Morphological and physiological studies of the genus Spirogyra

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Authors Rickert, Francis Brilon, 1914-

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/347460 MORPHOLOGICAL AMD PHISIOLOGICAL STUDIES OF THE GEMS SPIROGYRA'

by Francis B. Rickert

A Thesis submitted to the Faculty of the DEPARTMENT OF BOTANY

In Partial; Fulfillm ent of the Requirements | For the Degree of " . MASTER OF SCIENCE

In the Graduate College

THE .UECiTERSITI OF ARIZONA STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­ quirements for an advanced degree at the University of Arizona and is deposited in The University Library to be made available to bor­ rowers under rules of the Libraiy. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made* Requests for permission for extended quotation from or reproduction of th is manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in their judgment the proposed use of the material is in the interests of scholarship. In a ll other instances, however, permission must be obtained from the author.

APPROVAL BY THESIS DIRECTOR

This th esis has been approved on the date shown below:

Robert W. Hoshaw z7 Date Professor of Botany AOIOOTIEDGEMEHTS

The author mould like to express Ms most sincere appreciation to Bro Hobert Ho Ho^iaw for suggesting this problem and for Ms help and encouragement during the investigation of the problem and the writing of this thesiso Many thanks are also expressed to the other members of the Department of Botany for th eir help and constructive criticismso

Deep appreciation is extended to Dr. Clarence E0 Taft for his work on the identification ©f species of Ssirogvra used in this investigationo

The author is also indebted to the Mational Science Foundation for the Summer Fellowships For Secondary. School Teachers which made the investigation possible<, Appreciation for technical assistance during the summer of 1963 is extended to Mr* Geoffrey Levinson #10 was working in Ihe Be sear oh Participation Program, of the national

Science Foundations Deep appreciation and gratitude is extended, to lfrs« Dorothy Riekerts w ife of the author,, for her patience and for typing this manuscripts > -. '; TABLE OF CONTENTS

;. . ": ; ' ' . Page

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REVXFIHT OF XiXFERATTJRHI © © © ©©©©©©©oooopp©. ©©©©©oo©©©©©©©©©©©©©©©©©©©©©©© 3

Morphology 3*nd Is^conoiiy © © © © © © © © © © © © ©■ © © © © © © © © © © ©©©©©©©©pp©©©©© © 3

Isolation andl Otilttxro©o©©©©©©©©©©©©©©©©©©©©©© © © © © © © © ©© ©©© © © & ©

M ineral N u tritio n and C ulture Mediaoo <> © © © © ©©»©©© ©© ©»© ©©©©©© ©© © 7

Sexuality andi Conju^ation©©<»o©©©©©©©©©©©©©©©© ©©©©©o©©©©*©©©©©© ^

%y^.ote Germination© © © © © © © © © © © © © © © © © © © © © © © © © ©> © © © © © © © © © © © © © © © © © 12

MATMIAIiS AMD li^FTHODS© © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © llj.

N atu ral C o lle ctio n s © © © © © © © © © © © © © © © © © © © © © ©©©©©©©©©©©o© ©©©©©©op© Ilf.

I s o la tio n Frocedure © © © © © ©( © © © © © © © © © © © © © © © © © © © ©©©©©«©© © © ©©©©©©©p llj.

Growth and Maintenance' of C u ltu res © © © © © © © © © © © © © © © © © © © © © © © © © © © © 17

In d u ctio n of Confutation© © © © © © © © © © © © © © © © © © © © © © © © © © © ©©©©©©©©©©© 20

Zygospore Ge ritimat ion©©©©©©©©©©©©©©©©©©©©©©©©©©©©©©©©©©©o©©©© © 21

■ Microscopic and Photographic Equipment © © ©«©«©© © © © © © © © © © © © © © * © © 22

IffiSUITS d © © © © © ©•© •© © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © 23

. - C o lle ctio n and I s o la t ion © © © © * © ©©©o©©©©©©©©©©©©©©©©o©©©©©©©©©©© 2 3

Growth and M aintenance o f C u ltu re s © © © © © © © ©© © © © © © © © © © © © © © © © © © © © 23

Conjugation© © © © © © © © ©' © © © © © © © ©. © © © © © o'© © ©. © © ©,© © © © © ©'©©©’©©© © © © © ©©©©.© © 2^

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Moarphology. and Taaconomy© © © o© © © © o,© © ©© © ©© © o,© ©© © © ©© © ©© © © © © © © ©© ©© ©o 30

DISCUSSION o O © <9 © © © © O ©60 ;©_ © O © O o O © © © 60© 60© © © © © © o © © o’© © .© oo o o oooooooooooooo 57

Growth and Maintenance of Cultures© © © ©©© ©©©o© © ©o oo© © ©©o© ©©©©©oo 57 Iso la tio n .Procedures o © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © ©«© © © o 59

ConQU^a11ono © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © & © © © © 59

2y*^ospor0 Germination© ©©©©o© ©o© © © © © © © © © © © © © © © © © © © © © © © © ©»© © © © © © © 60

Morphology and Taxonomy © © © © © © ©♦© © © © © © © ©©©©©o©©©©©© © © © © © © ©©©©©©© 61

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V LIST OF TABLES

Table ' Page 1 Sources for clonal cultures of Splrogyra used in this

, ■ ; 0 8 1 igation. Q « • o o o « o 6 s ^ op e o o

Average on a 0”^ basiso ® o««oo oo p o o © o o p o o ® © o © © © © © © © © © © © © © © 26

3 , ■" Clorox treatment for/zygospores tokill bacteria with ; percent of germination of zygospores on desmid-agar after treatment©©© © © © © © © © ©©o©©©©©©©*©©©©©© ©©©©©©©©o©©©©©©©©©©©© 29 A Comparison of morphology for vegetative c e lls using

10 or more filaments each from 10 strains©©©.©©©..©©©o©©© 31

5 Comparison of zygospore characteristics with ranges

shown by 10 or more zygospores ©...© © © © © ©.©««©©..©o©©©©©.© 33

VI " MST OF FIGURES' , '

^ ; , . ■ .

1= Spirogyra sp0 (Upper Sabino) vegetative cells3 3 2 $ 0 . 36

.2* ' Spirogyra spe (Gardner Canyon) vegetative c e lls s X250oOoO<,o 36

3o Spirogyra spo (Organ Pipe) vegetative cellss JZ$0e 0»=«„ <, „»6 36

Ii.o Spirogyra sp» (Sycamore Canypn) vegetative c e lls5 X230»»«««, 36

5» Spirogyra sp.o (Snowllake) vegetativp .cells,X 2 3 0 . . 38

6 0 Spirogyra spo (Tanque Verde) vegetative cells, X230«o«««o, 0 38

7o Spirogyra sp0 (Eiggs lake) vegetative cells,X 2 3 0 .*> 38

8 0 Spirogyra sp. (Rose Garyon) vegetative c e lls , X230

9» Spirogyra sp. (1.B913) vegetative c e lls , .X230o,««<,<,«<,»...Co = itO

10o Spirogyra sp0 (LB91i|.) vegetative c e lls , X250»»«=».».»»»»,«» lj.0 11o Spirogyra spo (Snowflake) vegetative c e lls , showing tightly coiled chromotaphdres and lhrgevpyrehoids, X530d>oooodooooo i|.0 12o Spirogyra spo (Upper Sabino), natural collection, several . filament widths, cells with papillae forming, ' X123®»«,«o.«» 1|.2 13. Spirogyra spo (Upper Sabino), two cells showing the • beginning of papillae formation, 5 XU0..».«....«..«...0»«,.. kZ lit. Spirogyra sp. (Upper Sabino), cells show conjugation tubes formed, protoplasts about to make contact, starch accum­ ulation appears and female gametangium is swollen, X^itO.... itij. 15. Spirogyra Spe (Upper Sabino) > cohjugation tubes formed, prbtoplasts about to make contact, X51tO...... itit

16. Spirogyra sp. (Upper Sabino), natural collection with zygospores, X9G. o.,...,,...... ,,...... ,...... ,,.....*,... 1^6.

17. Spirogyra sp. (Upper Sabino), zygospores, tubes and-empty

Cells , X5itO OO.OOOOOOOOOO. 0,0 O'.OOO 0,0. 0.0 0 0. 00 OO.OOOO, 0 0.0 0 0 1^6 .

