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Chromosomal behaviour during meiosis in mosses 1964

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CHROMOSOMAL BEHAVIOUR DURING MEIOSIS IN MOSSES by FREDERICK JOHN DILL B.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of B i o l o g y and Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1964 In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference and study. I further agree that per- mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that, copying or publi- cation of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission* Department of Biology and Botany The University of B r i t i s h Columbia, Vancouver 8 . Canada Date August 15. 1964. i i ABSTRACT The primary purpose of the present work was to examine meio s l s i n mosses, c o n c e n t r a t i n g on the d e t a i l of prophase I, a m e i o t i c stage which has "been d e s c r i b e d i n d e t a i l only i n Pleurozium s c h r e b e r i * 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 under- taken to study the e f f e c t of heat s t r e s s on m e i o s l s . Hypnum c i r c i n a l e Hook, and Brachythecium f r i g i d u m (CM.) Besch. were used to study m e i o s i s . The procedure of p r e p a r i n g spore mother c e l l squashes was s i m i l a r t o t h a t used by Steere et a l . (1954), w i t h m o d i f i c a t i o n s 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 t o ensure against p r o d u c t i o n of heat induced anomalies. Except f o r l a t e prophase I, meiosis i n both s p e c i e s c o n f o r med to t h a t found i n P. s c h r e b e r i . In H. c i r c i n a l e and B. f r i g i d u m d i p l o t e n e was followed by chromosome e l o n g a t i o n and r e s u l t e d i n a d i f f u s e stage. T h i s stage i s m o r p h o l o g i c a l l y analogous t o the d i c t y o t e n e stage of the growing oocytes of many animals, and appears to have been 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 t h a t the stage occurs 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 s i g n i - f i c a n c e i s unknown. In the heat s t r e s s experiments, p l a n t s of H. c i r c i n a l e were e i t h e r maintained under l a b o r a t o r y temperatures while being s t u d i e d or they were t r e a t e d w i t h a heat shock over a p e r i o d of f o u r or s i x hours w i t h the maximum temperature i n the general ranges of 25°C , 31°C, and 36°C The maximum temper- ature was maintained f o r 4 hours i n the 25°C experiment and i i i 1/2 hour i n the remaining experiments. The heating and cooling gradients were almost equivalent (l°C./5 min.), and the star t i n g and f i n i s h i n g temperature was 14°C. The temperature of the natural environment during the study ranged between 7-ll°C Severe anomalies, including chromosome clumping and multiple association, precocious disjunction, chromosome contraction, spindle breakdown and i n h i b i t i o n , premature meiotic induction and meiotic abortion 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 accelerates prophase I stages of H. c i r c i n a l e . The time available for these stages appears to be too b r i e f f o r synthesis of necessary products leading to --active stages, thus causing severe abnormalities which result i n abortion of meiosis. On the basis of these r e s u l t s , i t i s apparent that cyto- l o g i s t s working with moss material should take care i n handling the plants p r i o r to f i x a t i o n to ensure against heat induced meioticanomalies. v i i ACKNOWLEDGEMENTS I wish to extent my g r a t i t u d e t o my d i r e c t o r , Dr. W. ,B. S c h o f i e l d f o r h i s c r i t i c i s m and d i s c u s s i o n o f the r e s e a r c h and f o r a s s i s t a n c e i n p r e p a r i n g the. manuscript; to Dr. K. I . Beamish f o r r e a d i n g the manuscript and f o r many d i s c u s s i o n s r e g a r d i n g 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 the manuscript; and to t h e Depart- ment of B i o l o g y and Botany f o r the use of equipment and f a c i l - i t i e s . I wish to thank Dr. J . R. M i l l e r of the Department of P a e d i a t r i c s , U n i v e r s i t y of B r i t i s h Columbia, f o r the use of h i s Z e i s s Photomicroscope, and Mr. L. Hanic who suggested u s i n g i r o n - p r o p r i o n i c carmine to 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 wish acknowledge r e c e i p t of a Queen E l i z a b e t h S c h o l a r s h i p ( U n i v e r s i - t y of B r i t i s h Columbia), 1961-1962, which was donated by H. R. MacMIllan; and of a N a t i o n a l Research C o u n c i l Studentship, 1962, which was waived. F i n a l l y , I wish to express my a p p r e c i a t i o n to my wif e f o r t y p i n g the manuscript. i v TABLE OF CONTENTS ABSTRACT' i i TABLE OF CONTENTS i v ACKNOWLEDGEMENTS v i i INTRODUCTION 1 CHAPTER 1, M e i o s i s i n Hypnum c i r c i n a l e Hook, and Brachytheclum f r i g i d u m (C.M.) Besch 3 METHODS ' 4 The Handling and P r e p a r a t i o n of M a t e r i a l f o r C y t o l o g i c a l Study 4 M a t e r i a l 6 OBSERVATIONS 7 Me i o s i s i n Hypnum c i r c i n a l e 7 . M e i o s i s i n Sporangium #6, P o p u l a t i o n #2463 12 M e i o s i s i n Brachythecium f r i g i d u m 13 DISCUSSION .16 Leptotene and Zygotene 17 Pachy t ene-Diplo t ene 18 The E l o n g a t i o n Stage 19 D i a k i n e s i s and the Remainder of M e i o s i s 22 CHAPTER 2, The Effect of Heat S t r e s s on Mei o s i s i n Hypnum c i r c i n a l e Hook 25 METHODS 29 Methods of T r e a t i n g the P l a n t s 29 Methods of A n a l y s i n g Treated P l a n t s 31 Sampling 31 A n a l y s i s 32 M a t e r i a l s 33 V RESULTS. .34 Experiment #1 .' 34 Experiment #2 38 Experiment #3 40 Experiment #4 44 Experiment #5 47 Experiment #6 51 Experiment #7.... 54 DISCUSSION 57 (1) Chromosomal Behaviour of Mosses under Laboratory C o n d i t i o n s 58 (2) The S e n s i t i v i t y of Moss SMC to S l i g h t Heat Stress....59 (3) N a t u r a l O c c u r r i n g Heat S t r e s s as a Mechanism of Inducing Mutation or S t r u c t u r a l / N u m e r i c a l A l t e r a t i o n s of the Chromosomes 61 (4) The Mode of Response of the SMC to Heat S t r e s s 62 Clumping and M u l t i p l e A s s o c i a t i o n of the Bivalents...63 Extreme Condensation or C o i l i n g of the B i v a l e n t s 65 Precocious D i s j u n c t i o n 66 S p i n d l e D e s t r u c t i o n or I n h i b i t i o n , Prophase Asynchrony and Premature M e i o t i c I n d u c t i o n 66 SUMMARY. 