Zygospore formation, germination, and theontogeny of the chloroplast of Sirogonium

melanosporum (Randhawa) Transeau

Item Type text; Thesis-Reproduction (electronic)

Authors Dennis, Arthur Eldon, 1931-

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 27/07/2018 16:39:54

Link to Item http://hdl.handle.net/10150/551764

ZYGOSPORE FORMATION, GERM INATION,. A ND THE

ONTOGENY OF THE CHLOROPLAST OF SIROGONIUM

M ELANOSPQRUM (RANDHAWA) TRANSEAU

by

A rthur Eldon. D ennis

A T h e s is Subm itted to the F a cu lty o f the

DEPARTM ENT OF BOTANY

In .P a r tia l F u lfillm en t of the R eq u irem en ts For the D e g re e of

MASTER O F SCIENCE

In th e G raduate C o lleg e

THE UNIVERSITY O F ARIZONA

1 9 6 5

STATEM ENT BY AUTHOR

T his th e s is has been su bm itted in p artia l fu lf illm en t of req u irem en ts for an advanced d eg ree at The U n iv ersity of A rizon a and is d ep o sited in the U n iv ersity L ib ra ry to be m ade a v a ila b le to b o rro w ers under r u le s of the L ib rary .

B r ie f quotations from th is th e s is a re a llow ab le w ithout sp e c ia l p e r m iss io n , p rov id ed that a ccu ra te acknow ledgm en t of so u rce is m ad e. R eq u ests for p e r m is s io n for extended quotation from or rep rod u ction of th is m a n u scr ip t in w hole or in part m ay be granted by the head of the m ajor departm en t or the Dean of the G raduate C o lleg e when in h is judgm ent the p ro p o sed u se of the m a ter ia l is in the in te r e s t s of sch o la rsh ip . In a ll other in s ta n ce s , how ever, p e r m is s io n m u st be obtained from the author.

A PPR O V AL BY THESIS DIRECTOR

T his th e s is has been approved on the date shown below:

ROBERT W. HOSHAW P r o fe s so r of Botany

2 7 !?cs~Date

ACKNOW LEDGM ENTS

The author w ish e s to e x p r e s s h is d eep est a p p rec ia tio n and

tn ost s in c e r e gratitu d e to Dr.. R ob ert W= Hoshaw for su g g estin g th is

p rob lem and for h is contin ued su g g estio n s and gu id ance throughout the

in v e s tig a tio n s and the w ritin g of th is m a n u scr ip t, I w ould a lso lik e to

thank him for the photographs for F ig u r e s 3 -7 , 16, and 18,

A p p rec ia tio n i s extended to E liza b e th P» F ra n tz and her

h o u seg u est from M unich, G erm any, F ra u U se H e lle re r , w h ose tr a n s la ­

tion s of p o rtio n s of De B ary (1858) and T rondle (1907) p ro v ed m o st

va lu ab le for the co m p letio n of th is w ork ,

. I am in d eb ted to R ich ard D, W aer, fe llo w graduate student in

the B otany D epartm ent, for the technique, of inducing m ovem en t of the

m a le g a m ete in S irogonium m ela n o sp o ru m .

Thanks a r e extended to D r, C h a r les T, M ason, D r. R ob ert S.

M ellor, and. Dro, W alter S., P h illip s for read in g and c r it ic iz in g the

m a n u scr ip t.

TA BLE O F CONTENTS

P age

LIST OF ILLUSTRATIONS „ . o „ „ „. , o „ . . „ „ . . . „ v

LIS T OF TjA.BLES © © © © © © © © © © © © © © © © © © © © © v n

ABSTRAC T © © © © © © © © © © © © © © © © © © © © © © © © v m

INTRODUCTION A ND REVIEW OF LITERATURE © © . © © . © 1

M ATERIALS AND METHODS © © © © ©,. © © © © © © © © © © © 13

(So 1.Lectioti- S ite © © © © © © © © © © © © © © © © © © © © 13IS OiatlOn O © O O'© © © 0 o © © © O 0- 0 0-0 o o o © o o 16JN/I e di a o © © © © © © © © © © © © © © © - © © © © © © © © *19JLiigtlto o o o o o © o o o o o o o o o © b o © © © © o o .2 0T exnp er atimr e © © © © © © © © © © © © © © © © © © © © © 21Culturing., T ra n sferr in g , and M anipulatory T ech n iq u es © © .2 1BL eag ent s © © © © © © © © © © © © © © © © © © © © © © © 24P hotography © © © © © © © o © © © o © © © © © © © © © 2 5

OBSERVATIONS AND RESULTS© © © © © © © © © . • © © © © © . 27

V eg eta tiv e C ell and the Chloroplasts© © . © © © © © © © © © 27C y to k in esis © © © © © © © © © © © © © © © © © © © © © © ̂ 29G am etic F orm a tio n and the R o le of the C hlor oplasts© © © © 30Z ygote and C h lo ro p la stic O rganization© © © © © © © © . © © 31Z ygosp ore © © © © © © © o © © © © © © © © © © © © © © 32G erm ination of Z y g o sp o re © © © © © © © © © © © © © © - © 34G erm lin g and R eo rg a n iza tio n of the C h io ro p ia sts © © © © © 36

DISC USSION o o © o o o o o o o o o o o o o o o o © o o © 55

S U 3V1 iMAeRj *Y" o o o o o © o o o o o o o o o o o o o o © o o © 64

LITERATURE GIT EX) © © © © © © © © © © © © © © © © © © © 66

LIST OF ILLUSTRATIONS

F ig u re P age

I O C* O Lie C tlOU Site O O .O O O O -O *0 O "O e O O • O O O O O o 14

2o ^la^i. of coILectiou* siteo @ @ o o @ o o o @ o ■ o o @ >o 15

3« , G am etan gia l fo rm a tio n 0 » . o . « « « . » « . «. » » 47

4 o ^̂3a m e te s o o o o © © © © © © © © © © © © © © © © © 47

5© . A zygote 30 min© a fter the fu sio n of the gametes© © © 47

6= A young z y g o sp o re contain ing ch lo r o p la s tic fra g m en ts © 48

7© . A m atu re zy g o sp o re . © © © » , © - . © © « « » © . . © « 48

8© A sm a sh ed z y g o sp o re show ing th e tra n sp a ren t in n er c e l lw a ll w ith in the outer w a lls © © © © . . © . © © © © 49

9© ■ A m atu re z y g o sp o re sm a sh ed in IKI . © © © © ©. © . © 49

10© S irogonium m elan osp oru m from M ex .22 so il show ing ac e l l in the p r o c e s s of c e l l d iv is io n © © . © © © © © © © - 49

II© O n e -c e lle d germ ling from cu ltu re © . © © © © © © © © 50

1 2© S am e o n e -c e lle d germ lin g . as F ig u r e 11 but at g r ea term a g n ifica tio n © ©. © © © © © ©. © ©. ©. © © © © © © © 50

13. T w o -c e lle d sta g e of g erm lin g in F ig u r e 11 © = © . © © 50

14© Four - c e l le d s ta g e of g erm lin g in F ig u r e 11 © © ,© © © © 50

15© S ix - c e l le d s ta g e of g erm lin g in F ig u r e 11 © ©. © . © © © © 50

16© O n e -c e lle d g erm lin g w ith in fe m a le gametangium© © © ,© 51

17© T h r e e -c e l le d g erm lin g © © © ©. ». ©. © © © » » © ©. © © 51

v

v i

LIST OF ILLUS THAT IONS ■ - C ontinue d

F ig u re P age

18o . B a sa l portion of s e v e n - or e ig h t -c e l le d g erm lin g 0 o 51

19o - F o u r -c e lle d g e r m lin g » o o » o o o Q » o o o » o » 0 52

20o . Four - c e l le d germ lin g o o o ® - . o o » o •. o o ®. 0 o • 52

21o F o u r -c e lle d n atural g erm lin g o. o o . o o 0 o @ o o o 52

22o B a sa l p ortion of a 2 0 -c e l le d natural g erm lin g » o o o o 53

23o . M iddle p ortion of th e sa m e g erm lin g shown in F ig u r e 22 53

24o - A p ica l p ortion of the sa m e g erm lin g shown in F ig u r e s2 2 g 2 3 o e o o o o o o e o o o o b o o o o o o o o 53

25o F ourth c e l l of 2 0 -c e l le d g erm lin g shown in F ig u r e s 22,2 3, 24 O O O O O 0 - 0 o o 'o o o o o o o o o o o o © 5 4

260 F ifth c e l l of g erm lin g shown in F ig u r es 22, 23, 24 0 © © 54

27o E ighth c e l l and p art of the ninth c e l l of the g erm lin gshown in F ig u r e s 22, 23, 24 © o © © o o © © © o © © 54

28o . A p ica l c e l l of the germ lin g shown in F ig u r es 22, 23, 24 54

BIST O F TABLES

T able

1.

20' .

3=

. 4 0

5„

60

7o

8.

9o

P age

Q u alita tive d if fe re n c e s betw een the gen era Sp irogyra andSirogonium a s co m p iled from the lite r a tu r e <> « o » o . o 5

G eograp h ica l d istr ib u tio n of 14 s p e c ie s of S iro g o n iu m 0 0 o 7

A v e ra g e m axim um , m in im um , and m ean te m p era tu res (in d e g r e e s F a h ren h eit) for the Sonoran D e se r t sta tio n at G uaym as,, S o n ., M exico from 192 3 -1 9 3 8 < , . . < > o . „ 17

A v era g e m onthly, s ea so n a l, and annual r a in fa ll at G uaym as,S o n . , M exico , in in c h e s . . . . . . . . . . . . . . o 17

A v era g e sea so n a l and annual r a in fa ll in e a s t and w e s tUttar P rad esh ,. India, in in c h e s „ « . . = o . . . . . 18

A v e ra g e m onthly, sea so n a l and annual r a in fa ll at G reen ­v i l le , M iss is s ip p i, in in c h e s . . . . . . . . . . . . . . . 18

D im en sio n s of cu ltu ra l z y g o sp o re s 2 hr a fter rew ettin g . . 34

C ell len g th s, a v e ra g e c e l l len gth s, and g r e a te s t w idths at s u c c e s s iv e s ta g e s of grow th of a cu ltu ra l gerrrding of

. S irogonium m elan osp oru m . . . . . . . . . . . . . . 45

S e le c te d c e l l len g th s, a v e ra g e c e l l length s in clu d in g a ll d e le ted c e l l le n g th s ,. and g r e a te s t w idths at s u c c e s s iv e s ta g e s of grow th of a natural germ ling of S irogon ium m elan osp oru m . . . - ., . . . . . . . . . . . . . . . 46

v ii

ABSTRAC T

Two h o m o th a llic c lo n e s of S irogonium m elan osp oru m (Randhawa)

T ra n sea u (1944) w e r e is o la te d from a s ite 7» 5 m ile s (120 I km ) w e st

n orth w est o f Guay m a s, Sonora, M ex ico 0

The ob ject of th is in v e stig a tio n w as to d e v ise a m eth od by

w hich the co m p le te life c y c le of th is Z ygn em atacean a lga cou ld be

fo llow ed , and to o b se r v e and r e c o r d th e accom panyin g ev en ts of th is

c y c le e S p ec ia l em p h a sis w as g iven to the p ro b lem s co n cern ed w ith th e

d iso rg a n iza tio n and reo r g a n iz a tio n of the chi or op la s ts during g a m eto -

g e n e s is and e a r ly g erm lin g develop m en t, r e s p e c t iv e ly , and w ith the

germ in ation of the z y g o sp o re .

A llow ing the f ila m e n t-n e ts of S irogonium m elan osp oru m , c o n ­

tain ing m atu re z y g o sp o re s , to dry s lo w ly at room tem p era tu re and

rem a in dry for two to four w eek s b e fo re w etting w ith supernatant from

T h in gsh eim s o il-w a te r m edium and p la c in g them under f lu o r e sc e n t

lig h tin g .o f about 3, 200 lux, p ro v ed to be a s e ffe c t iv e for inducing

g erm in ation as any of the s e v e r a l tech n iq u es tr ie d . U sing the p r e c e d ­

ing m eth od only lim ite d germ in ation of the z y g o sp o re s from cu ltu re

w a s obtained .

. F ra g m en ta tion of the c h lo r o p la s ts o ccu rs in both the m a le and

fe m a le gam etangia during g a m e to g e n e s is and the ch lor o p la sts rem a in

v i i i

in fra g m en ts throughout g a m etic union3 z y g o sp o r ic m aturation^ and in ­

to the e a r ly s ta g e s of the developm ent of the germ lingo The f ir s t

sta g e s of c h io r o p la s tic r eo rg a n iza tio n a r e f in g e r - lik e p ro jec tio n s of

ch lo r o p la stic m a te r ia l extending from both s id e s of a c en tr a l band of

c h io r o p la stic fragm entso A s reo rg a n iza tio n con tin u es, th e cen tra l band

d im in ish es in s iz e and th e fin ger - lik e p ro jec tio n s b eco m e the stra ig h t

r ib b on lik e c h io r o p ia s ts of the m atu re v e g e ta tiv e c e l l of S irogon iu m 0

The a fo rem en tio n ed p r o c e s s e s occu r co n cu rren tly w ith c e ll d iv isio n up

to about the th r e e - to s ix - c e l le d sta g e of the develop m en t of the germ -

lingo The a p ica l c e l l r e ta in s d iso rg a n ized c h io r o p la stic m a ter ia l until

a m uch la ter s ta g e in the grow th of the germ lingo

INTRODUCTION A ND REVIEW OF LITERATURE

The genus S irogonium K uetzing (1843) nom=. c o n s , i s one of the

13 gen era p r e se n t ly in c lu d ed in th e fa m ily Z y g n em aceae (Randhawa,

I9 5 9 )9 or, a s i t i s known in the U nited S ta te s , the fa m ily Z ygn em ataceae

(T ran seau , I9 5 1 )0 T his sp e llin g d iffe re n c e i s a lso r e f le c te d in the nam e

of the ord er, Z y g n em a les (Randhawa, 1959), and Z ygn em a ta les (Sm ith,

1933),, In a cco rd a n ce w ith S ilv a (1962);the sp e llin g e s ta b lish e d by Sm ith

(1933) w ill be u sed throughout th is m a n u scr ip t. . The m e m b ers of th is

order can be d istin g u ish ed from a ll other g reen a lg a e by th eir la ck of

fr e e sw im m in g, f la g e lla te d g a m etes and sp o res; sex u a l rep rod u ction i s

a cco m p lish ed by the m ovem en t of am oeb oid g a m etes through, or w ith in ,

a tube, p e c tic sheath , or an envelop in g p e c tic m a s s (T ra n sea u , 1951)»

No. W ille in 1897, acco rd in g to Randhawa (1959), c la s s i f i e d the green

a lg a e into two m ajor grou p s, C onjugatae an d .C h lorop h yceae, b eca u se

of the v e ry d is t in c t iv e m eth od of rep rod u ction , conjugation , in the

fo r m e r . The C onjugatae, h ow ever, h ave as m uch in com m on w ith other

o rd ers in the C h loroph yceae a s th e s e o r d e r s have w ith one another.

T h ere fo re the C onjugatae a r e p r e se n tly c o n s id ered a s on ly an order

w ith in the la r g er group, C h lorop h yceae, by m any p h y c o lo g is ts (F r itsc h ,

1935; Sm ith, 1950; B old, 1957; S ilva , 1962). F . F . B lackm an and A . G.

T a n sley in 1902, a ccord in g to Randhawa (1959), u sed the term Akbntae

in r e fe r r in g to the Z y g n em a ta les b eca u se of the a b se n c e of f la g e lla on

the g a m e te s . A nother com m on nam e for th is ord er , u sed by F r its c h

a s la te as 1935, i s C onju gates.

The m em b ers of the fa m ily Z y g n em ataceae a r e m o re or l e s s

p erm an en tly fila m en to u s , u su a lly unbranched and la ck p o r e s in th eir

w a lls of c e l lu lo s e (Sm ith, 1950; T ran seau , 1951; Randhawa, 1959).