-V: v i i : ''1 v:;;; ; ■ .. , IS,, gpirogyra sp, (Sabino Cargron), germling just emerging5

132^77777, 0,000 000000 0 0 00, 6. OO, ,0,000 0 , 0, 0,0, ,0, 00'oe, ee,,o, e lj.8

. 19.0 Splrogyra sp, (Sabino Canyon) 5 germling fu lly emerged, one c e ll, -^.325ooo,,,,,,,,,,, ooo,,,,,,,-,,,,,,,,,,,,,,,,,,,, i^.8 20„ Splrogyra sp, (Sabino Canyon)s 'germling,. two cell stage, X325 , OOOO ,0, 00,0 00 00 O 0,0 O OO 00 OOO, 0,0, 00,0000000000000000000 pS 21, Splrogyra sp0 (Organ Pipe), vegetative filaments show rep 13-0 at e end w alls, iX!600, 000, 0,00 o,,, 00, o,,,, ,, 00,000 000,0 50

22, SplrOgyra sp, (Organ Pipe), normal zygospore,600 X ,....,,,, 50 23, Splrogyra sp, (Organ Pipe), lenticulate zygospore, X600,„,,$0

2ii, Splrogyra sp, . (Organ Pipe), various stages of conjugation,

X250 O 06000 0000 O 00 6, O OOO, OOO,,, 00, OOOOOOO 00 000000 00,000 0 0 , 0 0 52

25- 'Splrogyra sp, (Organ Pipe), gametes in one c e ll aborting, X320000,0,,,,6,000 0,000,000000000,0000000000,0000000OOOOOOO 52 26, Splrogyra sp, (Organ Pipe), one cell conjugating at the end w all, in one c e ll gametes have failed to fuse, X320,»«.,»,, 52

27, Splrogyra sp, (Organ Pipe), tubes have just fused, no con­ tact between protoplasts, starch plates prominent, X625,,,, 54 28 , Splrogyra sp, (Organ Pipe), two c e lls with tubes formed, one gamete rouuded, X625,ooo,,,,,,,,,,,0,00, 00,00000,ooo,,, 54

29, Splrogyra sp. (Organ Pipe), both gametes rounded, male gamete moving through the tube, IBS25.r e dope 000 d 000.000 o 000000

30, Splrogyra sp, (Organ Pipe), zygote in cell, shows rep! I.c a te end w a ll, ,X600, o-o-, o , , , , ,o o o , , oo o , , , ,0 o , , o o , , o 0 0, , 56

31, Splrogyra sp, (Gardner Canyon), zygospore,X575,. . , • 56

viii IHCRODUCriOH

Hie genus SpiroOTra is one of the more familiar of the filamentous algae. Nearly everyone who has studied high school biology has heard Spjrogyra mentioned as a filament with sp iral ehloroplastsc, Hie most common form of sexual reproduction in

Spirogyra/ sealarifofm Gonjugation^ is also; mentioned in many high school textbooks. Many people, for many years, have studied this ■' = member of the family Zygnemataceae. Spirogyra is by far the most common member of this family Wich also includes other filamentous, algae such as Zygnema, Mougeotia and Sirogonium.

Hie family Zygnemataceae is characterized by having unbranched filaments, by complete lack .of motile reproductive elements, by axial or parietal chloroplasts which may be stellate, ribbonlike or platelike bodies (Tramseau, 19^1)., Reproduction in,the Zygnemataceae - may be asexual, by cell divisions or by the formation of akinetes, aplanosporss or parthenospores; or sexual,by lateral or by scalariform conjugation involving fus ion of ame bo id garnet e s (Fritsch,135 9 )° Collections of "pond scums" from lakes, ponds, slow moving streams and drainage ditches are likely to contain Spirogyra and frequently wij.1 contain Zygnema or other members of the Zygnemataceae, as th is family is represented, wdrld wide. The slimy' feeling of many of the - Zygnemataceae is due to the presence of a pectose mucilaginous sheath which is formed outside the cellulosic cell wall.

: .. :/ ■■ ' v ; : 1 : ' . : ' Transeau (19^1) described 275 species of Spirogyra3 and landtiawa (1959 ) described 289 specieSc A great many papin the older literature described various species of Spirogyra and their life cycles as Observed in natureo SH in all, hcfwever, very little is actually known about Snirogyra beyond the descriptive stage®

Few investigators have cultured this gems in the laboratoyr under controlled conditions or have studied this gems in cultureo

A number of investigators have pointed out the need of studies in culture (Coleman, 1961) from the standpoints of taxonomy, morphology, physiology and genetics = Godward (I95tiia) pointed out the confusion in the taxonomy of the genus Spirogyra and suggested the use of cytological criteria, including chromosome studies to aid in properly classifying the various species® V The present investigation has involved collecting the alga . Spirogyra from various lakes* ponds and streams in Arizona, and the isolation of clones from these collections through selecting single filament pieces and transferring them' into test tubes of soil^irater e From, these tube cultures, larger cultures irere groin in milk bottles of soil-water for use in various experiments concerning culture media, conjugation and production of zygospores* pure cultures, and studies of the morphological characteristics necessary for classification0

Greatest emphasis in th is study was on methods of inducing conjugation and techniques for obtaining zygospore germinationo ■ • ' swisir of m s w u E E

studied £pr mangr years and a mmber of investigators have described the characteristics of the, genus from the viewpoint of general Y morphology (Fritsehs 1935j Smith3 1950s Transeaus 19$1; Randhawa5 1959)o Ihile the oharacteristios of the gems are rather elear=cuts the taxonomy of the species is difficult and often confusing. It is virtually impossible to classify a species in the gems Spirogyra without zygospores: or asexual spores in addition to vegetative • materiala : The works of Transeau (1951) and Randhawa (1959) are extremely helpful in species'identifications as both of these . workers describe the morphological and anatomical characteristics essential to species Classif ications as well as supplying keys to species and descriptions of each species found in the keys* Transeau (1951) listed 275 species of Spirogyras and Randhawa (1959) listed 289 species* The taxonony is centered on the conjugation apparatus» Transeau (1951) pointed out that of 53U species of

Zygnemataceae 5 one reproduces only by akinetes3 38 by aplanospores9 . , ’ h9h by zygospores and one has been found only in the vegetative condition* He also showed that of the k9k zygosporic species, scalariform conjugation is found in hOO species, 72 species have

3 both lateral and sealarifprm corgugationj,. aM 22 species usually conjugate only laterally0 The confusion in the species taxonomy of the genus Spirogyra was discussed by Godward (1^53ia)> who suggested the use of cytological criteria, including chromosome studieSj, to improve classification of species in th is genus* Godward (195>i|bs. 195kc) has studied the diffuse centromeres or polycentrie chromosone s in Spirogyra, and showed that eytologieal'criteria, if available, mi^it help to . eliminate; some of the confusion in classificationo Allen (1958) found that different sized filaments from clonal cultures of Spirogyra were apparently the result of poly­ ploidy, which would seem to indicate that we probably are describing organisms as separate species when in a l l likelihood they are polyploids of the sane speeieso Oltmanns (1922) discussed the morphology of the genus

Spirogyra, with excellent descriptions of chloroplasts and other cellular characteristics» ■ Isolation and Oultureo The cla ssic work in the area of isolation and culture is that of Pringsheim (I9ii.6) who summarized and described the best methods up to that time * He discussed: the use of pipettes and glass hooks as means of selecting single cells or filaments for isolation, various devices for obtaining pure cultures, and culture media for both unialgal and pure cultures o

■ Levin (1959) described various methods of isolation for development of clonal cultures * / > v ./V' ■ i ' Bold (191*2) summarized the work which had been done on the cultivation of algae from 1928 to 191*0° He also clarified the terminology referring to cultures and stated that no conclusions concerning algae in organic material can be valid unless the algae are in pure cultures ° Bold (195*0) has also pointed out that the real criteria of taxonomic delim itation must be obtained from prolonged morphological and physiological studies? and that com­ parative cultural studies under controlled conditions w ill help to provide a satisfactory solution to the taxonomic problem. He has indicated that while much has been accomplished through studies of Ohlamydomonas? more might be learned through similar studies of the filamentous genera. Tiffany (190) stated that algae are ideal organisms for many-kinds of research and that;culture collections in the United States and in Europe are able to supply the investigator with unial- gal or pure cultures of many species of algae°

Brandwein (19ii.b) successfully cultured a hardy species of Spirogyra by placing i t in a jar in which he was growing a culture of Daphnia, Bits of egg yolk had been added to pond water for the Daphnia culture, Johansen (191*0) stated that it i s probably impossible to duplicate under artificial conditions the exact combination of environmental factors necessary to produce conjugation? and that i t is " d ifficu lt to keep species of Zygnematales in culture vegatatively and is scarcely worth attempting"o ' v. ■/Goldstein ■(.i96i}'- sbopi4;'ti^:;:E^tTiiiral'populations' of Eudorina possess sexaal potential not evident in natwe bat observable in culture with proper environmental conditions o

Krauss (1958) pointed out that algae are used, because of the simplicity with which they canbe cultured, to study mechanisms common to a ll plants< He believed that while ■much of the previous work was oriented, toward problems of general physiology of a ll plants, that a.great deal of information about the algae, was also learned in the process«,. : "While only a few culture studies of morphology, physiology and l if e cycles have been made on gSpirogyra (Allen, 19583 Czurda, 1933) quite a mmber of investigators have worked with other algal genera®