69 LITERATURE CITED 70 APPENDIX PLATES 1-9 v i LIST OF TABLES Table I , Tetrad a n a l y s i s o f sporangia of experiment #1.....35 Table I I , Tetrad a n a l y s i s of c o n t r o l sporangia of po p u l a t i o n #1363 which were used i n experiments 1 and 2 .36 Table I I I , Tetrad a n a l y s i s of sporangia of experiment #2...39 Table IV, Tetrad a n a l y s i s of sporangia of experiment #3....41 Table V, Tetrad a n a l y s i s of c o n t r o l sporangia of p o p u l a t i o n #2267 which were used i n experiment #3 42 Table VI, Tetrad a n a l y s i s of sporangia of experiment #4....46 Table V I I , Tetrad a n a l y s i s of sporangia from p o p u l a t i o n # 2463 which were used i n experiments 4-7 46 Table V I I I , Tetrad a n a l y s i s o f sporangia of experiment #5..49 Table IX, Tetrad a n a l y s i s of sporangia of experiment #6....52 Table X, Tetrad a n a l y s i s of sporangia of experiment #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 Table XI, Te t r a d a n a l y s i s of sporangia of experiment #7, p l a n t s under normal photoperiod 56 Table X I I , A general summary i n d i c a t i n g the mode of 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 of heat s t r e s s on the SMC of H. c i r c i n a l e 64 1 INTRODUCTION The volume of l i t e r a t u r e p e r t a i n i n g to chromosomal and n u c l e a r behaviour i n mosses i s small when compared to that concerning h i g h e r p l a n t s and animals. I t i s s i g n i f i c a n t t h a t many of the e a r l i e s t works i n the f i e l d of c y t o l o g y and c y t o - 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 the study of p l a n t c y t o l o g y and c y t o g e n e t i c s i n g e n e r a l . Outstanding among these c o n t r i b u t i o n s was the work of A l l e n (1917, 1919) who i n i t i a t e d the study of sex chromosomes i n p l a n t s and that of the Marchals (1907 et seq., c i t e d by A l l e n , 1935) who f i r s t produced a r t i f i c i a l p o l y p l o i d s i n mosses and supplemented t h e i r work with c y t o l o g i c a l evidence. Bryophyte m a t e r i a l was used by H e i t z (1928, c i t e d by Steere et a l . , 1954) i n h i s s t u d i e s on n u c l e a r behaviour i n which he introduced the term "hetero- chromatin", and f u r t h e r e d the study of sex chromosomes i n p l a n t s . S e v e r a l other authors have a l s o made notable c y t o l o g i c a l c o n t r i - butions u t i l i z i n g bryophytes. T h i s e a r l y work, which i s mainly g e n e t i c a l , i s adequately reviewed by A l l e n (1935, 1945) and more r e c e n t l y by Lewis ( i 9 6 0 ) . In the l a s t twenty years, c y t o l o g i c a l study of w i l d p o p u l a t i o n s of mosses has developed s u b s t a n t i a l l y and most p u b l i c a t i o n s have been concerned w i t h e s t a b l i s h i n g chromosome numbers and s t u d y i n g chromosomal behaviour. Mehra and Khanna (1961) have reviewed t h i s p e r i o d q u i t e c r i t i c a l l y and have i n d - i c a t e d t h at although the trend has been s h i f t e d to the study of chromosome number and i n some cases behaviour, there are r 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 taxonomic problems. I t i s noteworthy that although most of the chromosome 2 counts reported f o r mosses have been obtained from the study of meiotic metaphases, only rarely has the d e t a i l of the meiotic process been studied. One of the e a r l i e s t studies of meiosis i n a moss was that of Wilson (1909), who presented a f a i r l y detailed account of meiosis i n Mnium hornum. He was l i m i t e d , however, by poor techniques and apparently did not accurately observe some of the early prophase stages. In 1932, Scheuber attempted to describe meiosis i n Timmia cucullata 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 (especially those of prophase I) well documented and i n his description of meiosis i n Pleurozium schreberi. 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 primarily to provide a detailed account (especially of prophase I) of meiosis i n one or more mosses. The need f o r such a study i s obvious because many workers have conducted cytotaxonomical studies with a limited know- ledge of meiosis i n the plants they were studying. A secondary purpose grew from observations made during the preliminary i n v e s t i - gation to t h i s study and also as a consequence of. several observa- tions made during a previous study ( D i l l , unpublished data). These observations had suggested trhat bringing mosses into the laboratory, even f o r a short period, may cause anomalous chromosomal behaviour. There was also some ind i r e c t evidence from the l i t e r a t u r e which supported these observations. Since several authors (Steere et a l . , 1954; Bryan, 1956 et seq.) had suggested bringing mosses into the laboratory to speed maturation of the capsules, i t was f e l t that an investigation of the effects of temperature stress on mosses was warranted. This matter i s discussed i n Chapter 2. 3 CHAPTER 1 Meiosis i n Hypnum c i r c i n a l e Hook, and Brachythecium frigidum (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 sectioning 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. Heitz (1928, cited by Vaarama, 1949) developed the squash technique f o r mosses, a technique which has been used almost exclusively since that time. Early i n the present investigation, attempts were made to study meiosis i n several moss taxa. I t became apparent that not a l l would be suitable, as i n many the prophase I chromosomes did not s t a i n well or they were seldom observed. This was especially true of members of the Grimmiaceae and some other acrocarpous fa m i l i e s . 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, especially the Hypnaceae (sensu-lato), had prophase I stages that could be easily stained. The chromosome number was also an important factor to be considered, as obviously a low number would permit much more accurate observations of the in 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. Neither species had been cy t o l o g i c a l l y studied. 