T h ese c h a r a c ter s d is tin g u ish th is fa m ily from the other two fa m ilie s .

M eso ta en ia cea e and D e sm id ia c ea e , in th is o rd er . M ost m em b ers of the

Z ygn em ataceae have a p e c tic la y er o u tsid e the c e llu lo s ic c e l l w a ll w hich

m ak es them s lim y or s lip p ery ; S irogon iu m , how ever, la ck s th is outer

la y er of p e c to se (W est and F r its c h , 1927; Sm ith, 1950; T ran seau , 1951;

: Randhawa, 1959). • Sm ith (1950) s ta ted that the c e l l w a lls w ere c y lin d r i­

ca l in the Z ygn em ataceae but T ran seau (1951) w as m o re sp e c if ic and

sta ted that c y lin d r ic a l c e l l s o ccu rr e d on ly during v e g e ta tiv e growth,

w h erea s rep ro d u ctiv e s tr u c tu r es m ay be quite v a r ia b le in s iz e and fo r m .

C onjugation i s u su a lly through a tube, but th is i s not the c a se in the

genus S iroc lad iu m nor i s it sa id to be the c a se in the genus, S irogonium

(Randhawa, 1959)° - C onjugating c e l l s in the Z y g n em ataceae do not have

th e ir p ro top lasts, e sca p in g from the e n c lo s in g w all during gam etic union

(Sm ith, 1950), and the z y g o sp o r e s a r e fo rm ed e ith er in th e conjugation

tube or w ith in one of the gam etan g ia (W est and F r its c h ,. 1927; Randhawa,

1959). - A ccord in g to Randhawa (1959) the c h lo ro p la sts can be c la s s i f ie d

into one of four g en era l ty p es , (1) a s in g le a x ile p late,, (2) two p a r ie ta l

p la te s , (3) one to s e v e r a l s te lla te s tr u c tu r es and (4) one to m any sp ir a l

rib b on s. The genera. S irogonium and S p irogyra have the fourth type of

ch lo r o p la s tic s tru c tu re .

In a p u b lica tion tit led P h y co lo g ia G en era lis , K uetzing (1843)

e r e c te d the genus Sir ogonium basing h is b r ie f d escr ip tio n upon a s in g le ,

s t i l l va lid , and the on ly p r e se n tly r ep o rted E uropean sp e c ie s ,. Sir ogonium

Istic ticu m (E ngl, B ot, ) K uetzing, K u etzin g‘s d e scr ip tio n a s g iven by

2. B ew is (1925) i s a s fo llo w s; "S irogonium K uetz, 1843; C e llu la e v e g e -

ta tivae c y lin d r ic a e , sp o r ife r a e su b in fla tae o r c u lifo r m e s . F a s c ia e

chlorophylloSoie lo n g itu d in a les , p a r ie ta le s , le v ite r f le x u o sa e , nod osae

(p lerum que 2 -3 , r a r iu s 4 in quaque ce llu la ), granula a m y la cea 7 -8

in vo lu ta e , C opulatio gen u flexa , s in e tubo con n ex ivo , " It i s u n d erstan d ­

able, w ith such a b r ie f and g en era l d escr ip tio n , that the genus S irogonium

h as not been a ccep ted a s d istin ct from the genus S p irogyra by m any

p h y c o lo g is ts . The l i s t of co m p a r iso n s b etw eem S p irogyra and S irogonium

1, , R e fe r s to S ow erb y 's (J a m es B eC a rl) E n g lish B otany; or, . coloured. f ig u r e s of B r it ish P la n ts , w ith th e ir e s s e n t ia l c h a r a c te r s , syn on ym s, and p la c e s o f growth; to w hich w ill be added, o cca s io n a l r em a rk s by J am es E dw ard Sm ith , V o l, 35 (1813) of 36 V o ls , p, 1463 and the fa c in g c o lo r e d p la te . W hite and. Co, London, 1 7 9 0 -1 8 1 4 ,

2, L ew is (1925) has the fo llow in g footnote; "G rayJs (1821) d escr ip tio n of C h oasp is an ted ates K u etz in g ^ by 22 y e a r s , G rayl s d ia g n o sis i s a s fo llo w s; ‘C hoappis, T hallu s th read lik e , s im p le , tubu­la r , jo in ted , kneebent; coupling a t the bend, by a p er fo ra tio n in each jo in t, w hich tr a n sm its the gra n u les from one p lant to th e o th er, w h ere th ey form an e llip t ic spore; g ra n u les sc a tte r e d , - -N o t s l ip p e r y ,1"

4

(T ab le 1} i s co m p iled from De B ary (I8 5 8 )s L ew is (1925), F r its c h

(1935), Sm ith (195% T ran seau (1951 and Randhawa (1959)= The f ir s t

th ree c h a r a c te r s , . m orp h o lo g ica l an iso g a m y , sc a tte r e d conjugation , and

the a b sen ce of a p e c tic sheath , a r e probab ly the m o st r e lia b le in d i s ­

ting m shin g Sixogonium from S p iro g y ra . . N one of th e se c h a r a c te r s i s

m en tion ed s p e c if ic a lly in K u etzin g 's d escr ip tio n of the genus S irogonium

and only the th ird , a b sen ce of a p e c tic sheath , i s g iv e n .in G ray's

d e scr ip tio n of the genus C h oasp is w h ere he sta ted that the plant body i s

"not s lip p er y . "

T ra n sea u (1951) had the fo llow in g to sa y about the p r o c e s s of

conjugation in S irogon iu m ;

The d evelop m en t of th e g am etan g ia tak es p la c e on ly from c er ta in v e g e ta tiv e c e l l s sc a tte r e d s in g ly or in .p a ir s a long the f ila m e n ts . U su a lly the. progam etan g ia d iv id e in to two unequal gam etangia - - one sh ort and one long - -a n d food su b sta n ces accu m u la te in th em . . T h ere m ay be two d iv is io n s r e su lt in g in a la r g er gam etangium betw een two sh ort c e l l s . . D e B ary s ta ted that the f ir s t type of d iv is io n r e s u lte d in fe m a le and the la tter in m a le g a m etes; he c a lle d the sh o rt c e l l s " s te r ile . " H ow ever, the d evelop m en t of gam etan g ia i s h igh ly v a r ia b le in so m e c o l le c ­tio n s . A pp arently p roga m eta n g ia m ay conjugate w ithout d iv is io n . A ny of the sh ort c e l l s m ay b ecom e m a le gam etan gia and d iv isio n in to th re e c e l l s b e fo re conjugation i s far le s s freq u en t than d iv i­sion in to two unequal c e l l s . . A s a r e s u lt of the fle x in g of the gam etan g ia at the beginning of a conjugation, s u c c e s s iv e conju­gation s in a p a rticu la r fila m en t a r e each w ith a d ifferen t f i l a ­m ent. C onjugated f ila m en ts thus form a tan g led n et.

The actu a l m ovem en t of the m a le g am ete through the p ore

betw een the gam etangia has,, ev id en tly , not been ob served ; at le a s t i t

has not been rep o rted in the lite r a tu r e . The next d e sc r ip t iv e m a te r ia l

5

T able 1. . Q u alita tive d iffe re n c e s betw een the g en era S p irogyra and Sirogonium a s co m p iled from the lite r a tu r e .

S p irogyra S irogonium

l . y M orp h olog ica l iso g a m y i s 1exh ib ited by m o st sp ecies*

2* In sca la r if or m conjugation it 2=is com m on for long se c tio n s of the two fila m e n ts to have n ea r ly ev er y c o n sec u tiv e c e l l conjugating*

3* A m u cou s or p e c tic sheath i s 3*present*

4* L a tera l co n ju g a tio n .is com m on 4*in m any sp ec ies*

5* G enuflection of th e f ila m en ts 5*at th e t im e of conjugation i s uncommon*

6* The ch lo r o p la s t(s ) i s (are) . 6* u su a lly sp ir a lly arranged*

7* P rogam etan g ia a r e not p r o - 7*duced p r ior to the fo rm a tio n of the gam etangia*

8* '(S terile" c e l l s a re u su a llylacking*

9* T h ere i s a w e ll d evelop edconjugation tube present*

M orp h olog ica l a n iso g a m y is exh ib ited by a ll d e scr ib e d sp ecies*

S ca la r ifo rm conjugation o ccu rs s in g ly or o c c a s io n a lly in p a ir s s c a tte r e d along the f ila m e n ts .

The f ila m en ts la ck a m ucous or p ec tic sh eath .

No la te r a l conju gation h as been rep o rted for any sp ec ies*

G en u flection of th e f ila m en ts at the tim e of conju gation i s quite com m on.

The c h lo r o p la s ts a r e u su a lly stra ig h t or n ea r ly so*

P roga m eta n g ia a r e often p r o ­duced p rior to the form ation of gam etan g ia .

8* " Sterile" c e l l s a r e often found ad jacent to the gam etan gia .

9* A w e ll d eve lop ed conjugation tube i s u su a lly absen-tS ..s

on the life c y c le of S irogon ium m en tion ed by m o st au th ors i s co n cern ed

w ith the z y g o sp o re . The z y g o sp o r e s a r e u su a lly e ll ip s o id but so m e

sp o r es in any p a r ticu la r c o lle c t io n a re a ls o ovoid a cco rd in g to T ra n sea u

(I951)o . Randhawa (1959) l is te d o n esp p ec ies S. ph acosporum with, le n t ic u ­

lar z y g o sp o r e s . . In a ll but two of the 15 r e c o r d e d s p e c ie s of S irogon ium ,

the sp o re w a ll is co m p o sed of th ree la y e r s but in S. hui and S.

v a n d a lu ren sis th e m edian w a ll i s d iv id ed into two la y e r s . It i s the

m edian w a ll w hich con ta in s the p igm en t and m ay be sm ooth or exh ib it

the c h a r a c te r is t ic orn am en tation . Both of th e se c h a r a c te r s a re im p o r ­

tant in defin ing s p e c ie s in : S irogon ium (T ran s eau, 1951). The co lor of

the m edian w a ll v a r ie s from y e llo w to brown or b lack .

The h igh ly v a r ia b le j). s t ic tic u m i s the m o st w id e ly d istr ib u ted

s p e c ie s of th e gen u s, being found in ! E u rop e , A s ia , A fr ic a , A u stra lia ,

South A m e r ica , Canada, and the U nited S ta te s . T able 2 i s co m p iled

fro m T ra n sea u (1951 )j Randhawa (1959), and r e c o r d s of c o lle c t io n s by

m em b ers of the B otany D ep artm en t at the U n iv ersity of A r iz o n a . It

su m m a r iz e s the d istr ib u tion of the other 14 s p e c ie s of S irogon ium .

T his ta b le i l lu s tr a te s the w id e d istr ib u tion of the gen u s S irogon ium but

i t a lso r e f le c t s the fa c t that th is genus has been found e x te n s iv e ly only

in th o se reg io n s w hich h ave been m o st thoroughly sea r c h e d . . It is quite

prob ab le that th is genus is m uch m o r e com m on than th e p r e se n t, known

d istr ib u tion w ould in d ica te .

7

T able 2<. G eograp h ica l d istr ib u tio n of 14 s p e c ie s of S irogon iu m ,

South N orthS p ec ie s A m e r ica A m e r ica A fr ica A s ia

cey lan icu m C eylon; Bom bay, & Madhya P „ , India

floridanum sou th w estF lo r id a

C apetown So A fr ica

hui K iangsi,. China

il l in o ie n s e L erna , 111,; e a ste r n O kla3

indicum Gorakhpur, Uttar P rad esh , India

in f latum

m egasp oru m near Quito, B astrop , Tex= E cuador

P ettad , north Gujerat,. India

Szechw an,China

m elan osp oru m G reen v ille ,M is So; near Guay m a s, Son. M exico

F yzab ad (F a iz a - bad), Uttar P r a ­d esh ,. India

phacosporum near M orelia , M ich, j M exico

near R angoon, B urm a

p seu d o - florid anum

V ila s county, W isco n sin

r e t ie ulatum K arnal D is tr ic t , Eo Punjab, India

tenu ius Cordoba, . O kla», T ex a s , A rgentina; A rk«, & F la . Mato G rosso,B ra z il

8

T able 2 --C o n tin u ed

SouthS p ec ie s A m e r ic a

N orth Am e r ic a A fr ic a A s ia

v a n d a lu ren sis V andalur, M ad­r a s State, India

v en ter sicu m V enter s - dorp, Trans v a a l, . South A fr ic a

Gorakhpur,- Uttar P. &

Ho shangabad M adhya P . , India

In O ctober of 1961 Bra. R ob ert Hoshaw of the B otany D ep a rt­

m ent, U n iv ers ity of A r izo n a , c o lle c te d an a lga near G uaym as, Sonora,

M exico , w hich he la ter is o la te d in to c lo n a l cu ltu re . Two of th ese c lo n a l

cu ltu res su rv iv ed and m a te r ia l w as sen t to D r.-C o E„ Taft at Ohio

State U niver s ity . He id e n tif ie d th is a lga a s S irogonium m elan osp oru m

(Randhawa) T ran seau (1944}0 Randhawa (1938) c o n s id e re d th is org a n ism

to be only a su b sp e c ie s of S. v en ter sicu m but T ra n sea u (1944) r a is e d it

to fu ll sp e c if ic standing on the b a s is of i t s o v e r -a ll la r g er d im en sio n s

and it s darker c o lo r e d (b lack) z y g o sp o r e s . Randhawa (1959) a ccep ted

th is change.

Both T ra n sea u (1951) and Randhawa (1959) g iv e m uch the sa m e

d e scr ip tio n of th is s p e c ie s of S irogon iu m . S. m elan o sporum h as v e g e ­

ta tiv e c e l l s from 7 0 -9 0 m ic r o n s in w idth and from 1 4 0 -2 6 0 m icro n s

long, each contain ing from 6 -9 n ea r ly stra ig h t c h lo r o p la s ts . The c e l l s

a re sa id to conjugate d irec tly , i , e„ w ithout form ation of a conjugation

tube, and the gam etan g ia in fla te up to 12 0 -1 6 6 m ic r o n s . The m a le

g a m ete m o v e s through a p ore in the tube to fu se w ith the fe m a le g a m ete .

The young zygote d ev e lo p s in to a zy g o sp o re w hich has a brown to b lack

and v e r r u c o s e m ed ian sp o re w a ll. The m atu re zy g o sp o re i s u su a lly

e llip so id and 9 0 to 110 m ic r o n s w ide by 140 to 160 m ic r o n s long.

P r io r to the p r e se n t c o lle c t io n of S. m elan osp oru m from

M exico th is s p e c ie s had been rep o rted only from the r eg io n around

Fyzabad, Uttar P ra d esh , India, and near G reen v ille , M is s is s ip p i. In

the Indian habitat at the c lo s e of the sou th w est m o n so o n -b o rn e ra in s ,

th is a lga b eco m es abundant and p ro d u ces m atu re z y g o sp o r e s at the end

of Septem ber and duriiig the d r ier m onths of O ctober and N ovem ber

(Randhawa, 1959).

Som e authors have sta ted that it i s d ifficu lt or im p o ss ib le to

m ain ta in m em b ers of the Z ygn am ataceae in the la b o ra to ry (C ham berla in ,

1932; Johansen, 1940; B old, 1942) but s in c e the advent of P r in g sh e im 1 s

so il-w a te r m edium (P r in g sh e im , 1946) and i t s m o d ifica tio n by Starr

(1964) the cu ltu rin g of m any kinds of a lg a e has been g r e a t ly fa c ilita ted .

It w as the u se of th is m edium that en ab led A llen .(1958) to m ain ta in her

cu ltu res of S p irogyra for m any m onths during her study of a s p e c ie s

co m p lex in that gen u s. M any kinds of a lg a e , including m em b ers of the

Z ygn em ataceae, a r e m ain ta in ed co n tin u ou sly at the Indiana C ulture

C o llectio n of A lgae (S tarr, 1964).

