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Chromosomal behaviour during meiosis in mosses Dill, Frederick John 1964

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CHROMOSOMAL BEHAVIOUR DURING MEIOSIS IN MOSSES  by  FREDERICK JOHN D I L L B.Sc,  The U n i v e r s i t y  o f B r i t i s h C o l u m b i a , 1961  A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department of B i o l o g y and B o t a n y  We a c c e p t t h i s required  thesis  as c o n f o r m i n g t o t h e  standard  THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t , 1964  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the requirements for an advanced degree at the U n i v e r s i t y  of  B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference  and  study.  I f u r t h e r agree that  per-  mission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the Head of my Department or  his representatives.  I t i s understood that, copying or p u b l i -  c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not without my  written  Department of  permission*  Biology and Botany  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8. Canada Date  by  August 15. 1964.  be  allowed  i i  ABSTRACT  The  primary purpose  o f t h e p r e s e n t work was  t o examine  m e i o s l s i n mosses, c o n c e n t r a t i n g on t h e d e t a i l o f p r o p h a s e  I,  a m e i o t i c s t a g e w h i c h has "been d e s c r i b e d i n d e t a i l o n l y i n Pleurozium schreberi*  S e c o n d a r i l y , an i n v e s t i g a t i o n was  taken to study the e f f e c t  of heat  Hypnum c i r c i n a l e Hook, and  s t r e s s on m e i o s l s .  Brachythecium  B e s c h . were u s e d t o s t u d y m e i o s i s . The s p o r e mother c e l l et a l .  (1954),  squashes  was  under-  frigidum  procedure  (CM.)  of preparing  s i m i l a r t o t h a t u s e d by  Steere  with m o d i f i c a t i o n s i n handling the plants  to f i x a t i o n t o ensure  a g a i n s t p r o d u c t i o n o f heat  prior  induced  anomalies. Except med  to that  f o r l a t e prophase f o u n d i n P.  f r i g i d u m d i p l o t e n e was resulted i n a diffuse analogous  I, meiosis i n both species c o n f o r  s c h r e b e r i . I n H.  circinale  B.  f o l l o w e d by chromosome e l o n g a t i o n and stage.  T h i s stage i s m o r p h o l o g i c a l l y  t o the d i c t y o t e n e stage o f the growing  many a n i m a l s , and  and  appears  oocytes  of  t o have b e e n d e s c r i b e d , i n p l a n t s ,  only i n Balsamina h o r t e n s i s .  I t i s probable that  the  stage  o c c u r s i n many moss f a m i l i e s ;  at present i t s f u n c t i o n a l  signi-  f i c a n c e i s unknown. In the heat  stress  experiments,  p l a n t s o f H.  circinale  were e i t h e r m a i n t a i n e d u n d e r l a b o r a t o r y t e m p e r a t u r e s w h i l e b e i n g s t u d i e d o r t h e y were t r e a t e d w i t h a h e a t period  shock  over  o f f o u r o r s i x h o u r s w i t h t h e maximum t e m p e r a t u r e  g e n e r a l ranges a t u r e was  of  25°C , 31°C,  maintained f o r  4  hours  and  36°C  i n the  a i n the  The maximum t e m p e r -  25°C  experiment  and  iii  1/2 hour i n the remaining experiments. g r a d i e n t s were almost equivalent  The h e a t i n g and c o o l i n g  (l°C./5 min.), and the  s t a r t i n g and f i n i s h i n g temperature was 14°C.  The  temperature  of the n a t u r a l environment d u r i n g the study ranged between 7-ll°C Severe anomalies, i n c l u d i n g chromosome clumping and m u l t i p l e a s s o c i a t i o n , precocious d i s j u n c t i o n , chromosome c o n t r a c t i o n , s p i n d l e breakdown and i n h i b i t i o n , premature m e i o t i c i n d u c t i o n and meiotic a b o r t i o n were observed to some extent i n spore mother c e l l s from a l l treatments except the ones from the 25°C. heat shock  experiment.  Room temperature a c c e l e r a t e s prophase I stages of H. circinale .  The time a v a i l a b l e f o r these stages appears to be  too b r i e f f o r s y n t h e s i s of necessary products l e a d i n g to --active stages, thus causing severe a b n o r m a l i t i e s which r e s u l t i n a b o r t i o n of m e i o s i s . On the b a s i s of these r e s u l t s , i t i s apparent that c y t o l o g i s t s working w i t h moss m a t e r i a l should take care i n h a n d l i n g the p l a n t s p r i o r to f i x a t i o n to ensure against heat induced meioticanomalies.  vii  ACKNOWLEDGEMENTS  I  w i s h t o e x t e n t my g r a t i t u d e t o my d i r e c t o r , D r . W. ,B.  Schofield f o r h i s criticism  and d i s c u s s i o n o f t h e r e s e a r c h a n d  for  a s s i s t a n c e i n p r e p a r i n g the. m a n u s c r i p t ; t o D r . K. I . B e a m i s h  for  r e a d i n g t h e m a n u s c r i p t and f o r many d i s c u s s i o n s  regarding  heat induced m e i o t i c anomalies i n n a t u r a l p o p u l a t i o n s o f p l a n t s ; to  Dr. J . K u i j t  f o r r e a d i n g t h e m a n u s c r i p t ; and t o t h e D e p a r t -  ment o f B i o l o g y a n d B o t a n y ities.  f o r t h e u s e o f equipment  I w i s h t o t h a n k D r . J . R. M i l l e r  and f a c i l -  o f t h e Department o f  P a e d i a t r i c s , U n i v e r s i t y o f B r i t i s h Columbia, f o r t h e use o f h i s Z e i s s Photomicroscope, iron-proprionic  using  carmine t o s t a i n m e i o t i c n u c l e o l i . I a l s o  acknowledge r e c e i p t ty  a n d Mr. L . H a n i c who s u g g e s t e d  o f a Queen E l i z a b e t h S c h o l a r s h i p  wish  (Universi-  o f B r i t i s h C o l u m b i a ) , 1961-1962, w h i c h was d o n a t e d b y H. R.  M a c M I l l a n ; and o f a N a t i o n a l R e s e a r c h C o u n c i l S t u d e n t s h i p , 1962, to  w h i c h was w a i v e d . F i n a l l y ,  I w i s h t o e x p r e s s my  my w i f e f o r t y p i n g t h e m a n u s c r i p t .  appreciation  iv  TABLE OF CONTENTS  ABSTRACT'  i i  TABLE OF CONTENTS  iv  ACKNOWLEDGEMENTS  v i i  INTRODUCTION CHAPTER 1 ,  1  Meiosis frigidum  i n Hypnum c i r c i n a l e Hook, and B r a c h y t h e c l u m (C.M.) B e s c h  3  METHODS The  '  4  H a n d l i n g and P r e p a r a t i o n o f M a t e r i a l f o r C y t o l o g i c a l  Study  4  Material  6  OBSERVATIONS  7  Meiosis  i n Hypnum c i r c i n a l e  Meiosis  i n Sporangium  Meiosis  i n Brachythecium f r i g i d u m  #6,  7.  Population  #2463  12 13  DISCUSSION  .16  L e p t o t e n e and Z y g o t e n e  17  P a c h y t e n e - D i p l o t ene  18  The  19  E l o n g a t i o n Stage  D i a k i n e s i s and t h e Remainder o f M e i o s i s CHAPTER  2,  The  Effect  o f Heat S t r e s s on M e i o s i s  c i r c i n a l e Hook  22 i n Hypnum 25  METHODS  29  Methods o f T r e a t i n g t h e P l a n t s  29  Methods o f A n a l y s i n g T r e a t e d  31  Plants  Sampling  31  Analysis  32  Materials  33  V  RESULTS.  .34  E x p e r i m e n t #1  .'  34  E x p e r i m e n t #2  38  E x p e r i m e n t #3  40  E x p e r i m e n t #4  44  E x p e r i m e n t #5  47  E x p e r i m e n t #6  51  E x p e r i m e n t #7....  54  DISCUSSION (1)  57  Chromosomal B e h a v i o u r o f Mosses u n d e r  Laboratory  Conditions (2) The  S e n s i t i v i t y o f Moss SMC  (3) N a t u r a l  Occurring  Inducing of the (4)  The  58 t o S l i g h t Heat  Stress....59  Heat S t r e s s as a Mechanism  Mutation or Structural/Numerical  of  Alterations  Chromosomes  61  Mode o f R e s p o n s e o f t h e  C l u m p i n g and  SMC  t o Heat S t r e s s  M u l t i p l e A s s o c i a t i o n of the  62  Bivalents...63  Extreme C o n d e n s a t i o n o r C o i l i n g o f the B i v a l e n t s  65  Precocious  66  Spindle  Disjunction  Destruction  A s y n c h r o n y and  or I n h i b i t i o n ,  Premature M e i o t i c  Prophase  Induction  66  SUMMARY.  69  LITERATURE CITED  70  APPENDIX PLATES  1-9  vi  L I S T OF TABLES T a b l e I , T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #1.....35 Table I I , Tetrad analysis o f control sporangia o f p o p u l a t i o n #1363 w h i c h were u s e d i n e x p e r i m e n t s  1 and 2  .36  T a b l e I I I , T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #2...39 T a b l e I V , T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #3....41 T a b l e V, T e t r a d a n a l y s i s o f c o n t r o l s p o r a n g i a o f p o p u l a t i o n #2267 w h i c h were u s e d i n e x p e r i m e n t #3  42  T a b l e V I , T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #4....46 Table V I I , T e t r a d a n a l y s i s o f sporangia from p o p u l a t i o n # 2463 w h i c h were u s e d i n e x p e r i m e n t s 4-7  46  T a b l e V I I I , T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #5..49 Table IX, T e t r a d a n a l y s i s o f s p o r a n g i a o f experiment  #6....52  T a b l e X, T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #7, p l a n t s under l a b o r a t o r y c o n d i t i o n s  55  T a b l e X I , T e t r a d a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t #7, p l a n t s under normal p h o t o p e r i o d  56  T a b l e X I I , A g e n e r a l summary i n d i c a t i n g t h e mode o f a c t i o n of d i f f e r e n t  c a t e g o r i e s o f h e a t s t r e s s on t h e  SMC o f H. c i r c i n a l e  64  1 INTRODUCTION The  volume o f l i t e r a t u r e  p e r t a i n i n g t o chromosomal  and n u c l e a r b e h a v i o u r i n mosses i s s m a l l when compared t o c o n c e r n i n g h i g h e r p l a n t s and many o f t h e e a r l i e s t  animals.  works i n t h e f i e l d  It i s significant  that that  o f c y t o l o g y and  cyto-  g e n e t i c s i n mosses made v a l u a b l e c o n t r i b u t i o n s t o t h e s t u d y o f plant  c y t o l o g y and  cytogenetics i n general.  t h e s e c o n t r i b u t i o n s was initiated  (1917, 1919)  t h e s t u d y o f sex chromosomes i n p l a n t s and  the Marchals produced  t h e work o f A l l e n  O u t s t a n d i n g among  (1907  e t s e q . , c i t e d by A l l e n , 1935)  artificial  p o l y p l o i d s i n mosses and  work w i t h c y t o l o g i c a l  evidence.  Bryophyte  by H e i t z (1928, c i t e d  by S t e e r e e t a l . , 1954)  on n u c l e a r b e h a v i o u r i n w h i c h he chromatin",  and  furthered  who  that  who  first  supplemented  m a t e r i a l was  genetical,  i n his studies  i n t r o d u c e d the term  "hetero-  t h e s t u d y o f sex chromosomes i n p l a n t s .  bryophytes.  In the l a s t  twenty  developed  s u b s t a n t i a l l y and  with establishing  s t u d y i n g chromosomal b e h a v i o u r .  (1961) have r e v i e w e d  this period  and  most  chromosome  Mehra and  quite c r i t i c a l l y  a l t h o u g h t h e t r e n d has  chromosome number and  been s h i f t e d  Khanna  and have  i s noteworthy  that  ind-  to the study  i n some c a s e s b e h a v i o u r , t h e r e a r e  a p p l i c a t i o n s of these f i n d i n g s to c l a r i f y It  (1935, 1945)  years, c y t o l o g i c a l study of wild  p u b l i c a t i o n s have been c o n c e r n e d  icated that  by A l l e n  (i960).  p o p u l a t i o n s o f mosses has  numbers and  contri-  T h i s e a r l y work, w h i c h i s m a i n l y  i s adequately reviewed  more r e c e n t l y by L e w i s  their used  S e v e r a l o t h e r a u t h o r s have a l s o made n o t a b l e c y t o l o g i c a l butions u t i l i z i n g  of  taxonomic  of  rare  problems.  a l t h o u g h most o f t h e chromosome  2 counts r e p o r t e d f o r mosses have been obtained from the study of m e i o t i c metaphases, only r a r e l y has the d e t a i l of the m e i o t i c process been s t u d i e d . a moss was  One  of the e a r l i e s t s t u d i e s of meiosis i n  that of Wilson (1909), who  account of m e i o s i s i n Mnium hornum.  presented a f a i r l y  detailed  He was l i m i t e d , however, by  poor techniques and apparently d i d not a c c u r a t e l y observe some of the e a r l y prophase stages.  In 1932,  Scheuber attempted  to d e s c r i b e  meiosis i n Timmia c u c u l l a t a but f a i l e d to observe many of the stages i n prophase I .  Not u n t i l the work of Vaarama (1954a,1954  b) were the d e t a i l s of meiosis i n mosses ( e s p e c i a l l y those of prophase I) w e l l documented and i n h i s d e s c r i p t i o n of meiosis i n Pleurozium s c h r e b e r i . Vaarama (1954b) has presented the most complete  d e t a i l s of meiosis i n mosses to date.  The purpose of the present study was d e t a i l e d account more mosses.  p r i m a r i l y to provide a  ( e s p e c i a l l y of prophase I) of meiosis i n one or  The need f o r such a study i s obvious because many  workers have conducted  cytotaxonomical s t u d i e s w i t h a l i m i t e d know-  ledge of meiosis i n the p l a n t s they were s t u d y i n g .  A  secondary  purpose grew from observations made d u r i n g the p r e l i m i n a r y i n v e s t i g a t i o n to t h i s study and a l s o as a consequence of. s e v e r a l observat i o n s made d u r i n g a previous study  ( D i l l , unpublished d a t a ) .  These  observations had suggested t hat b r i n g i n g mosses i n t o the l a b o r a t o r y , r  even f o r a short p e r i o d , may There was  cause anomalous chromosomal behaviour.  a l s o some i n d i r e c t evidence from the l i t e r a t u r e which  supported these o b s e r v a t i o n s . 1954;  Bryan, 1956  Since s e v e r a l authors (Steere et a l . ,  et seq.) had suggested b r i n g i n g mosses i n t o the  l a b o r a t o r y t o speed maturation of the capsules, i t was i n v e s t i g a t i o n of the e f f e c t s of temperature warranted.  f e l t that an  s t r e s s on mosses  T h i s matter i s d i s c u s s e d i n Chapter  2.  was  3  CHAPTER 1 Meiosis i n Hypnum c i r c i n a l e Hook, and Brachythecium f r i g i d u m (CM.) Besch. In the pioneering studies concerning meiosis i n mosses the workers were severely hampered by a l i m i t a t i o n i n techniques available.  A l l of the work was performed by the embedding and  s e c t i o n i n g technique; the f i x a t i o n was poor and many a r t i f a c t s were introduced. H e i t z (1928, c i t e d by Vaarama, 1949) developed the squash technique f o r mosses, a technique which has been used almost e x c l u s i v e l y since that time. E a r l y i n the present i n v e s t i g a t i o n , attempts were made t o study meiosis i n several moss taxa.  I t became apparent that not  a l l would be s u i t a b l e , as i n many the prophase I chromosomes d i d not s t a i n w e l l or they were seldom observed.  This was e s p e c i a l l y  t r u e of members of the Grimmiaceae and some other acrocarpous families.  Vaarama (1949) found t h i s to be true when he attempted  to study meiosis i n several members of the Grimmiaceae.  I t was  soon discovered that the pleurocarpous mosses, e s p e c i a l l y the Hypnaceae (sensu-lato), had prophase I stages that could be e a s i l y stained.  The chromosome number was also an important f a c t o r to be  considered, as obviously a low number would permit much more accurate observations of the i n d i v i d u a l chromosomes during prophase I.  For these reasons, Hypnum c i r c i n a l e Hook, and Brachythecium  frigidum (C.M.) Besch. were chosen. cytologically studied.  Neither species had been  4 METHODS The  H a n d l i n g and Preparing  s t u d y was  Preparation  slides  of M a t e r i a l f o r C y t o l o g i c a l Study:  o f s p o r e mother c e l l s  e s s e n t i a l l y the  technique of Steere  However, c a r e f u l c o n s i d e r a t i o n was  given  p r i o r to f i x a t i o n to prevent heat induced Chapter  the  l a b o r a t o r y as  the  p l a n t s t o as l i t t l e  were t h e n put biological  in flats  science  supplied. during  outside high  to the h a n d l i n g  of  abnormalities  (see  As  i n a shaded a r e a  building.  plants  An  most o f t h e  as t h e r e  was  was  and  LaCour  was  (i960).  This  page  13  the  the humidity  was  day. collection the  A small portion of t h i s material  was  any  m a t e r i a l brought i n from minutes.  s t a i n used f o r the  present  t o t h e method d e s c r i b e d  i s the  only  s t a i n t h a t has  f a c t o r y f o r chromosome work w i t h m o s s e s .  c f . footnote  coll-  i m m e d i a t e l y hung o u t s i d e  only  prepared according  s t u d y were  every  i f u n u s e d a f t e r 10-15  the  simulate  s t u d i e d , a p o r t i o n of the  u s e d when s l i d e s were t o be made and  Aceto-orcein  the  November o f 1963,  some p r e c i p i t a t i o n a l m o s t  discarded  They  Adequate water  plants f o r this  b r o u g h t i n t o t h e l a b o r a t o r y and  t h e b a s k e t was  made t o  between 7 - l l ° C , and  window i n a shaded b a s k e t .  r o o f on  to  subject  possible.  of the  a t t e m p t was  t h e months o f O c t o b e r and  were b r o u g h t  attempt t o  humidity conditions.  t e m p e r a t u r e was  and  hags and  t e m p e r a t u r e change as  . When p l a n t s were t o be was  meiotic  et a l . (1954).  q u i c k l y as p o s s i b l e , i n an  n a t u r a l t e m p e r a t u r e and  ected  for  2).  P l a n t s were c o l l e c t e d i n p l a s t i c  was  (SMG)  1  by  study  Darlington  proven  P r e - f i x a t i o n of  satisSMC  5 was  u s u a l l y done w i t h "3:1 a l c o h o l - a c e t i c  acid.  S q u a s h p r e p a r a t i o n s a r e made as f o l l o w s :  plants bearing  b r i g h t green mature-sized capsules are s e l e c t e d ection.  A sporophyte  from the  i s s e v e r e d f r o m t h e gametophyte and  ferred to a clean slide  on t h e s t a g e o f a d i s s e c t i n g  operculum  the capsule u s i n g s l i g h t a differentiated  rolled  toward  downward p r e s s u r e .  archesporium  this will  cause  and  t h e SMC  are spread s l i g h t l y  p e n e t r a t i o n of the f i x a t i v e spreading procedure  arid s t a i n .  almost  applied  i s set aside.  and t h e s l i d e  (about 5 minutes  Immediately  mass  i s generally  to the  SMC.  of s t a i n i s  Before the s t a i n begins to  A f t e r about  5 minutes, and  T h e • c o v e r s l i p i s then sealed with a (1:1)  s e a l i n g medium.  s a t i s f a c t o r y at t h i s  Although h e a t i n g the s l i d e  chromosomes ( A l - A i s h and  intensifies  Anderson,  pressure  spreading  semiStain  inten-  time.  there i s . evidence that h e a t i n g the s l i d e  found  rapid  after application) a clean coverslip i s  permanent g u m - m a s t i c - p a r a f f i n sity  from  following  a p p l i e d t o t h e c o v e r s l i p , t h u s f l a t t e n i n g t h e SMC  t h e chromosomes.  be  will  even and  i s applied  evaporated, the drop  g e n t l y a p p l i e d to the p r e p a r a t i o n .  to  t h e SMC  to ensure  a drop o f f i x a t i v e  When t h e f i x a t i v e has  is  the c o l u m e l l a to  B o t h t h e c o l u m e l l a and'empty c a p s u l e a r e removed  the s l i d e  dry  t h e mouth o f  In capsules possessing  e x t r u d e d t h r o u g h t h e mouth.of t h e c a p s u l e and  this  calyptra  a r e removed u s i n g a d i s s e c t i n g n e e d l e w h i c h i s t h e n  p l a c e d a t t h e b a s e o f t h e c a p s u l e and  follow.  trans-  microscope.  H o l d i n g t h e base o f the c a p s u l e w i t h f o r c e p s , b o t h the and  coll-  1961).  and  causes The  speeds  staining,  clumping  of the  present author  has  t h i s t o be t r u e w i t h some s p e c i e s , t h u s o m i t t e d t h i s  procedure  keep t h e chromosomes as c l o s e t o t h e i r n a t u r a l s t a t e as  possible.  Permanent s l i d e s  o f some o f t h e m a t e r i a l o f e a c h p o p u l a t i o n  6  were prepared according to the method of Darlington and LaCour (I960). A l l b r i g h t f i e l d observations were made using an Olympus b i n o c u l a r compound microscope with N.A. = 1.40 Ach. Condenser, a 125 mm.  o i l immersion o b j e c t i v e and X10 o c u l a r s .  Photographs  and phase contrast work were done on a Zeiss Photomicroscope. Material: M a t e r i a l of Hypnum c i r c i n a l e Hook, was c o l l e c t e d from three populations:  A l i c e Lake, B. C , D i l l #1363; U n i v e r s i t y of B r i t i s h  Columbia Endowment Lands, Vancouver, B.C., D i l l #2263; and Mt. Seymour, B.C. (2000 f t . ) , D i l l #2463.  Brachythecium frigidum  (CM.) Besch. was c o l l e c t e d from one population: B.C. (2000 f t . ) , D i l l #2563.  Voucher  Mt. Seymour,  specimens have been  deposited i n the U n i v e r s i t y of B r i t i s h Columbia Herbarium.  