4 METHODS The Handling and P r e p a r a t i o n of M a t e r i a l f o r C y t o l o g i c a l Study: P r e p a r i n g s l i d e s of spore mother c e l l s (SMG) f o r m e i o t i c study was e s s e n t i a l l y the technique of Steere et a l . (1954). However, c a r e f u l c o n s i d e r a t i o n was g i v e n to the h a n d l i n g of p l a n t s p r i o r t o f i x a t i o n t o prevent heat induced a b n o r m a l i t i e s (see Chapter 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 hags and were brought to the l a b o r a t o r y as q u i c k l y as p o s s i b l e , i n an attempt to s u b j e c t the p l a n t s to as l i t t l e temperature change as p o s s i b l e . They were then put i n f l a t s i n a shaded area of the r o o f on the b i o l o g i c a l s c i e n c e b u i l d i n g . An attempt was made to simulate n a t u r a l temperature and humidity c o n d i t i o n s . Adequate water was s u p p l i e d . As most of the p l a n t s f o r t h i s study were c o l l - ected d u r i n g the months of October and November of 1963, the o u t s i d e temperature was between 7 - l l ° C , and the humidity was h i g h as there was some p r e c i p i t a t i o n almost every day. . When p l a n t s were to be s t u d i e d , a p o r t i o n of the c o l l e c t i o n was brought i n t o the l a b o r a t o r y and immediately hung o u t s i d e the window i n a shaded basket. A s m a l l p o r t i o n of t h i s m a t e r i a l was used when s l i d e s were to be made and any m a t e r i a l brought i n from the basket was d i s c a r d e d i f unused a f t e r 10-15 minutes. A c e t o - o r c e i n was the only s t a i n used f o r the present study and was prepared a c c o r d i n g to the method d e s c r i b e d by D a r l i n g t o n and LaCour ( i 9 6 0 ) . T h i s i s the only s t a i n t h at has proven s a t i s - f a c t o r y f o r chromosome work wit h mosses. 1 P r e - f i x a t i o n of SMC c f . f o otnote page 13 5 was u s u a l l y done wi t h "3:1 a l c o h o l - a c e t i c a c i d . Squash p r e p a r a t i o n s are made as f o l l o w s : p l a n t s b e a r i n g b r i g h t green mature-sized capsules are s e l e c t e d from the c o l l - e c t i o n . A sporophyte i s severed from the gametophyte and t r a n s - f e r r e d t o a c l e a n s l i d e on the stage of a d i s s e c t i n g microscope. Ho l d i n g the base of the capsule w i t h f o r c e p s , both the c a l y p t r a and operculum are removed u s i n g a d i s s e c t i n g needle which i s then placed at the base of the capsule and r o l l e d toward the mouth of the capsule u s i n g s l i g h t downward pressure. In capsules p o s s e s s i n g a d i f f e r e n t i a t e d archesporium t h i s w i l l cause the c o l u m e l l a to be extruded through the mouth.of the capsule and the SMC mass w i l l f o l l o w . Both the c o l u m e l l a and'empty capsule are removed from the s l i d e and the SMC are spread s l i g h t l y to ensure even and r a p i d p e n e t r a t i o n of the f i x a t i v e arid s t a i n . Immediately f o l l o w i n g t h i s spreading procedure a drop of f i x a t i v e i s a p p l i e d to the SMC. When the f i x a t i v e has almost evaporated, the drop of s t a i n i s a p p l i e d and the s l i d e i s set a s i d e . Before the s t a i n begins to dry (about 5 minutes a f t e r a p p l i c a t i o n ) a c l e a n c o v e r s l i p i s g e n t l y a p p l i e d t o the p r e p a r a t i o n . A f t e r about 5 minutes, pressure i s a p p l i e d to the c o v e r s l i p , thus f l a t t e n i n g the SMC and spreading the chromosomes. T h e • c o v e r s l i p i s then s e a l e d with a semi- permanent gum-mastic-paraffin (1:1) s e a l i n g medium. S t a i n i n t e n - s i t y i s g e n e r a l l y s a t i s f a c t o r y at t h i s time. Although h e a t i n g the s l i d e i n t e n s i f i e s and speeds s t a i n i n g , t here i s . evidence t h a t h e a t i n g the s l i d e causes clumping of the chromosomes ( A l - A i s h and Anderson, 1961). The present author has found t h i s t o be t r u e w i t h some s p e c i e s , thus omitted t h i s procedure to keep the 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 p o s s i b l e . Permanent s l i d e s of some of the m a t e r i a l of each 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 bright f i e l d observations were made using an Olympus binocular compound microscope with N.A. = 1.40 Ach. Condenser, a 125 mm. o i l immersion objective and X10 oculars. Photographs and phase contrast work were done on a Zeiss Photomicroscope. Material: Material of Hypnum c i r c i n a l e Hook, was collected from three populations: A l i c e Lake, B. C , D i l l #1363; University 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 collected from one population: Mt. Seymour, B.C. (2000 f t . ) , D i l l #2563. Voucher specimens have been deposited i n the University 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 Me i o s i s i n Hypnum c i r c i n a l e : Observations of meiosis i n each p o p u l a t i o n of H. c i r c i n a l e were made at the same time the r e s p e c t i v e populations were used as the experimental m a t e r i a l i n the heat s t r e s s experiments (Chapter 2 ) . I t was found, t h a t , with the exception of one spor- angium (a member of p o p u l a t i o n #2463, sporangium #6) a l l three 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 m o r p h o l o g i c a l l y i n d i s t i n g u i s h - a b l e . They d i f f e r e d only i n the maturation time of the c a p s u l e s . In g e n e r a l , members of p o p u l a t i o n #2463 matured two to three weeks l a t e r than those of #1363 and #2262. T h i s d i f f e r e n c e was a t t r i b u t e d t o the d i f f e r e n c e i n the a l t i t u d e s where the three p o p u l a t i o n s occurred i n nature. Number 2463 was c o l l e c t e d at 2000 f t . while #1363 and #2263 were c o l l e c t e d at sea l e v e l . For t h i s reason the d e s c r i p t i o n of the m e i o t i c process of these pop- u l a t i o n s i s combined and i s considered to be r e p r e s e n t a t i v e f o r the s p e c i e s . The a r c h e s p o r i a l l a y e r of H. c i r c i n a l e has d i f f e r e n t i a t e d when the capsules reach mature s i z e , but before there i s any i n d i c a t i o n o f browning of the annulus. When the spore mother c e l l s are f i r s t d i f f e r e n t i a t e d they are quadrate and s t a i n weakly wit h a c e t o - o r c e i n . No i n t e r n a l s t r u c t u r e i s d i s c e r n a b l e . M e i o s i s appears to be i n i t i a t e d i n a l l the SMC of a capsule simultaneously and, i n most sporangia, proceeds i n an almost synchronous manner u n t i l the i n i t i a t i o n of d i a k i n e s i s . Thus a l l SMC i n each prepared s l i d e appear to be at the same stage. On i n i t i a t i o n of meiosis 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 ced, weakly s t a i n i n g chromatin mass ( P L . 