10

■ A lthough it i s now m uch e a s ie r to grow a lg a e v e g e ta t iv e ly 9 it

i s s t i l l d ifficu lt to in d u ce th eir sex u a l c y c le for la b o ra to ry study. . One

of the le a s t un d erstood a sp e c ts of the l i f e c y c le of the Z y g n em a ta cea e

c en te r s around the zy g o sp o re , i t s v ia b ility and g erm in a tio n . A llen

(1958) had only lim ite d s u c c e s s in g erm in a tin g the z y g o sp o r e s of

s e v e r a l s p e c ie s of S p irogyra . T ron dle (1907) m ade e x te n s iv e stu d ies

of conjugation and g erm in ation in S p iro g y ra . He found that the z y g o ­

sp o r es of S. n eg lec ta en tered a " sta te of r e s t" andggerm inated only

after a p e r io d of about two m on th s. A cco rd in g to A llen (1958), H.

K lebahn in 1891 g erm in a ted z y g o sp o re s of two s p e c ie s of S p irogyra and

one of Z ygnem a after a llow in g them to overw in ter in the d ried condition ,

and g erm in a tio n of Z ygnem a s te llin u m w a s a cco m p lish ed by K urssanow

in 1 9 1 2 .by the add ition of f r e s h p eat w ater about one m onth after z y g o ­

sp ore fo rm a tio n . A cco rd in g to He B ary (1858) the g erm in a tio n of

Sirogonium z y g o sp o r e s (S. s t ic t ic u m ).w as f ir s t o b se rv e d in 1855 by

W ichura who sta ted that th eir g erm in a tio n w as c o m p le te ly analogou s to

the g erm in ation of the z y g o sp o r e s of S p iro g y ra . . He B a ry (1858) c o lle c te d

m atu re z y g o sp o re s and liv e f ila m en ts of S irogonium s tic tic u m in ea r ly

June, "cu ltured them in a room " and o b se rv e d the f ir s t g erm in a tio n s

in ea r ly O ctober, a p er io d of about four m onths a fter c o lle c t io n . . It i s

not c lea r w hether or not the cu ltu re w as kept in liquid m ediu m from the

tim e of c o lle c t io n through the tim e of g erm in a tio n of the z y g o sp o r e s .

11

M ore e x te n s iv e in form ation i s known about th e sex u a l cy b le s

of C hlam ydom onas (S tarr, 1949; L ew in , 1949, 1957; L ev in e and E b e r s -

old, 1958;: E ber so ld and L ev in e , 1959; L ev in e and F e ls o m e , . 1959;

G ow ans, I960 ),. C osm ariu m (S tarr, 1955, 1959), and Gonium (Stein,

1958)o . E rb en (1962) su m m a rized the p r e se n t sta te of kn ow ledge about

a lga l sp oru la tion and g erm in a tio n w ith the fo llo w in g w ord s: "By c o m ­

p a r iso n w ith the e x te n s iv e s tu d ies of the sp o r e s of fungi or b acter ia ,

h ow ever, th ere have been v e r y few c r it ic a l in v e s t ig a tio n s of the p h y s ic a l

and b io ch em ica l fa c to r s in v o lv ed in a lg a l sp oru la tion and germ in a tio n . "

T h ere a r e v e r y few r e fe r e n c e s in the lite r a tu r e w hich r e la te

the r o le p la y ed by the c h lo r o p la s t during th e life c y c le of any .m em ber

of the Z ygnem ataceae= . D e B ary (1858), Y ork ,(I913), and o th ers have

noted that th ere i s an accu m u lation of s ta r ch a s so c ia te d w ith the c h lo r o ­

p la s t p r io r to the p r o c e s s of conju gation . F r its c h (1935) sta ted that V.

C h m ieiev a sk y in 1890, and T rbndle in 1907 o b serv ed that the ch lo ro p la st

of the m a le gam ete , in S p irogyra , d is in teg r a te d at an e a r |y s ta g e .

A lthough De B ary (1858) im p lie d that the c h lo r o p la s ts of S irogonium

s tic ticu m w e re in. "shred s" which' " slow ly s tre tch out" during g erm lin g

grow th, he il lu s tr a te d a o n e -c e lle d g erm lin g w ith c h lo r o p la s ts fu lly

, d eve lop ed . A lle n 's (1958) figu res, a lso show fu lly d ev e lo p ed ch lo r o p la s ts

in o n e -c e lle d germ lin g s of S p iro g y ra . - A gain th is i s the c a s e w ith an

illu s tr a t io n by L ew is (1925) of h is T em n ogyra c o l l in s i i w h ich i s

12

c o n s id e re d by F r its c h (1935) and T ra n sea u (1951) to be only a s p e c ie s

of Spirogyrao

The p r e se n t study w as undertaken to e lu c id a te d evelop m en t

p rior to, w ith in , and im m ed ia te ly fo llo w in g the zy g o sp o re in

Sirogonium m ela n o sp o ru m .

M ATERIALS A ND METHODS

A d escr ip tio n of the c o lle c t io n s ite , the g en era l en v iron m en ta l

con d ition s and tech n iq u es of cu ltu rin g , and the sp e c if ic apparatus and

m eth ods of in v e stig a tio n a r e g iven below*

. C o llec tio n S ite

The o r ig in a l c lo n e s of S irogon ium m elan osp oru m u sed in th is

study w e re is o la te d by Dr * R obert W, Ho s haw from m a te r ia l c o lle c te d

on 18 O ct.T 96l from a sm a ll pond w h o se in term itta n ce i s d eterm in ed by

the ir r e g u la r su m m er and w inter ra in s o ccu rr in g in th is region* The

pond, w hich i s 7*5 m ile s (12* 1 km ) w e st n orth w est of G uaym as,. Sonora

M exico i s lo ca ted on the seaw ard s id e of the road lead in g from M exico

highw ay no. 15 to the r e s o r t a rea of San C a r lo s Bay about 3 m ile s (4 . 8

km ) w e st of the pond. . The pond i s 4 . 7 m ile s (7 . 6 km ) w e st of the tu rn ­

off to San C arlos B ay (Map, F ig . 2). . It i s only 150 to 250 y d s . south

and over the beach dunes to the sa lty w a te rs of the Gulf o f C a liforn ia

but the pond i t s e l f i s f i l le d w ith fr e s h w ater during the ra in y sea so n .

F ig u re 1 show s the c o lle c t io n s ite . The so il at th is s ite i s a fin e

tex tu red sandy c la y . The photograph, taken in. A p ril of 1963 p r io r to

the sum m er r a in s , show s the dry pond bottom . The car in the b ack ­

ground i s p ark ed on the r oad to S a n C a r lo s Bay w hich i s to the le ft (w es

F ig u re 1. . C o llec tio n s ite .

14

Fig. 1

Fig. 2

M AP OF COLLECTION SITE

to hermosillo 7 9 mi.

i ® " # fAmmC°15

ensenada san francisco

California

4

. 16

The a ir tem p era tu res p rev a ilin g at G uaym as a r e g iven in

Table' 3= The annual a v era g e p r ec ip ita tio n at G uaym as, by m onths and

s e a so n s a s co m p iled from the A m e r ica n A utom obile A ss o c ia t io n (1963)

i s g iven in T able 4„ • A footnote sta ted that the f ig u r e s r e p r e se n te d an

a v era g e over at le a s t a 5 -y e a r p er io d but the dates of th is p er io d w e re

not g iv en . The G uaym as reg io n i s a c o a sta l d e se r t w ith ra in s p r im a r ily

in the sum m er but th ese a r e quite sp orad ic and long p e r io d s of drought

la stin g through s e v e r a l s e a so n s a re not uncom m on (S h reve, 1964).

. The sea so n a l ra in fa ll in the other two h ab ita ts w h ere S irogonium

m ela h o sp o ru m .is known to occu r, i s g iven in T ab les 5 and 6 for c o m ­

p a r iso n and d is c u s s io n in a la ter sec tio n of th is m a n u scr ip t. T h ese

habitats a re Uttar P ra d esh , India (T ab le 5) and G reen v ille ,. M is s is s ip p i

(T able 6).

- Iso la tio n

With the u se of fin e g la s s p ip e tte s , p ie c e s of r e la t iv e ly c lea n

f ila m en ts about 4 or 5 c e l l s long w e re placed into w atch g la s s e s w hich

w e re f i l le d w ith supernatan t from ste a m ed so ii-w a te r m edium or s t e r i le

w a ter . The w atch g la s s e s w e re supported by tr ia n g le s of bent g la s s

tubing in p e tr i d ish e s . A ll the g la s sw a r e w as a u to c la v ed at 15 lb s .

p r e ssu r e for 15 m in . p r io r to u se . • A fter s e v e r a l tr a n s fe r s through

fr e s h liq u id contained in c lea n w atch g la s s e s and a g ita tio n to fu rth er

c lea n the f ila m e n ts , ea ch p ie c e w as f in a lly tr a n s fe r r e d to a sep a ra te

T able 3° A v era g e m axim um , m inim um , and m ean tem p era tu res in d eg rees F ah ren h eit for the Sonoran D e se r t sta tion at G uaym as, Son=, M exico from 1 9 2 3 -I9 3 8 0 C om piled from

. Sh reve (1964 )o

___________________ Jan* . Febo M ar». A p ro . May Jun.. Juh A ug. . Sep* . O ct. Novo . D ec. Y ear

M axim um 72 70 75 76 82 87 92 88 87 84 76 70 7 9 .5

M inim um 60 63 66 70 75 83 85 84 84 79 70 63 7 3 ,4

M ean 6 3 ,9 6.6, 0 69. 3 73, 0 7 7 ,9 83. 8 - 8 7 , 3 8 6 ,9 8 6 ,4 81, 1 72, 7 65, 5 7 6 ,1

T able 4 . A v era g e m onthly, s ea so n a l and annual ra in fa ll at G uaym as, .S o n ,, M exico in in c h e s .

D ec, Jan. F eb . M ar. A pr, . May Jun.11

Sep. O ct. Nov. T otal

1 .1 0 ,3 0 .2 0. 2 0. 1 0. 1 o o 1 .8 3 .0 2 .1 0 ,4 0 .4 9 .7

D ec. -F eb , - . M ar, -M ay Jun. -Sep , . O ct. -N ov.

1 .6 0 .4 6 .9

00 O -

o

T able 5 a A v era g e sea so n a l and annual ra in fa ll in g ast and w e st Uttar P rad esh , India in in ch es (Randhawa, 1959)°

Dec° -Febo Mar° -M ay Jun. -Sep° O ct. -N ov . T otal

Uttar P rad esh , e a st I , 53 I , 12 34. 44 2 .0 4 39° 13

Uttar P rad esh , w e st 2 .2 7 I ° 36 3 2 .9 8 0.97. 3 7 .5 8

T able 6. . A v era g e m onthly, sea so n a l and annual ra in fa ll at G reen v ille , (Uo, S.. D ept, of C om m erce , 1964)°

M is s is s ip p i in in ch es

D ec. . Jan. F eb . M ar. A pr. May Jun. . Jul. Aug. . Sep. O ct. Nov. T otal

5 .2 6 .4 5 .2 5. 7 5 .4 4. 0 3. 2 4 .4 2 .6 3. 2 2. 4 4. 6 52. 55

D ec. -F eb . , Mar . -M ay Jun. -Sep . O ct. -N ov .

. 1 6 .9 0 15,, 14 1 3 .4 9 7 .0 2

oo

19

s o il-w a te r tube (P rin gsh eirn , 1946)0 The tubes w ere p la ced in a .r a c k

in a con stan t tem p era tu re grow th cab in etc . Growth w as o b serv ed in

so m e of the tubes a fter s e v e r a l w eek s and from th e se tu b es the c lo n e s

w ere tr a n s fe r r e d to so il-w a te r bottles<> Only two c lo n e s , num ber 3 and

num ber 6, su rv iv ed in the s o il-w a te r b o ttle s , and th e se two c lo n es p r o ­

v id ed the so u r ce of a ll of the ex p er im en ta l m a te r ia l m a in ta in ed in cu ltu re .

M edia

The 18 x 150 m m p so il-w a te r te s t tubes w e r e p rep a red by

co v er in g , a p inch of CaCO^ w ith about on e-fo u rth inch of Indiana or

T e n n e sse e garden s o il, f il lin g th e tube w ith a p p ro x im a te ly 15 ml, of

deionized^, d is t i l le d w a ter , cover in g w ith s ta in le s s s t e e l cu ltu re tube

c lo su re s , and stea m in g for one hour on each of two c o n se c u tiv e days^

fo llow in g a m eth od d e sc r ib e d by Starr (1964)= Stock c u ltu r e s w e re

grow n in so il-w a te r b o ttle s rather than tu b es. For th is p u rp ose so ft

g la s s , o n e -h a lf pint m ilk b o ttle s w e re p rep a red in a m anner s im ila r to

that u sed in prep arin g the tu b es but u su a lly w ithout the CaCOg and w ith

a p ro p o rtio n a te ly la r g er am ount of so il and w a ter , . The pH of the m edium

w as c lo s e to 7 b e fo re in n ocu la tion ,

- S o il from the c o lle c t io n s it e w a s tr ie d in p rep a r in g so il-w a te r

b o ttle s , but the r esu lt in g m edium w as too turbid for u se .

Two in o rg a n ic m ed ia , g iven below , w e re a lso tr ie d but th e two

c lo n es of S irogonium u sed in th is study grew s lo w ly in both of them and■ y

did not con ju gate .

20

■ C zurda m edium (Allen^ 1958)» - -T o 995 m l of d e io n ized d is t i l le d

w ater w e r e added I m l of the fo llow in g so lu tio n s; . 10% KNO^.; 1 %

K 2 H B 04; 1% M gS04; 0„ 5% C a S 0 4 and 1 m l of iro n E D T A ( E th y len e -

d ia m in e te tra a ce tio a c id te tra so d iu m sa lt) so lu tion w hich i s d e scr ib ed

below . The pH w as ad ju sted to 6 „ 8 u sin g 0= 01 N HCi and 0, 01 N KOH»

. G odw afd m edium (p erso n a l com m u n ication to A llen , 1958),,'--

To 973 m l of d e io n ized d is t i l le d w ater w e r e added I m l of each

of the fo llow in g so lu tio n s: 25% KNO 3 ; 8 % M gS 04j 2% C alN O g^; 0 * 27%

K2 S i 0 3 ; . 2. 8 % K 2 HPi0 4 ; 1% CaCC^ and 1 m l of EDTA so lu tion . The pH

w as about 6 . 8 and w as not adjusted .

The fo llow in g ir o n E D TA so lu tio n i s m o d ified s lig h tly from the

one g iven by Starr (1964)?

Iron ED TA so lu tio n . - - T o 500 m l of d e ion ized *-d istilled w ater

w e r e added 2 . 61 g of EDTA; 2. 49 g of F e S 0 4 6 7H -,0 and 27 m l of 1 N

KOH.

Eight

A ll m e a su r em e n ts of ligh t in ten s ity w e re m ade w ith a W eston

Illum ination M eter m od el 756 contain ing a quartz f i l t e r . T h ese light

m ea su r em ents w e r e m ad e at the le v e l of the b a se s of th e so il-w a te r

cu ltu re b o ttle s but b eca u se so m e ligh t w as a b sorb ed or r e f le c te d by the

b o ttle s and the f ila m en ts shaded one another, the a c tu a l am ount of ligh t

reach ing, any p a r ticu la r fila m en t v a r ie d co n sid era b ly .

21

Banks of 40-w att,, c o o l-w h ite f lu o r e sc e n t lig h ts w ere u sed for

illu m in a tio n and, depending on th e d is ta n ce of the cu ltu re b ottle from

the so u rce , the in ten sity v a r ie d from 2, 6 9 0 -6 , 430 lux„ . Two light

r e g im e s w e re used , one w ith 1 2 hr of lig h t fo llo w ed by 1 2 hr of dark

and the other w ith 1 6 hr of ligh t fo llo w ed by 8 hr of dark. The cu ltu res

a lso r e c e iv e d in d ir ec t daylight through the la b o ra to ry w indow w hich

len g th en ed the to ta l lig h t ex p o su re t im e . A nother lig h t v a r ia b le w as

in trod u ced by the ja n ito r s c lean in g the ro o m s at n ight and students

w orking la te . . E x cep t for the daylight the am ount of ligh t r e c e iv e d from

so u r c e s other than the bank of. f lu o r e sc e n t lam p s w as n e g lig ib le , and

for the p u rp o ses of th is study a s tr ic t lig h t r eg im e w as not n e c e s s a r y .