This specimen was determined by Harold Robinson, Smithsonian I n s t i t u t i o n , Washington, D.C.  7 OBSERVATIONS M e i o s i s i n Hypnum  circinale:  Observations were made a t t h e as t h e  of meiosis  angium  2).  m a t e r i a l i n the heat  I t was  e x c e p t i o n o f one  #2463, s p o r a n g i u m #6)  p o p u l a t i o n s were c y t o l o g i c a l l y and They d i f f e r e d  s t r e s s experiments  found, t h a t , w i t h the  ( a member o f p o p u l a t i o n  able.  circinale  same t i m e t h e r e s p e c t i v e p o p u l a t i o n s were u s e d  experimental  (Chapter  i n e a c h p o p u l a t i o n o f H.  morphologically  only i n the maturation  those  o f #1363 and  #2262.  a l l three  indistinguish-  time of the  I n g e n e r a l , members o f p o p u l a t i o n #2463 matured two weeks l a t e r t h a n  to  capsules. three  This difference  a t t r i b u t e d t o t h e d i f f e r e n c e i n t h e a l t i t u d e s where t h e populations  occurred  i n nature.  2000 f t . w h i l e #1363 t h i s reason  and  the  Number 2463 was  #2263 were c o l l e c t e d  i s considered  t o be  was  three  collected  at  at sea l e v e l .  the d e s c r i p t i o n of the m e i o t i c process  u l a t i o n s i s combined and  spor-  of these  For pop-  representative for  species. The  a r c h e s p o r i a l l a y e r o f H.  when t h e  capsules  c i r c i n a l e has  r e a c h mature s i z e ,  but  before  differentiated there i s  i n d i c a t i o n o f browning of the annulus.  When t h e  cells  q u a d r a t e and  with  are f i r s t  aceto-orcein.  a p p e a r s t o be and,  differentiated No  initiated  i n most s p o r a n g i a ,  until  the  slide  a p p e a r t o be On  initiation  initiation  they  are  spore  mother  stain  1,  i n a l l the  SMC  of a capsule  Meiosis  simultaneously  p r o c e e d s i n an a l m o s t s y n c h r o n o u s manner  of d i a k i n e s i s .  at the  same  of meiosis  f i g . 1-3)•  weakly  internal structure i s discernable.  Thus a l l SMC  i n each  prepared  stage. SMC  become s p h e r i c a l and  are  c h a r a c t e r i z e d by an a c e n t r i c a l l y p l a c e d , w e a k l y s t a i n i n g mass ( P L .  any  At t h i s  stage,  which i s  probably  chromatin  8 l e p t o t e n e , the chromatin The  i s a mass o f v e r y e n t a n g l e d  threads are d e f i n i t e l y  s i n g l e and i n r a r e c a s e s chromonemata  c a n be d i s c e r n e d ( P L . 1, f i g . 3 ) . e n t i r e nucleus in  some c e l l s  the width  I t i s q u e s t i o n a b l e whether t h e  o r only the chromatin threads  of the c e l l  o f chromatin  i s a c e n t r i c a l l y placed, f o r  a p p e a r t o t r a v e r s e up t o  ( P L . 1, f i g . l ) .  While  a c e n t r i c a l l y p l a c e d s y n a p s i s o c c u r s ; o f t e n SMC which the threads to  those  threads.  are double  but c e l l s  of the leptotene stage.  the chromatin i s c a n be o b s e r v e d i n  are otherwise  Evidence  o b t a i n e d by phase c o n t r a s t o b s e r v a t i o n .  2/3  identical  f o r synapsis  was  One SMC was n o t e d i n  w h i c h two s i n g l e t h r e a d s p a i r e d f o r a s h o r t p o r t i o n o f t h e i r length, while the unpaired fig.  2).  Other  cells  s e c t i o n s were w i d e l y s e p a r a t e d  ( P L . 1,  o f t h e same s l i d e h a d m o s t l y s i n g l e - t h r e a d e d  chromatin. When s y n a p s i s i s c o m p l e t e t h e chromosomes become s h o r t e r and  thicker.  migrates  A t t h e same t i m e , t h e c h r o m a t i n  to the center of the c e l l  during this  time  ( P L . 1, f i g . 4-9).  As p a c h y t e n e p r o c e e d s ,  t h e chromosomes  and t h i c k e n , and t h e d i p l o t e n e phase a p p e a r s (PL.  1,  f i g . 10,11;  of  homologs becomes e v i d e n t  In  r a r e SMC, a l l s i x b i v a l e n t s a r e c l e a r l y  (PL. 2, f i g . 2)  Chromosomes  assume a t y p i c a l p a c h y t e n e m o r p h o l o g y , ' b u t no  n u c l e o l i are evident. ten  mass l o o s e n s and  as t h e r e p u l s i o n  PL2.  fig.  i s heterochromatic,  ( P L . 1, f i g . 11).  1,2).  discernible at diplotene  b u t u s u a l l y o n l y one o r two a r e s e p a r a t e d  t h e chromosomal mass  shor-  from  The s m a l l e s t b i v a l e n t ,  which  o f t e n becomes h i g h l y c o n d e n s e d d u r i n g p a c h y -  t e n e and d i p l o t e n e ( P L . 1, f i g . 10; P L . 2, f i g . 2 ) .  In addition,  t h e r e a r e c e r t a i n segments o f o t h e r chromosomes w h i c h a r e h e t e r o chromatic fig.  11).  and s t a i n d e e p l y d u r i n g some p e r i o d o f d i p l o t e n e , ( P L . 1, However, i t was n o t p o s s i b l e t o i d e n t i f y t h e chromosomes  9 by these regions as they were seldom w e l l enough separated from the mass to make accurate observations then.  Occasionally  n u c l e o l i could be observed during diplotene (PL. 2, f i g . 2 ) , but generally they stained e i t h e r too poorly t o be seen or were obscured i n the chromosomal mass. On the basis of the diplotene stage as shown i n PL. 2, f i g . 2, i t i s apparent that the nucleol a r organizing chromosome i s one of the l a r g e r b i v a l e n t s , (probably the second l a r g e s t ) .  I t i s possible that the small  heterochromatic  b i v a l e n t i s also associated with the nucleolus. The degree of c o i l i n g of the chromosomes during diplotene i s not as great as that found i n most p l a n t s .  The b i v a l e n t  separated from the mass shown i n PL. 1, f i g . 11, represents the highest degree of c o i l i n g observed during diplotene.  The b i v a l e n t  shown i n PL. 2, f i g . 1 (highly squashed c e l l ) i s probably the l a r g e bivalent which has such a d i s t i n c t i v e morphology at metaphase I . Diplotene was the most commonly observed prophase stage during the time the populations were studied.  I t was d i f f i c u l t  to estimate the period of time the diplotene p e r s i s t s under n a t u r a l conditions, but from the large number of sporangia found i n t h i s stage during the period of study (about 2 weeks) i t appears that t h i s stage may p e r s i s t as long as a week. Hear the end of diplotene the b i v a l e n t s become l e s s w e l l defined (PL. 2, f i g . 3 ) , and chromosomes gradually lengthen and l o s e t h e i r discreteness, at which time the nuclear  contents  assume an i n t e r p h a s e - l i k e appearance (PL. 2, f i g . 4,5). Under phase contrast, f i n e chromatin threads and what appear to be chiasmata were v i s i b l e .  During t h i s phase some short sections  of b i v a l e n t members were seen (PL. 2, f i g . 4 ) , suggesting that  10  the d i p l o t e n e s t r u c t u r e and at  o f t h e chromosomes had  been  t h a t t h e chromosomes were i n an u n c o i l e d s t a t e . this  body.  s t a g e u s u a l l y c o n t a i n one As i n t h e e a r l i e r  large deeply s t a i n i n g  s i n c e t h e c h r o m a t i n was  to the p e r i p h e r y of the c e l l  slightly Up  s m a l l e r t h a n t h e SMC t o and  divisions  cells chromatin  o f one  A l t h o u g h most s p o r a n g i a had  t h e n u c l e u s was  distingspread  probably  only  itself.  including this  o f t h e SMC  The  s t a g e s , no n u c l e a r membrane was  u i s h a b l e d u r i n g t h i s phase, but almost  maintained  chromosome e l o n g a t i o n s t a g e ,  capsule are almost  the  synchronous.  some t r a n s i t i o n s t a g e s  (eg.  pachytene-  d i p l o t e n e or d i p l o t e n e - e l o n g a t i o n stage) the m a j o r i t y o f the appeared is two  lost. days,  i n t h e same s t a g e . Each c e l l  t h e n each may  o r d e r by a r a p i d some SMC  may  From t h i s  remain  s t a g e on, however,  synchrony  i n t h e e l o n g a t i o n s t a g e an  estimated  proceed  t o the next  s t a g e i n a random  condensation of the b i v a l e n t s .  p o s s e s s i n g condensed b i v a l e n t s  o t h e r c e l l s were e i t h e r i n l a t e r the elongation stage.  Only  d i p l o t e n e and  In sporangia with  (ie. diakinesis), a l l  s t a g e s o f m e i o s i s o r were i n  i n extremely  r a r e cases d i d  s t a g e s o f p r o p h a s e o c c u r a f t e r c o n d e n s a t i o n had observation c l e a r l y  SMC  begun.  earlier This  i n d i c a t e s that the e l o n g a t i o n stage f o l l o w s  t h e r e f o r e cannot  be i n t e r p r e t e d  as a p r e d i p l o t e n e  stage. Condensation  or c o i l i n g o f the b i v a l e n t s c o u l d not  f o l l o w e d a c c u r a t e l y s i n c e t h i s appears In  some c e l l s ,  t h e c h r o m a t i n was  (PL. 2, f i g . 6 ) . s t a g e and  diffuse  and  i n one  rapidly.  l a r g e mass  T h i s s t a g e o c c u r r e d between t h e e l o n g a t i o n  diakinesis,  o c c u r r e n c e i t may  to proceed very  be  b u t b e c a u s e o f i t s i r r e g u l a r and i n f r e q u e n t  r e p r e s e n t an a b n o r m a l o r p o o r l y f i x e d e l o n g a t i o n  11  stage.  W i t h o u t f u r t h e r s t u d y i t c a n n o t be a c c e p t e d as a  occurrence i n the meiotic Diakinesis  i s characterized  h i g h l y condensed b i v a l e n t s the  cell.  bivalents  sequence o f Hypnum  In conjunction  circinale.  by a g e n e r a l  clumping o f the  ( P L . 2, f i g . 7,8) i n t h e c e n t e r with spindle  configuration.  Metaphase I o f Hypnum c i r c i n a l e i s c h a r a c t e r i z e d i n c l u d i n g f o u r w h i c h a r e medium i n s i z e , as s m a l l e r  l a r g e and m o r p h o l o g i c a l l y  ( P L . 2, f i g . 9 ) .  by s i x b i v a l e n t s  one w h i c h i s u s u a l l y  t h a n t h e r e s t and one w h i c h i s v e r y distinct.  t h i s l a r g e b i v a l e n t was l o c a t e d plate  In the majority  of c e l l s  a t one s i d e o f t h e metaphase I  Disjunction  of the b i v a l e n t s  usually  occurred  quite  synchronously  bivalent  often  s l i g h t l y preceding the others to the poles.  completion of the f i r s t and  which u s u a l l y contains  ( P L . 2, f i g . 10) w i t h t h e l a r g e  division  assume a c o n f i g u r a t i o n  s i m i l a r t o prophase  appears a f t e r telophase  The chromosomes a t metaphase I I a r e a g a i n  a chromosome c o u n t be made a t t h i s  stage.  Only r a r e l y can A t anaphase I I t h e  chromosomes a p p e a r t o be s t i c k y and t e m p o r a r y b r i d g e s ( P L . 2, f i g . 1 2 ) .  abnormal s e p a r a t i o n or extra nuclear stage. l).  Although  a f t e r telophase I I ,  c o n d e n s e d and f o r m a clumped c o n f i g u r a t i o n .  formed  uncoil  ( P L . 2, f i g . 11)  one l a r g e h e t e r o c h r o m a t i c r e g i o n .  a c o n s t r i c t i o n of the cytoplasm frequently ( P L . 2, f i g . 1 1 ) .  On  t h e chromosomes p a r t i a l l y  c e l l w a l l f o r m a t i o n i s n o t complete u n t i l  I  of  f o r m a t i o n t h e clumped  s e p a r a t e and assume a metaphase I  distinguishable  usual  However, t h e s e a p p a r e n t l y  o f t h e chromatids as only  often  do n o t c a u s e  s e l d o m do f r a g m e n t s  c h r o m a t i n a p p e a r i n t e l o p h a s e I I and t h e t e t r a d  During telophase I I , wall formation begins  The c y t o p l a s m a p p e a r s a t f i r s t  ( P L . 3, f i g .  t o be c o n s t r i c t e d on t h e  12  plane  of the f i r s t  second d i v i s i o n .  d i v i s i o n and The  meiotic  I n o n l y one circinale  l a t e r than  the by  pro-  f i g . 2).  #2463:  Population  In t h i s  differ distinctly  s p o r a n g i u m SMC  of  H.  f r o m what  were p r e s e n t  in a l l  diakinesis.  I n a b o u t 40$  of those  s m a l l e s t b i v a l e n t had  i n metaphase  disjoined precociously.  o n l y a s i n g l e member o f t h e p a i r was  separated  from  m a i n chromosomal mass (PL. 3,  f i g . 3-6).  some metaphases i n w h i c h b o t h  t h e u n i v a l e n t s were e v i d e n t  o t h e r s i n w h i c h t h e b i v a l e n t had The  not  L a t e r stages  had  a n o t i c e a b l y high frequency  the  SMC  in first  and  d i v i d e d (PL. 3,  (PL. 3,  occurred with a frequency sporangia.  of d i v i s i o n  one  i n the  and  fig.  F u r t h e r m o r e , i n many o f t h o s e  l a g g a r d the  some c a s e s letely  small bivalent.  (PL. 3,  omitted  f i g . 9)  from the  c a u s i n g an u p s e t  These cells  s m a l l b i v a l e n t was  amount o f p a r t i c i p a t i n g  s i z e of the  On  with  the  normal i n bridge  involved  (PL,  bridge  greater  3,  or  than  o t h e r hand, t h e r e were s m a l l b i v a l e n t was  s p i n d l e apparatus.  d e m o n s t r a t e d on m o r p h o l o g i c a l  anomalies  o n l y one was  of  chromatin  of the  not  chromatin  i n which the  i n meiosis  a  sporangium  About 15$  bivalent participated  and  8).  u s u a l l y the  f i g . 8,9).  o f 2-3$  O f t e n more t h a n  contained  formation  be  were  fig.7).  in this  of abnormalities.  second t e l o p h a s e  b r i d g e and/or l a g g a r d s  was  However, t h e r e  the  r e m a i n d e r o f t h e b i v a l e n t s a p p e a r e d t o a c t as t h e y d i d i n  'normal' s p o r a n g i a .  the  was  d i p l o t e n e of prophase I w i t h the m a j o r i t y i n  d i a k i n e s i s the  Usually  of  sporangium o f the t h r e e p o p u l a t i o n s  normal.  metaphase I and I and  (PL. 3,  examined d i d m e i o s i s  considered stages  #6,  i n Sporangium  on t h e p l a n e  sequence i s c o n c l u d e d  d u c t i o n of a t e t r a d of spores Meiosis  then  comp-  E v i d e n t l y something  i n t h i s p l a n t but  this  grounds. A s t r u c t u r a l  could  not  aberration  13  m i g h t p o s s i b l y have been d e t e c t e d  at d i p l o t e n e or  but u n f o r t u n a t e l y n e i t h e r o f t h e s e slide.  was  t r e a t e d i n the  possibility  discussed  Meiosis  slight.  in detail  of meiosis  o f B.  The  main purpose o f t h i s  unique to that s p e c i e s .  results., a study Essentially  two  are  study  was  observed a f t e r d i p l o t e n e T h u s , on t h e  p o p u l a t i o n was  pattern of meiosis circinale.  to  in  basis  of  sufficient.  i n B.  frigidum  However t h e r e were  sequence which, a l t h o u g h  was certain  seemingly d i f f e r e n t  in  more c l e a r l y d e m o n s t r a t a b l e i n  frigidum. first  of these  e a r l y prophase. the  i s the  presence of the n u c l e o l u s  E a r l y p r o p h a s e o f B.  a c e n t r i c l o c a t i o n of a nucleus  chromosomes. the  stage  s p e c i e s , were p r o b a b l y  The  by  the  o f one  same as t h a t o f H.  f a c e t s of the  making  f r i g i d u m were made f o r  H.  c i r c i n a l e was  others,  this  frigidumt  elongation  B.  the  consequences o f heat shock  determine i f the  the  in  later.  only a s i n g l e population.  the  present  same method as t h e  The  i n Brachytheeium  Observations  the  was  H e a t s h o c k m i g h t a l s o have c a u s e d s u c h a n o m a l i e s but  sporangia this  stages  pachytene  nucleolus  was  frigidum i s characterized containing  Phase c o n t r a s t o b s e r v a t i o n s  presence of a l a r g e nucleolus not  evident  (PL.  when t h e  during  3,  single-stranded  of t h i s  stage  f i g . 10,  11).  revealed  s l i d e s were i n i t i a l l y  This prepared  _ The a u t h o r has s i n c e o b s e r v e d l a r g e n u c l e o l i i n l e p t o t e n e t h r o u g h t o e a r l y d i p l o t e n e i n a number o f s p e c i e s v i z . R h a c o m i trium microcarpon. Claopodium c r i s p i f o l i u m , Isothecium s t o l o n i f e r u m , P l a g i o t h e c i u m u n d u l a t u m as w e l l as B. f r i g i d u m using proprionic-carmine with i r o n s t a i n . The s t a i n was made a c c o r d i n g t o t h e method o f D a r l i n g t o n and L a S o u r (i960) f o r a c e t o - c a r m i n e . However, p r o p r i o n i c a c i d r e p l a c e d t h e a c e t i c a c i d and t h e i r o n a c e t a t e was n o t a d d e d . I r o n was added by s u s p e n d i n g f o r c e p s  14  but  became e v i d e n t  only  a f t e r standing  migrating  p a c h y t e n e and  fig,  a p p e a r e d t o have no  1,2)  contrast  diplotene  or b r i g h t f i e l d  phases  illumination.  frigidum  o f B.  intensity  except f o r a h e t e r o p y c n o t i c  identified • u n c o i l as interphase threads  circinale  s p e c i e s was  One  This  and  the  (PL. 4,  SMC  chromosomes f o l l o w s t h e  s e c t i o n o f one  d i p l o t e n e , the  membrane i n B.  (PL. 4,  elongation (PL.  t h e d i a k i n e s i s i n H . c i r c i n a l e . but configuration i s followed  in  and  same of  by  evenly  the be  chromosomes an  frigidum  the  c i r c i n a l e during  f r o m page  two  f r i g i d u m was  clearly  Condensation of  p h a s e and  the  4,  f i g . 5).  i n B.  the  the  bivalents This  f r i g i d u m the  resembles clumped  a more n o r m a l d i a k i n e s i s i n w h i c h  i n the  of s i m i l a r s i z e are  stain  f i g . 3).  the h i g h l y condensed b i v a l e n t s are not  ^(continued  I n B.  4,  phase  c h r o m a t i n mass assumes  f i g . 3,4).  assume a clumped c o n f i g u r a t i o n  bivalents  using  in  PL.  During pachytene  t h a n t h o s e o f H.  that the n u c l e a r  quite  f i g . 12;  r e m a r k a b l e d i f f e r e n c e n o t e d between t h e  i n many o f t h e  distributed  3,  chromosome c o u l d n o t  Following  a p p e a r more c o a r s e  same s t a g e .  defined  f i g . 2).  appearance  (PL.  Cells  a l l t h e b i v a l e n t s have t h e  specifically. i n H.  months  v i s i b l e nucleolus  diplotene  chromosomes (PL. 4,  a few  SMC  clumped t o g e t h e r  (PL. 4,  present  f i g . 6).  a t metaphase I .  and  are  Six One  of  13)  a d r o p o f t h e s t a i n j u s t b e f o r e t h e s t a i n was u s e d . I t was f o u n d t h a t f r e s h m a t e r i a l as w e l l as m a t e r i a l f i x e d i n e i t h e r 1:3 a c e t i c a l c o h o l o r Newcomer's s o l u t i o n (Newcomer, 1953) stained well. G l a c i a l a c e t i c a c i d , f o l l o w e d by a w a s h i n g i n 1:3 a c e t i c a l c o h o l f i x c o u l d be employed a f t e r n o r m a l f i x a t i o n t o remove o i l d r o p l e t s . T h i s treatment d i d not i m p a i r the s t a i n a b i l i t y of the n u c l e o l i or chromatin.  15  t h e s e b i v a l e n t s i s r o d - s h a p e d and i s u s u a l l y l o c a t e d a t one o f t h e metaphase. p l a t e u s u a l l y very  ( P L . 4,  f i g . 7,8).  in this  bivalent i s  l o n g a t metaphase I and o f t e n d i s s o c i a t e s  Its!; b e h a v i o u r i s a n a l o g o u s t o t h a t circinale.  This  Anaphase I  species  s i m i l a r to that  ( P L . 4,  f i g . 9) was  circinale.  precociously.  of the large b i v a l e n t of remarkably  and t h e r e m a i n d e r o f t h e m e i o t i c i n H.  side  Abnormalities  were e x t r e m e l y r a r e and t e t r a d f o r m a t i o n  regular  sequence a t second  appeared  H.  normal.  was division  16 DISCUSSION  The  study of the  neglected.  For  the  meiotic  p r o p h a s e o f mosses has  most p a r t ,  been  s t u d e n t s o f moss c y t o l o g y  have  been c o n c e r n e d o n l y w i t h a c q u i r i n g chromosome c o u n t s and rather  superficially,  the  a t metaphase I .  The  20,000 d e s c r i b e d  species  (Mehra and  b e h a v i o u r and  o f mosses have now e s t i m a t e d 650  been  species).  the  a c c u m u l a t e d work has evolution  cytogenetics  and  The  o f mosses, t h e  genetics  most o u t s t a n d i n g  s i n c e 1948  i s that  has  been  c o n t r i b u t i o n to  I n an  attempt and  He  stated  duration.  However, d u r i n g  same a u t h o r d e s c r i b e d during  the  the  e a r l y prophase.  Although the  almost impossible c o n s p i c u o u s and negatively behaviour.  published  to study the  poor  behaviour of a  impossible.  eiliata,  and  had of  the  'special bivalent'  four  spherical  heteropycnotic  chromatin i n prophase I  to observe i n d e t a i l ,  persisted until  the  the  stainability  T h i s b i v a l e n t a p p e a r e d as a  r e s t of the  heteropycnotic At  meiotic  t h e s e s t a g e s were  h i s s t u d y o f H.  opaque n u c l e o l u s - l i k e v e s i c l e bounded, by bodies.  been  prophase stages l a t e r than pachytene  been r a r e l y observed, which suggested t h a t short  basic  at prophase I rendered d e t a i l e d examination (1949) t h a t  the  some members o f  G r i m m i a c e a e , Vaarama (1949, 1954a) f o u n d t h a t SMC  of  small.  