1, f i g . 1-3)• At t h i s stage, which i s probably 8 l e p t o t e n e , the chromatin i s a mass of very entangled threads. The 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 cases chromonemata can be d i s c e r n e d (PL. 1, f i g . 3). I t i s questionable whether the e n t i r e nucleus or only the chromatin i s a c e n t r i c a l l y placed, f o r i n some c e l l s threads of chromatin appear t o t r a v e r s e up to 2/3 the width of the c e l l (PL. 1, f i g . l ) . While the chromatin i s a c e n t r i c a l l y placed synapsis occurs; o f t e n SMC can be observed i n which the threads are double but c e l l s are otherwise i d e n t i c a l to those of the l e p t o t e n e stage. Evidence f o r synapsis was obtained by phase c o n t r a s t o b s e r v a t i o n . One SMC was noted i n which two s i n g l e threads p a i r e d f o r a short p o r t i o n of t h e i r l e n g t h , while the unpaired s e c t i o n s were widely separated (PL. 1, f i g . 2). Other c e l l s of the same s l i d e had mostly s i n g l e - t h r e a d e d chromatin. When synapsis i s complete the chromosomes become s h o r t e r and t h i c k e r . At the same time, the chromatin mass loosens and migrates to the cente r of the c e l l (PL. 1, f i g . 4-9). Chromosomes d u r i n g t h i s time assume a t y p i c a l pachytene morphology,'but no n u c l e o l i are evident. As pachytene proceeds, the chromosomes shor- ten and t h i c k e n , and the d i p l o t e n e phase appears as the r e p u l s i o n of homologs becomes evident (PL. 1, f i g . 10,11; PL2. f i g . 1,2). In r a r e SMC, a l l s i x b i v a l e n t s are c l e a r l y d i s c e r n i b l e at d i p l o t e n e (PL. 2, f i g . 2) but u s u a l l y only one or two are separated from the chromosomal mass (PL. 1, f i g . 11). The s m a l l e s t b i v a l e n t , which i s heterochromatic, o f t e n becomes h i g h l y condensed d u r i n g pachy- tene and d i p l o t e n e (PL. 1, f i g . 10; PL. 2, f i g . 2). In a d d i t i o n , there are c e r t a i n segments of other chromosomes which are h e t e r o - chromatic and s t a i n deeply d u r i n g some p e r i o d of d i p l o t e n e , (PL. 1, f i g . 11). However, i t was not p o s s i b l e to i d e n t i f y the chromosomes 9 by these regions as they were seldom well 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 either too poorly to 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 nucleo- l a r organizing chromosome i s one of the larger bivalents, (probably the second l a r g e s t ) . I t i s possible that the small heterochromatic bivalent 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 plants. The bivalent 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 bivalent shown i n PL. 2, f i g . 1 (highly squashed c e l l ) i s probably the large bivalent which has such a d i s t i n c t i v e morphology at meta- phase 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 persists under natural 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 persist as long as a week. Hear the end of diplotene the bivalents become less well defined (PL. 2, f i g . 3), and chromosomes gradually lengthen and lose t h e i r discreteness, at which time the nuclear contents assume an interphase-like appearance (PL. 2, f i g . 4,5). Under phase contrast, fin 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 bivalent 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 of the chromosomes had been maintained and that the chromosomes were i n an u n c o i l e d s t a t e . The c e l l s at t h i s stage u s u a l l y c o n t a i n one l a r g e deeply s t a i n i n g chromatin body. As i n the e a r l i e r stages, no n u c l e a r membrane was d i s t i n g - u i s h a b l e d u r i n g t h i s phase, but s i n c e the chromatin was spread almost to the p e r i p h e r y of the c e l l the nucleus was probably only s l i g h t l y s m a l l e r than the SMC i t s e l f . Up to and i n c l u d i n g t h i s chromosome e l o n g a t i o n stage, the d i v i s i o n s of the SMC of one capsule are almost synchronous. Although most sporangia had some t r a n s i t i o n stages (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 of the SMC appeared i n the same stage. From t h i s stage on, however, synchrony i s l o s t . Each c e l l may remain i n the e l o n g a t i o n stage an estimated two days, then each may proceed to the next stage i n a random order by a r a p i d condensation of the b i v a l e n t s . In sporangia w i t h some SMC possessing condensed b i v a l e n t s ( i e . d i a k i n e s i s ) , a l l other c e l l s were e i t h e r i n l a t e r stages of meiosis or were i n the e l o n g a t i o n stage. Only i n extremely r a r e cases d i d e a r l i e r stages o f prophase occur a f t e r condensation had begun. T h i s obser v a t i o n c l e a r l y i n d i c a t e s t hat the e l o n g a t i o n stage f o l l o w s d i p l o t e n e and 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 of the b i v a l e n t s could not be followed a c c u r a t e l y s i n c e t h i s appears to proceed very r a p i d l y . In some c e l l s , the chromatin was d i f f u s e and i n one l a r g e mass (PL. 2, f i g . 6 ). T h i s stage occurred between the e l o n g a t i o n stage and d i a k i n e s i s , but because of i t s i r r e g u l a r and i n f r e q u e n t occurrence i t may represent an abnormal or p o o r l y f i x e d e l o n g a t i o n 11 stage. Without f u r t h e r study i t cannot be accepted as a u s u a l occurrence i n the m e i o t i c sequence of Hypnum c i r c i n a l e . D i a k i n e s i s i s c h a r a c t e r i z e d by a gen e r a l clumping of the h i g h l y condensed b i v a l e n t s (PL. 2, f i g . 