T em p erat ure

C u ltu res w e r e kept e ith er in a con stan t tem p era tu re cab in et

m ain ta in ed at 2242 C or in the la b o ra to ry w ith a v a r ia b le tem p era tu re

of from 21 to 25 C. T em p eratu re flu ctu a tio n s of 3 or 4 C o ccu rred a s

the ligh ts w ent on and off. The cu ltu res grew about eq u a lly as w e ll at

25 C a s th ey did at 21. C and perhaps even a l i t t le b etter at the higher

tem p era tu re .

C ulturing, T ra n sferr in g , and M anipulatory T echn iqu es

No attem p t w as m ade to e lim in a te the b a c te r ia l f lo r a from the

c lon a l c u ltu r e s . T h is study did not in v o lv e any b io ch em ica l exp erim en tation .

22

in w hich p u re c u ltu re s a r e e s s e n t ia l , though it i s a lw a y s d e s ir a b le to

w ork w ith -axen ic c u ltu re s i f p o s s ib le .

U sin g ,a fla m ed in n ocu latin g n eed le and s c i s s o r s , sm a ll p ie c e s

of f ila m en ts w e re tr a n s fe r r e d to fr e sh ly s tea m ed so il-w a te r b ottles

ev ery 3 to 6 w eek s thus m ain ta in in g the c lo n e s . The fila m en ts in the

b o ttle s , from w hich the tr a n s fe r s w e r e m ad e, w e re a llo w ed to continue

conjugating for one w eek or lon ger until m ature z y g o sp o r e s w ere p r o ­

duced or the w h ole net d ied w ithout producing z y g o sp o r e s . , In the la tter

c a s e the f ila m en ts w e r e d isca rd ed . In the fo rm er c a s e the n ets w ith

m atu re-look in g . z y g o sp o re s w e re rem o v ed .fro m the b o ttle s and p la c e d

in s te r i le p la s t ic p e tr i d ish e s and a llo w ed to dry at room tem p era tu re ,

after w hich th ey w e r e s to r ed in the dark for la ter g erm in a tio n e x p e r i­

m en ts . M atu re- looking z y g o sp o re s w e r e d eterm in ed by s iz e , shape,

and the nature of the in tern a l con ten ts a s v iew ed through th e sp o re w a lls .

To in d u ce zy g o sp o re g erm in a tio n so il-w a te r supernatant or

o c c a s io n a lly Godward m edium w as added to the d r ied fila m en ts in the

p e tr i d ish e s w hich w e re then p la ced under a bank of f lu o r e sc e n t lig h ts .

S ev era l other p r o c ed u r es w e r e u sed as fo llo w s: (1) the f ila m en ts w e re

h eated to 40 G for 48 to 72 hr in an oven p r io r to w ettin g , ( 2 ) the f i l a ­

m en ts a fter being flo o d ed w ith liqu id w e re then h eated to 40 C for about

24 hr, (3) the f ila m en ts w ere p la ced in a r e fr ig e r a to r at about 5 C for

24 hr p r io r to hydration , (4) the inundated fila m en ts w e r e p la c e d in the

dark for 72 hr b efo re being pla.ced under the lig h ts .

' . 23

N atu ra lly p rod u ced z y g o sp o r e s w e re d er iv ed from a so il s a m ­

p le , c a lle d h e r ea fter M ex 2 2 , c o lle c te d by the author on 19 Septem ber

1962 from the o r ig in a l c o lle c t io n s ite , near Guay m a s, w h ich w as at

that tim e a dry pond bottom (F ig .. 1)= T his sa m p le w as k ep t in a tigh tly

c o v er e d g la s s ja r at room tem p erature,, In subsequent s e c t io n s o f th is

m a n u scr ip t "natural" w ill r e fe r to m a te r ia l d er iv ed from the M ex 2 2

so il sa m p le and "cultural" w ill r e fe r to m a te r ia l d e r iv ed from the

clon a l la b o ra to ry c u ltu re s .

- S m all p in ch es of M ex 22 s o il w e r e w et w ith supernatant from

so il-w a te r in order to obtain natural g e r m lin g s for co m p a r iso n w ith

th o se p rod u ced in the la b o ra to ry .

The grow th of in d iv id u al g e r m lin g s w as fo llo w ed by tr a n s ­

ferr in g them to sep a ra te 1 0 0 x 2 0 m m g la s s p e tr i d ish e s to w hich w as

added fr e sh so il-w a te r supernatant e v ery 2 to 3 days in order to lim it

the grow th of b a c ter ia a s m uch a s p o s s ib le .

O b serv a tio n s of the v e g e ta tiv e p h a se , conjugation , form ation

of the zy g o sp o re , germ in ation , and grow th of the g e rm lin g w ere c o n ­

ducted w ith B ausch and.L om b s te re o zo o m d is se c t in g and binocular m ic r o ­

sco p es and an A m erica n O ptical m ic r o sc o p e w hich had an e sp e c ia lly

u sefu l 2 OX o b jec tiv e a s w e ll a s the stan d ard I OX and 43X o b jec tiv e s .

O b servation s at low m a g n ifica tio n w e r e p er fo rm ed in the o r ig in a l p e tr i

d ish es but for o b serv a tio n s req u ir in g 2 OX and 43X o b jec tiv e s th e m a te ­

r ia l w as tr a n s fe r r e d v ia g la s s c a p illa r y p ip ette s or d is s e c t in g n e e d le s

24

to m ic r o sc o p e s l id e s . U sing the 10X and 2 OX o b jec tiv e s o b serv a tio n s

w e re m ade and photographs taken w ith the object s im p ly im m e r se d in

an open drop of flu id thus p reven tin g any dam age to th e organ ism fro m

the p r e s s u r e of th e co v er g la s s . A nother m ethod em p lo y ed to p reven t

in jury to the l iv e m a te r ia l being photographed w as the u s e of sm a ll

p ie c e s of no. I co v er g la s s p la ced under the g la s s c o v e r .

Z y g o sp o res w e re sm a sh ed by p lac in g the cover g la s s d ir e c t ly

on the sp o re and applying p r e s s u r e w ith a d is se c tin g n eed le near one

end of the sp o r e .

A ll m e a su r em e n ts w e r e m ad e w ith a m ic r o m e ter d isc w hich

w as p la ced in one of the 15X o cu la rs and c a lib ra ted w ith a sta g e

m ic r o m e te r .

Z y g o sp o res and young g e rm lin g s w ere d is s e c te d out of the

e n c lo s in g gam etan g ia l w a ll using d is s e c t in g n e e d le s , suturing n e e d le s ,

and g la s s n e e d le s m ade by drawing out g la s s tubing in to c a p illa r y tu b es,

breaking, and fla m in g the end to form a bead.

, R ea g en ts '

R eagen ts u sed in th is study in c lu d e io d in e -p o ta ss iu m -io d id e

so lu tion , ruthenium red , p r o p io -ca r m in e , a c e to -c a r m in e , and ch ry so id in

Y.

The io d in e -p o ta ss iu m -io d id e so lu tion w as p rep a red as fo llo w s:

T hree g ra m s of p o ta ss iu m io d id e and 1 g of iod in e c r y s ta ls w e re d is so lv e d

25

in 300 m i of w a te r » T his so lu tion w as u sed to te s t for s ta r ch in the

v e g e ta tiv e p h ase , ga m eta n g ia l form ation , and in th e sm a sh ed z y g o sp o re .

. R uthenium red , p rep a red by d is so lv in g 0 .5 g in 500 m l of

w a ter , w a s u se d to te s t for th e p r e se n c e of p ec tic com pounds in th e

v e g e ta tiv e p h a se .*

Both p ro p io - and a c e to -c a r m in e w e re u sed to gain so m e in s ig h t

into ev en ts w hich o ccu rred w ith in the zy g o sp o re w h ere m e io s is is

a ssu m ed to o ccu r . T h ese r ea g e n ts w e re p rep a red as fo llo w s: O ne-

half gram of ca rm in e w as added to 1 0 0 m l of b o ilin g 50% p rop ion ic or

a c e tic a c id and the r esu lt in g so lu tio n f i l te r e d . Z y g o sp o res w ere p la ced

d ir e c tly in to a drop or two of the so lu tion and sm a sh ed a s p rev io u s ly

d escr ib ed .

C h ryso id in Y g iv e s a y e llow co lo r in the p r e se n c e of fa ts and

o ils and w as u sed to tr a c e th e se com pounds through the l ife c y c le of

Sirogonium m ela n o sp o ru m . . It w as p rep a red by adding 0. 5 g of the dye

to 500 m l of w a ter .

Photography

A ll photographs w e re taken on typ e A K odachrom e II film and

d evelop ed by Kodak, L os A n g e le s . . C olor n eg a tiv es w e r e m ade from

the s l id e s and the p r in ts w ere then m ad e from th e se n e g a tiv e s .

P h otographs of liv in g as w e ll a s sta in ed m a te r ia l w e r e m ade

w ith 11 OX,. 2 OX, and 43X ob jectives, and a I OX ocular using, a C arl Z e is s

A ttachm ent C am era for p h otom icrograp h y w ith b a sic body I, and a

P h otovolt m od el ZOO M p h o to m eter . L ight ex p o su res w e re m ade by

holding the se a r c h unit of the ph otom eter a g a in st the fo c u s in g e y e p ie c e

of the Z e is s a ttach m en t. In order to d e term in e the b e s t exp osu re

t im e s and ligh t in te n s it ie s a te s t r o ll w a s ex p osed . Two exp osu re

t im e s and ligh t m eter rea d in g s w e re used , I sec at a read in g of 7 and

1 /5 sec a t a read in g of 23. Both of th e se rea d in g s w e r e m ad e using

the g r e a te s t s e n s it iv ity of the ph otom eter and r e p r e s e n t I . 5 lux and

4 .9 lux, r e s p e c t iv e ly . The s lo w er tim e and low er ligh t in ten sity gave

b etter depth of f ie ld and w as u sed in m o st in s ta n c e s . L ight quantity

w as ad ju sted by opening or c lo s in g the ir i s diaphragm a s s o c ia te d w ith

the con d en ser len s of the m ic r o sc o p e .

. A ll l iv e m a te r ia l w hich w as photographed w as m ounted in

supernatant from so il-w a te r m ed iu m .

OBSERVATIONS AND RESU ETS

Sirogon ium m elan osp oru in i s a h om oth a llic o rg a n ism in w hich

clon a l cu ltu res conju gate to p rod u ce v ia b le z y g o sp o r e s .

V eg eta tiv e C ell and the C h lo ro p la sts

A ty p ica l m atu re v e g e ta tiv e c e l l of S» m elan osp oru m has 6 to

9 r ib b on lik e c h lo r o p la s ts each w ith m any sca tter e d p y ren o id s . The

c h lo r o p la s ts a r e stra ig h t or s lig h tly s p ir a lle d from one end of the c e l l

to the o th er . The p a r ie ta l c h lo r o p la s ts a r e fla t and h ave sm ooth ,

s lig h tly w avy, or h igh ly ir re g u la r m a r g in s . The p y ren o id s protrude

from both s id e s of th e f la t c h lo r o p la s ts (F ig . 25). The n u cleu s is

su sp en ded by stran d s of cy to p la sm tow ard the m id d le of the c y lin d r ic a l

c e ll and i s u su a lly le n s -sh a p e d (F ig s . 24, 26). The end w a lls a re p lan e

and t e s t s w ith ruthenium r e d in d ica te th e p r e se n c e of v e r y l it t le p e c tic

m a ter ia l excep t in th ese end w a lls . The d iam eter of th e c e l l s in the

two c lo n e s stu d ied a v era g ed about 76 'm ic r o n s , but so m e cu ltu res c o n ­

ta in ed fila m en ts of g r ea ter than 80 or l e s s than 71 m ic r o n s in d ia m eter .

The c e l l s on e ith er s id e of the gam etan g ia in F ig u re 3 a r e ty p ica l v e g e ­

ta tiv e c e l l s . The length of v e g e ta tiv e c e l l s i s h igh ly v a r ia b le and c e l l s

have been m e a su r ed w hich a re both sh o rter than the 140 m icro n s and

lon ger than the 260 m ic r o n s g iven by T ra n sea u (1951) a s the l im its for

27

28

c e l l length s of th is s p e c ie s . If, h ow ever, one ta k es the a v era g e of f iv e

or m o re co n sec u tiv e c e l l s th ey fa ll e a s i ly w ithin the 1 4 0 -2 6 0 m icro n

ra n ge . The c e l l to the le ft of the m a le gam etangium (F ig . 3) i s g rea ter

than 200 m icro n s in length . The c e l l s on e ith er s id e of the fem a le

gam etangium (F ig.- 3) m e a su r e about 130 m ic r o n s and il lu s tr a te the

fa c t that c e l l s m ay be sh orter than the d im en sio n s in d ica ted by T ra n sea u

(1951). Short c e l l s a r e not a lw ays a s s o c ia te d w ith s tr u c tu r es of r e p r o ­

duction; h ow ever, th ey a re often prod u cts of r e c e n t c e l l d iv is io n a s w ill

be shown im a la ter sec tio n of th is m a n u scr ip t dealin g w ith the d ev e lo p ­

m ent of the germ lin g .

. De B ary (1 858), w orking w ith S. s tic ticu m , ' o b se rv e d that the

c h lo r o p la sts m ay sp ir a l in e ith er d irec tio n and th is i s a ls o the c a se

w ith S irogonium m elan osp oru m (F ig . 3). . In fa c t the c h lo r o p la s ts in a

s in g le c e l l m ay sp ira l one d irectio n for a portion of the length of the

c e ll , then bend and l ie p a r a lle l w ith the w a lls of the c e l l or sp ira l in

the o p p osite d irec tio n for the rem a in d er of the d is ta n ce . Other c h lo r o ­

p la s ts m ay be only sh ort p ie c e s not extending the fu ll length of the c e l l .

T h ere is , in fact, c o n s id e ra b le v a r ia tio n from the ty p ica l c e l l d e scr ib e d

above. T his v a r ia tio n , though it o c c u r s in new ly tr a n s fe r r e d , a c t iv e ly

grow ing cu ltu re s , i s m uch m o re pronounced in older cu ltu res w hich

m ay have a lte r e d the grow th m ed iu m .

C y to k in esis

V eg eta tiv e c e l l d iv is io n in Sjrogonium m elan osp oru m is

app arently v e r y s im ila r to v e g e ta tiv e c e l l d iv isio n in S p irogyra as

d e scr ib e d by A llen (1 9 5 8 ) 0 An annular grow th w h ich fo r c e s the c h lo r o -

p la s ts aw ay from the c e l l w a ll i s the f ir s t in d ica tion of c e l l d iv is io n .

The ring b eco m es th ick er a s i t g row s in w ard and c o n s tr ic ts the c h lo r o -

plastso The c h lo r o p la s ts extend through the opening in the in co m p le ted

septum a s show n in F ig u re 10= The c h lo r o p la s ts a r e fin a lly cut in two

by the advancing sep tu m . . The new tr a n s v e r s e w a ll ap p ea rs to be n e a r ly

co m p le te by the tim e the septum is c lo s e d .

One c e l l d iv is io n in a g erm lin g from M ex 22 s o il w as o b serv ed

ju st a s the septum w as beginning to fo r m . D uring continued o b se r v a ­

tion th e annular ingrow th of the septum p ro ceed ed up to th e point shown

in F ig u re 1 0S in about 10 to 15 m in u tes and then stopped . O b servation

for over one hour d is c lo s e d no further d evelop m en t and o b serv a tio n

w a s term in a ted b e fo re the septum w as co m p leted . The n u cleu s (n u cle i)

• w as (w ere) not seen during th is p er io d of o b serv a tio n . Sh ortly a fter

the com p letion of c y to k in e s is the n u c le i of the new c e l l s a r e v is ib ly

c lo se r to the new ly fo rm ed , p lan e septum but th ey m ig r a te to the c e n te r s

of the new (daughter) c e l l s in le s s than 24 h r .