prophase.of Hedwigia c i i i a t a Br. & Sch.  of the  the  Although  l i m i t e d knowledge t o  o f t h e work t h a t has  o f Vaarama.  of  O v e r 90$  e s t a b l i s h e d numbers have been p r e s e n t e d s i n c e 1948,  taxonomy and  bivalents  established  the  contributed  determining,  morphology of the  chromosome numbers o f a t l e a s t 700  Khanna, 1961,  severely  t h e v e s i c l e was  was  quite  d i a k i n e s i s , whereupon i t became  underwent u n u s u a l  t i m e , Vaar.ama s t a t e d t h a t  segregation  the r e l a t i o n s h i p  17  between t h e n u c l e o l u s - l i k e v e s i c l e and. t h e o r d i n a r y n u c l e o l u s not  known.  I t i s i n t e r e s t i n g to note that d u r i n g a p i l o t  using propionic-carmine I o f s e v e r a l mosses  ( c f . footnote  page 13) t h e p r e s e n t  (Hedw.) B r y . E u r .  separate  author Plagiothecium  In a d d i t i o n to the n u c l e o l u s - l i k e  v e s i c l e t h e r e was a n o r m a l n u c l e o l u s entirely  study  as a s t a i n f o r n u c l e o l i i n t h e p r o p h a s e  observed a s i m i l a r s p e c i a l b i v a l e n t i n prophase I o f undulatum  was  present  which appeared  from the s p e c i a l b i v a l e n t .  Also, the v e s i c l e  o f t h e s p e c i a l b i v a l e n t s t a i n e d i n a manner s i m i l a r t o t h a t o f the n u c l e o l u s ,  i n d i c a t i n g that the v e s i c l e  i s possibly nucleolar  in structure. The  first  and o n l y d e t a i l e d s t u d y  o f t h e chromosomes a t m e i o t i c by Vaarama schreberi  (1954b).  p r o p h a s e i n mosses was  behaviour reported  He f o u n d t h a t t h e chromosomes o f P I e u r o z i u m  ( B r i d . ) M i t t , s t a i n e d w e l l d u r i n g p r o p h a s e I and t h a t ,  b e c a u s e t h e y were few i n number  (5), o b s e r v a t i o n s  chromosomes d u r i n g t h e e a r l y s t a g e s the  of the general  only published  to the present  were p o s s i b l e .  of individual He  presented  f i g u r e o f t r u e d i p l o t e n e i n mosses known  author.  The e a r l y p r o p h a s e o f  Pleurozium  s c h r e b e r i compares q u i t e c l o s e l y w i t h t h e e a r l y p r o p h a s e o f H. c i r c i n a l e  and B. f r i g i d u m .  or d e p i c t the elongation stage two s p e c i e s p r e s e n t e d  will  that followed diplotene i n the  h e r e and he i n d i c a t e d t h a t a clumped  kinesis d i r e c t l y followed unusual stage  However, Vaarama d i d n o t d e s c r i b e  diplotene.  dia-  F u r t h e r mention o f t h i s  be made i n t h e d i s c u s s i o n o f e a c h p r o p h a s e  stage. leptotene The  and Z y g o t e n e : a c e n t r i c a l l y l o c a l i z e d nucleus  at the e a r l i e s t  prophase I  18  s t a g e i s o f wide o c c u r r e n c e has  observed  (Vaarama, 1 9 5 4 b ) .  The  present  t h e phenomenon i n a l l o f t h e a p p r o x i m a t e l y  s p e c i e s o f mosses examined d u r i n g t h e p a s t t h r e e y e a r s data).  The  chromosomes c a n be  p a r t l y double  stranded  completely double  observed  ( a s i n H.  c i r c i n a l e . PL.  n u c l e a r membrane has  t o say whether the chromatin  H.  side.  As n o t e d  was  mass and mass.  was  was  with the  threads  of  the c e l l  by  one  t h e mass.  remainder  the  a f t e r t h i s migration begins,  to the  remains l o c a l i z e d  of the c e l l .  p o s s i b l y u s i n g f l u o r e s c e n t microscopy help to elucidate t h i s Paehytene-Diplotene:  the  center  o f t h i s movement i s from  f o r some  i n d i c a t i n g that the nucleus  past the middle  chromatin  than  o f t h e chromosomes w h i c h become l o o s e n e d  Most o f t h e c h r o m a t i n  t i m e must e x t e n d  initiation  the  propionic-carmine  l o c a t e d c l o s e r to the center of the c e l l  o r a few  The  chromatin  o f t e n - q u i t e separate from  i n early'pachytene,  chromatin  Furthermore,  I n a d d i t i o n , when t h e chromosomes m i g r a t e  of  on a l l  on r a r e , o c c a s i o n s i n  acentrically localized.  f o o t n o t e page 13)  the  stage, i t i s  o f t h e SMC)  n u c l e o l u s o f some o f t h e s p e c i e s s t u d i e d w i t h (cf.  takes Since  w e l l past the center of the c e l l while the  the chromatin  stranded,  i s bound c l o s e l y  earlier,  c i r c i n a l e l o n g , presumably s i n g l e ,  extended of  a t one  (unpublished  i s actually quite large  extending to or past the middle  localized  at t h i s  thirty  f i g . 2), or  polarization.  not been observed  s i d e s by i t o r w h e t h e r t h e n u c l e u s (ie.  1,  stranded, i n d i c a t i n g that synapsis  place d u r i n g t h i s p e r i o d of chromatin  difficult  either single  author  at  time this  Further studies,  with unfixed material  may  matter.  :  m i g r a t i o n o f t h e p a i r e d chromosomes t o t h e c e n t e r o f  19  t h e SMC, is  as observed  i n b o t h H.  c i r c i n a l e and  B. f r i g i d u m  s i m i l a r t o t h a t d e s c r i b e d by Vaarama (1954b) i n P l e u r o z i u m  schreberi. The  t r a n s i t i o n between p a c h y t e n e and  precise.  I n f a c t , Vaarama p r e f e r r e d t o lump t h e two  many c a s e s and  referred  to pachytene-diplotene  a f t e r t h e chromosomes have m i g r a t e d , begin to repel in  one  e a c h o t h e r but  stages i n  stages.  Soon  s e c t i o n s o f the b i v a l e n t s  o n l y seldom can t h i s  be  followed  chromosome as u s u a l l y some p a r t o f e v e r y chromosome i s  entangled w i t h the o t h e r s . short  d i p l o t e n e stage i s not  enough t h a t one  o r two  r e p u l s i o n i s completed contact points.  However, when t h e b i v a l e n t s a r e are e a s i l y  presented  evidence  t h a t the contact p o i n t s observed but m e r e l y  the  a l o n g t h e whole chromosome e x c e p t  T h e s e would u s u a l l y be d e s i g n a t e d , as  Vaarama (1954b) has  chiasmata,  separated from  areas  Elongation The  i n H.  the  i n h i s s t u d y on P. s c h r e b e r i  d u r i n g d i p l o t e n e are not  . f o r m a t i o n was  true  No  special  made i n t h i s  i n v e s t i g a t i o n so t h e q u e s t i o n must r e m a i n open f o r t h e  The  at  chiasmata.  of s t i c k y heterochromatin.  s t u d y o f t h e method o f chiasma"  rest,  present.  Stage:  most r e m a r k a b l e  circinale  and  B.  aspect  o f t h e m e i o t i c sequence  f r i g i d u m was  the occurrence  of  observed  the  chromosome e l o n g a t i o n s t a g e w h i c h f o l l o w e d d i p l o t e n e .  Vaarama  (1954b) d i d n o t d e s c r i b e o r d e p i c t t h i s s t a g e f o r P l e u r o z i u m schreberi. is  present  mosses. H.  However i t r e m a i n s q u i t e p p o s s i b l e t h a t t h i s i n Pleurozium  The  circinale  present and  B.  stage  s c h r e b e r i as w e l l as i n many o t h e r  author has,  s i n c e the i n i t i a l  s t u d i e s on  f r i g i d u m , examined m e i o s i s i n C l a o p o d i u m  crispifolium occurred  (Hook.) R. & C.  i n t h a t s p e c i e s as w e l l .  o f permanent s l i d e s date  and f o u n d  (Dill,  t h a t the e l o n g a t i o n stage  Furthermore,  an  examination  o f t w e l v e moss t a x a s t u d i e d a t an e a r l i e r  unpublished  data), suggests  t h a t a l l have  elongation stage.  Unfortunately this  conclusively u n t i l  t h e c o m p l e t e s e q u e n c e o f m e i o s i s i s examined  be e s t a b l i s h e d  for  each o f t h e s p e c i e s .  and  C. c r i s p i f o l i u m r e p r e s e n t t h r e e moss f a m i l i e s  that  The f a c t  cannot  this  t h a t H. c i r c i n a l e .  the stage i s probably widespread  B. f r i g i d u m .  indicates  also  i n t h e mosses.  A p o s t - d i p l o t e n e e l o n g a t i o n s t a g e h a s been r e p o r t e d on r a r e o c c a s i o n s i n t h e p l a n t kingdom. a stage, termed s t r e p t s i t e n e , (Impatiens  balsamina)  i n the dicot^Balsamina hortensis  s i m i l a r t o t h a t observed  a u t h o r i n Hypnum and B r a c h y t h e e i u m . osomes was a l s o  V i o l a n t e (1929) d e s c r i b e d  The e l o n g a t i o n o f t h e chrom-  accompanied b y n u c l e a r g r o w t h .  t h a t l e w i t s k y (1927) and M o d i l e v s k y  had  artifact.  observed  this  had a p p a r e n t l y  (1927)  b u t exam-  pachytene stage  i n the developing  phase o c c u r s b e f o r e d i p l o t e n e  ( C a r r and O l i v e r 1958) .• W i l s o n  (1952) h a s r e p o r t e d t h a t t h e mature r e s i s t a n t Allomyces  considered  paper i n d i c a t e s that.this i s not t h e case.  o f many a s c o m y c e t e s , b u t t h i s i s a typical  similar  V i o l a n t e a l s o mentioned t h a t S z a k i e n  There i s a l s o a p o s t - s y n a p t i c e l o n g a t i o n stage  and  indicated  s t a g e i n t h e f e r n Osmunda r e g a l i s .  i n a t i o n o f Szakien's  asci  He  (1918) h a d o b s e r v e d  s t a g e s i n o t h e r p l a n t s and t h a t L e w i t s k y the stage  by t h e p r e s e n t  sporangia of  i s i n a s t a t e o f suspended prophase I o f m e i o s i s , but  he was u n a b l e  t o determine  which stage  o f prophase I .  The  photo-  g r a p h s do n o t r e s e m b l e t h o s e o f t h e e l o n g a t i o n s t a g e r e p o r t e d here.  21  According to Pratt  and  Long  (1917), von W i n i w a r t e r  (1900)  d e s c r i b e d an e l o n g a t i o n s t a g e i n p r o p h a s e I w h i c h f o l l o w e d d i p l o t e n e i n the maturing Winiwarter  gave t h e t e r m  oocytes  of rabbit  'Noyoux d i c t y e '  and man.  (sic) to this  More r e c e n t l y Ohno, Maki.no, K a p l a n and K i n o s i t a K l i n g e r and A t k i n ( 1 9 6 2 ) , have r e a f f i r m e d v o n original that  o b s e r v a t i o n s on o o g e n e s i s  the d i c t y e stage  recent papers are h i g h l y The  (termed  )^ i s a  extended  oocytes remain  Ohno,  Winiwarter's  and have  stage i n which the  i n this  accumulate  phase u n t i l  indicated stage i n  chromosomes  o v u l a t i o n when t h e m e i o t i c  During the i n t e r v e n i n g p e r i o d the nutrient u t i l i z e d  Furthermore,  cells  by t h e f u t u r e embryo.  some i n s e c t s , t h e l a m p b r u s h  i s homologous w i t h t h e d i c t y o t e n e s t a g e .(Seshachar a c c o r d i n g to C a l l a n  a n i m a l s have a s t a g e w h i c h c o r r e s p o n d s  and  stage Bagga,  (1963) p r o b a b l y a l l  to the d i c t y o t e n e or  lampbrush stage i n t h e i r d e v e l o p i n g o o c y t e s . that  and  and m a i n t a i n t h e i r d i p l o t e n e s t r u c t u r e .  I n many a m p h i b i a n s and  1963).  phase.  (1961),  dictyotene or d i c t y a t e  'resting'  s e q u e n c e i s resumed. grow and  i n man  Von  He  also  indicates  t h e l a t e r a l p r o j e c t i o n s o f t h e d i p l o t e n e chromosome o f many  primary  spermatocytes  behaviour,  may  also represent a lampbrush-like  b u t t h a t t h e chromosomes a r e n o t g r e a t l y e x t e n d e d  l e n g t h as t h e y a r e i n t h e o o c y t e s .  Although  the d i c t y o t e n e  chromosomes v a r y i n g r o s s m o r p h o l o g y among t h e animal groups i t i s apparent diplotene i n structure  Wilson  that  different  t h e chromosomes a r e  basically  ( R i s , 1945).  (1925) r e f e r s t o t h i s  in  s t a g e as t h e d i c t y o t i c  stage.  22  Aside from the few i n s t a n c e s mentioned i n the f u n g i , which probably do not represent the d i c t y o t e n e stage, the only f a c t o r y evidence  f o r the occurrence  of such a stage i n p l a n t s to  my knowledge has been r e p o r t e d i n Balsamina h o r t e n s i s Balsamina).  satis-  (impatiens  In t h i s case the sporocyte n u c l e i apparently i n c r e a s e d  In s i z e d u r i n g t h i s phase, i n a manner s i m i l a r to the growth found i n oocytes.  However, the growth was  not so marked.  At  the present time, i t i s impossible to say i f the r e l a t i o n s h i p between the animal d i c t y o t e n e and t h a t found  i n mosses and i n  Balsamina i s s u p e r f i c i a l or i s based on s i m i l a r s t r u c t u r e . . The SMC  of mosses do not enlarge d u r i n g d i c t y o t e n e and the d u r a t i o n  of the stage i s not l o n g i n r e l a t i o n to the other m e i o t i c as i t i s i n oocytes. Thetometabolism of the SMC  events,  d u r i n g meiosis  i s v i r t u a l l y unknown and i t i s q u i t e p o s s i b l e t h a t a high r a t e of metabolic  a c t i v i t y may  i n s i z e of the  take place without  a p p r e c i a b l e growth  cells.  I t appears probable t h a t the d i c t y o t e n e stage has some f u n c t i o n a l s i g n i f i c a n c e i n the m e i o t i c sequence of mosses, but u n t i l d e t a i l s of the metabolic  events t a k i n g p l a c e d u r i n g t h i s  stage are d i s c o v e r e d l i t t l e can be s a i d about t h i s matter. D i a k i n e s i s and the Remainder of MeiosisI The clumped d i a k i n e s i s observed i s commonly found i n mosses.  i n both species s t u d i e d here  Vaarama (1954b) i n d i c a t e d that a  s i m i l a r s i t u a t i o n occurred i n P. s c h r e b e r i and many other mosses. U s u a l l y b i v a l e n t s at d i a k i n e s i s i n h i g h e r p l a n t s are d i s t r i b u t e d about the nucleus  evenly  or are near the n u c l e a r membrane.  Steere, et a l . (1954) have r e p o r t e d making many counts from  23  diakinesis  stages i n which.the  b i v a l e n t s were w i d e s p r e a d .  t h e prometaphase s t a g e o b s e r v e d this  same s t a g e .  In t h i s  i n B. f r i g i d u m c o r r e s p o n d s  c a s e , H.  circinale  s c h r e b e r i would n o t have a c l a s s i c a l clumped  s t a g e cannot  Possibly  be r e f e r r e d  and  Pleurozium  t y p e o f d i a k i n e s i s and  t o as d i a k i n e s i s .  b e f o r e p r o m e t a p h a s e ; i t may  the  However,  B. f r i g i d u m and many o t h e r s p e c i e s have t h e clumped s t a g e d i c t y o t e n e and  to  be a n o t h e r  following  distinctive  f e a t u r e o f t h e m e i o t i c s e q u e n c e i n mosses. Metaphase I c o n f o r m s w e l l w i t h t h a t i n t h e two  species studied here.  which i s u s u a l l y l o c a t e d p l a t e has  1961).  bivalent  a t t h e p e r i p h e r y o f t h e metaphase I  d i s p l a y e d d u r i n g prophase I i n e i t h e r  species discussed here.  pond t o t h e l a r g e  'H'  (1957a,b,c).  Hypnum c i r c i n a l e  The  S t u d i e s on s o m a t i c o  tissue  P o s s i b l y these b i v a l e n t s  chromosomes o f m i t o t i c  small heterochromatic bivalent  perhaps  falls  a category that these authors a,b,c) h e t e r o c h r o m a t i c behaviour,  remainder  s t u d i e d here and  would  found  in  i n t o the c l a s s of " m o r p h o l o g i c a l l y  say corresponds  (1961),  t o Yano's  'h' chromosomes o f m i t o s i s .  (1957  Its*; of  r e m i n i s c e n t o f the minute b i v a l e n t ' s  the behaviour.  o f t h e m e i o t i c sequence i n b o t h t h e s p e c i e s  i s unremarkable.  B o t h have n o r m a l s e c o n d  normal t e t r a d f o r m a t i o n , i n d i c a t i n g the presence  balanced  of  corres-  ( i e . p r e c o c i o u s d i s j u n c t i o n ) i n s p o r a n g i u m #6  p o p u l a t i o n # 2464 was  no  c e l l s d e s c r i b e d by  i n d i s t i n g u i s h a b l e m i n u t e b i v a l e n t s " o f Mehra and Khanna  The  Khanna,  They a r e u s u a l l y r e f e r r e d t o as h e t e r o c h r o m a t i c , but  help to e l u c i d a t e t h i s matter.  Yano  l a r g e rod-shaped  been d e s c r i b e d i n many moss s p e c i e s (Mehra and  e v i d e n c e f o r t h i s was t h e two  The  of the h i g h e r p l a n t s  g e n e t i c system.  The  time  and  divisions  of a  well-  method o f w a l l f o r m a t i o n  24  during meiosis this  i n mosses has  i s done one  begins  cytoplasm  a light  end  of the  s t a i n i n g and  which t r a v e r s e s the  first  The  higher  Until  cells  wall This i s  c o n s t r i c t e d area  f o l l o w i n g telophase  I.  of  Quite  mosses as i t does i n  plants.  chromosome c o u n t s  f r i g i d u m have n o t  division.  apparently  t h i s mechanism v a r i e s i n d i f f e r e n t  different  and  been s t u d i e d c l o s e l y .  o n l y say t h a t i t appears t h a t the  t o form toward the  i n d i c a t e d by  likely  can  not  (n=6)  f o r H.  circinale  been p r e v i o u s l y r e p o r t e d .  and  B.  B o t h Hypnum  B r a c h y t h e c i u m have a g r e a t v a r i e t y o f chromosome numbers  reported, w i t h i n the  a v a r i a t i o n t h a t l i e s not same s p e c i e s .  to inaccurate  o n l y between s p e c i e s but  Much o f t h i s v a r i a t i o n may  observations  and  until  the  counts are  also  be a t t r i b u t a b l e clearly  e s t a b l i s h e d d i s c u s s i o n s o f t h e i r taxonomic s i g n i f i c a n c e are relatively  menaingless.  I n c o n c l u s i o n , i t c o u l d be the  dictyotene  stage,  said that, with  the m e i o t i c  sequence i n these  c o n f o r m s c l o s e l y t o t h a t f o u n d i n most h i g h e r dictyotene may  be  stage,  although  o f common o c c u r r e n c e  the  exception mosses  plants.  an u n u s u a l f e a t u r e o f p l a n t i n many moss  taxa.  of  The meiosis,  25 CHAPTER 2  The E f f e c t of Heat S t r e s s on M e i o s i s i n Hypnum c i r c i n a l e Hook.  I t i s well-known that m e i o t i c stages i n most mosses are s t a i n e d most e f f e c t i v e l y i f the capsules are immersed i n a c e t i c a l c o h o l f o r a very b r i e f p e r i o d .  With most mosses a 24 hour  f i x a t i o n p e r i o d w i l l reduce the chromosome s t a i n a b i l i t y . t o an extent that d e t a i l e d observations are impaired. reason, many.authors (Steere et a l . , 1954; Anderson and Crum , 1959)  such  For t h i s  Bryan, 1956  et seq.;  have brought the p l a n t s d i r e c t l y to  the l a b o r a t o r y , and have made the p r e p a r a t i o n s i n the l a b o r a t o r y from l i v i n g m a t e r i a l .  I t has been suggested  t h a t p l a n t s which  are not mature enough f o r meiosis at the time of c o l l e c t i o n can be grown i n the l a b o r a t o r y u n t i l they mature. p l a n t s are kept i n covered p l a s t i c d i s h e s . authors a l s o s t a t e t h a t maturing order to slow down m e i o s i s .  U s u a l l y the  The above mentioned  p l a n t s can be r e f r i g e r a t e d i n  Although there are presumably  a b n o r m a l i t i e s induced by use of e i t h e r of these techniques, c o n t r o l l e d experiments have been conducted  no no  to s u b s t a n t i a t e t h i s  claim. These same"authors note one p a r t i c u l a r s t r i k i n g f e a t u r e of meiosis i n w i l d populations of mosses:  many chromosomal a b e r r -  a t i o n s occur and o f t e n some b i v a l e n t s tend to d i s j o i n i o u s l y at metaphase I .  precoc-  To quote Steere et a l . , (1954):  "In the present i n v e s t i g a t i o n , i t i s observed that c the sporocytes of mosses brought i n from the f i e l d e a r l y i n the day were o f t e n i n l a t e prophase or t e t r a d stages. Exposure to l i g h t and warmth, however, induced the young sporocytes to resume m e i o s i s . In view of the many chromosomal a b e r r a t i o n s noted at the f i r s t and second m e i o t i c d i v i s i o n s , a .careful  study of prophase behaviour i s u r g e n t l y e s p e c i a l l y of pachytene stages." It  i s puzzling that  two as  f a c t s and  The  t h i s phenomenon had on  arrival.  w a t e r e d and had  stored  n o r m a l meiosis..  abnormalities  treatment given  may  have  the  arisen  clumping of chromatin i n  r e l a t e d to the  material  she  She  She  water  content  that p a r t i c u l a r l y and  mentioned t h a t  at reduced  the  low  studied  been m a i l e d t o h e r  the  i f the  (unspecified)  capsules plants  showed  were  were  temperatures,  they  (1956a) o b s e r v e d s i m i l a r c l u m p i n g  u n u s u a l metaphases i n B r u c h i a the  attempt t o connect  treatment.  seemed t o be  cells.  