Stein (1958 a) studied the morphological characteristics and the reproductive cycles of Gonium pectorale Miller under controlled laboratory conditions, and was able to control various phases of the life cycle by manipulations of environmental conditions®

She was,::as a result,: able to make further studies of the organism* including genetic studies of meiosis and of populations® Stein

(1958b) also studied Astrophomene gubernaculifera Pocock and

Yolvulina steinii Playfair in culture0 She found G0o of. no significance in sexual, reproduction in Astrophomene where Starr

(1955a) found it valuable in the placedem desmids® Starr (195W, 195Ub) also studied heterothallism and inheritance of mating type and a leth al factor in Gosmarium botrytis yar® subtimidum under controlled laboratory: conditions® ' . / ,99:: Holleriberg (1958) was able to study the cycle of the marine alga Porphyra perforata in culture s and was able to follow the development of earpospores into filamentous plants which discharged asexual spores which grew directly to form new blades of Porphyra

plantso It was suggested by Coleman (1961} that algal culture methods would be improvedby the adoption of synchronizing techniques

which are available for algae0 She suggested further that these • techniques would be put to a valuable use if physiological studies of the processes of c e ll development and growth were made«

©ther studies of algae in culture include those of Gerloff, Fitzgerald and Skoog (1950) who were able to produce bacteria-free

cultures of Nostoe by the use of ultraviolet light. Machlis 1962 ( )

,succeeded in obtaining pure cultures for his studies of Oedogonium . by a process in vtoich he used penicillin and a washing procedure»

Levine and Ebersold (1960)5 in a review of literaturej, reported on studies of three species of Chlamydomonas which involved problems? in’physiologys genetics and cytology of the organisms e Allen (1958) made a very thorough study of a species complex of Spirogyra^, with emphasis on the conjugation and genetics

of the species as well as culturing methods»

Mineral Hutrition and Culture Mediae Ih ile numerous studies have been made on algal nutrition, both from the standpoints of mineral needs and metabolism, only a few studies have dealt directly with Spirogyra (Allen, 1958)» Bold (19^2) summarized the work Gtone on the eultivation of algae to 19^0° He suggested the use of inorganic culture solutions for obtaining, pure cultures <> and pointed out that confusion exists in the use of named culture mediae

Bold, (I9ii.2) also stated that phosphoruss magnesium and sulfur ions are included in nearly all media in addition to a nitrogen source ' such a.s nitrate or ammonium ions» Iron is probably introduced into most media as impurities in other s a lts . Ih ile Bold (I9l|2) discussed at some length such items as nutrition^, pH, temperature and use of solidifying substances such as agar^ he made no mention of any filamentous alga. He did point out that maiyr organisms which do not do well in ordinary media can be cultivated by addition of soil-water and also :recommended the use of the micro-nutrient elements needed by higher plants. -

Hunter (1961)9 using techniques of histochemistry9 showed the presence of a number of enzymes in Spirogyra that are also found in higher plants9 although he was not certain as to the location of soma of the enzyme activities in the c e llo :

Meyers (195)1) used growth as a measure of overall nutrition and metabolism in his studies. He supplied COg. by a bubbling technique, and provided sources of potassium, magnesiums sulfates phosphate, and nitrogeno His trace elements were supplied by so il- water. While he made ho study of filamentous Gonjugatae, he found the nitrogen content of the algae studied to be considerably higher than in most of the higher plants, and found that some algae use nitrate, some ammonium, and some both, as sources of nitrogen. . ' V :: : .... . ' . 9 Meyers (1951) also pointed to the need for thiamine and cobalamin as growth factors for many algae5 and suggested a possible need of IAJL in someo Maehlis (1962) showed a need for vitamin and other growth factors in Oedogoniumo .. ; Gerloffa Fitzgerald and Skoog (19^0) found the necessary concentration Of phosphate and sulfate to be low by comparison with nitrates and also found the need for magnesium and calcium to be low by comparison with potassimno These investigators also determined that iron availability was better when they used ferric citrate rather than ferric chloride« Frovasoli (1958) stated that f ie ld studies of major salt and trace metals are essential for establishing laboratory cultures and later pure cultures, and that laboratory media should be as close to the natural environment as possibleo He found the most important factors to be ( l) total so lid s, (2) ratio of monovalent to bivalent ions, (3) predominant anions and cations, and (it) the ratio of Ca/Mg0

Frovasoli (1958) also found that excesses as well as deficiencies of a number of substances can be inhibiting factorso Starr (I960) listed the algae which are available at the

Culture Collection of Algae at Indiana University, and described a number of media * ich are useful in maintaining cultures in the laboratory« /d : y"''v.. ■ ' Sexuality and Conjugationo Trondle (190?) described conjugation in several species of Spirogyra and noted a number of differences among them* He reported a breakdown of the male chromatophores in the zygote and the formation of carotenoid crystals0 Benecke (1925) also found: that conjugation varied with species^ and that more conjugation took place when the pH of his solutions was slightly alkaline* He found a temperature of 26C to be best for conjugation in most of the species he studiedo Czurda (1933) tabulated the results of his experiments on conjugation in Spirogyrao This led him to the conclusion that the vegetative cells had to reach a state of ,?Kopulationsdispositioni5

before conjugation would take place, He also found a considerable variation in conjugation among different species. Fritsch (1935)

.described the process of conjugation generally and that of the Zygnemataceae more sp ecifically „ Allen (1958) worked with a number of species of Spirogyra

but her major efforts concerned a species identified as Spirogyra pratensis Transeau. The main form of conjugation in this species is lateral although it may also conjugate ty the scalariform method.

She tried to determine the effects of lig h t> temperature., pH and nutrition on the conjugation of Spirogyra pratensis Transeau. Ihile Allen (1958) found no significant results as to the effect of nitrogen

depletion on conjugation in Spirogyra^. . Coleman •(.1962). indicated this to be important in sexuality in some other algae» This points out that

there are great differences in requirements for different species, •

however, a grpwth curve for Ghlamydomonas (Coleman, 1962) is similar to the curve for higher plants, and may apply to many algae. This

would seem to reinforce the idea of Czurda (1933) that the alga must reach the stage of ’’Kopulationsdisposition'1 before it w ill conjugate 0

& number of instances of abnormal or unusual conjugation have been found in Spirogyrao Brown (1918) found instances of double

and triple connections in species displaying scalarifom conjugatien®

Gates (1932 ) noticed a doable zygospore in Spirogyra fluviatilis : H ilse, formed when two male gametes joined one female from an extra long cell® Eritsch (I93f>) pointed out that brief exposure to freezing

temperatures or addition of small quantities of anaesthetic to the water would bring about abnormal c e ll d ivision s« Moner (195>U) found evidence that a substance is-produced by Pediastrum duplex Meyer which stimulates swarming® He found that a . medium in which swarming had occurred would induce swarming prematurely

in'other colonies® Raper (1952) stated that chemical regulation of

sex is illustrated by simultaneous formation of conjugation tubes and their attraction to each others howevers proof of chemical regulation by sex hormones had been found only in two groups of fungi® -Smith (I9j?l) discussed sexual substances of gametes and believed,them to be sex hormones which could determine a relative sexuality between isogametes0

Starr (1955a) studied sexual strains of placoderm desmids and found that lig h t, composition of the medium and COg appeared to be lim iting factors® Coleman (1959) studied sexual iso latio n in Pandorina

morum Bory® Grell (1956) reviewed the literature of recent years oh . sex, reproduction and cytogenetics o f protozoa and algae®

Hoshaw (1961 ) identified a number of terms used in discussions

of algal reproduction and suggested the use of living material in - W ' V ' ’ V - ' ■ . 12 teaching about the algae0 He explained the controlled sexual cycles of species of Chlamydomonas» Cosmarium and Oedogppiuma "vdiichs he stated^ can easily be used as teaching materials because the cycles can

be controlled at -will in the laboratory» •

Zygote Germination0 Fritseh (1935) described the mature zygospore of the Zygnemataeeae as having a thick wall, commonly three

layered, with the outer often being cutieularized and the middle one often showing d istin ctive markings. He stated that before germination, a large amount of fat is converted back to starch and the ehloroplasts become more distinct» As the zygospore wall bursts at one end, the contents emerge s t i l l surrounded by the innermost layer = Division soon occurs and the lower cell then contains scanty chlorophyll while

the upper c e ll proceeds to divide to form the new filam ent0

lewin (I9 l|.9 ) Was able to secure nearly 100$ germination of

zygospores of Chlamydomonas by keeping newly formed zygotes in continuous light for 2k hours and then transferring them to the darko He found that the zygote walls were thinner and that the contents would

divide in 5-6 days and by the end of £>-=6 more days, nearly a ll zygo­

spores would have produced ij. or 8 zoospores 0 He found that the

initial light must exceed II4.-I8 hours, but that if the light exceeded

26-30 hours, a hard walled zygospore developed^ : Starr (1955b) found that while ho single method is applicable to a l l algae, the zygospores of Cosmarium botrytls yaro subtimidum ,/ would germinate after a period of dormancy, either dry- or. in liquid medium, by transferring them to fresh medium. He also found that ■ ' ' - v V ' ■ ■ ; ' , ' v '13-: zygospores Tfaieh had been dried and then frozen would germinate within