30

G am etic F o rm a tio n and the R o le of the C hlor op Lasts

The f ir s t in d ica tio n s of conjugation a re the a d h eren ce of the

gam etan g ia l c e l l s (u su a lly w ith the prod u ction of p a p illa e ) and a d ark en ­

ing of the c e l l con ten ts a s an abundance of ph otosynth ate i s produ ced

and-accum ulatedo The a ccu m u la ted food m a te r ia l i s co m p o sed p r im a r ily

of sta rch , a s in d ica ted by IKI te s t s , but so m e o il g lob u les, a re p r e se n t

a lso a s in d ica ted by t e s t s w ith ch ry so id in Yo . The p y ren o id s , a s s o c ia te d

w ith s ta r ch sy n th es is ,, appear to b ecom e m o re n u m erou s and som ew hat

la rg er than in the v e g e ta tiv e c e l l s of th e filam ento The n u cleu s w hich

i s r ea d ily v is ib le in v e g e ta tiv e c e l l s b eco m es o b scu red by the a ccu m u la ted

food m a te r ia ls (Fig= 3), , A lthough the fe m a le gam etangium i s la rg er in

s iz e than that of the m a le , th ere i s no apparent d iffe re n c e in the ev en ts

w hich occur in each up to th is point in th e sex u a l cycle* . In the w ord s of

De B ary (1 858) who d e sc r ib e d th is p r o c e s s in S* stic ticu m ,. "* , » both

c e l l s a cq u ire a d a rk -g reen , c o a r s e -g r a in e d ap p earan ce w hich d if fe r ­

en tia tes them from the v e g e ta tiv e ce lls* "

The p ro to p la st co n tra c ts aw ay from the gam eta n g ia l w a lls and

rounds up to form the g a m e te s (Fig* 4)*. It w ill be noted that the g reen

co lo r of the ch lorop h y ll i s ev en ly d istr ib u ted throughout the gam ete

(excep t a g a in st the c e l l m em b ran e) and th ere i s no in d ica tio n that the

c h lo r o p la sts a r e s t i l l in the form of ribbons* The only g a m etic m o v e ­

m ent o b se rv e d w as in d u ced by poking th e m a le gam etan gium w ith a

31

d is se c t in g n eed le w hich ca u sed the m a le ga m ete to m ove .through the

p ore in the conju gation tube (F ig . 4}.. . The g am etic m a te r ia l w hich

m oved through the p o re ap p eared to be co m p o sed of a v is c o u s flu id

contain ing granular and globular p a r t ic le s . T his type of gam etic

m a ter ia l and the flow ing m ovem en t i s not c o n s is te n t w ith a continuous

r ib b o n -lik e c h lo r o p la st but w ould be quite p o s s ib le w ith a frag m en ted

one. . W hether th is i s a ty p ica l type of g a m etic m ov em en t i s not known

s in c e it o ccu rred only a fter stim u la tio n w ith a d is s e c t in g n eed le and

not sp on tan eou sly .

- Z ygote and. C h lo ro p la stic O rgan ization

In .F ig u re 5 a young zy g o te (30. m in u tes a fter g a m etic union) is

show n. The c h lo r o p la s tic m a ter ia l, a s in d ica ted by th e g reen co lo r ,

app ears to be eq u ally d istr ib u ted throughout the zy g o te ex cep t at the

m a rg in s w h ere the g lobu lar fra g m en ts of the c h lo r o p la s ts can be seen .

T h ese fra g m en ts a re p rod u ced during g a m etic d evelop m en t and c o m ­

pacted , a fter g a m etic union, in the zy g o te . The sp h e r ica l nature of

the new ly fo rm ed zy g o te a lte r s w ith in s e v e r a l days to b eco m e e llip so id

in shape (F ig . 6 ). . T h is fig u re show s th e la s t sta g e of th e young zy g o te

b efo re the c o lo r e d and orn am en ted m ed ian w a ll d e v e lo p s . If .young

zy g o te s a r e gen tly sm a sh ed the ch lo ro p h y ll is. found in ir r e g u la r ly

shaped fra g m en ts of the c h lo r o p la s ts .

32

Z y g o sp o re

E ven ts w hich occu r w ith in the zy g o sp o re a r e even m o re d if f i­

cu lt to o b se rv e than th o se in th e g a m e te s and zygote b e c a u se of an

accu m u lation of starch , and o il and. the develbpm en t of the brown,

ornam ented , and ob scu rin g m edian sp o re w a ll (F ig s . 7, 8 ). Som e

in fe r e n c e s can be m ade fro m the ex p o sed con ten ts of sm a sh ed zy g o ­

s p o r e s . T h ere w e re no d is c r e te co n tra c ted or long r ib b o n -lik e c h lo r o -

p la s ts found in the z y g o sp o re s sm a sh ed in so il-w a te r supernatan t. In

fact, the ch lorop h y ll ap p eared in sc a tter e d , ir r e g u la r ly shaped fr a g ­

m en ts about 1 0 -2 0 m ic r o n s long. Z y g o sp o res sm a sh ed in a c e to - or

p r o p io -c a r m in e exh ib ited a s im ila r ap p earan ce.

The zy g o sp o re , a s w ould be exp ected , con ta in s m uch food

r e s e r v e in the form of o il g lo b u les and s ta rch g r a n u le s . The la tter i s

evident in F ig u re 9 w hich show s a zy g o sp o re sm a sh ed in io d in e -

p o ta ss iu m -io d id e rea g en t. The ir r e g u la r ly shaped s ta r ch g ra n u les

(appearing brown to b lack in th is photograph) a r e about 5 -1 0 m icro n s

in d ia m eter .

.A s has been r ep o r ted in the lite r a tu r e (T ran seau , 1951;

: Randhawa,, 1959), the zy g o sp o re has a th r e e - la y e r e d w a ll. The o u te r ­

m o st w a ll, surrounding the brown m ed ian sp o re w a ll, can be see n .in

F ig u re I I . The v e r y d e lic a te and tra n sp a ren t in n er sp o re w a ll is v is ib le

in F ig u re 8 . . A s has been r ep o rted for S p irogyra spp. (A llen , 1958)

th is inn er zy g o sp o re w a ll b e c o m e s the w a ll of the young g erm lin g (F ig .

33

11). . The m edian w a ils of z y g o sp o r e s of S irogonium raelanosporura a r e

ir r e g u la r ly v e r r u c o s e and. brown in co lo r (F ig s . 7, 8 }, I I , 16, 17, 18).

Randhawa (1938) in h is o r ig in a l d e scr ip tio n sta ted that th e co lo r of the

z y g o sp o r e s w as ". . . d e n se ly b lack w hen fr e sh . " The z y g o sp o r e s in

the cu ltu ra l m a te r ia l do not obtain su ch a dark co lo r a s can be seen in

the p r e v io u s ly m en tion ed ph otograp hs.

The s iz e and shape of the z y g o sp o r e s p rod u ced in cu ltu re a r e

quite v a r ia b le . M ost of the z y g o sp o re s a r e e ll ip s o id in sh ap e (F ig . 7)

but in ev er y cu ltu re b o ttle th ere a re a few zy g o sp o re s v a ry in g from

the ty p ica l shape tow ard s th e ovoid or e lon gate . F ig u r e 8 show s the

r em a in s of a zy g o sp o re w hich w as m o re ovoid than e l l ip s o id b efo re

sm a sh in g . T able 7, below , g iv e s the w id th and length d im en sio n s for

a num ber of z y g o sp o re s from each of two sep a ra te ly d r ied n e ts . T h ese

n ets w e re rew et b efo re m e a su r em e n ts w e r e m ad e.

. Only the d im en sio n s of th e la s t four z y g o sp o r e s , r ec o r d e d in

T able 7, fa ll e n tir e ly w ith in the l im its of the d im en sio n s g iven by

T ran seau (1951), 9 0 x 1 4 0 m ic r o n s , and m any a r e c o n s id era b ly s m a lle r .

One cu ltu ra l zy g o sp o re w h ose g erm in a tio n w as o b se rv e d m ea su red 8 8 x

135 m ic r o n s (F ig .- I I ) . Two z y g o sp o r e s from the M ex 22 so il sam p le

a ls o d isp la y ed the v a r ia b ility found in z y g o sp o r e s p rod u ced in cu ltu re .

One m e a su r ed 100 x 130 m ic r o n s (ovoid ) and the other 8 0 x 1 4 5 m icro n s

(e lo n g a te). Both of th e se z y g o sp o re s had germ in a ted . No sa t is fa c to r y

m ethod w as d e v ised for finding and sep a ra tin g th e se natural z y g o sp o re s

34

from the f in e c la y -sa n d s o il sam ple* . M easu rem en t, th er e fo re , of

sp o r es b efo re g erm in a tio n w as not p ossib le*

. T able 7* D im en sio n s of cu ltu ra l z y g o sp o re s 2 hr a fter rew etting*

. Width Length Width L ength

82 124 8 8 15482 124 72 12482 113 72 16593 134 82 14482 124 82 16582 113 78 13082 . 124 78 13577 144 80 13577 134 93 14482 165 93 14493 124 93 16582 103 93_ 16582 134

a v e ra g e 83 137

G erm ination of Z yg o sp o re

The trea tm en ts u sed in an attem p t to ind uce a c o n s is ten t

am ount of g erm in a tio n in cu ltu ra l z y g o sp o r e s , d e sc r ib e d on page 2 2

of th is m a n u scr ip t, w e r e la r g e ly u n su ccessfu l* L e s s than 5% g e r m i­

nation w as obtained from th o se sp o r es w hich , m o rp h o lo g ica lly , ap p eared

to be viable* O ften not a s in g le sp o re in a p articu lar net w ould g e r m i­

nate r e g a r d le s s of the trea tm en t it underwent* B ec a u se of th is v e r y

low and e r r a tic g erm in a tio n no s ta t is t ic a l c o m p a r iso n s cou ld be

m ade am ong the v a r io u s treatm en ts* G erm ination of one or m ore

zy g o sp o re s did occu r, h ow ever, in so m e d ish es contain ing nets g iven

a p r io r -to -w ettin g h eat trea tm en t, in n e ts g iven an a fter -w e ttin g h eat

trea tm en t, and in th o se n ets w hich w e r e s im p ly w et at room te m p e r a ­

tu re and p la ced under f lu o r e sc e n t ligh tin g . No g erm in a tio n o ccu rred

in any of the other d is h e s . The r ea so n for the p a r tia l e f fe c t iv e n e s s of

drying fo llo w ed by w etting for inducing germ in ation of th e z y g o sp o re s

has not been d eterm in ed . The g erm in a tin g z y g o sp o r e s d e h isc ed in a

regu lar fa sh io n o n e-h a lf to th r e e -fo u r th s the length of the sp o re .

, A lthough the d e h isc en ce w a s quite reg u la r , no lin e of d e h isc en ce w as

ever v is ib le in th e s e sp o r e s b e fo re the actu a l sp littin g at th e tim e of

germ in a tio n . Z y g o sp o res , w hether or not th ey germ in a ted , sw e lle d

som ew hat a s th ey im b ib ed w ater upon b ein g w e t te d .. A n o ticea b le change

in co lo r from brown to g reen o ccu rr e d in a ll th o se z y g o sp o r e s w hich

w ent on to germ in ate; how ever not a ll the z y g o sp o re s w h ich changed

co lo r germ in a ted . M ost g erm in a tio n o c cu rr e d w ith in 4 8 -7 2 hr a fter

w etting , but in a few c a s e s g erm in a tio n o ccu rr e d up to 14 days after

w ettin g . T w ice zy g o sp o re s g erm in a ted in n ets w hich had been w etted ,

r ed r ied , and w etted again . . Z y g o sp o res from the M ex 22 so il sam p le

a ls o d em o n stra ted th is d ifferen tia l g erm in a tio n .

G erm ination of z y g o sp o re s fro m cu ltu ra l m a te r ia l o ccu rred

w h ile the zy g o sp o re s w e r e s t i l l con ta in ed w ith in the o ld fe m a le

gam etan g ia l w a lls (F ig s . 16, 18). D is se c t in g the z y g o sp o r e s out of the

gam etan g ia did not in c r e a s e the p e r ce n ta g e of g erm in a tio n . . C ultural

36

z y g o sp o re s m o r e than one yea r o ld and n atural z y g o sp o r e s m o re than

two y e a r s o ld have g erm in a ted after rew ettingo

G erm ling and R eo rg a n iza tio n of the G h lorop lasts

The p ro to p la st of the z y g o sp o re , e n c lo se d by th e inner thin

e la s t ic c e l lu lo s ic sp o re w a ll, e m e r g e s through a rup tu re in the m ore

r ig id m ed ian and outer w a lls . T h ese w a lls a r e o c c a s io n a lly ir r e g u la r ly

torn at the end but m o st often they d e h isc e along a v e r y reg u la r lin e

(Figs<, 18, 21)o A s h as been m en tion ed e a r lie r , th is lin e of d e h isc en ce

i s not v is ib le p r io r to the g erm in a tio n of the z y g o sp o r e c . The lin e of

d e h isc en ce of the zy g o sp o re in F ig u re 11 w as som ew h at d is to r ted by

m an ipu lation s w h ich m oved the o n e -c e l led g erm lin g out of the sp ore

w a lls for photographic p u rp o se s .

The o n e -c e lle d g erm lin g con ta in s s ta rch and o il d ro p le ts , . In

cu ltu ra l m a te r ia l th e se su b sta n ces r a r e ly o b scu re the d is s o c ia te d

ch lo ro p la st in the young g erm lin g (F ig s , 11, 12, 17, 18), A c lu s te r of

orange g ra n u les i s often v is ib le in the o n e -c e lle d g erm lin g (F ig , 12), .

The author h as not d e fin ite ly d e term in ed w hat th e se g ra n u les r e p r e se n t

but a d is c u s s io n .o f th eir p o s s ib le o r ig in w ill be found in th e fo llow in g

sec tio n of th is m a n u scr ip t. The orange gra n u les occu r in young g e r m -

l in g s of both natural and cu ltu ra l z y g o sp o r e s , but th ey a r e not a lw a y s

r ea d ily v is ib le , . A s the g erm lin g in c r e a s e s in length , by c e l l d iv is io n ,

the gra n u les b eco m e le s s and le s s co n sp icu o u s and a r e even tu a lly lo s t

37

from v iew . , By the t im e the g erm lin g i s about 15 c e l l s long or e a r lie r

the gra n u les a r e no lon ger ev id en t.

E ven s in g le -c e l le d germ lin g s p r e se n t a v a r ie ty of a p p earan ces

w ith r e s p e c t to shape, w idth, and length . The c e l l s a r e u su a lly m o re

or l e s s e lon gate and th ey m ay or m ay not have a co n sp icu o u s bend (F ig s .