flaccid  the  (1956b) s u g g e s t e d t h a t  Phascum SMC of the  a u t h o r s d i d not  suggest t h a t  a r e s u l t o f the Bryan  the  needed,  but  no  m e n t i o n was  made  and  concerning  plants.  Khanna ( i 9 6 0 ) , i n a chromosomal s t u d y o f some H i m a l a y a n mosses, has  indicated that  carried  i n the  out  field  s a t i s f a c t o r y r e s u l t s , but factory.  On  as w e l l as  the  the  chromosomes.  This  because s t o r e d m a t e r i a l he  d i d not  hand, he  T h i s was  I n an  field,  had  study  abnormal weather meiotic  abnormalities  n o r m a l m e i o s i s i n c o o l wet  observation  concerning  were i n d u c e d by  (Dill,  in  weather.  bryophytes i n  environmental  unpublished data),  Mnium venustum, M i t t . 12  yield  t h e y were u n s a t i s -  other  t h a t b r i n g i n g mosses i n d o o r s  abnormal m e i o s i s . from the  only  abnormalities  earlier  a suggestion  the  d i d not  cause clumping of  chromosomal c l u m p i n g and  a p p e a r s t o be  were  e s p e c i a l l y apparent i n Barbula c o n s t r i c t a  d r y w e a t h e r , w h i l e i t had  which m e i o t i c  s a y why  indicated that  f i x a t i v e used could  M i t t , w h i c h had hot  other  a l l his c y t o l o g i c a l studies  there  t o r i p e n might , when examined  conditions. was cause directly  c l o s e l y p a c k e d b i v a l e n t s a t metaphase  I.  27  However, m a t e r i a l t h a t had c o n t a i n e r f o r 2-3 were l o o s e l y  been k e p t i n d o o r s i n a humid  days had metaphases i n w h i c h  spread throughout  the b i v a l e n t s d i s j o i n e d  the c e l l .  precociously.  c l a d i u m h e t e r o p t e r o i d e s B e s t was  went s e v e r e l y a b n o r m a l were o b s e r v e d .  I n a d d i t i o n , many o f  A c o l l e c t i o n of Hetero-  a l s o brought  b e f o r e t h e c a p s u l e s had m a t u r e d .  t h e chromosomes  i n t o the  A f t e r 2 weeks t h e WSMC u n d e r -  m e i o s i s (PL. 7, f i g . 3 ) .  No  good  S i n c e t h e c a p s u l e s were n e v e r f l a c c i d  u n l i k e l y t h a t h u m i d i t y was  related  to t h i s  anomalous  t h a t heat s t r e s s can s e v e r e l y a f f e c t  tetrads  i t seemed condition.  S t u d i e s u s i n g h i g h e r p l a n t s and v a r i o u s a n i m a l s demonstrate  laboratory  clearly  meiosis.  The  b u l k o f t h i s work, b o t h c y t o l o g i c a l and g e n e t i c a l , h a s  dealt  w i t h chiasma  Wilson,  frequency at v a r i o u s temperatures  b u t some h a s d e a l t  w i t h temperature  s p i n d l e f o r m a t i o n , centromere (Swanson, 1942, Henderson,  1943;  effects  b e h a v i o u r , and n u c l e o l i  Dorwick,  1957;  J a i n , 1957  of these f a c t s ,  an e x p e r i m e n t  a s c e r t a i n w h e t h e r o r n o t room t e m p e r a t u r e s h i g h t o cause  such anomalies.  c o n c e r n i n g h e a t and t h e room t e m p e r a t u r e  is  behaviour  et sea;  and  seemed j u s t i f i e d were  on m e i o s i s i n mosses.  First, i f  heat s t r e s s ?  Second,  i f the  sensitive  sensitivity  not n a t u r a l heat s t r e s s i n the environment  o f t h e chromosomes? stress?  to  sufficiently  c a u s e d m e i o t i c a b n o r m a l i t i e s , how  to s l i g h t  g r e a t , might  to heat  coiling,  Three major q u e s t i o n s arose  i t s effect  mechanism i n d u c i n g m u t a t i o n s  SMC  on chromosome  1959),  1962).  In view  a r e moss SMC  (Yanney  or structuralji'numerical  be  a  alterations  T h i r d , what i s t h e mode o f r e s p o n s e o f t h e  28  S e v e r a l e x p e r i m e n t s were d e s i g n e d of these  questions..  supplied  f o r a l l t h e q u e s t i o n s , c e r t a i n t r e n d s and some  c o n c l u s i o n s were  Although  t o answer some o r a l l  obtained.  c o m p l e t e answers were n o t definite  29 METHODS  Methods o f T r e a t i n g the P l a n t s : In the f o l l o w i n g experiments, s e v e r a l d i f f e r e n t types o f heat treatment were a p p l i e d .  I n some, s p e c i a l equipment  was  needed i n order t o a t t a i n a r e p r o d u c i b l e experimental procedure. The f i r s t  experiments i n v o l v e d t r e a t i n g the p l a n t s t o short  term heat s t r e s s ( i e . a p e r i o d o f about 4 h o u r s ) . p e r i o d d i d not seem r e a l i s t i c  A longer  s i n c e a l o n g e r p e r i o d o f abnormal  heat s t r e s s would not be expected under n a t u r a l c o n d i t i o n s . Furthermore, p i l o t sufficient  experiments showed that the s h o r t e r p e r i o d was  t o cause severe anomalies i n p l a n t s subjected t o the  h i g h e r ranges o f temperature used ( 3 0 - 3 7 ° C ) .  To ensure r e p l i -  c a t i o n i t was necessary t o c o n t r o l the r a t e o f temperature change when h e a t i n g as w e l l as when c o o l i n g the p l a n t s , thus a chamber p o s s e s s i n g a h e a t i n g plus a c o o l i n g u n i t , both o f which would g i v e approximately s i m i l a r r a t e s o f temperature change, was required. These requirements were p a r t i a l l y Warburg r e s p i r o m e t e r .  met by the use of the  The apparatus was used as a water bath  and was f i t t e d w i t h an aluminium c o o l i n g c o i l which was cooled with tap water. of  The top o f the bath was i n s u l a t e d w i t h b l o c k s  styrifoam. The p l a n t s were put i n t o covered 8" - 5" p l a s t i c cake d i s h e s  which were then p a r t i a l l y  submerged (to w i t h i n 1/2 " from the  upper rim) w i t h weights.  A hole was d r i l l e d  of the d i s h e s t o i n s e r t a thermometer.  i n the cover of one  The humidity o f the d i s h e s  was not c o n t r o l l e d but the p l a n t s were w e l l watered and the i n s i d e  30  of  the c o n t a i n e r s were kept damp so that moisture was never Since the humidity outdoors was c l o s e t o or a t 100$  lacking.  d u r i n g most o f the study, the humidity of the p l a s t i c d i s h e s probably approximated the c o n t r o l humidity q u i t e c l o s e l y . The h e a t i n g g r a d i e n t remained constant (l°C-C>  / 5 min.).  C o o l i n g was not as e a s i l y c o n t r o l l e d s i n c e the volume o f c o l d water p a s s i n g through the c o o l i n g c o i l s had t o be i n c r e a s e d as the  temperature approached room temperature.  Furthermore the  system could not c o o l f a s t enough at temperatures below that o f the  room.  I t was f e l t that t h i s was not c r i t i c a l s i n c e the  e r r o r was introduced a t temperatures which were not considered abnormal t o t h e p l a n t s . At 12-14 not  the beginning of each experiment the water bath was at 0.  I t was f e l t that any temperature under 15°C.  be d e l e t e r i o u s to the p l a n t s .  were placed i n t o the water bath.  Plants i n p l a s t i c  would  containers  The temperatures i n the c o n t a i n e r s  and the water bath were allowed t o e q u a l i z e before the h e a t i n g c y c l e was begun.  I n most o f the experiments performed u s i n g t h i s  apparatus, the p l a n t s were kept at the d e s i r e d maximum temperature for  30 minutes.  temperature  The water bath could be h e l d a t any d e s i r e d  by u s i n g the thermo-regulator.  D i f f i c u l t i e s were  encountered i n keeping p r e c i s e c o n t r o l o f the temperature w i t h i n the  p l a s t i c containers.  The temperature o f the p l a s t i c  containers  tended to l a g behind that o f the water bath at a constant r a t e u n t i l the At  d e s i r e d maximum temperature was reached i n the water bath. t h i s time the thermostat maintained the water bath temperature.  The r a t e o f i n c r e a s e i n temperature w i t h i n the c o n t a i n e r s dropped c o n s i d e r a b l y at t h i s time, so that t h e i r temperature never reached  that  of the water bath.  w i t h i n the p l a s t i c  For t h i s  temperature  container f l u c t u a t e d approximately  w h i l e the water bath remained maximum t e m p e r a t u r e of  r e a s o n t h e maximum  1°C.  a t a c o n s t a n t temperature-.  f o r a l l experiments  Thus,  a r e g i v e n w i t h an  error  1°C. The  c o o l i n g c y c l e was  begun by t u r n i n g o f f t h e h e a t i n g i  _eo.il and  s t a r t i n g a flow of tap water through the c o o l i n g  T h i s was  c o n t i n u a l l y r e g u l a t e d so t h a t t h e r a t e o f  change was was  s i m i l a r f o r c o o l i n g and  cooled to 1 4 ° C ,  f o r heating.  temperature  When t h e  t h e p l a n t s were removed and  t a k e n b a c k t o t h e r o o f where t h e y had  coil.  bath  immediately  been p r e v i o u s l y  stored.  Methods o f A n a l y z i n g T r e a t e d P l a n t s : The  methods f o r c y t o l o g i c a l s t u d y o f t h e s e p l a n t s  described  i n Chapter  Sampling—In  some t r e a t e d  a f t e r t h e i r removal attempt  was  1.  most o f t h e e x p e r i m e n t s  Warburg a p p a r a t u s  a t t h e end  time.  i n v o l v i n g the use  s p o r a n g i a were examined of the heat  shock  T h i s was  extremely  Many o f t h e s p o r o p h y t e s  of  difficult  o f any  selected  B e c a u s e more t h a n one  only  about  t i m e were s u i t a b l e f o r a n a l y s i s .  experiment  was  being  approximately  every second  day.  study  conducted  t h e same t i m e , p r o g r e s s i v e s a m p l i n g o f e a c h t r e a t m e n t  performed  one  clump o f p l a n t s  e s s e n t i a l t o l e a v e enough young c a p s u l e s f o r f u r t h e r  as w e l l . at  An  o n l y a l i m i t e d number o f s l i d e s c o u l d be p r e p a r e d a t  o n e - h a l f t h e s l i d e s made a t one was  immediately  period.  were e i t h e r t o o y o u n g o r t o o o l d f o r s t u d y , so t h a t  It  of the  made t o o b t a i n r e p r e s e n t a t i v e s o f a l l s t a g e s  m e i o s i s at each sampling time. because  are  was  However, day t o  day  32 s a m p l i n g was p e r f o r m e d when t i m e was a v a i l a b l e . E a c h s l i d e was examined a s soon a s i t was made and a n y meiotic  i r r e g u l a r i t i e s were n o t e d .  were n o t r e p r e s e n t e d  I f c e r t a i n stages  of meiosis  i n t h e g r o u p o f s l i d e s made a t one s a m p l i n g  t i m e t h e n f u r t h e r a t t e m p t s were made t o c h o o s e c a p s u l e s m i g h t be a t t h e d e s i r e d Analysis—Later  stage.  t h e s l i d e s were examined s y s t e m a t i c a l l y and  c a r e f u l observations  were made.  some q u a n t i t a t i v e d a t a  An a t t e m p t was made t o p r o c u r e  on t h e t r e a t m e n t s .  tabulating the abnormalities  a f t e r the treatment  t a b u l a t i o n was made c o n c e r n i n g  o f t h e SMC were i n l a t e  T h i s was done by  found i n c e l l s which had completed  o r h a d begun t o c o m p l e t e m e i o s i s This  which  slides  period.  i n which t h e m a j o r i t y  anaphase I I o r t h e t e t r a d s t a g e o f d e v e l -  opment.  F i v e hundred c e l l s  analysed  and t a b u l a t e d  from each s a t i s f a c t o r y s l i d e  as f o l l o w s , u s i n g  (0) n o r m a l t e t r a d s w i t h  four c e l l s ,  a laboratory  were  counter:  a l l o f which  have n u c l e i o f c o m p a r a b l e s i z e and w h i c h p o s s e s s no m i c r o n u c l e i ,  extra nuclear  f r a g m e n t s o f .chromosomes (1)  tetrads with one  four c e l l s  extra nuclear  chromosomes o r  ( P L . 3> f i g . 2). each w i t h  n u c l e i but p o s s e s s i n g  chromosome o r f r a g m e n t .  o f t h e f r a g m e n t was n o t p a r t i c u l a r l y  The s i z e  considered but  some were o b v i o u s l y l a r g e r t h a n any o f t h e b i v a l e n t s o f n o r m a l metaphase ( P L . 5, f i g . 3). (2) same a s ( l ) b u t p o s s e s s i n g (PL.  5,  fig.  chromosomes  4).  (3) same a s ( l ) b u t p o s s e s s i n g chromosomes  two e x t r a n u c l e a r  3 o r more e x t r a  ( P L . 5, f i g . 5).  nuclear  33  (4)  d e g e n e r a t e t e t r a d s o f SMC This  showing  i n c l u d e d t e t r a d s w h i c h had  aborted meiosis.  e i t h e r more t h a n , o r  l e s s than, four c e l l s with n u c l e i . had  Almost  ( P L . 5,  e x t r a n u c l e a r chromosomes,  a l l of these  fig.  6-8).  C o n t r o l p l a n t s were examined and a n a l y s e d f r o m t h e same p o p u l a t i o n u s e d i n t h e e x p e r i m e n t s , and a t t h e same t i m e t h e e x p e r i m e n t s were c o n d u c t e d .  Observations presented  earlier  ( C h a p t e r l ) c o n c e r n i n g Hypnum c i r c i n a l e were d e r i v e d f r o m controls.  The  tetrad  the  a n a l y s i s o f t h e c o n t r o l s , however, i s  included with the r e s u l t s  of the  experiments.  Materials: Pilot  e x p e r i m e n t s were p e r f o r m e d i n t h e s p r i n g o f  1963  w i t h p l a n t s o f Hypnum subimponens L e s q . f r o m p o p u l a t i o n s c o l l e c t e d in  t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a Endowment L a n d s ,  B.C.  (Dill  #5163).  Experiments  conducted i n the f a l l  u s i n g t h e same p o p u l a t i o n s o f H. the  preparations  that  from w i l d  t h e s p o r a n g i a f r o m any  the  same.  not  be g e n e t i c a l l y i d e n t i c a l .  of  Certainly  gametophytes  a large  o f H.  o f 1963  circinale  described i n Chapter  S i n c e t h e m a t e r i a l was said  performed  t h a t were u s e d i n  1. p o p u l a t i o n s i t c a n n o t be '  one p o p u l a t i o n were  s p o r a n g i a from d i f f e r e n t  circinale  were  genetically  p o p u l a t i o n s would  However, s i n c e t h e d e n s e p o p u l a t i o n s p r o b a b l y have been p r o d u c e d ,  to  e x t e n t , by a s e x u a l means, i t i s s u s p e c t e d t h a t t h e >;•-  gametophyte members a r e q u i t e u n i f o r m g e n e t i c a l l y . as t h e sp-ecies i s monoe&ious and t h e gametophytes fertile,  Vancouver,  t h e s p o r o p h y t e s o f one  In  addition,  probably  self-  clump, o r , s m a l l p o p u l a t i o n ,  also  33  would be g e n e t i c a l l y quite uniform.  34  RESULTS The r e s u l t s o f p i l o t  experiments on Hypnum subimponens  i n the s p r i n g o f 1 9 6 3 i n d i c a t e d that the s p e c i e s was, d u r i n g i t s m e i o t i c c y c l e , extremely s e n s i t i v e t o short term heat s t r e s s ( 4 hour p e r i o d w i t h maximum temperature range at 3 2 , 3 6 , & 3 7 ° C).  Many unusual m e i o t i c a b e r r a t i o n s were observed, i n c l u d i n g  complete clumping of metaphase I chromosomes, p r e c o c i o u s d i s j u n c t i o n o f a l l or some of the b i v a l e n t s , abnormal t e t r a d a t i o n and complete a b o r t i o n o f m e i o s i s .  form-  However, i n s u f f i c i e n t  m a t e r i a l was a v a i l a b l e t o make a s a t i s f a c t o r y a n a l y s i s . f a l l o f 1 9 6 3 Hypnum c i r c i n a l e produced abundant  I n the  sporophytes and  was t h e r e f o r e s e l e c t e d as the m a t e r i a l with which to continue the work.  U n f o r t u n a t e l y , due t o the low y i e l d o f p e r t i n e n t  s l i d e s d u r i n g sampling even t h i s m a t e r i a l was i n s u f f i c i e n t t o i n d i c a t e more than trends ; i n abnormal behaviour. Experiment # 1 : Material—Hypnum Treatment—The  c i r c i n a l e , population #1363.  p l a n t s were maintained i n the water bath f o r a  p e r i o d of 4 1 / 2 hours w h i l e the temperature was r a i s e d t o 36.5 - 0.5°C  and h e l d a t that temperature f o r 3 0 minutes; then  returned t o 14°Q, were s t o r e d o u t s i d e  F o l l o w i n g the heat shock p e r i o d the p l a n t s (7-ll°C).  Observations—The f i r s t  2 0 s l i d e s were prepared 2 1 / 2 hours a f t e r  completion o f the heat shock p e r i o d . the SMC o f a sporangium.  Each s l i d e contained a l l  The m a j o r i t y of the sporangia had  completed meiosis and only young spores were seen.  These were  35 discarded.  The f i v e u s a b l e  categories: had  slides  fell  i n t o the f o l l o w i n g  one had o l d t e t r a d s } , two were m o s t l y  l e p t o t e n e , zygotene  and p a c h y t e n e ;  tetrads;  one  and one had l e p t o t e n e and  zygotene. A few metaphase I c e l l s were o b s e r v e d  i n the s l i d e s  t e t r a d s and a l l o f t h e s e were s e v e r e l y clumped those  shown i n PL.7, f i g . 7 ) .  e x t r a n u c l e a r chromatin, m e i o s i s was c o m p l e t e d of  with  (similar to  Some o f t h e t e t r a d s c o n t a i n e d  i n d i c a t i n g that the l a s t  d u r i n g the heat  part of  shock p e r i o d .  Only  t h e s l i d e s w i t h t e t r a d s were s u i t a b l e f o r a n a l y s i s ;  two  these  a r e s p o r a n g i a 1 and 3 i n T a b l e I . Table I :  T e t r a d a n a l y s i s o f s p o r a n g i a o f experiment  Sporangium No.  Time o f sampl i n g a f t e r the end o f t h e h e a t shock p e r i o d .  #1.  fo c e l l s o f e a c h a n a l y s i s c a t e g o r y o f 500 examined c e l l s p e r sporangium. 0  1  2  3  Comments  4  1  2.5 h r s .  88.4  8.4  1.6  0.6  3  2.5 h r s .  91.0  6.0  1.4  0.4  6  26 h r s .  65.8  11.2  1.2  1.0  19.8 tt  23  4 days  26.8  13.0  7.8  8.6  43.8 tt  The the heat  next  1.0 s l i d e h a d mostly tetrads 1.2 ti  12 s l i d e s were made 26 h o u r s a f t e r t e r m i n a t i o n o f  shock p e r i o d .  These f e l l  into the following categories:  f o u r c o n t a i n e d young spores!;, two had o l d t e t r a d s w i t h no stages  ; two had t e t r a d s w i t h some a c t i v e  active  s t a g e s ; two h a d  t h e t e r m " a c t i v e s t a g e s " i s u s e d to. d e n o t e a l l t h e s t a g e s from d i a k i n e s i s t o t e l o p h a s e I I .  mostly  36  Table I I :  T e t r a d a n a l y s i s o f c o n t r o l p l a n t s o f p o p u l a t i o n #1363 w h i c h were u s e d  Sporangium No.  i n .experiments  1 aand 2.  $ c e l l s o f each analysis category o f 500 examined c e l l s p e r s p o r a n g i u m . 0  1  2  3  4  Comments  1  94.0  4.0  0.0  1.0  1.0  mostly  5  93.6  5.0  0.0  0.8  0.6  it  7  89.0  5.6  4.6  0.4  0.4  11  96.8  2.6  0.0  0.3  0.3  ti  23  95.6  3.0  0.4  0.6  0.4  II  Average  93.8  4.0  1.0  0.6  dictyotene stages cells); in  0.5  +  tetrads  i»  +  (one o f t h e s e s l i d e s had some metaphase I  and two had l e p t o t e n e and z y g o t e n e .  a l l o f t h e s e s l i d e s were a b n o r m a l .  The a c t i v e s t a g e s "  Some o f t h e metaphase I  were clumped, b u t i n t h o s e t h a t were n o t clumped, o v e r 70$ had b i v a l e n t s that.were 10).  not oriented  on a p r o p e r s p i n d l e  ( P L . 4, f i g .  Anaphase I and I I c o n t a i n e d many l a g g a r d s and b r i d g e s .  A g a i n prophase  I s t a g e s c o u l d n o t be d i s t i n g u i s h e d f r o m  I stages observed for tetrad  i n the controls.  One s l i d e  suitable-  a n a l y s i s and i s i n c l u d e d i n T a b l e I (Page 3 5 ) .  S e v e n s l i d e s were made 48 h o u r s the heat  (#6) was  prophase  shock p e r i o d .  They f e l l  a f t e r the termination of  into the following  two  h a d young s p o r e s ; two had p r e d o m i n a n t l y  had  p r i m a r i l y m i g r a t i n g pachytene;  and  some p a c h y t e n e .  all  t h e s p o r e s were s p h e r i c a l and a p p e a r e d  active  categories:  s t a g e s ; one  and two h a d l e p t o t e n e , z y g o t e n e  I n one o f t h e s l i d e s  c o n t a i n i n g young s p o r e s , normal.  However i n  37 a second  s l i d e t h e r e were some  t h a t were h y a l i n e , These c e l l s  i n this  ( a p p r o x i m a t e l y 10$) l a r g e  r e s p e c t r e s e m b l i n g young  cells  spores.  c o n t a i n e d p y c n o t i c chromosomes i n v a r i o u s s t a g e s o f  meiosis;  s u c h c e l l s may be termed r e s t i t u t i o n  apparent  t h a t t h e s e c e l l s were SMC i n w h i c h m e i o s i s had a b o r t e d .  All fig.  active  cells.  It i s  ( P L . 4, f i g . 11, 12; P L . 5,  s t a g e s were a b n o r m a l  l ) . Metaphase I s t a g e s were sometimes p a r t i a l l y ,  completely  clumped.  on a p r o p e r  but never  Unclumped metaphaseswwere n o t o r g a n i z e d  s p i n d l e and u n i v a l e n t s and f r a g m e n t s were s c a t t e r e d  around t h e c e l l .  