7,8) i n the center o f the c e l l . In co n j u n c t i o n with s p i n d l e formation the clumped b i v a l e n t s separate and assume a metaphase I c o n f i g u r a t i o n . Metaphase I of 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 by s i x b i v a l e n t s i n c l u d i n g f o u r which are medium i n s i z e , one which i s u s u a l l y d i s t i n g u i s h a b l e as s m a l l e r than the r e s t and one which i s very l a r g e and m o r p h o l o g i c a l l y d i s t i n c t . In the m a j o r i t y of c e l l s 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 at one s i d e of the metaphase I p l a t e (PL. 2, f i g . 9 ) . D i s j u n c t i o n of the b i v a l e n t s u s u a l l y occurred q u i t e synchronously (PL. 2, f i g . 10) with the l a r g e b i v a l e n t o f t e n s l i g h t l y preceding the others to the p o l e s . On completion of the f i r s t d i v i s i o n the chromosomes p a r t i a l l y u n c o i l and 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 (PL. 2, f i g . 11) which u s u a l l y contains one l a r g e heterochromatic r e g i o n . Although c e l l w a l l formation i s not complete u n t i l a f t e r telophase I I , a c o n s t r i c t i o n of the cytoplasm f r e q u e n t l y appears a f t e r telophase I (PL. 2, f i g . 11). The chromosomes at metaphase I I are again condensed and form a clumped c o n f i g u r a t i o n . Only r a r e l y can a chromosome count be made at t h i s stage. At anaphase I I the chromosomes appear to be s t i c k y and temporary bridges o f t e n formed (PL. 2, f i g . 12). However, these apparently do not cause abnormal s e p a r a t i o n of the chromatids as only seldom do fragments or e x t r a n u c l e a r chromatin appear i n telophase I I and the t e t r a d stage. During telophase I I , w a l l formation begins (PL. 3, f i g . l ) . The cytoplasm appears at f i r s t to be c o n s t r i c t e d on the 1 2 plane of the f i r s t d i v i s i o n and then on the plane of the second d i v i s i o n . The m e i o t i c sequence i s concluded by pro- d u c t i o n of a t e t r a d of spores (PL. 3, f i g . 2). M e i o s i s i n Sporangium #6, P o p u l a t i o n #2463: In only one sporangium of the three p o p u l a t i o n s of H. c i r c i n a l e examined d i d meiosis d i f f e r d i s t i n c t l y from what was considered normal. In t h i s sporangium SMC were present i n a l l stages l a t e r than d i p l o t e n e of prophase I with the m a j o r i t y i n metaphase I and d i a k i n e s i s . In about 40$ of those i n metaphase I and d i a k i n e s i s the s m a l l e s t b i v a l e n t had d i s j o i n e d p r e c o c i o u s l y . U s u a l l y only a s i n g l e member of the p a i r was separated from the main chromosomal mass (PL. 3, f i g . 3-6). However, there were some metaphases i n which both the u n i v a l e n t s were evident and others i n which the b i v a l e n t had not d i v i d e d (PL. 3, f i g . 7 ) . The remainder of the b i v a l e n t s appeared to act as they d i d i n 'normal' sporangia. L a t e r stages of d i v i s i o n i n t h i s sporangium had a n o t i c e a b l y h i g h frequency of a b n o r m a l i t i e s . About 15$ of the SMC i n f i r s t and second telophase contained a chromatin bridge and/or laggards (PL. 3, f i g . 8,9). These anomalies occurred with a frequency of 2-3$ i n the c e l l s of the normal sporangia. Often more than one b i v a l e n t p a r t i c i p a t e d i n bridge formation and u s u a l l y the small b i v a l e n t was not i n v o l v e d (PL, 3, f i g . 8). Furthermore, i n many of those with only one b r i d g e or l a g g a r d the amount of p a r t i c i p a t i n g chromatin was g r e a t e r than the s i z e of the s m a l l b i v a l e n t . On the other hand, there were some cases (PL. 3, f i g . 9) i n which the small b i v a l e n t was comp- l e t e l y omitted from the s p i n d l e apparatus. E v i d e n t l y something was causing an upset i n meiosis i n t h i s p l a n t but t h i s could not be demonstrated on morphological grounds. A s t r u c t u r a l a b e r r a t i o n 13 might p o s s i b l y have been detected at d i p l o t e n e or pachytene but u n f o r t u n a t e l y n e i t h e r of these stages was present i n the s l i d e . Heat shock might a l s o have caused such anomalies but t h i s sporangia was t r e a t e d i n the same method as the others, making t h i s p o s s i b i l i t y s l i g h t . The consequences of heat shock are d i s c u s s e d i n d e t a i l l a t e r . M e i o s i s i n Brachytheeium f r i g i d u m t Observations of meiosis of B. f r i g i d u m were made f o r only a s i n g l e p o p u l a t i o n . The main purpose of t h i s study was to determine i f the e l o n g a t i o n stage observed a f t e r d i p l o t e n e i n H. c i r c i n a l e was unique to that s p e c i e s . Thus, on the b a s i s of the results., a study of one p o p u l a t i o n was s u f f i c i e n t . E s s e n t i a l l y the p a t t e r n of meiosis i n B. f r i g i d u m was the same as that of H. c i r c i n a l e . However there were c e r t a i n f a c e t s of the sequence which, although seemingly d i f f e r e n t i n the two s p e c i e s , were probably more c l e a r l y demonstratable i n B. f r i g i d u m . The f i r s t of these i s the presence of the n u c l e o l u s d u r i n g e a r l y prophase. E a r l y prophase of B. f r i g i d u m i s c h a r a c t e r i z e d by the a c e n t r i c l o c a t i o n of a nucleus c o n t a i n i n g s i n g l e - s t r a n d e d chromosomes. Phase c o n t r a s t observations of t h i s stage r e v e a l e d the presence of a l a r g e n u c l e o l u s (PL. 3, f i g . 10, 11). T h i s n u c l e o l u s was not evident when the s l i d e s were i n i t i a l l y prepared _ The author has s i n c e observed l a r g e n u c l e o l i i n l e p t o t e n e through to e a r l y d i p l o t e n e i n a number of species v i z . Rhacomi- tr i u m microcarpon. Claopodium c r i s p i f o l i u m , Isothecium s t o l o n - iferum, Plagiothecium undulatum as w e l l as B. f r i g i d u m u s i n g p r o p r i o n i c - c a r m i n e with i r o n s t a i n . The s t a i n was made ac c o r d i n g to the method of D a r l i n g t o n and La Sour (i960) f o r aceto-carmine. However, p r o p r i o n i c a c i d r e p l a c e d the a c e t i c a c i d and the i r o n a c e t a t e was not added. Ir o n was added by suspending f o r c e p s 14 but became evident only a f t e r standing a few months C e l l s i n m i g r a t i n g pachytene and d i p l o t e n e phases (PL. 3, f i g . 