11, 12, 16). The g erm lin g ssa t th is point m ay fo llow e ith er of two g e n ­

e ra l p lan s of d evelop m en t. The o n e -c e lle d g erm lin g m ay begin to d iv id e

im m ed ia te ly b e fo re e x te n s iv e c e l l e lon gation , producing a fila m en t of

sh ort c e l l s , or the o n e -c e lle d g erm lin g m ay e lon gate g r ea t ly b efore

the f ir s t c e l l d iv is io n producing a f ila m en t w ith a long tap erin g b asa l

c e l l and subsequent c e l l s w hich a re m o r e e longate tiian th o se of the f i r s t

p lan . The f ir s t p attern i s the one m o st often o b serv ed in g e rm lin g s p r o ­

duced from cu ltu ra l z y g o sp o r e s and the seco n d p attern i s m o st often

found in g e rm lin g s p rod u ced from natural z y g o sp o r e s . T h ese two

p a ttern s do not r em a in d is tin c t . A s the g e r m lin g s grow , the c e l l s of

the fila m en t, other than the b a sa l c e l l and th o se c e l l s ad jacent to it,

look v e r y m uch a lik e w hether the g erm lin g i s one from cu ltu ra l or

natural z y g o sp o r e s . A cu ltu ra l, th r e e -c e l le d g erm lin g of the f ir s t type

i s shown in F igur e 17 and two n atural four - c e l le d g e r m lin g s of the

secon d typ e a r e shown in F ig u r e s 19, 20. T h ese d is tin c tio n s a re not

ab so lu te . A r e la t iv e ly long b a sa l c e l l in a cu ltu ra l g e rm lin g i s shown

in F ig u r e 18 and a r e la t iv e ly sh o rt b a sa l c e l l in a natural g erm lin g i s

show n in F ig u r e 21. The b a sa l p ortion of the g erm lin g u su a lly r em a in s

38

w ith in the zy g o sp o re :" ca se" (F ig S o - 1 7 -2 2 ) . The g e r m lin g s shown in

F ig u r e s I I , 17 w e re te a se d out of the z y g o sp o re c a s e s in order to obtain

photographs of the en tire c e l l ( s ) . The zy g o sp o re c a s e s s e e n in th ese

f ig u r e s w e re both d is to r ted during th is p r o c e s s of teasingo

The n u cleu s w as not v is ib le in o n e -c e lle d g e r m lin g s , but w as

u su a lly v is ib le in both c e l l s of the tw o -c e lle d s ta g e . A fter the f ir s t

d iv is io n in th e g erm lin g , both c e l l s h ave the p oten tia l for further

d iv is io n and grow th but the b a sa l c e l l d iv id es only r a r e ly w h erea s the

a p ica l c e l l and it s d e r iv a tiv e s d iv ide freq u en tly . The b a sa l c e ll , w hether

or not it d iv id es a fter th e f ir s t d iv is io n , e lo n g a tes g r e a t ly and con tin u es

to do so ev en after the young germ lin g has d evelop ed to 35 c e l l s or m o re

in .len g th . The b a sa l c e l l m ay, in tim e , b ecom e d is to r te d and die, p r o b ­

ably due to in jury in f lic te d upon it by the m ech a n ica l s t r e s s e s of the

dividing and elongating c e l l s of the young fila m en t. G erm lin g s seld om

e m e rg e from their c a s e s a s sh ort s tra ig h t f ila m en ts co m p o sed of c e l l s

of uniform w idth . In young, g e rm lin g s ,. e sp e c ia lly th o se from natural

z y g o sp o r e s , the c e l l s grad u a lly in c r e a s e in w idth up to the filam en t

m axim um o c cu rr in g ,a t about the fourth to tenth c e l l . The m axim um

width, on ce it i s r ea c h e d by the young g erm lin g , r e m a in s r e la t iv e ly

constanitthroughout th e fu tu re grow th of the fila m en t. The fila m en t m ay

again taper a l it t le at the a p ica l end. The ap ica l c e l l i s n ea r ly a lw ays

I 1 /3 to 2X a s lo n g ,as any one of the next, few c e l l s c lo s e s t to it (F ig s .

22, 23, 24; T ab les 8, 9).

39

The bending of a fila m en t m ay be s lig h t as show n in F ig u re 13

or it m ay be m o re e x te n s iv e a s show n in F ig u r e s 17, I 8 0 The bends,

once they a r e form ed , a r e not sta tic but m ay, and often do change as

the c e l l s of the fila m en t d iv ide and e lo n g a te . The in it ia l bending of the

fila m en t is often the r e su lt of con fin em en t of the g erm lin g w ith in the

old w a lls of the gam etangium (F ig . 16), at le a s t in cu ltu ra l g erm lin g s .

N atural g e rm lin g s a r e not u su a lly con fin ed in the o ld gam etan g ia l w a lls

b eca u se th e se have d eca y ed aw ay from the zy g o sp o re p r io r to it s g e r m i­

nation. H ow ever, th ere i s u su a lly a tig h tly packed m a ss of so il p a r t ic le s

surrounding the z y g o sp o re s of the n atural g erm lin g s and th e se p a r t ic le s

m ay act in th e sa m e m anner a s the gam etan g ia l w a ll in o b stru ctin g the

ea r ly grow th of the g e rm lin g s (F ig s . 1 9 “22).

F ig u r e s 1 1 -15 show the even ts w hich o ccu rred in the sam e

cu ltu ra l g erm lin g during a p er io d of four d ays. Two su b sequ en t o b s e r ­

v a tio n s, one at the end of sev e n days and the other at the end of two

w eek s r e v e a le d only the slow d eath .of th is g e rm lin g . The c a u se of death

i s unknown. T h ere w as an accu m u lation of b a c ter ia a long the c e l l w a lls ,

a s can be see n in the photographs, and th e se b a c ter ia p lu s in jury due

to m an ip u lation s m ay have been contribu ting fa c to r s , at le a s t , in the

a r r e s te d grow th and su bseq u en t d e m ise of th is g e rm lin g . The even ts

of c h lo r o p la stic reo rg a n iza tio n w ere see n in a num ber of other g e r m ­

lin g s , h ow ever, w hich d eve lop ed in to f ila m en ts of m o r e than 1 0 0 c e l l s

and show ed no s ig n s of dying, up to the tim e of th eir being d isca rd ed .

40

A s has been poin ted out in a p r e v io u s sec tio n of th is m anu­

scr ip t, the c h lo r o p la s ts , at the tim e, of g a m etic form ation , begin to

fragm en t (Figo 3} and rem a in in th is cond ition throughout the p r o c e s s of

"conjugation and into th e m atu re z y g o sp o re until it b eg in s to g erm in a te ,

. A t th is tim e the a g g reg a ted fra g m en ts of the c h lo r o p la s tic m a ter ia l

appear in the o n e -c e ile d g erm lin g as m a s s e s of v a r io u s sh apes and

sizeso T h ese m a s s e s m ay or m ay not co m p le te ly f i l l the c e l l (FigSo

12, 1 6 ).o B ro w n ish -o ra n g e or r e d .ncaroten o id 11 g ra n u les a r e often seen

w ith in the s in g le -c e l le d g erm lin g (Figo- 12jo R em ark s about the p o s s ib le

or ig in of th e se g ra n u les w ill be found in the d is c u s s io n sec tio n of th is

m anuscripto T h ere i s a continuous s ta te of a c tiv ity w ith in the p ro to ­

p la s t A fter a p er io d of 30 m in u tes or m or e, rea rra n g em en ts! in the

c h lo r o p la stic fra g m en ts and the car otenoid g ra n u les b eco m e a p p a re n t

A s the c e l l e lo n g a te s , the c h lo r o p la s tic m a te r ia l b e c o m e s unequally

d istr ib u ted w ith in the ce ll; the la rg er am ount is found tow ard the a p ica l

end of the c e l t The b asa l c e l l d iv id es unequally in the reg io n of the

accu m u lation of the ch lo r o p la s tic m a te r ia l to g iv e r i s e to the secon d

c e ll of the f i la m e n t At about th is tim e one to s e v e r a l ir r e g u la r ly

shaped, r ib b on lik e s tr u c tu r es can be seen extending from both s id e s of

a m a ss of c h lo r o p la s tic m a ter ia L T h ese s tru c tu res r e p r e se n t the

beginning of the reo rg a n iza tio n of the c h lo r o p la s ts in the g erm lin g of

Sirogonium m elan osp oru m (com p are FigSo 11,. 12 w ith Figo 13)o

41

The carotenoid g ra n u les can be see n in the a p ica l c e l l of the

tw o -c e lle d germ ling in F ig u r e 13= T h ese gran u les a r e not a lw ays

tr a n s fe r r e d to the a p ica l c e l l a s a d is c r e te m a ss a s th ey have been in

th is germ ling but th ey m ay be ran d om ly d istr ib u ted in to the c e l ls of the

young fila m en t and even tu a lly lo s t from v ie w . The r ib b on lik e s tr u c tu r es

b eco m e m o re p rom in en t a s can be see n in F ig u re 14. . In fact, the

seco n d c e l l of th is fo u r -c e lle d germ ling has it s ch lor op la s t s su ffic ie n tly

reo r g a n iz e d to be r e c o g n iz e d as a c e l l in a .fila m en t o f Sirogonium u

The b a sa l and a p ica l c e l l s of the fo u r -c e lle d germ lin g show the le a s t

reo rg a n iza tio n of the c h lo r o p la s ts and the a p ica l c e l l w ill rem a in th is

w ay even after the g erm lin g i s m any c e l l s longo . F ig u r e 15 show s the

sa m e g e r m lin g £s d evelop m en t four days a fter the o n e -c e lle d stage*

The b a sa l, secon d , and th ird c e l l s o f th is s ix - c e l le d g erm lin g have

r e la t iv e ly w e ll d eve lop ed ch lor o p la s ts w h erea s the fourth and fifth c e l l s

a r e s t i l l in the p r o c e s s of reorgan ization * The c h lo r o p la s ts of the

a p ica l c e l l , a s has been m en tion ed e a r lie r , do not b eco m e o rg a n ized

for -some time*

G erm lin g s of 5 0 -1 0 0 c e l l s in length , i f they happen to bend

su ffic ie n t ly for one p ortion of the f ila m en t to co m e in to con tact w ith

another, w ill conjugate* C onjugation in th e young g e rm lin g s w as ob­

ser v e d only in th o se d er iv ed from cu ltu re and it i s not known w hether

g erm lin g s from the M ex 22 s o il w ill conju gate at th is v e r y ea r ly s ta g e

of developm ent*. The natural g e rm lin g s have never bent su ffic ien tly

42

for one p ortion of the fila m en t to com e in to con tact w ith another and

th is m ay fu lly accou nt for the lack of conju gation .

. F ig u re 21 show s a fo u r -c e lle d germ ling from a natural z y g o ­

sp o re in about the sam e sta g e of d evelop m ent and of c h lo r o p la s tic

reo rg a n iza tio n a s the fo u r -c e lle d cu ltu ra l germ ling in F ig u re 14. The

h e m isp h er ic a l bu lge betw een the b a sa l and seco n d c e l l of the fila m en t

w as the r e s u lt of in ju ry during th e rem o v a l of d irt adh ering to the

g erm lin g . A nother fo u r -c e lle d natural g erm lin g w h ich con ta in s r e o r ­

gan izin g c h lo r o p la s ts and m any o il d ro p le ts i s shown in F ig u re 20.

O nce the c h lo r o p la s ts of a c e l l of a g erm lin g have organ ized ,

any c e l l s r e su lt in g from d iv is io n of th is c e l l w ill a lso have o rgan ized

c h lo ro p la sts b eca u se of the nature of c e l l d iv is io n d e sc r ib e d on page 29

of th is m a n u scr ip t. , The c h lo r o p la s ts in the ap ica l c e l l do not fu lly

r eo rg a n ized until the g erm lin g i s 1 5 -2 0 c e l l s long or lo n g e r . In the 2 0 -

ce lled , natural g erm lin g of F ig u r e s 22, 23, 24 can be see n the p r o g r e s ­

s iv e u n sp ira llin g , narrow ing , and lengthenShg of the c h lo r o p la s ts from

the som ew h at u n organ ized c h lo r o p la s tic m a te r ia l of th e a p ica l c e l l to

the h igh ly o rg a n ized and stra ig h t c h lo r o p la s ts of th e sec o n d c e l l . T his

p r o g r e ss io n i s il lu s tr a te d even b etter in the four en la rg em en ts of s e c ­

tion s of the p reced in g g erm lin g (F ig s . 25,. 26, 27, 28).

T h ese f ig u r e s a lso i l lu s tr a te the v a ry in g num ber of c h lo r o p la s ts

a s so c ia te d w ith the d ifferen t c e l l s of the fila m en t at th is sta g e of th e

g e r m lin g £s d evelop m en t. The b a sa l c e l l m ay have from I to 4

43

chL oroplasts but u su a lly no m o re than 2 of them exten d in to the b asa l

I / 3 - 2 / 3 of the c e lh The seco n d c e l l m ay have from 3 to 6 c h lo r o p la sts

and the th ird c e l l from 4 to 7. . C e lls beyond the th ird one m ay have up

to 9 ch lorop lasts* T h ere a r e u su a lly few er c h lo r o p la s ts , 5 to 8 , in

c e l l s tow ard the ap ex of the filam ent* S in ce m o st of th e s e c e l l s w ill

in c r e a s e the num ber of th eir c h lo r o p la s ts up to 7, 8 , or 9, th ere m u st

be a m ethod of du p lication of the c h lo r o p la s ts w hich w as not o b serv ed

by the author * It i s not unusual to fin d sh ort p ie c e s of c h lo r o p la sts

w hich extend only a p ortion of the w ay from one end of the c e l l to the

other and it i s p o s s ib le that th ese sh o rt p ie c e s grow in length to

b ecom e th e f u l l - s iz e d c h lo r o p la s ts of the v e g e ta tiv e c e lls* , W here th e s e

p ie c e s co m e from i s not k n o w n ,. how ever*

Two d iffe re n c e s w ere noted betw een the grow th and d ev e lo p ­

m ent of natural v e r s u s cu ltu ra l g erm lin gs* F ir s t , r eo rg a n iza tio n of

the c h lo r o p la s tic fra g m en ts in to c h lo r o p la s ts w as u su a lly s lo w er in

cu ltu ra l g e r m lin g s , and secon d , th ey did not u su a lly h ave the v e r y

e lon gate and ta p ered b a sa l c e l l found in m o st of the n atural germ lin gs*

A s has been m en tion ed p r e v io u s ly , th e se a re only te n d en c ie s and not

a b so lu te d ifferen ces*

In T able 8 a r e found the c e l l len g th s, the a v e ra g e c e l l len gth s

in clu d in g .an d not inclu d ing the b a sa l c e l l , and th e g r e a te s t w idth of the

fila m en ts during s u c c e s s iv e s ta g e s of grow th of the cu ltu ra l germ ling

p ic tu red in F ig u r e s 1 1 -1 5* In T able 9 a re found the sa m e data for the

44

natural germ Ling, shown in F ig u r e s 2 2 -2 8 , ex cep t that so m e of th e c e l l

length S3 h ave been d e le ted in ord er to red u ce the ta b le to a m an ageab le

size* The b a sa l c e l l in e ith er g erm lin g i s co n s id era b ly longer than

any other c e l l in th e fila m en t and th e se c e l l s continued to e longate

throughout the p er io d of ob serv a tio n of the two germ lings* A fin a l

ob serv a tio n of the natural g erm lin g of T able 9 w as m ade 336 hr (14

days) a fter the f ir s t observation* The fila m en t of th is g erm lin g w as

co m p o sed of 72 c e l l s and m e a su r ed 14, 986 m ic r o n s in to ta l length*

The r e su lt in g .a v e r a g e of 208 m icro n s p er c e ll , includ ing the basa l

c e ll , i s su b sta n tia lly g rea ter than the 167 m icro n s per c e l l of the 51 -

ce lled , 208 hr ( 8 2 /3 days), s ta g e of grow th of the germ ling*

It w ill be noted that the b a sa l c e l l of the natural g erm lin g ( s e e

T able 9). d iv ided betw een the 1 0 - and 1 9 -c e l le d s ta g e s of grow th. The

sh orter c e l l s r e p r e se n t prod u cts of r e c e n t c e l l d iv is io n and a num ber

of them w e r e see n b efo re the septum w as co m p leted (Fig* 10)* The

p ro x im ity of n u c le i to the septum or tr a n s v e r s e w a ll i s an in d ica tion ofs

a s lig h tly la ter s ta g e in r e c e n t c e l l division* The n u cle i m ig ra te to

the cen ter of the c y lin d r ic a l c e l l s sh o r tly a fter c e l l d iv is io n i s co m p le ted .

45

T able 8 * . C ell len gth s,, a v era g e c e l l len g th s, and g r e a te s t w idths at s u c c e s s iv e s ta g e s o f grow th of a cu ltu ra l germ ling of Sirogonium m ela n o sp o ru m 0 A ll m e a su r em e n ts a r e g iven in m ic r o n s .