2-3 days when placed in fresh soil-water medinnio Starr (191+9) had found that with zygospores of a ghloroooccnm-lik e alga, the best method of semiring zygospore germination was to place the spores on

agar and put them in an incubator at 37 G for' 1+8 hours* Within

1+8 hours after remo-ral from the incubatorj, 70%. germination had occurred = • Cook (1962) found it necessary to place cultures of Bulbochaete hiloensis (Nordsto) Tiffany with oospores in tightly capped tubes for several weeks at warm temperatures, after which bacteria and mold had destroyed old filaments and part of the spore wall* The spores

then germinated when transferred to fresh medium and lig h t»

Hoffman (1961 ) tried a number of methods to secure germination

of zygotes and parthenospores of Oedogonium and Oedoclad iumo He ■ .

found that a period of dormancy was required by most, but that freezing could shorten the period of zygote dormancy* His best results were

from zygotes of Oedogonium plagiostomum var« gracillu so . He was able -

to get nearly 100$ germination of zygotes of this species by freezing them in the old medium for a day or more and then placing them in

fresh mediume Germination followed in 2-1+ dayso : F- , ' ' " ' ' F , mTEEiAis m ), iethods.

. Natural .CollectIons0 Experimental material vras collected

from variou regions throughout the state of Arizona for use in this investigat ion„ Small amounts of material were collected from lakess ponds? streams? drainage ditches or other water sources where Spirogyra might:be found = Members of the Zygnemataceae usually have a slimy ' feeling Titiich helps to identify the group in the field a t /

Since Spirogyra appears to be the most common genus in the •family Zygnemataceae? most collections contained one or more types of Spirogyra, and frequently contained Zygnema or Mougeotia as well*

The small amounts of material collected were placed in sterile plastic tubes with caps or in small clean bottles. These tubes or bottles were then placed on ice in ah ice chest until they could be taken -

into the laboratory» Table 1 shows the location, dates and collectors of the Spirogyra used in this study and the code letters used to identify the various collections as they w ill be used in this paper»

. Isolation Procedure„ Single filaments which appeared to be similar were selected from the natural collections for isolation., Examinations of the collections were made using a dissecting microscope

Single filaments were picked, up by use of a glass pipette which had been drawn out to a fine point and bent over at the end by placing it

quickly into the flame of a Bunsen burner« This closed the pipette., forming a small knob at the end (Pringsheimp 191^6) 0 Various so ils TABLE 1 Sources for clonal cultures of Spirogyra used in this investigation.

Date of Strain Code Collection Collector Source Location

Organpipe OP* 11-61 M. West Sonoyta River across from Rocker*s Well Organ Pipe National Monument, Arizona

Gardner Canyon GC 7-62 F. Rickert Gardner Canyon Creek, about 50 mi. S.W. of Tucson toward Patagonia, Arizona

Sycamore Canyon SC 3-63 A. Dennis Water tank, Sycamore Canyon, off Ruby Road 25 mi. N.W. of Nogales, Arizona

Blue Lake BL 7-62 N. Wilson Indian Reservation, White Mtns., N.E. Arizona

Snowflake SF 7-62 F. Rickert Gravel p it, 1 mi. N. of Snowflake, Arizona, State Highway 77

LB 913 LB913 3-53 M. A. Allen Culture Collection of Algae Indiana University

LB 914 LB914 3-53 M. A. Allen Culture Collection of Algae Indiana University

This clone has been identified as Spirogyra erassispina Jao and the species name will be used instead of the code letters for this species in the text of this thesis. Identification was not made until late during the investigation due to the need for conjugation and mature zygospores for species identification.

H vx TABLE 1 Sources of cultures of Spirogyra (continued)

Date of Strain Code Collection Collector Source Location

Upper Sabino US 11-62 E. Dobosh Stream, Upper Sabino Canyon, 15 mi. E. of Tucson

Lower Sabino LS 3-63 R. W. Hoshaw Dam at Lower Sabino Canyon, 15 mi. E. V of Tucson

Rose Canyon RC 3-63 R. W. Hoshaw Lake, Rose Canyon Lake, Catalina Mtns., 40 mi. N.E. of Tucson

Lower Molina LM 3-63 R. W. Hoshaw Lower Molina Basin, Catalina Mtns., 35 mi. N.E. of Tucson

Mexico 22 M22 9-62 A. Dennis Soil sample from pond near Guaymas, Sonora; Mexico

H. Martyr Dam M 7-63 F. Rickert Chiricahua Mtns., 2 mi. from S.W. Research Station

Tanque Verde TV 3-62 A. Dennis Tanque Verde Creek, 30 mi. E. of Tucson

Riggs Lake RL 5-62 F. Rickert Riggs Lake, Mt. Graham, Finaleno Mtns., near Safford, Arizona

Ft. Lowell FL 12-62 A. Dennis Pond, 5011 E. Ft. Lowell Rd., Tucson Arizona ■were used in an attempt to find one *ich would

support rapid growth. At least 10 isolations were made from each

y collection® Test tutes i/i/ith single'filaments were'placed near a

, white daylight f luopeseent light &t % - d ist ance which gave an illumin- -

ation of approximately 500 ft-c. Temperature under these lights

ranged from 22 C with the lights off to 26 G with the lights on® ,,

These tubes remained under the lights until a small mass of filaments

■ appeared» These masses of filaments were then transferred to half- .

pint milk bottles of soil-water medium for further growth to provide

sufficient material for experimentation^

Growth and Maintenance of Cultures® . ' :■

I® Media® Cultures for experimental use were maintained

in steamed or autoclaved soil-water medium in milk bottles. The

steamed soil-water medium was produced by covering the bottom of the

tube or bottle with, about -g- in Of garden soil. The tubes and bottles

•were then placed in a pressure. cooker with the pressure cap loose

so that ample steam would develop with out building up pressure.

The tubes.and bottles were steamed, for 1 hr on each of 2 successive

.days. For .autoclaved soil-water, the tubes or bottles were autoclaved

at 15 lb steam pressure for 15 min. The soil which appeared to give

the best growth was labelled 1^3: and wa.s secured from, the L» B. Bants

.-■'.y-/’■■farm,:/3^, ^ l e 's .; northw est ;;of. fenticbilO y/ylhdianao'

Other media used during this investigation and the methods

of preparing them were as follows s . ... , - . . ■ 18. Desmid Agar Medium (Starr5 I960)

ll.75 ml g la s s - d is tille d "water

, 25 ml so il-w ater supernatant

5 ml 0.1^ MgSOA : - ■ ■ ... / ' , 5 ml 0ol% K2 HPO^

5 ml 1.0% KN03 :

if. g Difco-Bacto agar ,

This medium was au toe laved at 19 lb steam pressure for lf> min and poured into sterile Petri dishes to a depth of 0.5 cm. Vitamin and biotin in concentrations of 10 ppm were added to this medium in some instances.

Czurda’s Medium—per lite r (Allen* 1958)

975 ml Pyrex-distilled water

1 ml 10.0% H 0 3

1 ml 1.0% K2HP0^

1 ml 1.0% MgSO^ ' : : . . , ; :

• 1 ml 0.2% FeSO^

1 ml 0.5% GaSO^ "• .

Adjust pH to 6.0.using 0.01 N H2S0^ and 0.01 N K0H

Godward’s Medium—p e r l i t e r (A llen? 1958) ,

973 ml P y re x -d istille d water

1 ml 25.0% KN03 . .

i mi 8.0% Mgso^ , : : •: , 5

1 ml 5=8% NagSO^

1 ml 2.0% Ga(M03 )2 . 5 s:, . 1 ml 0.27% KSi03

’ ' 1 ml 2 . 8% KgHPO^

; ' i mi i 0o% oaco3

Since no KSiO^ was available, NaSiO^ was substituted at the same p e rc e n t. The pH was not a d ju ste d .

2. Light o , The • light sources used in a ll experiments were white daylight fluorescent tubes. Distances from light sources to cultures were arranged so that the intensity of the illumination -was from ItOO-oOO ft-c unless otherwise stated. Lighting was arranged to give various light and dark cycles. A cycle of 16 hr light followed by 8 hr dark was used for most experiments, but some work was done with continuous (2lt hr) light, and some was done with a "short day1* cycle of 10-hi.: light and lli hr dark.

3. Temperature. Temperature for most experiments was in the range of 22-26 C. Cultures tended to deteriorate inmost cases whenever the temperature exceeded'2^ 0.