Anaphases were s e v e r e l y d i s r u p t e d and i n  many, o f t h e c e l l s  i t was i m p o s s i b l e t o d e t e r m i n e  m e i o s i s t h e chromosomes were i n . i n t o g l o b u l a r masses. had  completed  Some o f t h e s e c e l l s ,  anaphase I w i t h o u t  outside the telophase I n u c l e i , suitable  f o rtetrad  lot  although  abnormal,  l e a v i n g l a g g a r d s and f r a g m e n t s ( P L . 5,  f i g . 2).  No s l i d e s  a d d i t i o n a l s l i d e s were made.  o n l y a few s u i t a b l e  of material.  I n u c l e i were clumped  were  analysis.  A f t e r f o u r days f i v e t h i s time  Telophase  what s t a g e o f  These s l i d e s  gories:  two c o n t a i n e d m o s t l y  tetrads;  one p o s s e s s e d  By  capsules remained i n t h e t r e a t e d fell  into the f o l l o w i n g cate-  r e s t i t u t i o n SMC and some b r o k e n  only tetrads;  one had m o s t l y d i c t y o t e n e ,  b u t had some d i p l o t e n e , m i g r a t i n g p a c h y t e n e and metaphase I cells;  and one h a d a l l d i p l o t e n e and p a c h y t e n e .  of the r e s t i t u t i o n (PL. 5, appeared and  f i g . 9),  SMC were i n d i c t y o t e n e and d i p l o t e n e s t a g e s  t h e remainder  abnormal.  About IQfo  were i n a c t i v e  stages that  One s l i d e was s u i t a b l e f o r t e t r a d  i s included i n Table I  ( s e e page  analysis  35).  A l l metaphase I s t a g e s were d i s r u p t e d and a p p e a r e d  unoriented  38 as t h e chromosomes were s p r e a d a l l o v e r t h e c e l l .  Generally-  many - u n i v a l e n t s were p r e s e n t . Two o b s e r v a t i o n s c a n be made a t t h i s p o i n t . synchrony  o f t h e prophase  stages appeared  d i s r u p t e d ; n e v e r were p a c h y t e n e , metaphase I s t a g e s o b s e r v e d Second, t h e f i r s t of  sampling.  F i r s t , the  t o be c o m p l e t e l y  d i p l o t e n e , d i c t y o t e n e and  i n one p l a n t  i n the control  d i p l o t e n e p h a s e s were s e e n a t t h e f o u r t h d a y  A l t h o u g h t h i s may be due t o s a m p l i n g  error i t  s u g g e s t s t h a t t h e d u r a t i o n o f d i p l o t e n e was p o s s i b l y immediately Experiment  material.  f o l l o w i n g t h e h e a t shock  shortened  period.  #2:  Material—Hypnum  circinale,  population  #1363  T r e a t m e n t — T h e p l a n t s were m a i n t a i n e d i n t h e w a t e r b a t h f o r a period  o f 3 1/2 h o u r s w h i l e t h e t e m p e r a t u r e  30.0- 0.5°  C., h e l d a t t h a t  t h e n r e t u r n e d t o 14°C. p l a n t s were s t o r e d Observations—The  t h e end o f t h e h e a t classes: had  active  f o r 30 m i n u t e s and  F o l l o w i n g t h e heat  outside first  temperature  was r a i s e d t o  shock p e r i o d t h e  (7-ll°C.).  8 s l i d e s were p r e p a r e d i m m e d i a t e l y  shock  p e r i o d and f e l l  after  into the following  one had m o s t l y t e t r a d s w i t h some a c t i v e s t a g e s ; one s t a g e s and d i c t y o t e n e s t a g e s ; two had m o s t l y  d i c t y o t e n e w i t h some d i p l o t e n e ; two had m i g r a t i n g p a c h y t e n e and d i p l o t e n e ; and two were i n l e p t o t e n e and z y g o t e n e . Many metaphase I s t a g e s were o b s e r v e d About  20$ were clumped  i n this  material.  (PL. 5, f i g . 10) w h i l e t h e r e m a i n d e r  v a r i e d f r o m p a r t i a l l y clumped  (PL. 5,  f i g . 11,12) t o t h o s e  39  a r r a n g e d on a n a p p a r e n t l y  normal s p i n d l e  ( P L . 6, f i g . 1-3)•  R a r e metaphase I s t a g e s were o b s e r v e d w i t h u n o r i e n t e d ided bivalents  (PL. 6£ f i g . 4 ) .  metaphase I c e l l s  with oriented  and d i v -  The chromosomes o f many o f t h e b i v a l e n t s a p p e a r e d more  highly  c o n d e n s e d and more g l o b u l a r t h a n t h o s e o f t h e c o n t r o l s . i n many o f t h e c e l l s  However,  t h e chromosomes were i n d i s t i n g u i s h a b l e f r o m  those i n the c o n t r o l s .  Unfortunately  only  one s l i d e  was  s u i t a b l e f o r t e t r a d a n a l y s i s , and i s s p o r a n g i u m 4 i n T a b l e I I I .  Table I I I :  Sporangium No.  Tetrad  analysis of sporangia  Time o f sampl i n g a f t e r the end o f t h e h e a t shock p e r i o d .  o f experiment  % c e l l s o f each a n a l y s i s c a t e g o r y o f 500 examined c e l l s p e r sporangium. 0  1  2  3  4  #2.  Comments  4  0 hrs.  85.6  6.8  0.6  0.6  6.4  mostly tetrads  13  72 h r s .  57.8  22.0  6.6  5.6  8.0  II  Of  s e v e n s l i d e s made 24 h o u r s a f t e r t h e t e r m i n a t i o n  heat shock p e r i o d ,  f o u r were d i s c a r d e d  much t o o young f o r m e i o t i c the  following classes:  and  one had young The  stages.  of the  because t h e p l a n t s  The r e m a i n d e r f e l l  two were i n p a c h y t e n e , and  were  into  diplotene;  spores.  p r o p h a s e I s t a g e s and t h e young s p o r e s a p p e a r e d  normal.  No m a t e r i a l was a v a i l a b l e f o r t e t r a d a n a l y s i s . T h r e e s l i d e s were made 72 h o u r s a f t e r t e r m i n a t i o n heat shock p e r i o d .  These f e l l  of the  i n t o the f o l l o w i n g classes:  one  40  had o l d t e t r a d s w i t h about 1$ a c t i v e stages and two were i n l e p t o t e n e and pachytene. The s l i d e w i t h t e t r a d s was analysed and i s sporangium 13 i n Table I I I (page 39). (PL.  6, f i g .  The t e t r a d s contained many a b n o r m a l i t i e s  7-9). The a c t i v e stages, which were a l l anaphases,  contained laggards or e x h i b i t e d abnormal s e g r e g a t i o n .  Prophase  I stages appeared normal. Experiment #3: Material—Hypnum Treatment—The  c i r c i n a l e , p o p u l a t i o n #2267  p l a n t s were maintained i n the water bath f o r  3 3/4 hours, while the temperature was r a i s e d t o 31.5 - 0.5° C , l e f t at that temperature f o r 30 minutes, and then lowered t o 14° C.  F o l l o w i n g the heat shock p e r i o d the p l a n t s were s t o r e d  outside  (7-ll°C.)  O b s e r v a t i o n s — F i v e s l i d e s were prepared immediately a f t e r the heat shock p e r i o d .  These f e l l  i n t o the f o l l o w i n g c l a s s e s :  one  had mostly t e t r a d s w i t h some a c t i v e stages; one had d i c t y o t e n e and d i p l o t e n e ; and three had e a r l i e r stages o f prophase I . All  metaphase I stages observed had some degree o f clumping,  although i t was not as severe as t h a t i n experiment #1. A l l observed anaphases had laggards and prophase I c e l l s appeared normal.  One s l i d e was s u i t a b l e f o r t e t r a d a n a l y s i s and i s  sporangium 3 i n T b l e IV (page 4 1 ) . a  E i g h t s l i d e s were made f i v e hours a f t e r the end of t h e heat shock p e r i o d , and f e l l  into the following classes:  one had  t e t r a d s ; one had d i c t y o t e n e ; one had t r a n s i t i o n a l stages between  41  Table IV: Tetrad a n a l y s i s of sporangia from experiment #3.  Sporangium No.  Time of sampl i n g a f t e r the end of the heat shock period.  $ c e l l s of each a n a l y s i s category of 500 examined c e l l s per sporangium. 0  1  2  3  4  Comments  3  0 hrs.  93.0  1.8  0.8  0.8  3.6  mostly tetrads  7  5 hrs..  73.0  11.6  3.0  3.0  8.4  II  17  20 h r s .  87.0  3.0  0.0  0.0  10.0  18  20 h r s .  63-0  7.8  1.2  0.2  27.2  19  20 h r s .  45.6  13.0  3.2  21  5 days  56.6  14.2  8.4  9.0  16.0  22  5 days  54.8  17.8  9.8  8.4  9.2  26  5 days  68.0  12.0  2.2  0.8  17.0  31  7 days  77.0  13.6  1.4  0.8  7.2  32  7 days  65.2  1.8.-6::L 5.8  2.4  . 1.2 27.0  young tetrads many a c t i v e stages old tetrads mostly tetrads tetrads with many a c t i v e stages mostly tetrads old tetrads  a c t i v e stages with young tetrads 8.0 .old tetrads  diplotene and d±$fgrotene ; four had leptotene t o pachytene; and one had diplotene and migrating pachytene. No a c t i v e stages were observed i n the sampling. The prophase I stages appeared normal, except that synchrony of d i v i s i o n w i t h i n each sporangium was l o s t .  I n contrast t o the f i n d i n g s i n  experiment 1 and 2, some diplotene f i g u r e s were observed.  One  42  s l i d e  was  Table  I V  Table  V :  s u i t a b l e (page  f o r  T e t r a d  a n a l y s i s  #  which  2263  Sporan-  % of  No.  c e l l s 500  0  4 8  99.2  10  94.0  o l d  s l i d e s  p e r i o d .  t e t r a d s  t e t r a d s ; metaphase prophase  I  Most  and  of  c o n t r o l used  each  i n  a n a l y s i s  per  3  0 . 0  4.4  were They  0 . 8  f e l l  some  had  20  hours  a f t e r  i n t o  the  made  a c t i v e  m o s t l y and  d i c t y o t e n e two  had  the  f o l l o w i n g  stages;  one  w i t h  m o s t l y  t e t r a d s ;  s t a i n i n g would J not a l l o w ] .accurate observ a t i o n s of V m i c r o n u c l e i  0.6  end  of  the  heat  c l a s s e s :  two  a c t i v e  stages,  had  d i a k i n e s i s d i c t y o t e n e  and  had young  r a r e  w i t h  e a r l y  s t a g e s .  twelve  of  hundred  appeared  to  somewhat  abnormal  of  laggards  d u r i n g  have  frequency the  of  s p o r a n g i a  abnormal. l a t t e r w h i l e  f i c i a l l y  / o l d  0.2  n o r m a l ;  shock  Comments  0.4  appeared  the  #3.  4  i n  were  i n  p o p u l a t i o n  category  stages  i n  7  sporangium  2  1.0  of  experiment  a c t i v e  ever,  sporangium  s p o r a n g i a  the  The  i s  0 . 8  stages;  I  were  3.6  w i t h  one  of  c e l l s  1  95.6  shock  a n a l y s i s  41).  glum  S i x  t e t r a d  T h i s  s p o r a n g i a the  w i t h  most  o r i e n t a t i o n  anaphases  o l d e r  t e t r a d s may  were  a c t i v e  that  at  ones the  the were  heat  a c t i v e  i n  be  l a t e  shock  sporangium  metaphases  d i s c r e p a n c y  former  suggests  one  the  was a l l  on  the  q u i t e  low.  by  the  d u r i n g  prophase.  a f f e c t e d  s p i n d l e .  anaphases  e x p l a i n e d stages  counted  l e s s  the  T h i s  Howobserved f a c t  t h a t  heat super-  s e v e r e l y  43 the l a t e later  stages o f prophase I than  i t d i d t h e metaphase I and  stages. The  p r o p h a s e s t a g e s a g a i n showed l a c k o f s y n c h r o n y b u t  otherwise tetrad  appeared  normal.  T h r e e s p o r a n g i a were s a t i s f a c t o r y f o r  a n a l y s i s and a r e s p o r a n g i a 1 7 , 18, and 19 i n T b l e I V a  (page 4 1 ) . F i v e d a y s a f t e r t h e end o f t h e h e a t were p r e p a r e d . had  They f e l l  stages);  stages  In  (one o f t h e s e had o n l y r a r e  observed  prophase I s t a g e s .  s t a g e s were a b n o r m a l .  bivalents.  fig.  V e r y few metaphases were  clumped o r t h e r e were  The b i v a l e n t s a p p e a r e d  10).  on t h e s p i n d l e .  ( P L . 6,  d i c t y o t e n e and  s t a g e s , t h e r e were many metaphase I s t a g e s . (71 o f 100 c e l l s  Anaphases  unoriented  I n t h e sporangium w i t h predominantly  t h e most p a r t  disjoined  h i g h l y condensed.  many l a g g a r d s and b r i d g e s , o r a p p e a r e d  active  s t a g e s , most  and most o f t h e s e were o r i e n t e d on a s p i n d l e , b u t o f t e n  t h e chromosomes were p a r t i a l l y  had  active  s t a g e s ; and f o u r h a d  t h e s p o r a n g i a w i t h t e t r a d s and some a c t i v e  the active  one  one h a d o l d t e t r a d s ; two had  one had d i c t y o t e n e and many a c t i v e  various unsynchronized  of  into the following classes:  o l d t e t r a d s and y o u n g s p o r e s ;  t e t r a d s and a c t i v e  shock p e r i o d 9 s l i d e s  These, f o r  c o u n t e d ) had n o r m a l a r r a n g e m e n t  I n t h e metaphase c e l l s w i t h a b n o r m a l  bivalent  a r r a n g e m e n t , t h e b i v a l e n t s were u s u a l l y h i g h l y c o n d e n s e d . T h r e e s p o r a n g i a were s u i t a b l e f o r t e t r a d  a n a l y s i s and a r e  s p o r a n g i a 21, 22, a n d 26 i n T b l e I V (page 4 1 ) . a  Seven days f o l l o w i n g t h e heat prepared one  and f e l l  s h o c k 6 more s l i d e s  into the following classes:  had o l d t e t r a d s ;  one h a d a c t i v e  were  one had s p o r e s ;  s t a g e s and y o u n g  tetrads;  44 two were i n d i c t y o t e n e and d i p l o t e n e w i t h r a r e p a c h y t e n e and  stages;  one was i n l e p t o t e n e . A g a i n t h e metaphase I s t a g e s r a n g e d  he n o r m a l  f r o m what a p p e a r e d t o  t o t h o s e t h a t were a b n o r m a l .  F i f t y - o n e o f t h e metaphase  I s t a g e s counted had a p p a r e n t l y normal  o r i e n t a t i o n on t h e s p i n d l e .  A l s o t h e r e were many c a s e s where i t was d i f f i c u l t w h e t h e r t h e o r i e n t a t i o n was n o r m a l o f t e n were condensed (PL.  o r abnormal.  from those o f t h e c o n t r o l s  the stages observed,  The chromosomes  s l i g h t l y more t h a n t h o s e i n t h e c o n t r o l s  6, f i g . 1 1 ) , b u t t h e r e were a l s o t h o s e t h a t  distinguished  to establish  c o u l d n o t be  (PL. 7, f i g . l ) .  a n a p h a s e s were t h e most a b n o r m a l .  chromosomes were n o t o r i e n t e d  (PL. 7, f i g . 2 ) .  Also,  commonly  Two s l i d e s were s u i t a b l e f o r t e t r a d  a n a l y s i s a n d a r e s p o r a n g i a 31 and 32  Experiment  The  and t h e r e were many l a g g a r d s .  n o n d i s j u n c t i o n o f one o r more chromosomes was most observed  Of  i n T a b l e IV (page 4 1 ) .  #4:  Material—Hypnum  circinale.  population  #2463  T r e a t m e n t — T h e p l a n t s were m a i n t a i n e d i n t h e w a t e r b a t h f o r a p e r i o d o f 6 1/2 h o u r s +  d u r i n g which t h e temperature  was  tfaaiseds  o  t o 25.0 - 0.5 C ,  kept a t t h a t temperature  lowered  The p l a n t s were p l a c e d o u t s i d e f o l l o w i n g t h e  t o 14°C.  treatment  (7-ll°C).  Observations—No treatment.  s l i d e s were p r e p a r e d i m m e d i a t e l y  A f t e r 24 h o u r s  into the following classes: tetrads;  f o r 4 h o u r s and  a f t e r the  6 s l i d e s were made and t h e s e one h a d a c t i v e  fell  s t a g e s and young  two had d i c t y o t e n e a n d a c t i v e s t a g e s ; and t h r e e h a d  45  m o s t l y d i c t y o t e n e w i t h some t r a n s i t i o n a l s t a g e s and  d i c t y o t e n e , and a l s o r a r e The  active  between d i p l o t e n e  pachytene.  stages observed d u r i n g t h i s  sampling  appeared  predominantly normal.  T h e r e was a n o c c a s i o n a l metaphase i n w h i c h  the b i v a l e n t s appeared  u n o r i e n t e d and were o v e r - c o n d e n s e d ,  in  g e n e r a l b e h a v i o u r was n o t s i g n i f i c a n t l y  control.  Prophase  s t a g e s were r a r e . is  sporangium  I stages exhibited  different  but  from t h e  some a s y n c h r o n y  and d i p l o t e n e  One p l a n t was s u i t a b l e f o r t e t r a d  a n a l y s i s and  6 i n T a b l e Y I (page 46).  T h i r t e e n s l i d e s were p r e p a r e d 72 d a y s a f t e r t h e end o f t h e treatment old  and t h e s e f e l l  into the following classes:  t e t r a d s w i t h few o r no a c t i v e  f o u r had  s t a g e s ; one had m o s t l y  w i t h some a c t i v e s t a g e s ; two had m o s t l y a c t i v e  tetrads  s t a g e s ; two  had m o s t l y d i c t y o t e n e w i t h r a r e d i p l o t e n e , d i a k i n e s i s and meta-^ phase I f i g u r e s ;  one had d i p l o t e n e - d i c t y o t e n e t r a n s i t i o n a l  two  had d i p l o t e n e and p a c h y t e n e ;  and  r a r e pachytene  stage;  and one had l e p t o t e n e , z y g o t e n e  stages.  Again the a c t i v e  stages appeared  predominantly  normal.  O n l y a few ( p r o b a b l y l e s s t h a n 5f°) o f t h e metaphases  appeared  s l i g h t l y unoriented with highly-condensed b i v a l e n t s .  The  prophase  from  stage exhibited  some a s y n c h r o n y  since stages  d i p l o t e n e t o metaphase I were o b s e r v e d i n two s l i d e s .  Pour  s l i d e s were s u i t a b l e f o r t e t r a d a n a l y s i s and a r e s p o r a n g i a 17, 12, 14 and 19 i n T a b l e VI (page 46). F i v e d a y s a f t e r t h e end o f t h e h e a t s h o c k s l i d e s were p r e p a r e d and t h e s e f e l l e i g h t had s p o r e s ; one had a c t i v e  p e r i o d 11 more  into the following  classes:  s t a g e s and y o u n g t e t r a d s ; one  46  Table V I :  Tetrad a n a l y s i s of sporangia of experiment #4,  Sporanglum No.  Time of sampfo c e l l s of each analysis l i n g a f t e r the category of 500 examined end of the heat c e l l s per sporangium shock period. n i ? ^ A  6  24 hrs.  83.0  7.6  1.4  1.0  7.0 a c t i v e stages and young tetrads  17  72 hrs.  90.2  3.4  0.2  0.4  5.8 o l d tetrads  12  72 hrs.  73.8  6.6  1.2  1.0  14  72 hrs.  87.4  7.2  1.8  0.2  3.4 "  19  72 h r s .  84.2  5.8  1.8  0.2  8.0 "  80.6 10.4  1.6  0.6  6.8 a c t i v e stages and young tetrads  21  5 days  Comments  17.4 mostly tetrads  Table V I I : Tetrad analysis of sporangia from population #2463 which were used i n experiments 4-'7. Sporangium No.  $ c e l l s i n each a n a l y s i s category of 500 examined c e l l s per sporangium 0 1 2 4 3  Comments  8  88.6  3-2  0.6  0.2  7.4  mostly tetrads  9  95.8  3.4  0.4  0.0  0.4  ii  10  94.6  3.8  0.4  0.0  1.2  n  12  94.0  2.6  0.2  0.2  3.0  II  13  93.6  2.8  0.4  0.0  3.2  II  Average  93.3  3.2"  0.4  0.1"  3.0  +  +  47  had  d i c t y o t e n e w i t h some metaphase I and r a r e d i p l o t e n e s t a g e s ;  and  one h a d m o s t l y The  dictyotene.  spores observed appeared normal,  as d i d t h e a c t i v e  s t a g e s , w i t h t h e e x c e p t i o n o f a few q u e s t i o n a b l e metaphase I stages.  Prophase  I stages appeared  s l i d e was s u i t a b l e f o r t e t r a d  slightly  analysis  asynchronous.  and i s s p o r a n g i u m  One  2 1i n  T a b l e V I (page 4 6 ) .  Experiment  #5:  Material—Hypnum Treatment—In  circinale,  an a t t e m p t  r a i s i n g t h e temperature  population #2463  t o d e t e r m i n e t h e immediate t o a l e v e l t h a t would  a n o m a l i e s , t h e p l a n t s were p u t i n t o t e m p e r a t u r e was r a i s e d  t h e water  induce severe b a t h and t h e  i n t h e n o r m a l manner t o 3 7 -  The m a t e r i a l was removed a t t h a t  temperature  effects of  0.2°C.  and s l i d e s  were  made a s s o o n a s p o s s i b l e . O b s e r v a t i o n s — O f t h e twenty treatment p e r i o d old. had  9 were d i s c a r d e d  The r e m a i n i n g 1 1 f e l l o l dtetrads;  s l i d e s made i m m e d i a t e l y a f t e r t h e  into  because  t h e s p o r a n g i a were t o o  the following classes:  two  f o u r h a d m o s t l y t e t r a d s and some a c t i v e s t a g e s ;  two had m o s t l y d i c t y o t e n e and some metaphase I s t a g e s ; t h r e e had d i p l o t e n e and l a t e All  pachytene  s t a g e s ; and one had l e p t o t e n e .  metaphase I s t a g e s o b s e r v e d were s e v e r e l y clumped; t h e  b i v a l e n t s h a d f u s e d i n t o ' a g l o b u l a r spheres o f c h r o m a t i n fig.  7 ) .  laggards,  O n l y a few o f t h e a n a p h a s e s i n d i c a t i n g perhaps t h a t  (PL. 7 ,  and t e l o p h a s e s t a g e s h a d  t h e s e s t a g e s were  completed  48  before t h e temperature otene  was c r i t i c a l l y  high.  s t a g e s had b e e n a f f e c t e d by t h e h e a t reduced  shock, f o r o f t e n t h e  chromatin  appeared  partially  condensed around t h e chromocentre.  prophase stages appeared  i n area.  Many o f t h e d i c t y -  normal  Chromosomes were a p p a r e n t l y The  earlier  ( P L . 7, f i g . 5 , 6 ) .  o c c a s i o n a l i n s t a n c e s o f abnormal p a i r i n g  T h e r e were  ( P L . 7, f i g . 4)  as t h i s anomaly was r a r e i t c o u l d n o t be d e t e r m i n e d was c a u s e d  by t h e h e a t  abnormality.  whether i t  s h o c k o r w h e t h e r i t was due t o n a t u r a l  F i v e s l i d e s were s u i t a b l e f o r t e t r a d  a r e s p o r a n g i a 4,  but  10, 2, 7, and 11 i n T a b l e V I I I  a n a l y s i s and  (page  49).  E l e v e n s l i d e s were made 4 h o u r s a f t e r t h e end o f t h e treatment mostly and  and t h e s e f e l l  into the f o l l o w i n g classes:  o l d t e t r a d s w i t h a few a c t i v e s t a g e s ; two had many a c t i v e  some t e t r a d s ;  one had d i c t y o t e n e and metaphase I s t a g e s ; and  s i x had e a r l y p r o p h a s e s t a g e s i n w h i c h d i p l o t e n e was Again cells  two had  rare.  t h e metaphase I s t a g e s were s e v e r e l y clumped.  