Hr from 1 st o b serv a tio n 0 c a . 24 c a . 48 ca .. 9 6

Length of fila m en t 1 - c e l le d 2 - c e l l ed 4 - c e l le d 6 - c e l le d

B a sa l c e l l (1st). 205 340 355 375

2 nd 170 105 135

3rd 40 2 0 0

4th 170 85

5 th 75

6 th 195

A vg. length of c e l l s w ithout b a sa l c e l l

— 170 105 136

A vg. length of a ll c e l l s of the fila m en t

205 255 167 178

T otal length of fila m en t 205 510 670 I , 065

G rea test w idth of fila m en t 73. 7 78. 0

o00 .

l> o00

l>

46

T able 9s. S e le c ted c e l l len g th s, a v era g e c e l l lengths in clu d in g a ll d e le ted c e l l len g th s, and g r e a te s t w idths at s u c c e s s iv e s ta g e s of grow th of a natural germ ling of S irogonium m e la n o - sporutrio A ll m e a su r em e n ts a r e in m icronso

6 -c e l le d 1 0 -c e l le d 1 9 - c e l le d 2 4 -c e lle d 2 8 -c e lle d 41 -c e l le d 51 - c e l le d0 hr 2 0 hr 44 hr 77 hr 1 0 1 hr 150 hr 208 hr

I Stc e l l 536 670 597 . 876 999 1 , 128 I , 2052 nd 1 2 6 154 2 1 6 371 402 340 5663rd 6 8 103 170 ■ 258 309 340 3354th 76 93 154 252 2 6 8 309 3195th 119 103 103 185 124 206 2476 th 156 103 >98 165 ' 113 137 1479 th 6 2 82 82 139 175 1031 0 th 103 82 72 98 154 11318th 6 2 103 103 154 10319th 113 103 108 124 11323rd 93 72 144 8224th 124 6 2 1 6 0 10327th 52 98 18528 th 93 93 10340 th 77 1544 1 st 93 15442nd 144 15450th 11351 st. 154A vg. length of c e l l s exclu din g b asa l c e l l

109 95 94 133 1 2 6 ■ 142 152

Avgfo length of a ll c e l l s of th e fila m en t180 152 120 164 158 166 167

Total length of the fila m en t1, 082 1 , 524 2, 281 3, 929 . 4, 411 6 , 973 8 , 523

i

G rea test w idth of th e fila m en t7 7 .2 76. 6 7 6 .2 - - 7 7 .3 7 7 . 3

F ig u r e s 3-5o - Sirogouiuiri rnelanosporum from cu ltu re .

F ig u re 3. G am etan gia l fo rm a tio n . N ote the fra g m en ted nature of th e c h lo r o p la s ts in both gam etan g ia . _ F e m a le gam etangium (a). M ale gam etangium (b).

F ig u re 4 . G a m etes . N ote the co n cen tra ted nature of the c h lo ro p ia stic m a te r ia l in the g a m e te s . The double conjugation shown i s not com m on in S. m e la n o sp o ru m . . "'Female gam etangium (a) contain ing the la r g e fe m a le g a m ete . M ale gam etangium (b) contain ing the sm a ll m a le g a m ete w h ich w ill m o v e through the p ore (c) to un ite w ith the fe m a le g a m ete .

F ig u re 5. A zy g o te 30 m in . a fter the fu sio n of the g a m e te s . N ote theco n cen tra ted nature of the ch lor o p ia stic m a te r ia l in the zy g o te .

, A lso note the conjugation tube. F e m a le gam etangium (a) contain ing the young zy g o te . The em pty m a le gam etangium

(b)o

\

47

Rg. 3

Fig. 4

Fig. 5

F i g u r e s 6-7o S iro g o u iu m m e ia n o sp o r u tn fr o m c u l t u r e 0

F ig u re 6 0 . A young zy g o sp o re contain ing g reen ch io r o p ia s tic fr a g m e n ts 0

F ig u re 7, . A m atu re z y g o sp o r e 0 N ote the brown co lo r and v e r r u c o s e nature of the m edian sp o re w alL The tra n slu cen t outer sp o re w a ll i s a ls o v is ib ie 0

F ig u r e s 8-9° • S irogonium xaelanosporiiim from culture,.

F ig u re 8» . A sm a sh ed zy g o sp o re show ing the tran sp aren t inn er c e l lw a ll w ith in the outer w a lls . N ote the ornam entation of the hr own m ed ian sp o re w a ll.

. F ig u r e 9» . A m atu re zy g o sp o re w h ich w as sm a sh ed in IKL N ote thecop iou s s ta rch g ra n u les w hich appear brown to b lack in th is i l lu s tr a t io n .

F ig u re 10 .. S irogonium m elan o sp o ru m , from M ex 22 so il show ing a c e l l in the p r o c e s s of d iv is io n . N ote how the p a r tia lly d evelop ed septum h as c o n s tr ic te d the c h lo r o p la s ts .

Ffg.8

Fig>9

Ffg.10

/

. F ig u r e s 11 - I5« , S irogonium m elan osp oru m from cu ltu re . T his s e r ie sof photographs r e c o r d s four s u c c e s s iv e s ta g e s of grow th in a s in g le germ lin g . C arotenoid g ra n u les (a).

„ F ig u r e s 1 3 -1 5 . The sh ort b lack lin e s ou tsid e the f ila m en ts in d ica te the p o s it io n s of the c e l l w a lls ; the b a sa l or f ir s t c e l l i s to the le ft .

F ig u re 11. O n e -c e lle d g erm lin g . N ote the fra g m en ted nature of the c h lo r o p la s tic m a te r ia l.

F ig u re 12. Sam e v iew a s F ig u r e 11. N ote the fra g m en ted nature of the c h lo r o p la s tic m a te r ia l.

F ig u re 13. . T w o^ celled s ta g e . , P hotograph taken about 24 hr a fter •' F ig u r e s 11, 12. N ote the d iso rg a n iza tio n of the c h lo r o ­

p la s t ic fr a g m e n ts .

F ig u re 14. F o u r -c e lle d s ta g e . . P hotograph taken about 48 hr a fterF ig u r e s 11, 12. N ote the beginning of o rg a n ized c h lo r o - p la s ts in the seco n d c e l l .

. F ig u re 1 5 .. S ix -c e l le d s ta g e . Photograph taken about 96 hr afterF ig u r e s 11, 12. N ote the r e la t iv e ly w e ll d evelop ed c h lo r o - p la s ts in th e seco n d and th ird c e l l s .

F ig u r e s 1 6 -1 8 . . S irogonium m elau osp oru m from cu ltu re .

F ig u re 16. O n e -c e lle d germ lin g w ith in fe m a le gam etan g iu m .

F ig u re 17. T h r e e -c e l le d germ lin g . N ote the d iso rg a n ized c h lo r o - p la s t ic m a te r ia l.

F ig u re 18. B a sa l p ortion of s e v e n - or e ig h t -c e l le d g e rm lin g . N ote the l in e of d e h isc en ce in the zy g o sp o re w a lls .

51

Fig. 16

1 0 0 /<

Fig.17

Fig.18

F ig u r e s 19-21« S irogoniu iu m elauosporurn. from M ex 22 so iL F ig u r es 19 9 21 o.. The b u lges betw een the b a sa l and secon d c e ll (a r r o w s),w e r e the r e s u lt of in jury during c lean in g of the f i la m e n ts »

F ig u re 19o Four - c e l le d natural germ lingo N ote the bend in the fila m en t and the long ta p ered b a sa l c e l l extending out from the z y g o ­sp o re c a s e .

F ig u re 20. F o u r -c e lle d natural germ lin g . N ote the d iso r g a n iz e d c h lo r o p la s tic m a te r ia l.

F ig u re 21=,.. F o u r -c e lle d natural germ lin g . N ote the f in g e r - l ik echi or o p la sts extending out from a d en se , c en tr a l m a ss of ch lo r o p la s tic m a te r ia l.

r 52

100/

Fig.19

100 /

Fig. 21

F ig u r e s 2 2 -2 4 , S irogouium .m elanosporurn frorn M ex 22 s o il , A s in g le , 2 0 -c e l le d natural g erm lin g . N ote the v e r y long b asa l c e l l . A s the c e l l s e lon gate , from the a p ic a l tow ard the b a sa l end, the c h lo r o p la s ts u n sp ira l and elon gate a ls o .

F ig u r e 22, F rom le ft to r igh t, zy g o sp o re c a se , b asa l and seco n d c e l l s , and part of the th ird c e l l . N ote the long, s tra ig h t, r ib b on ­lik e c h lo r o p la s ts .

F ig u re 23, - P a rt of the th ird c e l l , and fourth through eighth c e l l s . N ote the change from stra ig h t to sp ir a lle d c h lo r o p la s ts in the fourth and fifth c e l l s r e s p e c t iv e ly .

F ig u re 24, N inth through a p ica l (tw entieth).,-cells. N ote that the ch lp r o - p la s ts a r e m o re tig h tly sp ir a lle d . The a p ica l c e l l has the le a s t w e ll r eo r g a n iz e d c h lo r o p la s ts . M any of th e c e l l s have a co n sp icu o u s n u c leu s .

e s - B y

. F ig u r e s 2 5 -2 8 , . P o r tio n s of the sa m e n atu ra l g erm lin g of F ig u r e s 2 2 -2 4 ,

F ig u re 25, . F ou rth c e l l . N ote the fla t, stra ig h t c h lo r o p la s ts contain ing p y ren o id s .

F ig u re 26, F ifth c e l l . N ote the sp ir a lle d c h lo r o p la s ts and the n u cleu s su sp en d ed in the cen ter of the c e l l .

F ig u re 27, E ighth and p art of the ninth c e l l s . N ote the c lo s e ly sp a ced and h igh ly s p ir a lle d c h lo r o p la s ts w ith w avy m a r g in s .

F ig u re 28, , The a p ica l (tw entieth ) c e l l . N ote the p a r tia l d iso rg a n iza tio n of the c h lo r o p la s ts .

LZ ‘By

DISCUSSION

Sirogonium m elanosporurn has been found in th ree quite d if fe r ­

ent habitatso A t G r ee n v ille , M is s is s ip p i even the d r ie s t m onth,

O ctober, a v e ra g e s over two in ch es of r a in fa ll and the annual a v era g e

i s g r ea ter than 50 in ch es (T ab le 6 )« . In Uttar P rad esh ,. India the d r ie s t

m onth has le s s than o n e -h a lf of one in ch of ra in fa ll and th ere i s an

extended p er io d from O ctober through May. in w hich th e m onthly, aver -

age i s about one in ch or l e s s . The su m m er m onsoon b rin gs the g rea ter

p art of the y e a r ly a v era g e of over 35 in c h e s (T able 5). The G uaym as,

M exico s ite i s s im ila r to the Indian s ite in at le a s t one r e sp e c t; the

g r ea ter p art of the ra in fa ll c o m e s during the su m m er sea so n , but

G uaym as r e c e iv e s on ly o n e -f ifth as m uch ra in during th is sea so n as

d oes Uttar Pradesho , At G uaym as th ere a r e long p er io d s of l i t t le or no

ra in fa ll, and the m onth of June has so l i t t le that it a v e r a g e s l e s s than

0o 05 of an in ch . The f ir s t habitat i s m o is t a ll year long but it i s w e tte s t

in w inter and e a r ly sp rin g , N ovem ber through A p rih . The seco n d is

v e ry w et during the su m m er m onsoon , June through S ep tem b er, w ith a

m uch drier p er io d from O ctober through M ay. The la s t s ite i s quite dry

w ith only sp orad ic su m m er r a in s , from July through Sep tem b er and long

p e r io d s of drought from O ctober through June. The two c lo n es of

Sirogonium m e la n o sp o f um u sed in th is study have, at le a s t , four

55

56

a ttr ib u tes w hich w ould m ak e them "fit" for th e ir en v iron m en t at G uay-

m aso F ir s t , th is a lga grow s w e ll at tem p era tu res of 25 C and above.

T em p era tu res of th is m agnitude do p r e v a il during the su m m er grow ing

sea so n at G uaym as (T ab le 3), Second, th is a lga can p rod u ce m atu re

zy g o sp o re s in the r e la t iv e ly sh o rt p er io d s of a v a ila b le m o is tu r e . In

fa c t, the p r o c e s s of grow th from z y g o sp o re germ in ation to the p ro d u c­

tion of m atu re zy g o sp o re s m ay take only 2 -3 w e e k s . T hird , a r e s is ta n t

zy g o sp o re i s prod u ced w hich r e m a in s v ia b le throughout the long p er io d s

of drought, . Fourth,, zy g o sp o re s exh ib it d ifferen tia l germ in ation , i , e ,

th ey do not a ll g erm in a te during a s in g le w ettin g . T h is type of g e r m in a ­

tion i s advantageous in a llow in g th is a lg a to p e r s is t in th e h a rsh e n v ir o n ­

m ent near G uaym as, F o r exam p le , if a ll z y g o sp o re s g erm in a ted during

an e a r ly ra in and th e pond d ried up again b efore m atu re z y g o sp o res

could be produ ced then th is a lga w ould p e r ish from th is s i t e , , If a study

cou ld be m ade of the s im ila r it ie s and d if fe re n c e s of c u ltu re s of

Sirogonium m elan osp oru m from the th ree d ifferen t s i t e s , a better id ea

of the con cep t of a s p e c ie s in th is genus m igh t be ga in ed , . U nfortunately

cu ltu res from M is s is s ip p i and India h ave not been obtainab le up to the

p r e se n t t im e .

C ell d iv is io n , in th e two c lo n e s of S irogonium w hich w ere

stud ied , i s qu ite siraila-r to c e l l d iv is io n in the genus S p irogyra as

rep o rted by A llen (1958), . C y to k in esis exten ds over m o r e than an hour

in both g en era and it i s a cco m p lish ed by the annular ingrow th of a

57

septum w hich f in a lly cu ts the c h lo r o p la s ts in tw o 0 W hether the a c c o m ­

panying, p r o c e s s of k a r y o k in es is i s s im ila r in th e two g en era is not

known as it i s la r g e ly o b scu red by th e c h lo r o p la s ts of the liv in g m a te ­

r ia l , The n u c le i a re v is ib le only a fter cy to k in es is i s co m p le te .

The s iz e and shape of z y g o sp o re s i s h igh ly v a r ia b le in the

clon a l cu ltu res and th ere i s so m e v a r ia tio n in th o se from the natural

M ex 22 so il sa m p le a ls o . One p o s s ib le explanation for th is v a r ia b ility

i s that optim um growing, con d ition s m ay not a lw ays p r e v a il from one

a rea to another in a pond or from one b o ttle to another in the lab oratory

c u ltu r e s , • E n v iron m en ta l con d ition s m ay a lso change w ith tim e in e ith er

la b ora tory c u ltu re s or a pond as n u tr ien ts a r e a s s im ila te d from and

w a ste s a re e x c r e te d into the m ed iu m . A nother exp lanation i s in h eren t

v a r ia b ility w ithin, th is s p e c ie s . In a c tu a lity both of th e se fa c to r s p ro b ­

ab ly in te r a c t , The p o s s ib il ity of b a c ter ia and fungi in ter fe r in g w ith the

grow th of the c u ltu re s should not be o verlook ed .