12; PL. 4, f i g , 1,2) appeared to have no v i s i b l e n u c l e o l u s u s i n g phase co n t r a s t or b r i g h t f i e l d i l l u m i n a t i o n . During pachytene and d i p l o t e n e of B. f r i g i d u m a l l the b i v a l e n t s have the same s t a i n i n t e n s i t y except f o r a h e t e r o p y c n o t i c s e c t i o n of one of the chromosomes (PL. 4, f i g . 2). T h i s chromosome could not be i d e n t i f i e d s p e c i f i c a l l y . F o l l o w i n g d i p l o t e n e , the chromosomes •uncoil as i n H. c i r c i n a l e and the chromatin mass assumes an interphase appearance (PL. 4, f i g . 3,4). In B. f r i g i d u m the threads appear more coarse than those of H. c i r c i n a l e d u r i n g the same stage. One remarkable d i f f e r e n c e noted between the two s p e c i e s was that the n u c l e a r membrane i n B. f r i g i d u m was c l e a r l y d e f i n e d i n many of the SMC (PL. 4, f i g . 3). Condensation of the chromosomes f o l l o w s the e l o n g a t i o n phase and the b i v a l e n t s assume a clumped c o n f i g u r a t i o n (PL. 4, f i g . 5). T h i s resembles the d i a k i n e s i s i n H . c i r c i n a l e . but i n B. f r i g i d u m the clumped c o n f i g u r a t i o n i s f o l l o w e d by a more normal d i a k i n e s i s i n which the h i g h l y condensed b i v a l e n t s are not clumped together and are d i s t r i b u t e d q u i t e evenly i n the SMC (PL. 4, f i g . 6). S i x b i v a l e n t s of s i m i l a r s i z e are present at metaphase I . One of ^(continued from page 13) i n a drop of the s t a i n j u s t before the s t a i n was used. I t was found 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 or Newcomer's s o l u t i o n (Newcomer, 1953) s t a i n e d w e l l . G l a c i a l a c e t i c a c i d , followed by a washing i n 1:3 a c e t i c a l c o h o l f i x could be employed a f t e r normal f i x - a t i o n to remove o i l d r o p l e t s . T h i s treatment d i d not impair 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 these b i v a l e n t s i s rod-shaped and i s u s u a l l y l o c a t e d at one s i d e of the metaphase. p l a t e (PL. 4, f i g . 7,8). T h i s b i v a l e n t i s u s u a l l y very l o n g at metaphase I and o f t e n d i s s o c i a t e s p r e c o c i o u s l y . Its!; behaviour i s analogous to t h a t of the l a r g e b i v a l e n t of H. c i r c i n a l e . Anaphase I (PL. 4, f i g . 9) was remarkably r e g u l a r i n t h i s s p e c i e s and the remainder of the m e i o t i c sequence was s i m i l a r to that i n H. c i r c i n a l e . A b n o r m a l i t i e s at second d i v i s i o n were extremely r a r e and t e t r a d formation appeared normal. 16 DISCUSSION The study of the m e i o t i c prophase of mosses has been s e v e r e l y n e g l e c t e d . For the most p a r t , students of moss cy t o l o g y have been concerned only with a c q u i r i n g chromosome counts and determining, r a t h e r s u p e r f i c i a l l y , the behaviour and morphology of the b i v a l e n t s at metaphase I . The chromosome numbers of at l e a s t 700 o f the 20,000 d e s c r i b e d s p e c i e s of mosses have now been e s t a b l i s h e d (Mehra and Khanna, 1961, estimated 650 s p e c i e s ) . Over 90$ of the e s t a b l i s h e d numbers have been presented s i n c e 1948, Although the accumulated work has c o n t r i b u t e d l i m i t e d knowledge to the taxonomy and e v o l u t i o n of mosses, the c o n t r i b u t i o n to b a s i c c y t o g e n e t i c s and g e n e t i c s has been s m a l l . The most outstanding o f the work that has been publ i s h e d s i n c e 1948 i s that of Vaarama. In an attempt to study the m e i o t i c prophase.of Hedwigia c i i i a t a Br. & Sch. and some members of the Grimmiaceae, Vaarama (1949, 1954a) found that poor s t a i n a b i l i t y of the SMC at prophase I rendered d e t a i l e d examination i m p o s s i b l e . He s t a t e d (1949) t h a t prophase stages l a t e r than pachytene had been r a r e l y observed, which suggested that these stages were of short d u r a t i o n . However, d u r i n g h i s study of H. e i l i a t a , the same author d e s c r i b e d the behaviour of a ' s p e c i a l b i v a l e n t ' d u r i n g the e a r l y prophase. T h i s b i v a l e n t appeared as a s p h e r i c a l opaque n u c l e o l u s - l i k e v e s i c l e bounded, by f o u r h e t e r o p y c n o t i c bodies. Although the r e s t of the chromatin i n prophase I was almost impossible to observe i n d e t a i l , the v e s i c l e was q u i t e conspicuous and p e r s i s t e d u n t i l d i a k i n e s i s , whereupon i t became n e g a t i v e l y h e t e r o p y c n o t i c and underwent unusual se g r e g a t i o n behaviour. At the time, Vaar.ama s t a t e d that the r e l a t i o n s h i p 17 between the n u c l e o l u s - l i k e v e s i c l e and. the o r d i n a r y n u c l e o l u s was 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 study u s i n g propionic-carmine as a s t a i n f o r n u c l e o l i i n the prophase I of s e v e r a l mosses ( c f . footnote page 13) the present author 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 of Plagiothecium undulatum (Hedw.) Bry. Eur. In a d d i t i o n t o the n u c l e o l u s - l i k e v e s i c l e there was a normal n u c l e o l u s present which appeared e n t i r e l y separate from the s p e c i a l b i v a l e n t . A l s o , the v e s i c l e of the 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 that of the n u c l e o l u s , i n d i c a t i n g t h at the v e s i c l e i s p o s s i b l y n u c l e o l a r i n s t r u c t u r e . The f i r s t and only d e t a i l e d study of the general behaviour of the chromosomes at m e i o t i c prophase i n mosses was reported by Vaarama (1954b). He found that the chromosomes of PIeurozium s c h r e b e r i ( 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 prophase I and t h a t , because they were few i n number (5), observations of i n d i v i d u a l chromosomes duri n g the e a r l y stages were p o s s i b l e . He presented the only published f i g u r e of t r u e d i p l o t e n e i n mosses known to the present author. The e a r l y prophase of 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 the e a r l y prophase of H. c i r c i n a l e and B. f r i g i d u m . However, Vaarama d i d not d e s c r i b e or d e p i c t the e l o n g a t i o n stage that f o l l o w e d d i p l o t e n e i n the two sp e c i e s presented here and he i n d i c a t e d t h a t a clumped d i a - k i n e s i s d i r e c t l y f o l l o wed d i p l o t e n e . F u r t h e r mention of t h i s unusual stage w i l l be made i n the d i s c u s s i o n of each prophase stage. l e p t o t e n e and Zygotene: The 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 stage i s of wide occurrence (Vaarama, 1954b). The present author has observed the phenomenon i n a l l of the approximately t h i r t y s p e c i e s of mosses examined d u r i n g the past three years (unpublished d a t a ) . The chromosomes can be observed e i t h e r s i n g l e stranded, p a r t l y double stranded (as i n H. c i r c i n a l e . PL. 1, f i g . 