In  a t t e l o p h a s e I , t h e chromosomes o f t h e two n u c l e i were  clupped  i n t o g l o b u l a r spheres.  Anaphase s t a g e s were n o t  observed,  i n d i c a t i n g t h a t a c t i v e movement o f chromosomes h a d b e e n t e m p o r a r i l y arrested.  The d i c t y o t e n e s t a g e s h a d more h e t e r o c h r o m a t i c  than those  o f c o n t r o l s and t h e c h r o m a t i n  threads  t h i c k e r and s t u c k t o g e t h e r i n c e r t a i n a r e a s . s i z e o f the chromatin  for tetrad VIII  (page  appeared  In addition, the  mass a t d i c t y o t e n e h a d b e e n r e d u c e d .  prophase I stages appeared  normal.  Four  areas  s l i d e s were  Early  suitable  a n a l y s i s and a r e s p o r a n g i a 12, 17, 20 and 22 i n T a b l e 49).  T w e l v e s l i d e s were made 72 h o u r s f o l l o w i n g t h e end o f t h e  49  Table V I I I :  Sporangium No.  Tetrad a n a l y s i s of sporangia of experiment #5  Time of sampl i n g a f t e r the end of the heat shock period. n  $ c e l l s i n each a n a l y s i s category of 500 examined c e l l s per sporangium -. « ~ .  Comments  4  0 hrs.  91.4  5.2  1.0  0.6  1.8  old tetrads  10  0 hrs.  90.2  3.6  1.4.  0.2  4.6  tr  2  0 hrs.  85.0  6.8  3.0  1.4  7  0 hrs.  73.8  15.0  3.6  2.8  .3.8 a c t i v e s t a ges and young tetrads 4.8 ti  11  0 hrs.  67.0  14.0  4.6  5.0  9.4  it  90...8  5.6  0.8  0.0  2.8  old tetrads  8.4  1.2  1.2  4.6  mostly t e t rads  12  441  hrg.  17  41ihrs .  84.6  20  4 hrs.  65.0  14.0  4.0  4.0  13.0  a c t i v e stages and tetrads  22  4 hrs.  70.6  14.4  7.0  2.0  6.0  a c t i v e stages and young tetrads  25  72 h r s .  80.0  6.8  1.6  0.0  11.6  a c t i v e stages and o l d tetOrads  39  5 days  71.8  12.8  3.8  3.0  7.6  42  5 days  46.4  13.8  1.6  1.4  36.8  treatment.  These f e l l i n t o the f o l l o w i n g c l a s s e s :  old tetrads a c t i v e stages and tetrads  two had  young spores; one had a c t i v e stages and t e t r a d s , one had mostly a c t i v e stages; two had mostly dictyotene; f i v e had unsynchronized  50  e a r l y prophase stages l e p t o t e n e and  i n c l u d i n g d i c t y o t e n e ; and  one  had  zygotene.  I n t h e s l i d e s w i t h y o u n g s p o r e s , r e s t i t u t i o n SMC common, i n d i c a t i n g t h a t m e i o s i s had in  some c a s e s .  No  clumping  was  g e n e r a l l y s p i n d l e f o r m a t i o n was m e t a p h a s e s , examined a p p e a r e d (PL. 7,  f i g . 8;  PL.  8,  p h a s e s had  observed  a t metaphase I ,  abnormal.  t o be  but  F i f t y - t w o of  arranged  200  on a s p i n d l e  i n a l l cases the  chromosomes  d i s j u n c t i o n was.not s y n c h r o n o u s .  many l a g g a r d s but 88  e x h i b i t e d no  been c o m p l e t e l y a r r e s t e d  f i g . 12), but  were h i g h l y c o n d e n s e d and  were  abnormalities.  Ana-  o f IQO examined t e l o p h a s e I  stages  1  There are at l e a s t  explanations f o r t h i s discrepancy.  One  two p o s s i b l e  i s t h a t the l a g g a r d s  anaphase were i n c l u d e d i n t h e t e l o p h a s e n u c l e i d e s p i t e  their  apparent  l a c k o f movement; t h e s e c o n d  i s t h a t m e i o s i s had  arrested  soon a f t e r the treatment  that these  and  cells  been  would  e v e n t u a l l y become r e s t i t u t i o n SMC  .  entirely  satisfactory.  explanation i s contrary to  findings  elsewhere  first  while r e s t i t u t i o n  i n p l a n t s w i t h young s p o r e s . a n a l y s i s and  Eight fell  Only  s l i d e was  were  a  one  had  d i p l o t e n e , and  t e t r a d s and  three  had  two  had  observed  (page  young s p o r e s ;  a c t i v e s t a g e s ; one  had  v a r i o u s e a r l y prophase I  s l i d e s w i t h y o u n g s p o r e s had  had  suitable for  i s s p o r a n g i u m 25 i n T b l e V I I I  i n t o the f o l l o w i n g c l a s s e s :  The  one  SMC  the  i s puzzling  s l i d e s were made 5 d a y s a f t e r t h e t r e a t m e n t  old tetrads; and  the second  been a r r e s t e d f o r t h r e e d a y s t h e SMC  gone i n t o r e s t i t u t i o n ,  tetrad  Neither explanation i s  i n t h e s e s t u d i e s and  b e c a u s e i f m e i o s i s had not  The  at  49).  and one  these had  dictyotene stages.  many r e s t i t u t i o n SMC  and  51 abnormally  shaped s p o r e s  ( P L . 8, f i g . 3-5)•  Often  spores  were s p h e r i c a l b u t were j o i n e d t o g e t h e r by t h r e a d s o f c e l l w a l l m a t e r i a l (PL. 8 , f i g . 4). restitution 5).  SMC were clumped i n t o l a r g e a g g r e g a t e d  ( P L . 8, f i g .  The a c t i v e s t a g e s were m a r k e d l y a b n o r m a l and p r o b a b l y h a d  been a r r e s t e d .  Prophase s t a g e s appeared  were s u i t a b l e f o r t e t r a d i n Table VIII  Experiment  Two  slides  (page 49).  #6:  Treatment—In  this  temperature  population  experiment  l a b o r a t o r y and put i n t o was 22- 2°C.  purpose o f t h e treatment meiotic  normal.  a n a l y s i s and a r e s p o r a n g i a 39 and 42  Material—Hypnum c i r c i n a l e ,  #2463  t h e p l a n t s were b r o u g h t  covered (normal  plastic dishes.  The l a b o r a t o r y  indoor conditions).  was t o d e t e r m i n e  into the  The  the behaviour o f  chromosomes u n d e r l a b o r a t o r y c o n d i t i o n s .  Observations—The had  I n many c a s e s t h e s p o r e s and  been b r o u g h t  first  s l i d e s , made 24 h o u r s a f t e r t h e p l a n t s  i n d o o r s , were m a r k e d l y a b n o r m a l .  I n 50-  60$ o f t h e metaphase I s t a g e s t h e r e was no s p i n d l e o r i e n t a t i o n , and  i n almost  condensed. ped  a l l the c e l l s  t h e metaphase I b i v a l e n t s were h i g h l y  M o r e o v e r , t h e r e were many c e l l s w i t h p a r t i a l l y  clum-  b i v a l e n t s a s w e l l as t h o s e w i t h p r e c o c i o u s l y d i s j o i n i n g b i -  valents.  T h e r e were a l s o t h o s e  c o n t a i n i n g a mixture  of the var-  i o u s a b n o r m a l i t i e s . Anaphases h a d many l a g g a r d s and a p p e a r e d organized.  P r o p h a s e s t a g e s were a p p a r e n t l y n o r m a l .  f a c t o r y d i p l o t e n e was o b s e r v e d ;  only t r a n s i t i o n a l  d i p l o t e n e and d i c t y o t e n e were p r e s e n t .  No  dis-  satis-  s t a g e s between  Two s l i d e s were  suitable  52 f o r t e t r a d analysis and are sporangia 1 and 2 i n Table IX, Table IX: Tetrad a n a l y s i s of sporangia of experiment 6. Sporangium No.  Time of sampfo c e l l s of each analysis ^JJnts were category of 500 examined brought indoors, c e l l s per sporangium. 0 1 2 3 4  1  24 n r s .  14.8  5.2  1.2  0.0  2  24 hrs..  27.2  2.0  0.4  0.2  6  30 h r s .  18.0 10.0  0.0  0.0  Comments  78.8 mostly tetrads 70.2 "  10  j.30 h r s .  39.6  9.2  2.2  0.4  72.0 a c t i v e stages and tetrads 48.6 o l d tetrads  12*  72 h r s .  1.2  0.0  0.0  0.0  98.8 a c t i v e stages and  17*  72 h r s .  0.0  1.0  0.0  0.0  99.0 "  18*  72 hrs.  0.8  0.0  0.0  0.0  99.2 "  *  i n these three sporangia no normal a c t i v e stages were observed and i n most of the SMC meiosis had aborted before second d i v i s i o n began. T h i r t y hours a f t e r plants were brought indoors 6 more s l i d e s were prepared.  These f e l l i n t o the f o l l o w i n g c l a s s e s :  two had o l d tetrads; one had a c t i v e stages and tetrads; one had dictyotene and a c t i v e stages; and two had pachytene stages. Again the a c t i v e stages were abnormal; over 60$ of the metaphases had abnormal spindle formation (PI. 8, f i g . 6-9). Even c e l l s w i t h some metaphase o r i e n t a t i o n had h i g h l y condensed b i v a l e n t s , one or more of which u s u a l l y displayed disjunction.  precocious  A l l anaphases had laggards and i n many cases the  53  chromosomes were s p r e a d a l l t h r o u g h stages appeared  normal.  the c e l l .  The p a c h y t e n e  Two s l i d e s were s u i t a b l e f o r t e t r a d  a n a l y s i s and a r e s p o r a n g i a 6 and 10 i n T b l e I X (page 5 2 ) . a  S e v e n t y - t w o h o u r s a f t e r t h e p l a n t s were b r o u g h t l a b o r a t o r y 9 s l i d e s were p r e p a r e d classes:  one had y o u n g s p o r e s  s t a g e s and t e t r a d s ; and in  a c t i v e stages  and t h e s e f e l l  and o l d t e t r a d s ;  into the  into the following f o u r had a c t i v e  one h a d a c t i v e s t a g e s ; two h a d d i c t y o t e n e  ( t h e r e were a l s o some e a r l y p r o p h a s e I s t a g e s  one slide)'; and one h a d d i c t y o t e n e . It  was n o t e d  t h a t t h e p l a n t s were m a t u r i n g  r a p i d l y by t h i s t i m e .  Many o f t h e c a p s u l e s t h a t were n o t f u l l  s i z e had begun m e i o s i s The  extremely  prematurely.  s p o r a n g i u m w i t h young s p o r e s h a d about 80$ r e s t i t u t i o n .  SMC and many d e g e n e r a t e were c o m p l e t e l y  tetrads.  aborted  ( P L . 9,  many c a s e s t h e c o m p l e t e t e t r a d  The a c t i v e s t a g e s  of meiosis  f i g . l ) and i t a p p e a r e d s t a g e would n e v e r  that i n  be r e v e a l e d .  P r o p h a s e I s t a g e s were a s y n c h r o n o u s and no d i p l o t e n e s were observed.  The d i c t y o t e n e chromosomes were o f t e n c o n d e n s e d  the chromocentre  ( s i m i l a r t o t h a t i n P L . 9,  c e l l s were o b s e r v e d  f i g . 4)  around  and many  i n w h i c h t h e chromosomes were clumped  a t i g h t l y packed b a l l  into  ( s i m i l a r t o t h a t i n P l l 9» f i g . 5 ) .  This  s t a g e c o u l d n o t be a c c u r a t e l y i n t e r p r e t e d , b u t was p o s s i b l y an aborted pachytene o r d i p l o t e n e . tetrad  T h r e e s l i d e s were s u i t a b l e f o r  a n a l y s i s and a r e s p o r a n g i a 12, 17 and 18 i n T a b l e I X  (page 5 2 ) .  These t e t r a d  s t a g e s v e r y s e l d o m had f o u r c e l l s  m e i o s i s had a b o r t e d a t o r b e f o r e s e c o n d 3). had  In addition,  i t was o f t e n n o t e d  division  ( P L . 9,  that i n d i v i d u a l  f i g . 2,  chromosomes  formed m i c r o n u c l e i . Observations  since  were made a g a i n a t 4 and 6 d a y s a f t e r t h e  54  p l a n t s were b r o u g h t completely  into the laboratory.  a b o r t e d ; b y 4 d a y s many o f t h e SMC h a d u n d e r g o n e  restitution.  Those t h a t h a d n o t u n d e r g o n e r e s t i t u t i o n  either aborted a c t i v e closely  M e i o s i s had  clumped  stages  (PL. 9 , f i g .  o r p r o p h a s e s t a g e s w h i c h were 5)t a s p r e v i o u s l y m e n t i o n e d .  6 d a y s t h e c a p s u l e s w i t h any s i g n o f m e i o t i c a c t i v i t y extremely  s m a l l (about l / 2 f u l l  a few a r c h e s p o r i a l c e l l s .  Experiment  were  s i z e ) and t h e s e had o n l y  developed  M e i o s i s had a b o r t e d .  p o p u l a t i o n #2463  T r e a t m e n t — T h e p l a n t s were b r o u g h t open t r a y s .  photoperiod.  purpose o f t h i s the humidity  i n t o t h e l a b o r a t o r y and p u t  One o f t h e t r a y s was p u t i n t o a  d u r i n g the evenings outdoor  At  #7:  Material—Hypnum c i r c i n a l e ,  into  possessed  cupboard  and a t n i g h t i n o r d e r t o s i m u l a t e t h e n o r m a l The o t h e r was l e f t  experiment  i n the p l a s t i c  counterbalancing the effect  was t w o f o l d :  o u t on a b e n c h .  The  (a) t o determine  whether  d i s h e s was c o n t r i b u t i n g t o o r o f the indoor temperature,  demonstrate whether a p h o t o p e r i o d  response  (b) t o  was c o n t r i b u t i n g t o  the a b n o r m a l i t i e s . O b s e r v a t i o n s — S l i d e s were made f r o m a f t e r t h e p l a n t s were b r o u g h t The in had  a b n o r m a l i t i e s observed experiment  times  i n d o o r s , much a s i n e x p e r i m e n t  6.  were s i m i l a r t o t h o s e d e s c r i b e d  6, and m e i o s i s had a b o r t e d 72 h o u r s a f t e r t h e p l a n t s  been brought Tetrad  both t r a y s at c e r t a i n  indoors.  a n a l y s i s o f s p o r a n g i a from both t r a y s are i n c l u d e d  i n T a b l e s X and X I .  The a n a l y s e s i n d i c a t e d  that the severity  55  o f t h e a n o m a l i e s was  comparable  t o those.observed i n experiment  6.  T a b l e X:  Tetrad  a n a l y s i s o f s p o r a n g i a o f e x p e r i m e n t 7,  under l a b o r a t o r y  plants  conditions.  Sporangium No.  Time o f samples a f t e r the plants were b r o u g h t  $ c e l l s o f each a n a l y s i s c a t e g o r y o f 500 examined c e l l s p e r sporangium  7  36 h r s .  71.2  5.0  0.0  0.0  23.8  12  36 h r s .  68.8  4.2  0.0  0.0  27.0  9  36 h r s .  56.0  8.6  1.2  0.0  34.4  Comments  old tetrads 11  mostly tetrads  11  36 h r s .  49.6  4.6  0.4  0.0  45.4  "  13  36 h r s .  47.6  8.6  1.4  0.6  41.8  "  14  36 h r s .  42.2  9.6  0.2  0.0  48.0  "  16  72 h r s .  2.6  1.2  0.4  0.2  95.0  arrested a c t i v e stages and t e t r a d s  56  Table XI:  T e t r a d a n a l y s i s o f s p o r a n g i a o f experiment under normal  7,  plants  photoperiod.  Sporangium No.  Time o f samp$ c e l l s i n each a n a l y s i s Comments ling after ' c a t e g o r y o f 500 examined c p l a n t s were c e l l s p e r sporangium. "brought i n d o o r s . Q ^ 2 5 4  1  24 h r s .  78.4  0.6  0.0  0.0  21.0  very o l d tetrads  4  24 h r s ,  86.0  8.2  0.0  0.0  5-8  "  2  24 h r s .  30.2  9.6  1.2  0.0  58.8  "  18  72 h r s ,  4.2  0.0  0.0  0.0  95.8  old tetrads  19  72 h r s .  2.2  0.4  0.0  0.0  97.4  11  20  72 h r s .  0.0  0.8  0.0  0.0  99-2  active s t a g e s and tetrads  22  72 h r s .  3.0  0.0  0.0  0.0  97.0  mostly tetrads  23  72 h r s .  0.8  0.2  0.0  • 0.0  99.0  active s t a g e s and tetrads  57  DISCUSSION  It  i s realized  suggest l i t t l e  that  sample s i z e s had  experiments  T h i s was  i n which the heat  the p o s s i b i l i t y suggested  shock was  of recovery a f t e r from the t e t r a d  especially  analysis  In a d d i t i o n i t i s obvious that  be r e f i n e d  by u s i n g c o n t r o l l e d  relatively mild  the experiments  temperature,  3,  could  h u m i d i t y , and  photo-  p r o b a b l y more  Although the i n t e r p r e t a t i o n  possess these l i m i t a t i o n s ,  super-  (see experiment  p e r i o d rooms w h i c h w o u l d g i v e more a c c u r a t e and meaningful r e s u l t s .  evident i n  s u c h shock was  page 4 0 ) .  be  that  i n f o r m a t i o n c o u l d have been d e r i v e d i f t h e  been l a r g e r .  those experiments  ficially  of these  more t h a n t r e n d s o f a b n o r m a l b e h a v i o u r and  much more m e a n i n g f u l  and  the r e s u l t s  of the  experiments  some i n f o r m a t i v e c o n c l u s i o n s c a n  drawn. I t was  heat  shock  stated  e a r l i e r that  experiments  (1) t o d e t e r m i n e  was  the purpose  of conducting the  fourfold:  whether e r r o r s  chromosomal b e h a v i o u r would be  i n interpretation  of  i n t r o d u c e d i f mosses were  brought  i n d o o r s t o mature t h e s p o r a n g i a , (2) t o d e t e r m i n e  the s e n s i t i v i t y  o f moss SMC  to s l i g h t  heat  stress, (3) t o i n d i c a t e t h e l i k e l i h o o d the environment  might  to determine  stress i n  be a mechanism o f i n d u c i n g m u t a t i o n s  structural/numerical alterations (4)  that n a t u r a l heat  o f t h e chromosomes,  t h e mode o f r e s p o n s e  o f t h e SMC  or  and to heat  stress These f o u r c a t e g o r i e s a r e d i s c u s s e d u n d e r s e p a r a t e  headings.  58  ( l ) Chromosomal B e h a v i o u r  o f Mosses u n d e r L a b o r a t o r y  Conditions.  Experiment undertaken  6  51)  (page  7  and  to i n v e s t i g a t e the e f f e c t s  om  o f mosses u n d e r l a b o r a t o r y c o n d i t i o n s . • indicate that  s e v e r e a b n o r m a l i t i e s may  mosses i n d o o r s t o r i p e n . humid c o n t a i n e r s had and  relatively  arid  •Furthermore,  tray.  chromosomal The  be  o f the a n a l y s i s of experiment  results  clearly  i n t r o d u c e d by  p l a n t s kept  6 and  dishes. the f i r s t  i n the very  l a b o r a t o r y temperature  In fact, part  of  second  relative  may  account  dishes.  This  variation i n  part  o f experiment  7,  day-to-day In  had  i n w h i c h t h e p l a n t s were  p h o t o p e r i o d s i n open t r a y s , was  influence' of m e i o t i c response  s u p p o s i t i o n was  borne  of the t e t r a d  (H. c i r c i n a l e )  not  expected  t o day l e n g t h .  out by o b s e r v a t i o n s o f m e i o s i s and analysis  , i t certainly  the  only f o r the species emphasizes  '  the  o f g r e a t c a r e when h a n d l i n g mosses p r i o r t o f i x a t i o n . t h a t mosses m a t u r i n g  by  This  56).'  ( T a b l e X I , page  Although these r e s u l t s are v a l i d  quite l i k e l y  evidence  f o r these d i f f e r e n c e s .  t o i n d i c a t e any  tested  comparison  a b o r t i o n of meiosis.  s u b j e c t e d t o normal  results  a  experiment  e v e n t , a f t e r 72 h o u r s , b o t h o f t h e s e e n v i r o n m e n t s  The  open  w i t h s e v e r e l y a b n o r m a l m e i o t i c b e h a v i o u r more  i s n o t c o n c l u s i v e , however, s i n c e n o r m a l  induced complete  bringing  t h e y were i n much d r i e r  s l o w l y t h a n d i d t h o s e i n t h e humid p l a s t i c  any  behaviour  p o s s i b l y the p l a n t s under the lower  humidity responded  primarily  A l t h o u g h the. s p o r a n g i a o f t h o s e i n  c o n d i t i o n s than those i n the p l a s t i c  suggests that  were  a b n o r m a l i t i e s s i m i l a r to those i n the  t h e open t r a y d i d n o t become f l a c c i d ,  7  54)  (page  i n warmer c l i m a t i c  a  necessity It i s  r e a s of the  59  continent o r i n l a t e s p r i n g o r summer i n the c o o l e r c l i m a t i c regions are not as s e n s i t i v e t o s l i g h t heat s t r e s s as a r e those maturing d u r i n g c o o l e r temperatures.  Steere et a l . (1954)  s t a t e d that most o f the C a l i f o r n i a n mosses s t u d i e d matured i n winter and e a r l y s p r i n g . heat s e n s i t i v e .  Many o f these s p e c i e s might w e l l be  However, i n t h e i r study they d i d not i n d i c a t e  how i n d i v i d u a l p l a n t s were handled so i t i s impossible to evaluate any a b n o r m a l i t i e s they r e p o r t e d .  I t i s l o g i c a l , c o n s i d e r i n g the  way many o f the mosses must have been handled by many authors (Steere et a l . , 1954; Bryan, 1956a et seq.; Anderson and Crum, 1959) that at l e a s t some o f the unusual f e a t u r e s d e s c r i b e d f o r meiosis i n mosses ( i e . clumping, m u l t i p l e a s s o c i a t i o n and precocQ ious d i s j u n c t i o n of c e r t a i n b i v a l e n t s ) , can be a t t r i b u t e d to the ~ i n f l u e n c e o f heat shock p r i o r t o f i x a t i o n . (2)  The S e n s i t i v i t y o f Moss SMC t o . S l i g h t Heat  Stress  The prresent r e s u l t s i n d i c a t e that H. c i r c i n a l e i s extremely s e n s i t i v e t o heat s t r e s s .  However, the temperature l e v e l at  which no a b n o r m a l i t i e s were produced l e v e l ) was not determined.  ( i f there i s an absolute  I t i s s i g n i f i c a n t that even t h e  treatment a p p l i e d i n experiment 4 (heat s t r e s s w i t h a maximum temperature o f 2 5 ° C  f o r 4 hours) was s u f f i c i e n t t o cause some  r e d u c t i o n i n normal t e t r a d f o r m a t i o n .  In experiments 6 and 7,  the s u s t a i n e d room temperature caused complete c e s s a t i o n o f meiosis a f t e r t h r e e days i n such an environment.-  T h i s suggests  that l o n g p e r i o d s at temperatures between 15-20°C. may  produce  abnormalities. I t i s d i f f i c u l t t o compare the s e n s i t i v i t y o f H. c i r c i n a l e to other organisms and t o h i g h e r p l a n t s i n p a r t i c u l a r  because  60  a wide v a r i e t y stress. Barber Li  o f methods have been u s e d  Heat s h o c k (1941,  experiments  1942),  Emsweller  and  experiments  e t sea..), Yanney W i l s o n those  experiments  results  because  (1943)  Brierley  and  of E l l i o t  (1962).  (Yanney W i l s o n , 1 9 5 9 ) .  