' Gontrolled-environment chambers. Four controlled- ' environment chambers were used during the investigation.

a. A refrigerator was modified to give continuous light at 200-300 ft-c from fluorescent tubes and a.constant temperature o f 16 G.

b. A growth: cabinet was used which gave an illumination of 300-^00 ft-c and a temperature of 22± 2 C.

c. A growth chamber with a light intensity of 300-300 ft-c, a temperature of 27 C and a relative humidity of 30% was- used f o r some experim ents« -A ■ a' : ' ■ do A grovrth. room which maintained light intensities

(50-60D ft-c) "which could be varied by moving cultures to more

distant shelves and a temperature of 22 ir 2 0 was used for maintaining

cultures in soil-water0

5o Purif icat ion0; Two methods were used in attempts to

produce pure cultures« ; '

Bo A modification of the procedure described by Machlis

(1962) for obtaining pure, cultures of Oedogonium was used0 This

procedure invplyed washing a wad of ■filaments with a strong stream

of distilled water and dragging the filaments through a penicillin-

agar medium» ; :)■ ■ 1 ; ■ _ ' ;

bo A method of treating zygospores with a solution of

Clorox and germinating the treated zygospores on sterile desmid-agar was used (Table 2)0

Induction of Gonjugation0 A number of'investigat ions were

carried out to study conjugation in goirogyra* These investigations

. involved a variety of species in .unialgal cultures, and a variety of media. Media used included both liquid, and agarized forms of Ozurda’s medium5 Godwardf s medium and desmid medium as described earlier in

. this paper,: ■soil-water supernatant, and distilled-w ater agara .

The following methods were used to induce conjugation^

1. Watch "glasses containing fresh soil-water supernatant

inoculated with Spirogyra'filaments from soil-water cultures were placed on glass triangles in Petri dishes. The bottoms of the Petri dishes were covered with a %% solution of NaHCO^ to increase the 00^ i;:;'. -} /...... v. v 21 content of the surrounding air. Some of the Petri dishes Here placed under light and some were placed in the dark*

. . 2 o The distilled=water. agar nethod described by Allen (1958) was usedo Petri plates of distilled-water agar were inoculated by

spreading a small wad of filaments over the surface of the agar =.

The inoculated plates were then placed,under, light0

3o Petri plates' containing: desmid agar with soil-water

supernatant were inoculated with a small wad of filaments spread

over the agar surface. These plates were then placed under light„

During these experiments, light and temperature were variedo

The early experiments which involved growth media and conjugation^ while not conclusive, led to later, .experiments' using these methods

(Table 3)o

Zygospore Germination. A number of methods of inducing ,

zygospore germination suggested in the literature were tried (Dewin, '

19k9i Starr, 1955b| Allen, 1958j Cook, 1962).

1. Material containing zygospores which had matured in the light were allowed to ■dzy for periods of 2 weeks to5 months. Portions

of the dried material were placed in Petri plates and covered with fresh soil-water supernatant, and placed in the light.

2. Material containing zygospores was removed from the light within a few days after conjugation and allowed to mature in the dark

for 2 weeks before drying. After drying, these materials were covered with fresh soil-water supernatant and placed in the light.

3. Dried agar containing zygospores was wet down with fresh soil-water. supernatant arid placed in an -iricribator at 37 0 for U8 hrs -

then removed from the incubator and placed, in the light o

lio" Dried agar containing zygpsppres. was wet dovsn with fresh

soil-water supernatant and frozen for lj.8 hr. After thawingj, the

material was placed in the light, ' : '

■ 5, Jhe above conditioning treatments were used and:then the

material was placed on desmid-agar with soil-water supernatant in Petri

plates and the plates were put in the light. This was varied by placing

some of th e p la te s in th e dar k,

, Microscopic and Photographic Equipment, A Bausch and Eomb

Stereozoom dissecting microscope with sub-stage lighting was used for

. isolating filaments and for examination of Petri plates for conjugation

' or for germination of zygospores, ■

When closer observation was required, ma-terials were placed

on glass slides and examined with a Deitz compound microscope with

10Xa bbX and 100X (oil immersiori) objectives. This microscope was

fitted with an eyepiece micrometer, which had been calibrated with a

stage micrometer to measure in microns. Photographs were taken during

the,, 1962- 63.-academic y e ar with- a L eitz compound m icroscope w ith a

Eeitz conical’ adapter (MIKAS) and a 35. mm Leica'camera body. Film

used for the above photographs was Kodak Panatomic X and outdoor

type/Kodachrbme II, used with a blue light filter. Photographs

■ taken during the summer of 1963 were taken with an American Optical

phase-light microscope with a Zeiss adapter and camera body. Film

used in this case was Kodak Plus X and Kodachrome II T’ype Ac. EESWS

Collection and Isolationo Natural collections of algae containing Spirogyra were made from-li). locations in Arizona^, and

2 culturess LB913 and LB91I1.5 were secured from the Culture Collection of Algae at Indiana University „ Single. filament isolations were, made from each collection and were transferred into tubes of soil-water medium and grown in the laboratoiy under a controlled environmentc

At least 10 isolations were made from each collection and only rarely did more than half of these grow into a vigorous culture.

Filaments of Spirogyra seem to be injured easily in handling and by exposure to air during transfer unless a.sizeable filament mass is handled at one time. As soon as suitable cultures developed in the tubes, transfers were made into milk bottles of soil-water medium.

Of all the natural collections, three were lost as a result of death of the cultures, those from lower Molina Basin, Mexico and Ft. Lowell

(see Table l). Fortunately the Mexico culture.was from a soil sample and can probably be reisolated after wetting down another aliquot of soil, and the other two can probably be replaced by additional collections.

' Growth and Maintenance of Cultures. All early attempts to culture Spirogyra in artificial medias with or without agar, failed.

Only in soil-water medium could cultures be maintained. “While attempts were made to grow Spirogyra bn desmid-agar, with and without soil**

V .a, - ^ .. ' ' ■ water' supernatants in desmid medium without agar, in Czurdars liquid medium and in Godward1s liquid medium, in a ll cases the cultures died within,2-3 days after placing them in these media0;

Yjheh 10 ppm of vitamin a.nd 10 ppm of biotin were added to desmid agar with soil-water, luxuriant gpowth occurred on agar slants.

Slants were • then set up with only added to the desmid medium, slants with only biotin added to desmid medium, and with only soil- water supernatant added to the desmid medium, all solidified with agar at the same percent« Groups of filaments added to all these various slants; grew very wello It is not Imown at this time why these cultures grew when previous attempts had all failed, nor why the alga grew on agar but not in the same medium without agar. Failure to aerate the medium may have been a factor. It does seem, however, that better growth occurs in. all cases when groups of filaments, rather than single filaments are transferred to the desmid agar,

Growth bn. desmid agar with soil-water .supernatant w ill frequently last for a month to six weeks before the culture deteriorates.

Temperatures above 2l$.-23 C also seem to have a deleterious effect on

Spirogyra in culture. All cultures can be maintained for longer periods.without transferring them to fresh medium if the light itiiensity and the temperature are lowered. More rapid growth appears to occur under a light cycle of at least 16 hr of light and is not hindered by continuous.light if the temperature .is kept at or below

2k C, ‘ ■ ' ' , ' : : ' '■ ' 25 Conjugationo The first conjugation took place in the

laboratoiy, n\ihen cultures grotdng on agar were transferred onto fresh

agar medium. Conjugation took place to varying'degrees on all slants

within a period of 3 weeks after transfer.

The first trial with water agar plates as a means of promoting

conjugation was negative. Within 10 days after inoculation? the algae

had disintegrated on all the plates. The use of watch glasses with

soil-water supernatant on glass triangles in Petri plates seemed

at first to be a better method of producing conjugation. At least

some conjugation occurred in a few dishes by this method, with best

results coming from plates under continuous light. The first attempt with this method produced a few zygotes .fpom the combination of

LB913 and LB9-ll|. in the watch glasses. . •

The results of early experiments showed that some of the

strains would conjugate more easily than others. In all parts of

this experiment conjugation was found in Spirogyra crassispina Jao.