2), or completely double stranded, i n d i c a t i n g that synapsis takes p l a c e d u r i n g t h i s p e r i o d of chromatin p o l a r i z a t i o n . Since the n u c l e a r membrane has not been observed at t h i s stage, i t i s d i f f i c u l t to say whether the chromatin i s bound c l o s e l y on a l l s i d e s by i t or whether the nucleus i s a c t u a l l y q u i t e l a r g e ( i e . extending to or past the middle of the SMC) with the chromatin l o c a l i z e d at one s i d e . As noted e a r l i e r , on rare, occasions i n H. c i r c i n a l e l o n g , presumably s i n g l e , threads of chromatin extended w e l l past the c e n t e r of the c e l l w h i le the remainder of the chromatin was a c e n t r i c a l l y l o c a l i z e d . Furthermore, the n u c l e o l u s of some of the s p e c i e s s t u d i e d w i t h propionic-carmine ( c f . f o o tnote page 13) was o f t e n - q u i t e separate from the chromatin mass and was l o c a t e d c l o s e r t o the center of the c e l l than the mass. In a d d i t i o n , when the chromosomes migrate to the c e n t e r of the c e l l i n early'pachytene, i n i t i a t i o n of t h i s movement i s by one or a few of the chromosomes which become loosened from the mass. Most of the chromatin remains l o c a l i z e d f o r some time a f t e r t h i s m i g r a t i o n begins, i n d i c a t i n g t h a t the nucleus at t h i s time must extend past the middle of the c e l l . F u r t h e r s t u d i e s , 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 w i t h u n f i x e d m a t e r i a l may h e l p to e l u c i d a t e t h i s matter. Paehytene-Diplotene: : The m i g r a t i o n of the p a i r e d chromosomes to the c e n t e r of 19 the SMC, as observed i n both H. c i r c i n a l e and B. f r i g i d u m i s s i m i l a r to t h a t d e s c r i b e d by Vaarama (1954b) i n Pleurozium s c h r e b e r i . The t r a n s i t i o n between pachytene and d i p l o t e n e stage i s not p r e c i s e . In f a c t , Vaarama p r e f e r r e d to lump the two stages i n many cases and r e f e r r e d to pachytene-diplotene stages. Soon a f t e r the chromosomes have migrated, s e c t i o n s of the b i v a l e n t s begin to r e p e l each other but only seldom can t h i s be f o l l o w e d i n one chromosome as u s u a l l y some part of every chromosome i s entangled w i t h the o t h e r s . However, when the b i v a l e n t s are short enough that one or two are e a s i l y separated from the r e s t , r e p u l s i o n i s completed along the whole chromosome except at the contact p o i n t s . These would u s u a l l y be designated, as chiasmata. Vaarama (1954b) has presented evidence i n h i s study on P. s c h r e b e r i t h a t the contact p o i n t s observed d u r i n g d i p l o t e n e are not t r u e chiasmata, but merely areas of s t i c k y heterochromatin. No s p e c i a l study of the method of chiasma" . formation was made i n t h i s i n v e s t i g a t i o n so the q u e s t i o n must remain open f o r the present. The E l o n g a t i o n Stage: The most remarkable aspect of the m e i o t i c sequence observed i n H. c i r c i n a l e and B. f r i g i d u m was the occurrence of the chromosome e l o n g a t i o n stage which f o l l o w e d d i p l o t e n e . Vaarama (1954b) d i d not d e s c r i b e or d e p i c t t h i s stage f o r Pleurozium s c h r e b e r i . However i t remains q u i t e p p o s s i b l e that t h i s stage i s present i n Pleurozium s c h r e b e r i as w e l l as i n many other mosses. The present author has, s i n c e the i n i t i a l s t u d i e s on H. c i r c i n a l e and B. f r i g i d u m , examined meiosis i n Claopodium c r i s p i f o l i u m (Hook.) R. & C. and found t h a t the e l o n g a t i o n stage occurred i n that s p e c i e s as w e l l . Furthermore, an examination of permanent s l i d e s o f twelve moss taxa s t u d i e d at an e a r l i e r date ( D i l l , unpublished d a t a ) , suggests that a l l have t h i s e l o n g a t i o n stage. U n f o r t u n a t e l y t h i s cannot be e s t a b l i s h e d c o n c l u s i v e l y u n t i l the complete sequence of meiosis i s examined f o r each of the s p e c i e s . The f a c t t h a t H. c i r c i n a l e . B. f r i g i d u m . and C. c r i s p i f o l i u m represent three moss f a m i l i e s i n d i c a t e s a l s o that the stage i s probably widespread i n the 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 stage has been r e p o r t e d on r a r e occasions i n the p l a n t kingdom. V i o l a n t e (1929) d e s c r i b e d a stage, termed s t r e p t s i t e n e , i n the dicot^ B a l s a m i n a h o r t e n s i s (Impatiens balsamina) s i m i l a r to that observed by the present author i n Hypnum and Brachytheeium. The e l o n g a t i o n o f the chrom- osomes was a l s o accompanied by n u c l e a r growth. He i n d i c a t e d t h a t l e w i t s k y (1927) and Modilevsky (1918) had observed s i m i l a r stages i n other p l a n t s and t h a t Lewitsky had apparently considered the stage a r t i f a c t . V i o l a n t e a l s o mentioned t h a t Szakien (1927) had observed t h i s stage i n the f e r n Osmunda r e g a l i s . but exam- i n a t i o n of Szakien's paper i n d i c a t e s that.this i s not the case. 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 i n the developing a s c i of many ascomycetes, but t h i s phase occurs before d i p l o t e n e and i s a t y p i c a l pachytene stage (Carr and O l i v e r 1958) .