seldom  A constant c r i t e r i o n  i s also d i f f i c u l t  and  (1957  Even  exhibit  t h e l e n g t h o f t i m e needed t o c o m p l e t e was  Bao  (1955), J a i n  (1959) and H e n d e r s o n  temperature  by  compared t o t h e  u s i n g s u s t a i n e d temperatures  sequence a t a s p e c i f i c  sensitivity  heat  s u c h as t h o s e p e r f o r m e d  (1940) a r e uncommon i n t h e l i t e r a t u r e  s u s t a i n e d temperature  i n applying  conflicting the m e i o t i c  taken i n t o  account  on w h i c h t o b a s e  to e s t a b l i s h .  Most w r i t e r s  deal  w i t h chiasma  f r e q u e n c y a t metaphase; o n l y r a r e l y i s m e n t i o n  of the f i n a l  outcome o f m e i o s i s .  made  Chiasma f r e q u e n c y i s v e r y  difficult  t o s t u d y i n moss b i v a l e n t s bedause t h e chromosomes a r e  s m a l l and  seldom  a c c u r a t e chiasma effects he was that  have a s u f f i c i e n t l y  distinct  morphology f o r  counts.  e t seq.)  has  (1957  on m e i o s i s o f a v a r i e t y not  interested  restitution nuclei  produced  Henderson  of  ( l ) 35-  f o r 72 h o u r s , and  (1962) showed t h a t  gregaria,maintained of the spermatocytes pycnotic.  and no v i a b l e  2°C.  f o r 86  and  hours. Schistocerca  a t 4 0 ° C . f o r t h e d u r a t i o n o f m e i o s i s , many failed  t o u n d e r g o c l e a v a g e and  Both of these treatments  circinale.  chromosomes  f o r 58 h o u r s ,  i n the desert l o c u s t ,  became  a r e much more s e v e r e  prolonged than those n e c e s s a r y t o cause H.  observed  p o l l e n g r a i n s were  2°C.  (3) 33-  heat  Although  p r o d u c t o f m e i o s i s he  a b o r t e d a t metaphase, t h e  a f t e r treatments  (2) 34 - 3°C.  studied  o f L o l i u m perenne L .  i n the f i n a l  p o l l e n mother c e l l s  formed  Jain  and  a b o r t i o n of meiosis i n  I t i s not s u r p r i s i n g t o f i n d  t h a t H.  circinale  61  and  p r o b a b l y many o t h e r mosses a r e p a r t i c u l a r l y  t o temperature  i n c r e a s e because  sensitive  t h e optimum m e i o t i c  temperature  i s much l o w e r t h a n i t i s f o r h i g h e r p l a n t s . w h i c h f l o w e r i n t h e summer.  I t i s i n t e r e s t i n g t o n o t e t h a t Pao and L i (1940) o b s e r -  ved a h i g h degree Vicia  o f u n i v a l e n c e i n p o l l e n mother c e l l s o f  c r a c c a L. immediately  f o r 30 m i n u t e s . temperature presumably  a f t e r a heat  T h e s e p l a n t s were s t u d i e d  was 7°C. adapted  Fall  i n J a n u a r y when t h e  t o these lower temperature  d i f f e r e n c e between t h i s t e m p e r a t u r e  I t seems o b v i o u s t h a t  between v a r i o u s l a t e  In Lolium  and room temp-  t o produce  comparison  The  o f heat  anomalous sensitivity  s p r i n g - m a t u r i n g mosses and f a l l / w i n t e r -  m a t u r i n g mosses w o u l d p r o b a b l y significance  ranges.  may be between 15-20 ° C .  e r a t u r e c o u l d be c o n s i d e r e d i n s u f f i c i e n t meiosis.  as m i l d a s 2 5 ° C .  and w i n t e r - m a t u r i n g mosses a r e  t h e optimum m e i o t i c t e m p e r a t u r e relative  shock  o f heat  a i d i n determining the adaptive  stress.  (3) N a t u r a l O c c u r i n g Heat S t r e s s a s a Mechanism o f I n d u c i n g Mutation or Structural/Numerical A l t e r a t i o n s  of the  Chromosomes. This question i s only a matter f o r s p e c u l a t i o n at t h i s The  present experiments  specifically  directed  were n e i t h e r s u f f i c i e n t l y  refined nor  t o answer t h i s q u e s t i o n .  A b n o r m a l i t i e s i n d u c e d by n a t u r a l h e a t  stress similar to  t h o s e i n d u c e d e x p e r i m e n t a l l y have been o b s e r v e d ulations  i n wild  i n b o t h h i g h e r p l a n t s and mosses. B e a m i s h  1961b) h a s o b s e r v e d  time.  severe clumping  pop-  (1961a,  o f metaphase I s t a g e s and  h i g h l y a b n o r m a l a n a p h a s e s i n p o l l e n mother c e l l s  of Saxifraga  species under f i e l d  (Beamish,  conditions.  I t i s suspected  1961b)  62  that  these abnormalities r e s u l t e d  Khanna ( i 9 6 0 ) has  observed  c o n s t r i c t a M i t t . One less  severe heat  isolated  from h i g h  a similar  effect  be p r o d u c e d  s t r e s s may  induce l e s s  ( i e . mutations,  s e v e r e and  However, t h e  that  types  that  are p o s s i b l y  produced  t h e o t h e r hand, i t i s r e a s o n a b l e t o c o n s i d e r  chromosomal a b e r r a t i o n s o f e i t h e r t h e s t r u c t u r a l o r n u m e r i c a l  t y p e m i g h t be p r o d u c e d . a t metaphase o b s e r v e d  From t h e h i g h f r e q u e n c y  i n these experiments  and  of univalency  others  (see  Henderson 1962), n o n d i s j u n c t i o n , l e a d i n g t o a n e u p l o i d y , be t h e most common anomaly p r o d u c e d be  significant  observed present  experiments.  (4) The  counted  a c c u r a t e l y , so no of t h i s  response  o f t h e SMC  bivalents;  To  Nondisjunction  generally as:  was  i n the  estimate could  t o Heat  Stress.  The  was  different categories  metaphase I c l u m p i n g  and m u l t i p l e  extreme c o n d e n s a t i o n o r c o i l i n g o f t h e  prophase asynchrony performed  reliable  to i n c r e a s e d temperatures  precocious disjunction;  experiment  types  anomaly.  i n s e v e r a l i n t e r r e l a t e d ways.  a s s o c i a t i o n of bivalents;  I n no  also f e r t i l e .  Mode o f R e s p o n s e o f t h e SMC  c a n be c l a s s i f i e d  and  stress.  However, o n l y i n r a r e a n a p h a s e s c o u l d a l l  be made o f t h e f r e q u e n c y  manifest  heat  o c c a s i o n a l y i n t h e t r e a t e d moss m a t e r i a l u s e d  chromosomes be  The  by s l i g h t  might  i n the p o p u l a t i o n , of course, the aneuploid  must o f n e c e s s i t y be v i a b l e and  ion;  could  For instance, i t i s currently  i m p o s s i b l y t o say whether p o i n t mutations On  possibly  chromosomalaaberrations)  i s p r e s e n t l y unknown.  by t h i s mechanism.  i n t h e moss B a r b u l a  might s p e c u l a t e from t h e s e o b s e r v a t i o n s t h a t  o c c u r r e n c e o f anomalous m e i o s i s .  of anomalies  temperatures.  and  spindle d e s t r u c t i o n or  premature m e i o t i c  inhibit-  induction.  h e r e , however, were a l l o f  these  63  c a t e g o r i e s of response observed. d i v e r s i t y o f t i m e and i n t e n s i t y XII  (page 64)  different  This simply r e f l e c t s of heat s t r e s s used.  the Table  i n d i c a t e s i n g e n e r a l t h e mode o f a c t i o n o f  c a t e g o r i e s o f h e a t s t r e s s u s e d on t h e SMC  of  H.  circinale.  Clumping  and M u l t i p l e A s s o c i a t i o n o f t h e  S e v e r e c l u m p i n g was very h i g h temperature experiments  2(page  t e m p e r a t u r e was  c l u m p i n g was  observed o n l y under  38)  and 3 (page 40)  i n w h i c h t h e maximum  immediately a f t e r the t r e a t m e n t , but  not c o n s i d e r e d s e v e r e .  With  temperatures and  S e v e r e c l u m p i n g i s a p r i m a r y r e s p o n s e t o h i g h tempere n t e r i n g metaphase I  t h e p l a n t s have been r e t u r n e d t o n o r m a l  temperatures  experiments 1  .  (page 34)  confirmed that  to completion.  and 5 (page 47)  s e v e r e l y clumped  c h r o m a t i n mass  M u l t i p l e a s s o c i a t i o n o f t h e b i v a l e n t s was except experiment  r e l a t e d t o the clumping e f f e c t I n experiment  above,  c l u m p i n g t o a l m o s t no m u l t i p l e b i v a l e n t Experiments  4  may  restitution. observed  (page 44)  consisten-  and i s  observed at h i g h temperatures.  2 and 3, m e n t i o n e d  a f t e r treatment.  (see  c e l l s do n o t c o n t i n u e m e i o s i s  R e l a x a t i o n o f t h e clumped  i n a l l experiments  after  I t i s expected, although  o c c u r however, but t h e c e l l s p r o b a b l y undergo  tly  this  c h r o m a t i n were g l o b u l a r  a t u r e s and does n o t o c c u r i n c e l l s  not  In  3 2 ° C , some o f t h e metaphase I  and  above 33°C. t h e a r e a s o f clumped shiny.  conditions of  ( i e . above a p p r o x i m a t e l y 3 3 ° C . ) .  between 30°  s t a g e s were clumped  Bivalents:  a s s o c i a t i o n ranged association  immediately  6 and 7 i n d i c a t e d t h a t  a s s o c i a t i o n , o c c u r s a t room t e m p e r a t u r e a f t e r a day  from  multiple  indoors.  64  Table X I I :  A g e n e r a l summary i n d i c a t i n g t h e mode o f a c t i o n o f different H.  Heat S t r e s s  36 ± 1 ° C . Shock  c a t e g o r i e s o f heat  s t r e s s on t h e SMC o f  circinale.  Primary  Effect  -metaphase I c l u m p i n g -arrest of active stages -condensation of dictyotene -premature m e i o t i c i n d u c t i o n (not manifest) -prophase asynchrony  Secondary  Effect  - s p i n d l e breakdown, o r inhibition - c o n d e n s a t i o n o f metaphase I b i v a l e n t s -asynchrony o f a n a p h a s e I , and l a g g a r d and b r i d g e f o r m a t i o n -restitution -prophase a s y n c h r o n y -severe r e d u c t i o n i n good t e t r a d f o r m a t i o n  3 1 -_il°C. Shock  -metaphase I p a r t i a l clumping - l a g g a r d and b r i d g e format!dsn a t a n a phase -normal a p p e a r i n g prophase -premature m e i o t i c i n d u c t i o n (not manifest) -prophase a s y n c h r o n y  -metaphase I b i v a l e n t association, precoci o u s d i s j u n c t i o n and condensation -partial spindle breakdown o r i n h i b i tion -prophase a s y n c h r o n y -significant reduction i n good t e t r a d p r o duction  25 i 1 ° C Shock  -not d e t e r m i n e d , b u t premature m e i o t i c induction i s presumed t o o c c u r  -metaphase I w i t h s l i g h t l y abnormal o r i e n t a t i o n on s p i n d l e -some anomalous a n a phases -slight reduction i n good t e t r a d f o r m a t i o n  -premature m e i o t i c induction -prophase asynchrony  -spindle i n h i b i t i o n and breakdown -disorientation at metaphase'I -abortion of meiosis  22 - 2 ° C Maintained  65  Multiple stress,  association, ( i e . heat  temperature  a primary  shock  stress.  in cells  period.  I t i s expected  been a f f e c t e d  T h i s may  or C o i l i n g one  traction  Pao  shock  experiments  metaphase I .  The  of the  Bivalents:  1942;  and L i (1940) o b s e r v e d  experiments  performed  Jain,  con-  circinale  mentioned  and u s u a l l y d e l a y e d a t  be  explained by.taking into  types of treatments u t i l i z e d .  there i s i n s u f f i c i e n t  In the heat  t i m e between t h e  treat-  f o r t h e s p o r a n g i a t o e x h i b i t , a t meta-  of d e l a y e d prophase  d u r i n g the treatment  p e r i o d . The  a t metaphase I o b s e r v e d  1957;  severe  by t h e o t h e r a u t h o r s  asynchronous  the examination  phen-  However, i n t h e s u s t a i n e d  phase I , the r e s u l t s  is  anomalies  Abnormal c o i l i n g  Swanson, 1942,  d i s c r e p a n c y may  the d i f f e r e n t  pro-  later.  i n several species.  above, t h e c o i l i n g was  ment and  to the  o f t h e b i v a l e n t s s i m i l a r t o t h a t n o t e d i n H.  temperature  shock  prophase  s t r e s s has b e e n n o t e d o f t e n i n o t h e r  (Pao and L i , 1940;  a f t e r heat  account  be r e l a t e d  e x c e p t #4.  omenon a s s o c i a t e d w i t h h e a t  and H e n d e r s o n , 1 9 6 2 ) .  was  t h e y must have  o f t h e most c o n s i s t e n t  i n a l l experiments  organisms  of  treatment  Although the  a f t e r the treatment,  which i s mentioned  T h i s f e a t u r e was  effect  t h a t t h e s e c e l l s were p r o b a b l y i n  i n some way.  Extreme C o n d e n s a t i o n  temperature  multiple association  a t metaphase I 5 days a f t e r t h e  normal  phase a s y n c h r o n y  observed  3,  d u r i n g the treatment.  s t a g e s appeared  o f moderate  t o 31°C.) i s a l s o a secondary  In experiment  observed  e a r l y prophase  effect  high condensation of  i n moderately  p o s s i b l y c a u s e d by a f a i l u r e  u n c o i l t o t h e e x t e n t t h e y do  c o i l i n g and  h e a t - t r e a t e d H.  asynchrony bivalents circinale  o f t h e metaphase I b i v a l e n t s t o  f o l l o w i n g t h e extreme  condensation  66  at the  clumped d i a k i n e s i s .  phase chromosomes do kinesis. may  be  This  not  failure  r e l a t e d to the  similar  In the  control material  a p p e a r as  of the  condensed as  the  those i n d i a -  " b i v a l e n t s t o r e l a x a t metaphase  anomalies i n the  The  s p i n d l e apparatus under  Disjunction:  precocious  d i s j u n c t i o n o f b i v a l e n t s a t metaphase I i s  c l o s e l y r e l a t e d to the material.  densation,  c o i l i n g anomalies found i n t r e a t e d  In a l l treatments except  disjunction  o f one  e x p e r i m e n t 4,  o r more b i v a l e n t s was  multiple  a s s o c i a t i o n , and  precocious  associated  t e r m i n a l i z a t i o n of the  a delay  points  c h i a s m a t e r m i n a l i z a t i o n may ( i e . s p e e d i n g up  the  i n spindle formation.  i n heat-treated  material  more c o n d e n s e d metaphase I b i v a l e n t s o f t h e  high  c h i a s m a f r e q u e n c y was  than i n the r e l a t i v e l y u n c o i l e d heat t r e a t e d m a t e r i a l .  caused  much h i g h e r controls  circinale  controls,  early disjunction.  Spindle  or I n h i b i t i o n , Prophase Asynchrony Induction:  of  the  bivalents  c o n t r i b u t i n g to the  Premature M e i o t i c  in  metaphase I b i -  caused chiasm t e r m i n a l i z a t i o n e a r l i e r than i n the  Destruction  that  asynchronous  It i s possible that  c o n t r a c t i o n n o t e d i n h e a t t r e a t e d H.  by  indicated  c o i l i n g was  the  of the  the  apart.  be  Swanson (1942) has  delayed.  valents  precocious  a d i r e c t e f f e c t of  p r o c e s s ) o r i t may  of Tradescantia  terminal  drift  and  Tradescantia  The  be  and  con-  formation.  c h i a s m a t a a t d i a k i n e s i s so t h a t  b i v a l e n t members l o s e a l l c o n t a c t Precocious  with  abnormal s p i n d l e  T h i s d i s j u n c t i o n o f b i v a l e n t s i s p r o b a b l y c a u s e d by  heat  I  conditions.  Precocious  the  meta-  and  thus  67  Spindle  d e s t r u c t i o n o r i n h i b i t i o n by h e a t s t r e s s c a n  be  i n f e r r e d f r o m t h e s e d a t a as  be  seen u s i n g  present  techniques.  o r i e n t a t i o n a t metaphase I and movement c a n spindle.  the  be u s e d as  degree of  functional structure.  (31  -  1°C.)  u s u a l l y the  the  p r o b a b l y by  However, a t t h e  s p i n d l e was  a p p a r a t u s was  of the  production  i n f e r r e d from observations  a t room t e m p e r a t u r e .  As  was  premeiotic  SMC  t o b e g i n m e i o s i s when t h e  size  evident  i n the  accelerated  days i n s t e a d  the  s y n t h e t i c mechanisms l e a d i n g t o t h e  that  a t room t e m p e r a t u r e . be  due  weeks.  satisfactorily  suspect  directly  an  normal  effective spindle main-  e a r l i e r the  prophase  (ie. stimulation  capsule  so  of 1/2  i s about  the  process  i n a period  a c t i v e stages of  i n such a short  In a d d i t i o n , the heat  that  At of of  I t would seem u n l i k e l y t h a t a l l  spindle production  to the  01  completed  1-2  take place  shock  as  of the m a t e r i a l  induction  chromosomal p a r t i c i p a t i o n was o f 1-2  the  of  p l a n t s k e p t a t room t e m p e r a t u r e .  same t i m e p r o p h a s e I was  l o g i c a l to  moderate h e a t  mentioned  premature m e i o t i c  ) was  a denaturation  never completely destroyed  a s y n c h r o n y and  not  the  abnormal o r i e n t a t i o n .  Complete i n h i b i t i o n  could  state of  metaphase I s t a g e s v a r i e d f r o m a l m o s t  o r i e n t a t i o n to s e v e r e l y  visible  anaphase  W i t h t e m p e r a t u r e s above 36°C., i t i s a p p a r e n t t h a t  its  the  bivalent  e f f e c t i v e n e s s of  c r i t e r i o n denoting the  s p i n d l e apparatus i s destroyed,  tained  a c t u a l s p i n d l e f i b e r s cannot The  the  only  time; thus i t i s  i s actually inhibited  spindle i n h i b i t i o n  s t r e s s but  meiosis  due  to a  may  reduction  i n t i m e a v a i l a b l e f o r c o m p l e t i o n o f n e c e s s a r y s y n t e h t i c mechanisms . A s y n c h r o n y o f d e v e l o p m e n t o f SMC  i n prophase w i t h i n  one  68  sporangium related  f o l l o w i n g v a r i o u s heat treatments  i s probably  t o t h e mechanism o f p r e m a t u r e m e i o t i c i n d u c t i o n  again r e f l e c t s  directly and  an a c c e l e r a t i o n o f t h e m e i o t i c p r o c e s s e s .  the time a v a i l a b l e  f o r normal  completion of s p e c i f i c  Reducing  stages  ( e s p e c i a l l y d i p l o t e n e ) i s apparently c o n t r i b u t o r y to the spindle  effectiveness  observed  s e v e r a l days f o l l o w i n g  abnormal  the  treatment. Heat i n d u c e d s p i n d l e breakdown o r i n h i b i t i o n has n o t e d many t i m e s i n p r e v i o u s s t u d i e s 1942;  and H e n d e r s o n , 1 9 6 2 ) .  experiments  based  activity  d u r i n g normal  a useful tool and  and  Failure  of the  induced spindle  A study i n v a r i a t i o n s heat  shocked  prophase  Swanson,  temperature  on o b s e r v a t i o n s o f c l e a v a g e  b i o c h e m i c a l mechanisms o f h e a t  have n o t b e e n i n v e s t i g a t e d .  et seq.;  These were c o n t i n u o u s  at r e l a t i v e l y h i g h temperatures.  s p i n d l e t o d e v e l o p was The  ( J a i n , 1957  been  of  may  failure. failure  synthetic prove  to  be  i n a c q u i r i n g a b e t t e r knowledge o f t h e p r o d u c t i o n  f u n c t i o n of the m e i o t i c s p i n d l e .  69 SUMMARY  1.  An  account  o f m e i o s i s emphasizing the prophase  I stages  has "been p r e s e n t e d f o r two  moss s p e c i e s ; Hypnum c i r c i n a l e Hook,  and B r a c h y t h e c i u m  (CM.)  frigidum  Besch.  In both  species,  m e i o s i s f o l l o w e d t h e g e n e r a l p a t t e r n o f e v e n t s f o u n d i n most plants  e x c e p t f o r a chromosome e l o n g a t i o n s t a g e w h i c h f o l l o w e d  diplotene.  E v i d e n c e was  i n many moss s p e c i e s .  p r e s e n t e d t h a t t h e s t a g e may  be  found  T h i s stage, which i s m o r p h o l o g i c a l l y  s i m i l a r to the d i c t y o t e n e stage of the developing oocytes of most a n i m a l s , has been d e s c r i b e d i n B a l s a m i n a h o r t e n s i s iens balsamina).  The  functional  significance  (impat-  of the dictyotene  s t a g e i n moss m e i o s i s i s p r e s e n t l y unknown.  2.  The  o f H.  effects  o f heat  s t r e s s o f m e i o t i c s p o r e mother  c i r c i n a l e has b e e n s t u d i e d .  temperature  B o t h h e a t shock  e x p e r i m e n t s were p e r f o r m e d .  as metaphase I c l u m p i n g , precocious disjunction, phase a s y n c h r o n y ,  bivalent  and  that  sustained  Severe anomalies  a s s o c i a t i o n and  such  contraction,  spindle destruction or i n h i b i t i o n ,  premature m e i o t i c i n d u c t i o n ,  circinale  pro-  and m e i o t i c  a b o r t i o n were o b s e r v e d i n most o f t h e e x p e r i m e n t s . i n d i c a t e t h a t H.  and  cells  The  results  i s extremely s e n s i t i v e to heat  stress  c y t o l o g i s t s w o r k i n g w i t h mosses s h o u l d be c a r e f u l i n  handling the p l a n t s p r i o r to f i x a t i o n to i n s u r e against  heat  induced m e i o t i c anomalies. The  effects  of heat s t r e s s under n a t u r a l c o n d i t i o n s ,  t h e mode o f r e s p o n s e  o f t h e SMC  t o heat  and  s t r e s s are d i s c u s s e d .  70 LITERATURE  CITED  A l - A i s h , M. and A n d e r s o n , L.E.. 1961. Chromosome s t u d i e s on some mosses o f t h e s o u t h e a s t e r n U n i t e d S t a t e s . The B r y o l o g i s t , 6 4 : 289-314. Allen,  C.E. 1917. A chromosome d i f f e r e n c e c o r r e l a t e d w i t h sex d i f f e r e n c e s i n S p h a e r o c a r p o s . S c i e n c e , U.S., 4 6 : 467. .  1919. carpos.  The b a s i s o f s e x i n h e r i t a n c e i n S p h a e r o Proc,. .Amer. P h i l . S o c , £ 8 : 298-316.  .  1935. The g e n e t i c s 269-291.  of bryophytes.  .  1945. The g e n e t i c s II: 260-287.  