Filaments of GC conjugated on both water-agar and desmid-agar plates,

but not in tubes or watch glasses,- Filaments of SF produced conjugation

tubes on water-agar, but failed to produce zygotes. In no case did

filaments of BB. produce conjugation tubes or zygotes. Only Spirogyra

crassispina Jao produced zygotes under conditions of "short days" with light for 10 hr and dark for -Up hr*

Since Spirogyra crassispina Jao appeared to conjugate best . under all conditions, it was.selected for quantification of zygote production. Results of this quantification are shown in Table 2= TABLE 2 Comparison of zygote formation under varying conditions for Spirogyra crassispina. Average on a 0-5 basis.*

Average zygote production Plates (10 per group) by day for 10 plates Conjugation response

Plates with Plates with no 10 0 's Medium Environmental conditions 2 4 6 8 L0 conjugation o f spores

Lights - 16 light - 8 dark 1. Water agar Temp. 26.6 C 0 .6 1 .1 1 .1 1 .4 L.4 2 1 In cabinet - humidity 50$

Lights - 16 light - 8 dark 2. Water agar Temp. 22-26c 0 .6 1 .7 2.3 2 .4 2.5 1 3 In laboratory - low humidity

Lights - 10 light - lk dark 3. Water agar Temp. 22-26 C 0 .4 0 .9 1.5 1 .9 L.9 0 0 In laboratory - low humidity

k, Desmid Lights - 16 light 8 dark agar w ith Temp. 22-26 C 0 .0 0 .1 0 .2 2.0 3 3 so il-w a te r In laboratory - low humidity

5. Desmid Lights - 16 light - 8 dark agar w ith Temp. 26.6 C 0 .0 0 .0 0 .2 0 .2 8 0 s o il-w a te r In cabinet - humidity 50$

* 0 - no zygospores 2 - 11-20 zygospores 4 - 31-^0 zygospores „ 1 - 1-10 zygospores 3 - 21-30 zygospores 5 - 100's of zygospores

ON The first zygotes were formed within 2 days after the water-agar plates were inoculated and the amount of conjugation gradually increased for several days5 followed by a sudden jump in zygote formation in several plates, and a stoppage of zygote formation in others. Of

30 water-agar'plates inoculated with Spirogyra crassispiha Jao under different conditions of light and temperature, only three failed to produce at least a few zygotes within 12 days. Best results came from the group under 16 hr of light and 8 hr of dark with a temperature of 22^26 C. Of the desmid-agar- plates5 only one showed conjugation a f t e r k days and only two after $ days, but a rapid jump in zygote production showed up after.8 days.- Plates placed in a controlled- environment cabinet at warmer temperature (26.6 C) and higher hum idity {$0%) were destroyed after 8 days by the growth of mold.

More mold developed in the plates containing nutrients than on water-agar plates. ISiHile conjugation is. slower to start on desmid-. . agar than on water-agar, it goes on more rapidly and appears to be more consistent throughout"the culture. Excellent growth Of all cultures took place in test tube slants with desmid-agar before any conjugation took place.

Zygospore Germination. Tn the attempt to produce a pure culture by treating the zygospores with Glorox before germination on desmid-agar plates, first germination appeared in about 3 weeks after the experiment, was started. Germlings appeared first from spores which had been treated with 0 .2 ^ Glorox for 5 min,

0.5$ for 15 min, and 1.0$ for 10 min. Within another 2 weeks. ■ V ; ' . : ' . " ' ' 2 8

germination had taken place on 9 of 19 plates, and the Clorox treatment

apparently -mas not harmful to the zygospores, as germination occurred

just as.frequently from spores treated at 2o0$ forV~> min as i t d id among

those treated only with'distilled water0 Best germination resulted,

. from■ th e‘spores treated for 10 min with 1,0% Glorpx. Germination

in this case was estimated at about $*0%} while from most of the other

treatments the germination appeared not to exceed 2*0%* No germination

appeared in the plates'which contained distilled water rather than

desmid-agar. Results are summarized in Table 3» Few bacteria

colonies appeared on these plates, but a lot of mold appeared« Three

germlings were taken out of the plates and transferred to fresh agar,

and here the algae outgrew the mold o This appears to be a method

worth further consideration for developing pure cultures of Spirogyra0

A fte r 3 weeks, the filaments grown from these germlings conjugated,

thus completing the life cycle on agar.

A tube of dried agar with zygospores was treated with a . •

1.0%; detergent solution in an attempt to k ill the mold. This tube,

along with another similar ,tube., was then: filled with distilled water

and frozen for lj.8 hr. After thawing, the contents of these tubes

were placed in plates of desmid-agar and coyered with, steamed soil- ...

water supernatant, and,.placed under lights. ., In 2 weeks, filaments

began to appear in the plate of zygospores that had not been treated

with detergent. After 3 weeks,, numerous filaments were found in this

plate, but no germination was apparent in the other plate. Hold was

present in both plates. Table 3 Olorox treatment for zygospores to kill bacteria with percent of

germination of zygospores on desmid agar after treatment

Clorox Time of Number Estimated. concentration treatm ent of germination • (minutes) p lates (%) v ' ' ■ . : : , ■ 0.25 > ■ : : :-1 ;' ' . . . 10 1 0

15 1 0 : ;v / V 0 , 5 0 . : ;i ' . 'v: . :- l . ■ :

. 10 . 1 . 2 ' " : 15 ■ 1 . 1

V :'V,- 0,75 ; 1 : . . 0 " ■■ X ■ > 10 1 : 0

< 15 1 0

- " ' o ■ :v > 1.0 . ■ -V s ... : 1 10 1 " 5 ■ 15 ■ ' u ; ' ; 0 , 2 . 0 ; ■■ /v ;V';i. y '■ 10 : 1 - 1 ■

. 15 1 j ; 3 :

Distilled water ; ; ;;\2 ; : i . : (control) '

Distilled water - no agar (control) ^ V/x'.; ' A plate with numerous zygosp ores on water-agar* after being

matured in the dark for 2 weeks following the peak of conjugation,

was wet down with steamed soil-water supernatant0 After 2 weeks,

no germination was .apparent, A similar plate with desmid-agar was

matured 2 weeks in the light and was then put in the dark for 2 weeks

before wetting,with soil-water supernatant0 • In 1 week, live filaments

began to appear, and in 2 weeks, numerous filaments were present., .

Careful .observation during this time failed to find any young germlingse

The fila m e n ts p re se n t seemed tb have re ju v e n ate d , r a th e r th a n new

• filaments having' formed from germination of zygospores. The agar may

have held enough moisture to keep the filaments alive, or akinetes

may have formed which produced the new filaments. This needs further

studyo ' ' '

Morphology and Taxonomy. • Careful observations and measure­ ment s were made of the cultures which grew well in the laboratory ?

Photographs were taken of vegetative cells, of conjugating cells, and

of matured zygospores. The characteristics which were studied were

those listed by Transeau. (I9$l) and Randhawa (195»9) as important in

the identification o f species = Cell width, cell length,' number of

chromatophores, number of turns of each chromatophore within a cell,

and type of end wall were determined for vegetative filaments of

10 different clones. The results of these determinations are shown -

in,Table U. 'Photographs of'vegetative cells are shown in Pig. 1-11.

End walls, chromatophores •and. pyrenoids can be seen in these photographs

and comparisons of cell sizes can be made. • . . . :: 31 Table ij. Comparison of morphology for vegetative cells using 10 or more filaments each from 10 strains0 .

Cell Cell No. of No. of End ' S train width length chroma- turns per , walls A tophores :chrpmat ophore

SF 50-52 167-312 3-4 .5-1.5 plane •

RL 27-31 113-207 2 2-4 plane . . BL3 37-42 123-224 3-4 3-4 plane ' • BL? 50-56 154-237 . 3-4 2-3 plane LB913: 27-30 66-141 1-2 . 2-4 plane

LBpllt ■ 30 84-114 1—2 3-4 plane OP 28-42 112-210 1-2 3-6 rep licate

LS 42 126-196 v y 1-..... 6-9' . plane GO 42 140-350 3 -4 2-3 plane

SC 28-42 45-268 1 2-5 rep lica te ' : ' 32 Studies of zygospores were made to determine width, length.

Shape and color of the zygospores« The spore walls were studied

' w ith the o i l immersion len s o f a compound microscope to determ ine th e

number of spore walls and the ornamentation of the walls» Color,

. number of walls and type of ornamentation are the most difficult to

determine« It was also determined in those cases where conjugation

had taken place whether the conjugation tubes were formed by one or '

both gametangia and whether the fertile female cell remained cylindrical,

• or whether it was enlarged or inflated on one or both sides» Results

of these determinations are sh own in Table 5®

A series of photographs was taken of the Spirogyra sp0

. from Sabino Canyon and of Spirogyra crassispina Jao0 Various stages

of conjugatioh from beginning papillary formation to mature zygospores

of the Sabino Canyon collection are shown in Fig. 12-17» Germinating

zygospores of this species are shown in Fig« 18-20» The nuclei of .

the germling, cells show plainly in Fig, 20. One can also see where.

the chromatophore was dissected by the septum from the continuation

of the spiral from the first cell to the second. Pig. 21-30 show

stages from the vegetative cell to the mature zygospore of Spirogyra

crassispina Jao. In Fig. 21, showing vegetative filaments, replicate

end walls can be seen, as well as other end walls that appear to be

plane. Two chloroplast's can be seen in some cells, while others

appear to have only pne. lihile''most zygospores of this clone appear

as ovoid to ellipsoid in shape (Fig. 22), an occasional one shows up

as lenticulate or elongated. This lentipulate form is shown in Fig. 23o Table 5 Comparison of zygospore characteristics with ranges shown by 10 or more zygospores

No. of Conjugation Female cell Strain Width Length Shape spore Ornamentation Color o f tubes. One c y lin d ric M w alls of median wall spore walls o r both en larg ed gam etangia in f la te d