The v e r y low p ercen ta g e of g erm in a tio n of z y g o sp o r e s from

cu ltu re i s s t i l l u n reso lv ed , . W hether th is low p ercen ta g e of g e r m in a ­

tion o ccu rred in natural z y g o sp o re s cou ld not be a sc e r ta in e d b eca u se

no sa tis fa c to r y m ethod of counting the num ber of z y g o sp o r e s in a m e a s ­

ured am ount of the M ex 22 so il w as d e v ised , A low p e r ce n ta g e of

g erm in a tio n w as a lso obtained in the gen u s S p irogyra by A llen (1958),

An explanation m ay in v o lv e a g en etic co n tro l m ech a n ism lead ing to

d ifferen tia l germ in ation w hich , a s h as been sta ted e lse w h e r e , m ay

58

confer an advantage on an a lga liv in g in r eg io n s of lim ite d and h igh ly

er r a tic ra in fa lLI '

By far the m o st co m p le te accou nt of the v e g e ta tiv e and sex u a l

p h a ses of th e genus S irogonium i s g iven by D e B ary (1 858} but h is

accou nt i s b a sed e n tir e ly on S irogonium stic ticu m w hich d iffers s o m e ­

what from So m elan osp oru m , e sp e c ia lly in the ev en ts p reced in g the

form ation of the g a m e te s . Both s p e c ie s begin the conjugation p r o c e s s

by the jo in ing to g e th er of two c e l l s w h ich a re s lig h tly bent tow ard one

anoth er. In S. m elan osp oru m th ere i s often produ ced a ra th er pronounced

conjugation tube w h ile in S. s tic ticu m th is tube i s r e p r e se n te d m e r e ly by

slig h t b u lg es . A lthough unequal c e l l d iv is io n g iv e s r i s e to the gam etan g ia l

c e l l s i,n both species^ th e .s m a ll c e l l i s m uch sm a lle r and r em a in s sm a ll

in S. s tic ticu m (the " ste r ile" c e l l of De B ary, 1858) w h erea s it ap p ears

to e lon gate in S. m elan osp oru m and in m o st c a s e s look s v e r y m uch lik e

an ord in ary v e g e ta tiv e c e l l . . In both: s p e c ie s the fe m a le gam etangium

b eco m es g rea tly in fla ted but the m a le gam etangium in f la te s only s lig h tly

or not at a ll . At the sa m e tim e the c h lo r o p la s ts b eco m e sh o rter , w id er ,

and appear to b eco m e lin ked one w ith the other by sh o rt p r o je c tio n s .

In S. m elan osp oru m th e se apparent lin k s a re fo rm ed a s the ch lo r o p la s ts

fragm en t and the p ie c e s b ecom e rea rra n g ed ( s e e F ig . 3). The r eg io n s

around the p y ren o id s b ecom e m o re and m o re d istin c t a s s ta rch g ra n ­

u le s a ccu m u la te . O il d rop lets a re a lso fo rm ed . The n u cleu s b eco m es

e n tir e ly ob scu red . The g a m etes round up b efo re fu sio n o c c u r s . In

59

both s p e c ie s a th r e e - la y e r e d w a ll i s fo rm ed around th e zy g o te but the

c o lo re d m edian w a ll i s sm ooth in So s tic ticu m and v e r r u c o s e in So

m elanosporum o . Both s p e c ie s a r e h o m oth a llic and conju gation tak es

p la ce in the v a r io u s p a r ts of the fila m en t at v e r y d ifferen t t im e s so

that one m ay fin d the e a r l ie s t s ta g e s of conjugation v e r y near m atu re

zygosp oreso

. 13e B ary (1858) gave the fo llow in g accou nt of g a m etic fo r m a -

tion s g erm in a tio n of the zygosp or ea and e a r ly d evelop m en t of the g e r m -

ling in S» stic ticu m (from a tra n s la tio n by F ra u U se H e lle re r ):

Hov?ever, in both, (gam etan g ia ) th e con ten ts gradu ally change th eir c o n s is te n c y . The chi or o p ia sts b eco m e w id er , freq u en tly lin ked by A n astom osen ; «... . «. The in n er c e l l w hich len gth en s into th e form of a tube b rea k s through the en c lo sin g sk in s in a deep v e r t ic le t e a r „ One end r em a in s stuck betw een th e se sk ins; the other w ith i t s f irm w a ll p ro tru d es ca rry in g w ith it the ch lorop h y ll content broken up into ir r e g u la r sh red s w h ich s lo w ly s tr e tc h out in to g reen fils/m en ts. The num erous o il g lo b u les a ttach ed to the ch lo ro p h y ll d isap p ear grad u ally .The l it t le heap s of r e d p igm en t rem a in d is tin c t long after the event of p a rtitio n . . I have not o b se r v e d any n u cleu s in o n e - c e lle d g e r m lin g s . Just a s w ith S p irogyra , h ow ever, a n u cleu s is a lw ays p r e se n t a fter the f i r s t d iv is io n .

. It i s in ter e st in g , to note that De B ary im p lie d at two p o in ts in h is accou n t

of the l ife c y c le of S irogonium stic ticu m that the c h ld ro p ia sts do not

rem a in a s d is tin c t e n t it ie s . F ir s t ,, he sta ted that the ch i or o p ia sts

b ecam e "linked by A n astom osen " during ga m etic fo rm a tio n and secon d ,

that the "ch loroph yll content" i s "bsoken up into ir r e g u la r sh red s"

w hich s tr e tc h out to form the c h lo r o p ia s ts during the grow th of the

g e rm lin g . H ow ever, as w a s m en tion ed in the in trod u ction (p,, II);

60

De B ary (1858) p ic tu red a o n e -c e l le d g erm lin g w ith fu lly d evelop ed

ch lo ro p la stso

, The "Little h eap s of r ed p igm ent" m ay be co m p a ra b le to the

"red-brow n bod ies" in S p iro g y ra d e sc r ib e d in 1852 by No. P r in g sh e im

(A llen , 1 9 5 8 ) 0 T ron d le (1907) c a lle d th e " red -b row n b od ies" of S p iro ­

gyra " K arotin k rista llen " from w hich the cu rren t nam e of ca ro ten o id

gran u les i s d er iv ed . T h a t.th ese b o d ies , in th e genus S p irogyra , a re

the r em a in s of the d is in teg ra ted m a le ch lo ro p la stid w as f ir s t d em on ­

s tr a ted by V® C h m ielev sk y in 1890 acco rd in g to F r its c h (1935)® A lle n

(1958) sta ted that the fe m a le p la s tid r em a in s a s a c o m p r e ss e d green

band® "The m a le p la stid , on the other hand, soon Loses i t s c o lo r ,

b eco m es gran u lar, and ev en tu a lly fra g m en ts into a few groups of

orange ‘c a ro ten o id 8 granules.® " T his p r o c e s s o ccu rs in the zygospore®

L ew is (1925) d e scr ib e d the fo llow in g p r o c e s s : "The m a le gamete® ® ®

does not in c r e a s e notably in d ia m eter , a s d oes the fe m a le g a m ete , and

i s fu rth er d is tin g u ish ed by the fa c t that the c h lo r o p la st d is in te g r a te s

co m p le te ly , lo s in g both co lo r and form® " D is in teg ra tio n of the m a le

p la s tid o c cu rs during g a m e to g e n e s is in th is species of S p iro g y ra a c c o r d ­

ing to Lewis® T ron d le (1907) had the fo llow in g to say about a s p e c ie s

of S p iro g y ra w hich con ta in ed th ree c h lo r o p la s ts per c e l l in the v e g e ta ­

t iv e p h a se (from a tra n s la tio n by F ra u U se H e llerer ):

Quite young zy g o te s con ta in ed s ix d is tin c t bands w hich Looked fu lly lik e th o se of the v e g e ta tiv e cells® The ed ge is n o rm a lly sca llo p ed , the p y ren o id s conta in cop iou s sta rch , and

61

the co lo r i s a beautifu l g reen . A fter so m e fo u rteen d a y s. » .. th ere i s a change. The fin e ind en tation of the ch lo r o p la s ts d isa p p ea rs , and the c h lo r o p la s ts th e m se lv e s b eco m e m o re or l e s s w o r m -lik e , sh o r ter , but th ick er . . . .. Not only the form , h ow ever, but a lso the co lor ch a n ges, at le a s t in p a r t. T hree c h lo r o p la s ts rem a in g reen , w h erea s the th ree o th ers b ecom e r e d d ish -y e llo w , s im ila r to a le a f som ew h at autum n -faded .The c h lo r o p la s ts of lik e c o lo r s each form a group, and, ind eed , th e one i s m a le , the other fe m a le . In a ll c a s e s , in fact, w h ere the conju gation can al w as c le a r ly v is ib le , for exam p le jo in ed la te r a lly to the c h lo ro p la st in p rep a ra tio n , I saw im m ed ia te ly opp osite it s opening, the th ree d is c o lo r e d ch lo ro p la sts ,- w h erea s the o th ers o ccu p ied the op p o site p o s i ­tion in the zygote; w hereupon I con clu ded that the f ir s t a re m a le , the la tter fe m a le . In s t i l l o ld er zy g o te s , the m a le bands d is in teg r a te in to s in g le p ie c e s w h ich form round to long m a s s e s , and under w eak pow er th e se appear d is tin c t ly gran u lar .O b servation under h igh pow er sh ow s'th a t n u m erou s l it t le y e llow c r y s ta ll in e -sh a p e d g ra n u les a re p r e se n t .

In S irogonium m elan d sp oru m , h ow ever, both the m a le and the fe m a le

c h lo r o p la stid s fragm en t during g a m e to g e n e s is and th ere i s no apparent

d is in teg ra tio n or d isc o lo r a tio n of p ia s t id fra g m en ts up to the tim e of

e a r ly zy g o sp o re form a tio n . The car c ten o id gra n u les in S. m e la n o -

sporum m igh t s t i l l be d er iv ed from the d is in teg r a te d fra g m en ts of the

c h lo r o p la s ts from e ith er the m a le or the fe m a le g a m ete or p ortion s of

both, but i f so , the d is in teg ra tio n o c cu rs a fter the o b scu rin g food

r e s e r v e s and z y g o sp o re w a ll have fo rm ed . The author h as no c o r r o b o r a ­

tiv e ev id en ce that the or ang e -r br own g ra n u les seen in m o st germ lin g s of

the Sir ogonium m elan o sporum cu ltu res a r e the r em a in s of c h lo r o p la sts

from e ith er the m a le or the fe m a le g a m e te s . The g ra n u les even tu a lly

b ecom e d isp e r se d by c e l l d iv is io n in th e germ lin g s and a r e no longer

v is ib le a fter 2 -1 0 m ito s e s . They a r e v is ib le , h ow ever, a s a d is c r e te

62

group in the a p ica l c e l l of the v a r io u s s ta g e s of grow th of the cu ltu ra l

germ ling shown in F ig u r e s 11, 12, 13, and 14. . In other germ lin g s ,

both natural and cu ltu ra l, th e se g ra n u les m ay be c o m p le te ly d isp e r se d

and not d is c e r n ib le from the other c e l l con ten ts in the 2 - , 3 or 4 -

c e lle d s ta g e s of grow th (F ig s .. 16, 19, 20).

, It i s p o s s ib le , though th is author has no ev id en ce for it, that

th ese gra n u les r e p r e se n t d is in teg ra tio n p rod u cts of n u clear m a ter ia l

produced during m e io s is in the zy g o te of S irogonium m elah osp oru m , if,

indeed , m e io s is o ccu rs h ere a s it does in s e v e r a l s p e c ie s of S p irogyra

(T ron d le , 1911; Godward, 1961). In any c a s e it i s c er ta in that m o re

w ork n eed s to be done in order to d e term in e the o r ig in and co m p o sitio n

of th e se o ra n g e-h r own g ra n u les in S irogon ium m elanosporum o

That c h lo r o p la s ts can fra g m en t and r e o r g a n iz e under a p p ro p r i­

a te con d ition s, such as grow th in the dark, h as been d em o n stra ted in

the genus E uglen a by W olken (1956,. 1961). It d oes not, th er e fo re , see m

so im p rob ab le that other co n d ition s, such a s th o se a r is in g from g a m e to -

g e n e s is , m ight ind uce ch lor op la s t fra g m en ta tion a lso , a s app arently

o ccu rs in the c u ltu re s of So m elan osp oru m u sed in th is study. . R e o r ­

gan ization of the ch lor o p la sts of E u glen a o ccu rs w hen th is a lga is

retu rn ed to the ligh t. In S. m elan osp oru m the r e o r g a n iz a tio n of the

c h lo r o p la s ts o ccu rs w ith the g erm in ation of the z y g o sp o re and the e a r ly

grow th of the g erm lin g .

63

The o r ig in of p la s tid s a cco rd in g to W eier (1963), h as not y et

b een fu lly so lv ed ev en w ith e lec tr o n m ic r o sc o p y « A study of the u ltr a ­

stru ctu re of S irogonium m elan osp oru tn throughout the p r o c e s s of

ch lo r o p la s tic fra g m en ta tion and reo rg a n iza tio n ;m ig h t w e ll exten d the

p r e se n t know ledge con cern in g the o r ig in of p la s t id s .

It i s in ter e st in g to note that although c r o ss -c o n ju g a tio n does

v e r y r a r e ly o c c u r ,' in d ica tin g that it i s p o s s ib le for ad jacen t c e l l s on a

fila m en t to d ifferen tia te into m a le and fe m a le gam etan g ia , no la te r a l

conju gations w e r e ev er o b serv ed . The p rob lem of s e x d ifferen tia tio n

in S irogonium m elan osp oru m or in the w h ole genus,, for that m a tter ,

n eed s e x te n s iv e study.

SUMMARY

lo . S irogon ium m elan osp oru m (Randhawa) T ra n sea u h as been

found in a pond near G uaym as, Sonora, M exico,,

2» The sex u a l p r o c e s s , conjugation , o c cu rs r e g u la r ly w ith in

the c lo n a l cu ltu res; th is a lga i s , th er e fo re , hom othallico

3o The width of the m atu re f ila m e n ts v a r ie s from 70 to over

80 m ic r o n s in a few in s ta n c e s .

4„ The sh ap es and s iz e s of z y g o sp o re s a r e h igh ly v a r ia b le .

5 . C ultural zy g o sp o re s can be in d u ced to g erm in a te by drying

them at room tem p era tu re for s e v e r a l w eek s and then w ettin g them w ith

s o il-w a te r supernatan t. L e s s than 5% g erm in ation o c cu rs u sin g th is

m ethod but no other m eth od w as d e v ise d w hich w ould in c r e a s e th is low

g erm in a tio n .

6 . O ils and e sp e c ia lly s ta rch a r e a ccu m u la ted during ga m etic

form ation and th e s e food r e s e r v e s a r e abundant in the zy g o sp o re and

the young g erm iin g a s w e ll.

7. F ra g m en ta tion of the c h lo r o p la s t o ccu rs during the fo r m a ­

tion of both of the g a m e te s . The c h lo r o p la s ts r em a in a s fra g m en ts

throughout the p er io d of ga m etic fu sio n , zygo te m atu ration and d o r­

m an cy , z y g o sp o re g erm in a tio n and into the e a r ly s ta g e s of d evelop m en t

of the g erm iin g .

64

65

8 o , R eo rg a n iza tio n of the c h lo r o p la s ts b eco m es apparent at

about the 2 - to 4 - c e l le d s ta g e of g erm lin g develop m en t,

9» The f ir s t s ta g e s in the reo rg a n iza tio n of the c h lo r o p la sts

a r e f in g e r - lik e p ro jec tio n s of c h lo r o p la s tic fra g m en ts extending from

both s id e s of a c en tr a lly lo c a te d m a ss of the sam e ch lor o p la stic m a te -

rialo A s reo rg a n iza tio n con tin u es the cen tra l m a ss d im in ish es in s iz e

and the r ib b on lik e c h lo r o p la s ts of th e m atu re c e l l of S irogonium a re

produ ced ,

10o . R eo rg a n iza tio n of th e c h lo r o p la s t i s quite s im ila r in both

the natural and the cu ltu ra l germ lin g s ,

11, The c h lo r o p la s ts a re re la tiv e ly , w ide w ith cren u la ted m a r ­

g in s and tightly, sp ir a lle d w hen they a r e f ir s t fo rm ed but a s the c e ll

e lo n g a tes the c h lo r o p la s ts un coil and appear to extend ,

12, . A grad ation from p o o r ly to h igh ly o rg a n ized c h lo r o p la sts

can be see n from the a p ica l tow ard th e b a sa l c e l l s , r e s p e c t iv e ly , in 1 0 -

to 5 0 -e e l le d g e rm lin g s ,

13, . O range-brow n gra n u les a r e p r e se n t in th e e a r ly s ta g e s of

the d evelop m en t of the g e rm lin g s in the two cu ltu res o f S irogonium u sed

in th is study. T hey m ay be co m p arab le to the " ca ro ten o id 11 gran u les

w hich in a few s p e c ie s of S p irogyra h ave b een shown to be the rem a in s

of the d is in teg r a te d m a le c h lo r o p la s ts ,

14, . C ell d iv is io n does o c c a s io n a lly occu r in the b a sa l c e l l of

Sirogon iu m ,

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