of bryophytes I I .  B o t . Rev., 1:  B o t . Rev.,  A n d e r s o n , L . E . and Crum, H. 1959. C y t o t a x o n o m i c s t u d i e s on mosses o f t h e C a n a d i a n Rocky M o u n t a i n s . N a t . Mus. Canada, B u l l . , 160: 1-89. B a r b e r , H.N. 1941. Chromosome b e h a v i o u r i n U n u l a r i a . G e n e t . , &2: 223-257. .  J.  1942. The e x p e r i m e n t a l c o n t r o l o f chromosome i n g i n F r i t i l l a r i a . J . G e n e t . , £3_: 359-374.  pair-  Beamish, K . I . 1961a. S t u d i e s o f m e i o s i s i n t h e genus S a x i f r a g a o f t h e P a c i f i c N o r t h w e s t . , Can. J . B o t . , 2%. 567580. •  1961b.  Personal  communication.  B r y a n , V.S. 1956a. Chromosome and s y s t e m a t i c p o s i t i o n o f t h e i n o p e r c u l a t e mosses, P l e u r i d i u m and B r u c h i a . Am. J . B o t . , 4J3: 460-468.  •  .  1956b. C y t o l o g i c a l and t a x o n o m i c s t u d i e s o f some s p e c i e s o f Astomum, A c a u l o n and Phascum. The B r y o l o g i s t , 5_9_: 118-129.  .  1957. C y t o t a x o n o m i c s t u d i e s i n t h e Ephemeraceae and Funariaceae. The B r y o l o g i s t , 60: 103-126.  C a l l a n , H.G. 1963. The n a t u r e o f l a m p b r u s h chromosomes. I n t e r n a t i o n a l R e v i e w o f C y t o l o g y , 15_: 1-34. C a r r , A.J.H. and O l i v e , L . S . 1958. Genetics of Sordaria famicola. I I . Cytology. Am. J . Bot.,4_5_: 142-150.  71  D a r l i n g t o n , C.A. and L a C o u r , L . E . I960. The h a n d l i n g o f chromosomes. George A l l e n & Unwin L t d . , London; 248 pp. D o r w i c k , G.J, 1957. The i n f l u e n c e o f t e m p e r a t u r e on m e i o s i s . H e r e d i t y , 11: 37-49. Elliot,  C.G. 1955. The e f f e c t frequency. Heredity,  o f t e m p e r a t u r e on 9.: 385-398.  chiasma  E m s w e l l e r , S.L. and B r i e r l e y , P. 1943. E f f e c t s of high t e m p e r a t u r e on metaphase p a i r i n g o f L i l i u m longiflorum. Bot.. Gaz., 105: 49-57. H e i t z , E.  1928. Das H e t e r o c h r o m a t i n d e r Moose. W i s s . B o t . , 6>9_: 762-85.8 ( n o t s e e n ) .  Henderson,  S.A. 1962. T e m p e r a t u r e and c h i a s m a f o r m a t i o n i n S c h i s t o c e r c a g r e g a r i a , I I . C y t o l o g i c a l e f f e c t s at 4 0 ° C . and t h e mechanism o f h e a t - i n d u c e d u n i v a l e n c e . Chromosoma ( B e r l . ) , 13_: 437^463.  J a i n , H.K.  1957. Lolium: 23-36.  •  Jahrh.  E f f e c t o f h i g h t e m p e r a t u r e on m e i o s i s i n nucleolar inactivation. H e r e d i t y , 11:  I960. I n d u c e d n e o - c e n t r i c a c t i v i t y i n chromosome ends. Chromosoma ( B e r l . ) , 12: 310-312. .  1962. Breakdown o f d i v i s i o n c y c l e and o r g a n i s a t i o n o f a t y p i c a l s p i n d l e s i n f u s e d p o l l e n mother c e l l s o f . L o l i u m . Chromosoma ( B e r l . ) , 12: 812-818. Khanna, K.R. I960. C y t o l o g i c a l S t u d i e s i n some H i m a l a y a n mosses. C a r y o l o g i a , 13_: 559-618. L e w i s , K.R. 1961. The g e n e t i c s o f b r y o p h y t e s . bryol. S o c , £: 111-130.  Trans. B r i t ,  L e w i t s k y , G.A. 1927. D i e B i l d u n g h i v a l e n t e r Chromosomen i n d e r Gonogenese v o n B e t a v u l g a r i s . P l a n t a , I I I (not seen); Mehra, P.N. and Khanna, K.R. 1961. Recent c y t o l o g i c a l i n v e s t i g a t i o n s i n mosses. R e s e a r c h B u l l e t i n o f t h e P a n j a h U n i v e r s i t y ( N . S . ) , 12: 1-29. M a r c h a l , E . a n d M a r c h a l , E . 1907, 1909, 1911. Aposporie et s e x u a l i t e c h e z l a mousses. I., I I . , I I I . Bull. A c a d . B l g . , C l . S c i . , 1 9 0 7 4 765-780; 1909: 12491288; 1911: 750-778 ( n o t s e e n ) ; e  72  M o d i l e v s k y , F . 1918. C y t o l o g i c a l and e m b r y o l o g i c a l on N e o t t i a n i d u s - a v i s L . A b h a n d l . N a t u r f . Kiew., XXVI ( n o t s e e n ) .  studies Gesellsch.  Newcomer, E.H. 1953* A new c y t o l o g i c a l and h i s t o l o g i c a l S c i e n c e , 118: 161.  fluid.  Ohno,S., M a k i n o , S., K a p l a n , W.D., and K i n o s i t a , R. 1961. Female germ c e l l s o f man. E x p . C e l l . R e s . , 24: 106-110. . Pao,  W.K.  K l i n g e r , H.P. and A t k i n , N.B. C y t o g e n e t i c s , 1: 42-//.  1962.  Human  oogenesis.  and L i , H.W. 1948. D e s y n a p s i s and o t h e r a b n o r m a l i t i e s . i n d u c e d by h i g h t e m p e r a t u r e s . J . G e n e t . , 4J3: 297-310.  P r a t t , 33.H. and L o n g , H.A. 1917. The p e r i o d o f s y n a p s i s i n t h e egg o f t h e w h i t e r a t , Mus n o r v e g i c u s a l b i n u s . , J . M o r p h o l o g y , 2 £ : 441-459. R i s , H.  1945. The s t r u c t u r e o f m e i o t i c chromosomes i n t h e g r a s s h o p p e r and i t s b e a r i n g on t h e n a t u r e o f "chromomeres" and l a m p b r u s h chromosomes. B i o l . B u l l . , 8 9 : 242-257.  S c h e u b e r , L M. 1932. C e l l u l e , 41:  A cytological 145-162.  study  o f Timmia c u c u l l a t a . L a  S e c h a c h a r , B.R. and S h a n t a Bagga. 1963* A cytochemical study of oogenesis i n the dragan-fly, Pantala f l a v e s c e n s . ( F a b r i c u s ) Growth, 21: 225-246. S t e e r e , W.C., A n d e r s o n , L . E . and B r y a n , V. 1954Chromosome s t u d i e s on C a l i f o r n i a mosses. Mem. T o r r e y B o t . C l u b , 20: 1-75. Swanson, C P . 1942. Gaz., 1 0 1 : .  Szakien,  Meiotic c o i l i n g i n Tradescantia. Bot. 457-474.  1943. The b e h a v i o u r o f m e i o t i c p r o p h a s e chromosomes as r e v e a l e d t h r o u g h t h e u s e o f h i g h t e m p e r a t u r e . Am. J . B o t . , 2 0 : 422-428. B. 1927. L a f o r m a t i o n des chromosomes h e t e r o t y p i q u e s dans 1 Osmunda r e g a l i s . L a C e l l u l e , X X X V I I I : 369-394. 1  Vaarama, A. 1949. M e i o s i s i n moss s p e c i e s o f t h e f a m i l y Grimmiaceae. P o r t u g . A c t a . B i o l . (A) R. B. G o l d s c h m i d t Volume, 1949: 47-48.  73  Vaarama, A. 1954a. S t r u c t u r e and b e h a v i o u r o f m e i o t i c b i v a l e n t s i n Hedwigia c i l i a t a . Arch. Soc. Zool.-Bot. Fenn. "Vanamo",.8: 195-206. _.  1954b. C y t o l o g i c a l o b s e r v a t i o n s on P l e u r o z i u m s c h r e b e r i , w i t h s p e c i a l r e f e r e n c e t o centromere evolution. Ann. B o t . S o c . "Vanamo", 28: 1-57.  V i o l a n t e , J.M. de S o u z a . 1929. l a P a r a s y n d e s e dans B a l s a m i n a h o r t e n s i s e t Campanula p e r s i c i f o l i a . . l a C e l l u l e , XXXIX: 235-268. Wilson,  C M . 1952. C l u b , 22:  W i l s o n , E.B. ed. W i l s o n , M.  Meiosis i n Allomvces. 139-160.  B u l l . Torrey Bot.  1925. The c e l l i n development and h e r e d i t y , 3rd M a c M i l l a n Company, N.Y.; 1232 pp.  1909. On s p o r e f o r m a t i o n and n u c l e a r d i v i s i o n i n Mnium ho mum, A n n a l s B o t . , 2J£: 141-157.  W i n i w a r t e r , H. v o n . 1 9 0 0 . R e c h e r c h e s s u r l ' o v o g e n d s e e t 1' o r g a n o g e n e s e de l ' o v a i r e de mammiferes ( l a p i n e t homme). A r c h . B i o l . , 17: 33-200 (not seen). Yanney W i l s o n , J . 1959. Chiasma f r e q u e n c y i n r e l a t i o n t o temperature. G e n e t i c a , 29_: 290-303. Yano, K.  1957a? C y t o l o g i c a l s t u d i e s on J a p a n e s e mosses. I . F i s s i d e n t a l e s , D i c r a n a l e s , Grimmiales, E u b r y a l e s . The K v o i d u - k a g a k u (Mem. F a c u l t y o f E d u c . N i i g a t a U n i v . ) , 6: 1-31.  •  1957b. C y t o l o g i c a l s t u d i e s on J a p a n e s e mosses. I I . Hypnobryales. Mem. T a k a d a B r a n c h , N i i g a t a U n i v ^ 1: 85-127.  .  1957c C y t o l o g i c a l s t u d i e s on J a p a n e s e mosses. I I I . Isobryales, Polytrichinales. I b i d . , 1: 129-159.  (to f o l l o w p a g e 73)  APPENDIX PLATES 1-9  (to face P l a t e l )  DESCRIPTION O F FIGURES O F PLATE 1  F i g u r e s 1-3, H. c i r c i n a l e ; s h o w i n g t h e a c e n t r i c a l l y p l a c e d c h r o m a t i n a t l e p t o t e n e and z y g o t e n e ; f i g . 1; l e p t o t e n e w i t h chromosome s t r a n d s e x t e n d i n g p a s t t h e m i d d l e o f t h e SMC ( s t r a n d s a p p e a r t h i c k b e c a u s e t h e s l i d e was o l d a t t h e t i m e t h e p h o t o g r a p h was t a k e n and t h e chromosomes have s w e l l e d ) ; f i g . 2; t r a n s i t i o n between l e p t o t e n e and z y g o t e n e , a r r o w i n d i c a t e s two s t r a n d s w h i c h a p p e a r t o be i n t h e p r o c e s s o f p a i r i n g (phase c o n t r s t ) ; f i g . 3; s t r a n d s w i t h chromomeres, a l l X2400. a  F i g u r e s 4-9,' H. c i r c i n a l e ; s h o w i n g v a r i o u s s t a g e s o f p a c h y t e n e ; f i g . 4,5; b e g i n n i n g o f m i g r a t i o n o f t h e c h r o m a t i n t o t h e c e n t e r o f t h e SMC; f i g . 7,8; m i d - p a c h y t e n e ; f i g . 9; l a t e p a c h y t e n e w i t h c h r o m a t i n c e n t r a l l y l o c a t e d , a l l  X2400.  F i g u r e s 10&11, H. c i r c i n a l e ; d i p l o t e n e ; f i g . 10; d i p l o t e n e showing the condensed h e t e r o c h r o m a t i c s m a l l b i v a l e n t ; f i g . 11; showing one o f t h e l a r g e r b i v a l e n t s w i t h a h e t e r o c h r o m a t i c segment (phase c o n t r a s t ) , X2400.  PLATE  1  ^ttr* N \ _ 4 l Ilk S  ,  1  * 9  10  (to  face Plate  2)  DESCRIPTION OF FIGURES OF PLATE 2  F i g u r e s 1&2, H. c i r c i n a l e ; d i p l o t e n e ; f i g . 1; a h i g h l y s q u a s h e d SMC s h o w i n g t h e d e t a i l e d s t r u c t u r e o f t h e l a r g e b i v a l e n t , X 4500; f i g . 2; l a t e d i p l o t e n e s h o w i n g a l l s i x b i v a l e n t s , i n c l u d i n g t h e h e t e r o c h r o m a t i c s m a l l b i v a l e n t and t h e n u c l e o l u s ( b o t h phase c o n t r a s t ) , X2400. F i g u r e 3, H. c i r c i n a l e ; t r a n s i t i o n a l s t a g e between d i p l o t e n e and t h e e l o n g a t i o n s t a g e (phase c o n t r a s t ) X&400. F i g u r e s 4 &5• H. c i r c i n a l e ; e l o n g a t i o n s t a g e ; f i g . 4; e l o n g a t i o n s t a g e Tarrow i n d i c a t e s a s e c t i o n where t h e chromosomes a r e s t i l l c l o s e l y p a i r e d ) ; f i g . 5; e l o n g a t i o n s t a g e showing t h e i n t e r p h a s e - l i k e a p p e a r a n c e o f t h e c e l l ( b o t h phase c o n t r a s t ) , X2400. F i g u r e 6, H. c i r c i n a l e ; chromatin. T h i s stage  a SMC w i t h d i f f u s e a p p e a r a n c e o f i s p r o b a b l y a r t i f a c t , X2400.  F i g u r e 7&8, H. c i r c i n a l e ; d i a k i n e s i s ; f i g . 7; d e n s e l y clumped b i v a l e n t s a t d i a k i n e s i s (phase c o n t r a s t ) ; f i g . 8; l a t e d i a k i n e s i s , X2400. F i g u r e 9, H. c i r c i n a l e ; metaphase I showing s i x b i v a l e n t s with the l a r g e b i v a l e n t i n a p e r i p h e r a l l o c a t i o n . The s m a l l e s t b i v a l e n t i s d i f f i c u l t t o i d e n t i f y i n t h i s case, X2400. F i g u r e 10, H. c i r c i n a l e ;  anaphase  I , X2400,  F i g u r e 1 1 , H. c i r c i n a l e ; i n t e r p h a s e o r p r o p h a s e I I s h o w i n g a s u g g e s t i o n of w a l l f o r m a t i o n between t h e two n u c l e i , X2400. F i g u r e 12, H. c i r c i n a l e ; anaphase I I showing c l o s e l y p a c k e d chromosomes and a t e m p o r a r y b r i d g e f o r m a t i o n , X2400.  PLATE '  :  2 j '  •••  *• W  ~"  (to  face Plate  3)  DESCRIPTION OF  FIGURES OF  F i g u r e 1, H. c i r c i n a l e ; t e l o p h a s e o f w a l l f o r m a t i o n , X2400. Figure  2, H.  circinale:  PIATE 3  I I showing the  beginning  a n o r m a l t e t r a d , X2400.  F i g u r e s 3-7, H. c i r c i n a l e ; s p o r a n g i u m #6; d i a k i n e s i s t o metaphase I , s h o w i n g t h e u n u s u a l b e h a v i o u r o f t h e s m a l l e s t b i v a l e n t ; f i g . 3; e a r l y d i a k i n e s i s w i t h a u n i v a l e n t s e p a r a t e f r o m t h e chromosomal mass; f i g . 4-6; l a t e d i a k i n e s i s o r e a r l y metaphase I , s h o w i n g a u n i v a l e n t o f t h e s m a l l e s t b i v a l e n t s e p a r a t e f r o m t h e mass. The o t h e r u n i v a l e n t of the s m a l l e s t b i v a l e n t i s c l o s e l y a s s o c i a t e d w i t h the r e m a i n d e r o f t h e chromosomes and i s n o t d i s t i n g u i s h a b l e ; f i g . 7; a metaphase I i n w h i c h t h e s m a l l e s t b i v a l e n t has n o t d i s j o i n e d p r e c o c i o u s l y , a l l X2400. F i g u r e s 8&9, H. c i r c i n a l e ; s h o w i n g b r i d g e and l a g g a r d f o r m a t i o n s a t t e l o p h a s e I ; f i g . 8; a b r i d g e and two u n i v a l e n t s i n v o l v i n g t h e l a r g e r chromosomes; f i g . 9; s h o w i n g t h e two u n i v a l e n t s o f t h e s m a l l e s t b i v a l e n t l e f t o f f t h e metaphase p l a t e , b o t h X2400. F i g u r e s 10&11, B. f r i g i d u m ; s h o w i n g t h e a c e n t r i c a l l y p l a c e d chromatin at leptotene-zytogene. The n u c l e o l u s i s i n a more c e n t r a l l o c a t i o n i n e a c h c e l l t h a n i s t h e c h r o m a t i n and i s o f l i g h t e r d e n s i t y t h a n t h e c h r o m a t i n , b o t h X2400 (phase c o n t r a s t ) . F i g u r e s 12, B. f r i g i d u m ; e a r l y p a c h y t e n e s h o w i n g t h e o f chromosome m i g r a t i o n , X2400.  beginning  ( t o f a c e p l a t e 4)  DESCRIPTION OP FIGURES OF PLATE  4  F i g u r e 1, B. f r i g i d u m ; m i d - p a c h y t e n e , X2400. F i g u r e 2, B. segment o f lower r i g h t section i s  frigidum; diplotene. The h e t e r o c h r o m a t i c one o f t h e chromosomes i s shown i n t h e hand c o r n e r o f t h e c e l l ; t h e o t h e r d a r k c a u s e d by o v e r l a p o f chromosomes, X2400.  F i g u r e 3&4» B. f r i g i d u m ; SMC i n t h e e l o n g a t i o n s t a g e . I n f i g . 3 t h e n u c l e a r membrane i s c l e a r l y e v i d e n t ; i n f i g . 4 i t i s l e s s w e l l d e f i n e d , b o t h X2400. Figure  5,  B. f r i g i d u m ; s h o w i n g a clumped d i a k i n e s i s ,  F i g u r e 6, B. f r i g i d u m ; X2400.  prometaphase showing s i x b i v a l e n t s ,  F i g u r e s 7&8, B. f r i g i d u m ; metaphase I . bivalent i s located at the periphery. common s i t u a t i o n , X2400. Figure  X2400.  In f i g . 7 the long T h i s i s t h e most  9, B. f r i g i d u m ; l a t e anaphase I , X2400.  F i g u r e 10, Sxp. 1, page 34; 26 h o u r s a f t e r t r e a t m e n t ; an u n o r i e n t e d metaphase I , X2400. F i g u r e s 10&11, exp. 1, page 34; 48 h o u r s a f t e r t r e a t m e n t ; f i g . 10; a p a r t i a l l y clumped and p o o r l y o r i e n t e d metaphase I ; f i g . 11; d i a k i n e s i s o r u n o r i e n t e d metaphase I , b o t h X2400.  PLATE  4  (to face P l a t e  5)  DESCRIPTION OF  FIGURES OF  PIATE 5  F i g u r e s 1&2, exp. 1, page 34; 48 h o u r s a f t e r t r e a t m e n t ; f i g . 1; anaphase I o r metaphase I w i t h u n i v a l e n t s ; f i g . 2; t e l o p h a s e I s h o w i n g g l o b u l a r n u c l e i , but no l a g g a r d s , b o t h X2400. F i g u r e s 3-8, exp. examples o f t h e c a t e g o r y 1; f i g 6,7,8; c a t e g o r y  1, page 34; 4 d a y s a f t e r t r e a t m e n t ; s h o w i n g 4 a b n o r m a l a n a l y s i s c a t e g o r i e s ; f i g . 3; . 4; c a t e g o r y 2; f i g . 5; c a t e g o r y 3; f i g . 4, a l l X2400*  F i g . 9, exp. 1, page 34, 4 days a f t e r t r e a t m e n t ; showing a r e s t i t u t i o n SMC t h a t was a r r e s t e d a t d i p l o t e n e , X2400. F i g u r e 10-12, exp. 2, page 38; i m m e d i a t e l y a f t e r t r e a t m e n t ; f i g . 10; h e a t i n d u c e d c l u m p i n g a t metaphase I ; f i g . 11; a p a r t i a l l y clumped metaphase I w i t h h i g h l y condensed b i v a l e n t s ; f i g . 12; n o r m a l s p i n d l e f o r m a t i o n w i t h m u l t i p l e a s s o c i a t i o n o f b i v a l e n t s , a l l X2400.  PLATE  5  ^  4  7  01  ^  ^  ^  [71 •••  •  ^  ^  ^  ^  (to face P l a t e  6)  DESCRIPTION OP  FIGURES OF  PLATE 6  F i g u r e s 1-3, exp. 2, page 38; i m m e d i a t e l y a f t e r t r e a t m e n t ; f i g . 1; metaphase I w i t h n o r m a l s p i n d l e f o r m a t i o n ; f i g . '2; metaphase I w i t h n o r m a l s p i n d l e f o r m a t i o n but w i t h h i g h l y c o n d e n s e d b i v a l e n t s ; f i g . 3; n o r m a l b i v a l e n t s a t metaphase I , o r i e n t a t i o n i s s l i g h t l y a b n o r m a l , a l l X2400.; F i g u r e s 4&5, exp. 2, page 38; i m m e d i a t e l y a f t e r t r e a t m e n t ; f i g . 4; metaphase I w i t h d i s s o c i a t e d and u n o r i e n t e d b i v a l e n t s ; f i g . 5; anaphase w i t h l a g g i n g chromosomes,- b o t h X2400. F i g u r e s 6 , exp. 2, page 38; 72 h o u r s a f t e r t r e a t m e n t ; s h o w i n g an anaphase I d i s p l a y i n g a b n o r m a l s e g r e g a t i o n , X2400. F i g u r e s 7-9, exp. 2, page 38; 72 h o u r s a f t e r t r e a t m e n t ; f i g . 7; a f i e l d o f a b n o r m a l t e t r a d s , X1650; f i g . 8; a t e t r a d o f c a t e g o r y 2; f i g . 9; a t e t r a d w i t h f i v e n u c l e i , b o t h X2400. F i g u r e 10, exp. 3, page 40J 5 days a f t e r t r e a t m e n t ; s h o w i n g a metaphase I w i t h good s p i n d l e a r r a n g e m e n t and s l i g h t l y o v e r - c o n d e n s e d b i v a l e n t s , X2400.  (to f a c e E l a t e  7)  DESCRIPTION OP FIGURES OP  PLATE 7  F i g u r e s 1&2, exp. 3, page 40; 7 d a y s a f t e r t r e a t m e n t ; f i g . 1; a n o r m a l a p p e a r i n g metaphase I ; f i g . 2; anaphase I e x h i b i t i n g n o n d i s j u n c t i o n of the l a r g e b i v a l e n t , both  X2400.  F i g u r e 3, H e t e r o c l a d i u m h e t e r o p t e r o i d e s ; a b n o r m a l a c t i v e s t a g e s and t e t r a d s a f t e r 2 weeks i n d o o r s , X2500. F i g u r e s 4-7, exp. 5, page 47; i m m e d i a t e l y a f t e r t r e a t m e n t ; f i g . 4; d i p l o t e n e s h o w i n g what a p p e a r s t o be a b n o r m a l p a i r i n g ; f i g . 5,6; a normal appearing l a t e d i p l o t e n e , a l l X2400; f i g . 7; a f i e l d o f SMC w i t h s e v e r e l y clumped metaphase I , X1300. F i g u r e 8, exp. 5, page 47; 72 h o u r s a f t e r t r e a t m e n t ; s h o w i n g a metaphase I w i t h some s p i n d l e o r i e n t a t i o n , b u t w i t h n o t a l l t h e b i v a l e n t s o r i e n t e d , X2400.  (to face P l a t e  8)  DESCRIPTION OP FIGURES OF PIATE 8  F i g u r e s 1&2, exp. 5, page 47; 72 h o u r s a f t e r t r e a t m e n t ; showing SMC w i t h s p i n d l e a r r a n g e m e n t , b u t w i t h highlycondensed b i v a l e n t s e x h i b i t i n g a s y n c h r o n o u s d i s j u n c t i o n o r f a u l t y o r i e n t a t i o n , b o t h X2400,. F i g u r e s 3-5, exp. 5, page 47; 5 days a f t e r t r e a t m e n t ; f i g . 3; a r e s t i t u t i o n SMC w h i c h has p a r t i a l l y d i v i d e d ; f i g . 4; p y c n o t i c s p o r e s and a r e s t i t u t i o n SMC ( d o t t e d l i n e s i n d i c a t e the f i n e t h r e a d s o f w a l l m a t e r i a l which j o i n two s p o r e s ) , b o t h X2400; f i g . 5; a l o w power f i e l d o f s p o r e s and r e s t i t u t i o n SMC w h i c h have c l u s t e r e d i n t o a l a r g e a g g r e g a t e , X450. F i g u r e s 6-9, exp. 6, page 51; 30 h o u r s a f t e r t h e p l a n t s were b r o u g h t i n d o o r s ; showing v a r i o u s a n o m a l i e s ( i e . no b i v a l e n t o r i e n t a t i o n , p a r t i a l c l u m p i n g and p r e c o c i o u s d i s s o c i a t i o n ) a t metaphase I , a l l X2400.  (to f a c e P l a t e  9)  DESCRIPTION  OF FIGURES OF PLATE 9  F i g u r e s 1-3, e x p . 6, page 51; 72 h o u r s a f t e r p l a n t s were b r o u g h t i n d o o r s ; f i g . 1; a b o r t e d m e i o s i s a t f i r s t d i v i s i o n ; f i g . 2; a b o r t e d m e i o s i s d u r i n g m i c r o n u c l e i f o r m a t i o n , b o t h XL300; f i g . 3; a t e t r a d formed f r o m a SMC a f t e r a b o r t i o n o f m e i o s i s ,  X2400.  F i g u r e s 4&5, exp. 6, page 51; 4 d a y s a f t e r p l a n t s were b r o u g h t i n d o o r s ; f i g . 4; d i c t y o t e n e w i t h t h e c h r o m a t i n p y c n o t i c and clumped a r o u n d t h e c h r o m o c e n t r e ; f i g . 5\ e a r l y p r o p h a s e i n w h i c h t h e c h r o m a t i n h a s clumped i n t o a b a l l , b o t h X2400.  

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