4 Outer - thin, Yellow w alls w rinkled. OP 35-42 49-70 Ovoid Median Inner - small Yellow- Both E nlarged w all o f variable sized brown 2 layers granules

Ovoid Reticulate to Yellow- GC 42-49 59-98 to 3 denticulately brown Both Enlarged denticulate r e tic u la te

V aried- Female cell SC 28-42 46-63 ovoid, 3 F oveolate Yellow- Both inflated on e llip s o id , brown tube sid e denticulate

LB913 V aried- X 31-35 43-64 ovoid, 3 Punctate to Yellow- Both V aried LB914 e llip s o id , fo v eo late brown denticulate

IS 47-58 74-89 E llip s o id 3 F oveolate Yellow- Both I n f la te d brown both sid es

V aried- sc 25-32 52-64 ovoid, 3 Foveolate Yellow- Both In f la te d e llip s o id brown tube sid e ■ ' ' ' : ; ' : ■ ■ : . * Cells of this species seem able to form conjugation tubes from all areas of the c e ll w all, including the end w all, A tube grown from an end wall is shown in Fig, 26, ■ The. Spirogyra from Organ'Pipe has been identified by

Dr, C, E. Taft of The Ohio, State University as a small variant of Spirogyra crassispina Jap, This species reportedly conjugated by both lateral and scalariform conjugation, however, no lateral con jug- ' ation has been seen in nearly 2 years of observation of this species. Dr, Taft (personal communicat ion) stated that Transeau did not have a slide of this species, but in his notes he had used the spore wall ; as the critical character, and the spore wall in this case is almost identical to that described as characteristic of this species. There are also many cells with single, chloroplasts although the species ' description indicates two chloroplasts.

Vegetative material and zygospores have also been sent to Dr. Taft of the clone from Gardner Canyon, and strain LB91u from the Indiana University Culture Collection, since these two strains appear to conjugate fairly well in culture. Since these materials were sent to Dr. Taft at a late date, sufficient time has not been available to complete the•identification, but the "Gardner Canyon clone has been tentatively identified as Spirogyra subreticulata Fritsch from the keys to species of Transeau (1951) and Eandhawa (1959)= The zygospore Of this species is shown in Fig. 31. -Fig, 2 shows the vegetative " cells of this species, . ’ v Figures l^s: Vegetative iilaments in Gultur'e= These figures show cdmparative cell wi(3th5 chromatophores and pyrenoids clearlyo (Magnification, X250)o

Fig. It Clone isolated fromcQllection from Upper Sabino Canyon

Fig. 2: Clone isolated from collection from Gardner Canyon.

Fig. 3* Clone isolated from collection from Organ Pipe National : : . Monument. ' . .

Fig. Ut Clone isolated from collection from Sycamore Canyon. 36

F i g . 3 Figures 5-8 ? Vegetative filaments in cultureo These figures show comparative cell width, chromatophores and pyrenoids clearly= (Magnification^ 1 '

Fig. 5s Clone isolated from collection from Showflake

Fig. 6; Clone isolated from collection from Tanque Verde.

Fig. tfs Clone isolated from collection from Riggs lakes

F ig ..8$ Clone isolated from collection from Rose Canyon* \ 38

Fig. 7 Fig. 8 ■■ Figures 9-11 ? Vegeta-bive filaanenfcs in culture e

Figo 9: Strain LB913 from Culture Collection of Algae5 Indiana Universityo (Magnification., X250)o

Figo 10$ Strain I1S9II4 from Culture Collection'of Algaes Indiana University, (Magnification, X250)o

Fig, 11$ Clone isolated from Snowflake collection. Notice ’ tightly wouixd ciiromatophores and large pyrenoidSo (M agnification, X5i).0)0

Figures 12-13s From natural collection from Upper Sabino Canyon*

F ig . 12 s S ev eral filam en t . w idths shown in n a tu ra l c o lle c tio n . :. ' , / Cells with papillae just starting and one cell containing a zygospore are shown. (Magnification* X12jp).

Fig. 13 i Two cells showing beginning of papillary formation. (Magnification* X51f.0)o , Fig. 13 Figures Hj-l*?*' Natural collection from. Upper Sabino Canyon0 (Magnification, X^ij.G)o Figo Iks Cells show conjugation tubes formed, protoplasts about to make contact, starch accumulation appears, and female garnetangium has swollenp Fig, 15$ Conjugation tubes have formed, protoplasts about to make contact. Female gametangia swollen. F i g . 15 Figures 16-17 s From natural polleetion from I%)per Sabino Cargron.,,

. , Fig,M 6s. String of,, zygoteso ■ This filament width was the only U>-- one conjugating: in the -natural collect ion* (Magnification's X90)o

V Fig. 17? Zygosporess tubes and empty cells. (Magnifications Xf&O). U6

F i g . 17 Figures 18-20t Germlings# from germinating zygospores from Sabino Canyon» Zygospore wall sp lits at one end3 contents emerge covered by the inner walle This cell divides to form 2 c e lls <. (Magnification) X325)°

. Figb l8t Germling just einergingo . :. ■ •

Figo 19s Germling fully emerged5 1 cell.

Fig, 20: Germling) 2 cell stage, Nuclei appear clearly in . these pellso Fig. 19 Fig. 20 Figures 21-23? Spirogyra erassispina Jao,, Clonal culture from Organ Pipe National Monument = . (Magnificati on3 Z600)» ' Figo 21: Vegetative filament showing replicate end walls6 ' Cells with one and. with two chromatophores-

Figo 225 Zygospdre, most common type, ovoid-ellipsoid in shapeo

F ig o 23? denticulate zygospores 1 : . So

Fig. 21

t ; ‘■if.

J r a

- 4 :

\

A I

F i g . 22 F i g . 23 Figures 2JEj.-26'* Spirogyra crassispina Jao0 Various forms and stages in conjugation of clonal cultures from Organ Pipe National Monument after .12 days on desmid agar- with soil=*water«, Fig, 2^: Various stages of conjugation, (Magnification, X25>0)o

Fig, 25J Various stages of conjugab ion. Gametes in one pair , of cells ,appear tP be aborting, (Magnification, %320), Fig, 26: Cell conjugating from the end. In one c e ll gametes . ■ have failed to.'fuse,: (Magnification, X320), F i g . 25 F i g . 26 Figures 27-29: Spirogyra erassispina jao. Clone from Organ Pipe National Monument on.desmid, agar fo r-12 days5 shawing various stages in conjugation. (Magnification, X625). Fig. 27t Tubes have .just fused, no contact between protoplasts. .Shows starch around pyrenoids. ... Fig. 28: Cells with tube formed and one gamete.has rounded up. Fig. 29: Both gametes have rounded up, and male gamete is moving . into tube. F i g . 28 F i g . 29 Figures 30-31s Zygospores. / Fig. 30 s Zygospore of Organ Pipe clone, Spirogyra crassispina Jao. Notice • replicate end ■mall. - (Magnification^ X600).

Fig. 31 1 Zygospore of Gardner Canyon clone» (Magnifications o Fig. 30

F i g . 31 DISCUSSION . ;

Growth and Maintenance of Culture So The need for unialgal and pure culture studies of algae has been recognized (Pringsheinu,,

I9I461 Cdleman5 196l)0 Some of the problems involved in maintaining filamentous algae in culture have, also been recognized (Johansen, 191*0$:

Bold, 191*2$ 1950)o Johansen (19I4.O) stated that it was scarcely worth attempting to keep the Zygriematales in culture vegeiativelyo■ The use of Pringsheim1 s soil-water' culture method has, however, made it possible to maintain cultures in the laboratory with little difficulty,..

While the maintenance of Spirogyra in soil-water medium is relatively easy, the maintenance of such cultures in defined inorganic media has been very difficult, ; v '

Alien (1959) found that all of the synthetic media tried were completely unsatisfactory for growth of Spirogyra and found it necessary to use steamed soil-water media for growth and maintenance of her cultures. The present investigation has largely supported her findings^ except that autoclaved soil-water medium has been almost as suitable as steamed soil-water medium in most instances. This invest­ igator has found that inoculation of new cultures with a group of filam ents, rather than single filaments, produces a more vigorous culture, Spirogyra cultures, have been maintained for as long as

6 weeks on desmid agar with soil-water by starting the culture with a small group of filaments, Several clones have been grown in this manner under fluprescent light of 300-000 ft-c and at temperatures

below 22-26 Go The growth pattern formed by Spirogyra on agar typically consists of many little whorls of filamenbso "While some species w ill

conjugate in 1-3 weeks on desmid agars other species continue in a

vegetative stage for several;weeks or u n til the agar begins to dry out© •The addition of vitamin B-^ and biotin to the desmid medium in concentrations of 10 ppm. appeared to be b en eficial3 but th is needs

further study® - ; . v • . Isolation Procedures. Whenever new cultures were collected in the fields they were kept refrigerated until they could be studied in the laboratory and single filaments could be isolated from them. The

methods of isolation described by Lewin (1959), with some modification^ have been satisfactory in developing clonal cultures of Spirogyra.

