexual reproduction of Pandosina charkowiensis and ob- servations on Volvulina steinii, Am. J. Bot. 41:1 Wants, A. D. 1958, Sexual isolation in Pandorina morum Bory. PD. thesis, Indiana Univ., BloomingtonThis document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.