The filaments of Spirogyra are large enough to be able to select

single filaments or pieces of single filaments with a glass or wire

■ hook. These filaments can be washed through two or three dishes of

distilled water, and can then be transferred immediately to test tubes of soil-water medium. Unlike Allen (1958), the present investigator has found a nichrome wire hook to be simpler bp use than the glass V

hook. The glass micropipette is very convenient, however, in picking up small filaments or zygospores® Conjugat ion® Studies of conjugation in Spirogyra crassispina

Jao have largely supported the findings of Allen (195>8) with Spirogyra , p raten sis•Transeau® Conjugabion proceeded more rapidly on d istille d water-agar than on other media. Zygospores were produced in watch 59

glasses placed on glass triangles in Petri plates, using either steamed or autoclayed soil-water medium, but the conjugation was neither as .■

rapid nor as uniform in the liquid media. Conjugation and zygospore formation appeared to be more uniform on desmid^-agar with soil-water

supernatant, but did not start as soon. Conjugation on desmid-agar was always‘ preceded by a period of c e ll division and growth,

Fritsch (1935) pointed out that the process of conjugation

occurs more frequently;in low-lying areas than in upland districts.

The findings in this investigation tend to confirm his report a Conjugation in this investigation occurred most readily in the culture from Organ Pipe National Monument, a relatively low area. The others which conjugated With considerable frequency were the clones from Gardner Canyon, Sabino Canyon and Sycamore Canyon which are areas of somewhat higher elevation. No conjugation was found, either in

natural collections or in culture, of the clones from Blue Lake in the llhite Mountain area where the elevation,is considerably higher0 Fritsch (1935) pointed out that conjugation usually occurs between two filaments in juxtaposition. This could be one reason why conjugation Is more abundant in the laboratory on agar than in watch

glasses of soil-water medium, as it is more difficult to keep filaments in juxtaposition in the l,i#id* % _ ■

, Allen (1958) found that conjugation occurred most rapidly

when cultures 2-k weeks old were transferred to water-agar. This has also been found in the.; present ■ investigation. This appears to support the theory that a climax of vegetative growth must occur before conjugation w ill take place« Czurda 1 ( 9 3 3) believed this to be the

case in the numerous species*-of Spirogyra -with "which he worked . If

the organism is in the proper stage of vegetative maturily, it may be

that the stress condition supplied by the distilled water-agar triggers

the conjugation, mechanism, whatever it may beo Zygospore Germinationo A number of different techniques have been suggested for inducing zygospore germination in various algae

(Lewin, 19U9; Starr, 19^9; Hoffman, 196l| Cook, 1962)= Starr (1955b) found that no single■method is applicable to all algae« Allen (1958)

allowed the zygospores of Spirogyra pratensis Transeau to mature for

10 days in the dark or in poor;ligh t and then dried them on agar in paper packets at room temperature= She then covered the material from a packet with fresh soil-water supernatant and placed them under a

light of 500-600 ft-c intensity and at a temperature of 20-23 Ov

She found that germinatid'n proceeded in about 3 days, but that results were unpredictable and the percentage'of, germination was very low. The present investigator has found results quite similar to those of

Allen (1958)= The percentage of germination was very low and was not predictable = The results obtained in one trial are not necessarily

obtained in another. Length of drying time, maturing in lig h t or in

darkness, heat treatments and freezing treatments had no significant effect on the germination of the zygospores of Spirogyra in this

investigation. This is an area which needs further study = I f the zygospores can be induced to germinate, treatment with a bactericidal

agent may be a good means of producing a pure culture = In the' present ■ , : : ' 6 1 .

investigation, the use of Glorox as a bactericide on the zygospores led

to development of cultures which appeared to be bacteria- free, but a

considerable amount of mold developed in the cultures, ilhen germlings were transplanted onto fresh media., they were able to outgrow, the mold, ' :

so that additional transfers might have produced cultures free of both mold and bacteria. The development of pure cultures of Spirogyra is another area vhich needs further work. .

While. Spirogyra was cultured in this study on an agar medium, i t was not grown for long enough periods to determine whether this environment would produce morphological changes in -the organism. ■■ Additional work needs to be done to find .out whether or not morphological changes would occur in pure culture or in agar culture over long periods. Once pure cultures have been.established, mineral nutrition studies, vitamin and other growth factor studies and genetic studies, can be made.

' Morphology and Taxonomy. Transeau (1951) and Randhawa (1959) pointed out the morphological characteristics essential for species determination in Spirogyra. . Alien (1958) listed many of these ' characteristics as qualitative characters to contrast with quantitative characters by which the strains with varying filament widths in her studies differed. The characteristics studied in this investigation included the qualitative characters, listed by Allen (1958) and some of the quantitative characters as well (Tables h-5) <• The type of septum, or end w all, is important in species determination. This was sometimes easy to see under the compound . microscope, and at other times was v - difficult to determine. Most of.the species studied in this invest­ igation appeared to have plane end w alls, but. the clone from Organ Pipe, Spirogyra crassispina JaOs and, the clone from Sycamore Canyon appear to have replicate end walls« Fritsch (1935) stated that fragmentation of species of Spirogyra with replicate end walls occurs more readily than species with plane end w alls« This appeared - to be the case with Spirogyra crassispina Jao3 as the filaments would break up much more

easily in this species than in those species with plane end walls that

were included in th is study.

Measurements of cell width and cell length, as well as

zygospore width and length was relatively easy with an eyepiece

micrometer, In the taxonomy of Spirogyra there is a great deal of

variance, however, in these quantitative characters. The species

..Used to the greatest exte'ht. in ihis. stu.dy wp.s identified by

Dr, G, E, Taft of The Ohio State University as ,ra small variant of

Spirogyra crassispina Jao,” The average cell width was considerably

smaller than the cell width listed by Transeau ( 1 9 5 1) in his description

of this species, and only a few of the larger cells of this culture

f e l l w ith in th e minimum o f th e range l i s t e d by T ranseau,

; , Gpuntihg ehromatophores presented some difficulty at times,

and there was a variance in chromatophore number within the cells of

a sin g le fila m e n t. The number of tu rn s made by a sin g le chromatophore

also varied a good bit within a filament. This is taken into account

in' species descriptions, .

This investigator found greatest difficulty with zygospore

characteristics, In the counting of . spore walls', it was necessary

to use oil-immersion, and with this, light parallax made it difficult : ' " : 'V''. ' ■ ■ ■ 63 to t e l l in some code's jHst how many layers made up the w all. It was also d iffic u lt to determine the type of ornamentation^ as the listed types shade from one to ahofherv Color of zygospore walls was also/

. a matter of shading from one to another«, By contrasty it was fairly easy to determine whether a female garnetangium was enlargeds or whether it was inflated on one or both

sideso The .difficulty here was due to the fact that considerable variance was. found within the species (Fig. 22-23)e It was easy$ after working with species identification in

Spirogyra, to understand why Godward (l95il-a ) spoke of the confusion in the taxonomy of Spirogyra and hoped that the use of cytological

characteristics and chromosome studies would aid in making the taxonomy. less confusingo ' . SUM6ERI

Clonal cultures of Spirogyra were produced and maintained in ;

Pringslieim's soil-water medium for collections from lit different sites iii Arizona. Two additional cultures, obtained from the

Culture .Collection of Algae- at Tndiana University, were maintained in soil«#rater medium.

A number of clones were established and maintained on agar slants of desmid medium wilh. soil-water supernatant. These clones were maintained in the laboratory for periods of A-6 weeks without transfer tofresh medium. lihen fresh, actively growing cultuies of Spirogyra crassispina

Jao were transferred from soil-water medium to distilled-water. agar, conjugation occurred within 2 days, in some cases, and continued for a period of 2 weeks. . Only scalariform conjugation .was found. Conjugation also occurred on desmid-agar with soil- water, but started after a longer period. Methods for effecting zygospore germination have been described. These methods included drying, heat treatment and freezing.

Zygospores were germinated) the germlings were grown and con­ jugated again to complete the life cycle of Spirogyra crassispina Jao on agar medium. The percentage of zygospore germination was low and unpredictable. ,

Morphological studies were made of a number of species and characteristics of-vegetative cells and of zygospores were determined„ . Identification of Spirogyra crassispina Jao was made during the period of this investigation*

A number of areas "which need further stucty have been described, including germination, of zygospores, purification of cultures, morphology of filaments in pure culture, physiology in pure culture, and chromosome studies, as an additional means of determination of species0 . LITBR&TUEE CITED

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• * 1962 * Sexuality, p* 711-729*/ In R. A* Lewin, (ed.), ' Physiology and biochemistry of algae * Academic Press, New York.

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