• Wilson (1952) has repo r t e d that the mature r e s i s t a n t sporangia o f Allomyces i s i n a s t a t e of suspended prophase I of me i o s i s , but he was unable to determine which stage of prophase I . The photo- graphs do not resemble those of the e l o n g a t i o n stage r e p o r t e d here. 21 A c c o r d i n g to P r a t t and Long (1917), von Winiwarter (1900) d e s c r i b e d an e l o n g a t i o n stage i n prophase I which f o l l o w e d d i p l o t e n e i n the maturing oocytes of r a b b i t and man. Von Winiwarter gave the term 'Noyoux d i c t y e ' ( s i c ) t o t h i s phase. More r e c e n t l y Ohno, Maki.no, Kaplan and K i n o s i t a (1961), and Ohno, K l i n g e r and A t k i n (1962), have r e a f f i r m e d von Winiwarter's o r i g i n a l observations on oogenesis i n man and have i n d i c a t e d t h at the d i c t y e stage (termed d i c t y o t e n e or d i c t y a t e stage i n recent papers )^ i s a ' r e s t i n g ' stage i n which the chromosomes are h i g h l y extended and maintain t h e i r d i p l o t e n e s t r u c t u r e . The oocytes remain i n t h i s phase u n t i l o v u l a t i o n when the m e i o t i c sequence i s resumed. During the i n t e r v e n i n g p e r i o d the c e l l s grow and accumulate n u t r i e n t u t i l i z e d by the f u t u r e embryo. In many amphibians and some i n s e c t s , the lampbrush stage i s homologous wi t h the d i c t y o t e n e stage .(Seshachar and Bagga, 1963). Furthermore, a c c o r d i n g to C a l l a n (1963) probably a l l animals have a stage which corresponds to the d i c t y o t e n e or lampbrush stage i n t h e i r developing oocytes. He a l s o i n d i c a t e s t h at the l a t e r a l p r o j e c t i o n s of the d i p l o t e n e chromosome of many primary spermatocytes may a l s o represent a lampbrush-like behaviour, but that the chromosomes are not g r e a t l y extended i n l e n g t h as they are i n the oocytes. Although the d i c t y o t e n e chromosomes vary i n gross morphology among the d i f f e r e n t animal groups i t i s apparent t h a t the chromosomes are b a s i c a l l y d i p l o t e n e i n s t r u c t u r e ( R i s , 1945). Wilson (1925) r e f e r s to t h i s stage as the d i c t y o t i c stage. 22 Aside from the few instances mentioned i n the fungi, which probably do not represent the dictyotene stage, the only s a t i s - factory evidence f o r the occurrence of such a stage i n plants to my knowledge has been reported i n Balsamina hortensis (impatiens Balsamina). In t h i s case the sporocyte nuclei apparently increased In size during t h i s phase, i n a manner similar 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 relationship between the animal dictyotene and that 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 similar structure.. The SMC of mosses do not enlarge during dictyotene and the duration of the stage i s not long i n r e l a t i o n to the other meiotic events, as i t i s i n oocytes. Thetometabolism of the SMC during meiosis i s v i r t u a l l y unknown and i t i s quite possible that a high rate of metabolic a c t i v i t y may take place without appreciable growth i n size of the c e l l s . It appears probable that the dictyotene stage has some functional significance i n the meiotic sequence of mosses, but u n t i l d e t a i l s of the metabolic events taking place during t h i s stage are discovered l i t t l e can be said about t h i s matter. Diakinesis and the Remainder of Meiosis I The clumped diakinesis observed i n both species studied here i s commonly found i n mosses. Vaarama (1954b) indicated that a similar s i t u a t i o n occurred i n P. schreberi and many other mosses. Usually bivalents at diakinesis i n higher plants are evenly distributed about the nucleus or are near the nuclear membrane. Steere, et a l . (1954) have reported making many counts from 23 d i a k i n e s i s stages i n which.the b i v a l e n t s were widespread. P o s s i b l y the prometaphase stage observed i n B. f r i g i d u m corresponds to t h i s same stage. In t h i s case, H. c i r c i n a l e and Pleurozium s c h r e b e r i would not have a c l a s s i c a l type o f d i a k i n e s i s and the clumped stage cannot be r e f e r r e d to as d i a k i n e s i s . However, B. f r i g i d u m and many other s p e c i e s have the clumped stage f o l l o w i n g d i c t y o t e n e and before prometaphase; i t may be another d i s t i n c t i v e f e a t u r e of the m e i o t i c sequence i n mosses. Metaphase I conforms w e l l with that of the higher p l a n t s i n the two species s t u d i e d here. The l a r g e rod-shaped b i v a l e n t which i s u s u a l l y l o c a t e d at the p e r i p h e r y of the metaphase I p l a t e has been d e s c r i b e d i n many moss species (Mehra and Khanna, 1961). They are u s u a l l y r e f e r r e d to as heterochromatic, but no evidence f o r t h i s was 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 of the two s p e c i e s d i s c u s s e d here. Studies on somatico t i s s u e would help to e l u c i d a t e t h i s matter. P o s s i b l y these b i v a l e n t s c o r r e s - pond to the l a r g e 'H' chromosomes of m i t o t i c c e l l s d e s c r i b e d by Yano (1957a,b,c). The small heterochromatic b i v a l e n t found i n Hypnum c i r c i n a l e perhaps f a l l s 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 i n d i s t i n g u i s h a b l e minute b i v a l e n t s " of Mehra and Khanna (1961), a category t h a t these authors say corresponds to Yano's (1957 a,b,c) heterochromatic 'h' chromosomes o f m i t o s i s . Its*; behaviour, ( i e . precocious d i s j u n c t i o n ) i n sporangium #6 of the p o p u l a t i o n # 2464 was reminiscent of the minute b i v a l e n t ' s behaviour. The remainder of the m e i o t i c sequence i n both the species s t u d i e d here i s unremarkable. Both have normal second d i v i s i o n s and normal t e t r a d formation, i n d i c a t i n g the presence of a w e l l - balanced g e n e t i c system. The time and method of w a l l formation 24 d u r i n g meiosis i n mosses has not been s t u d i e d c l o s e l y . U n t i l t h i s i s done one can only say that i t appears that the w a l l begins to form toward the end of the f i r s t d i v i s i o n . T h i s i s i n d i c a t e d by a l i g h t s t a i n i n g and apparently c o n s t r i c t e d area of cytoplasm which t r a v e r s e s the c e l l s f o l l o w i n g telophase I . Quite l i k e l y t h i s mechanism v a r i e s i n d i f f e r e n t mosses as i t does i n d i f f e r e n t h i g h e r p l a n t s . The chromosome counts (n=6) f o r H. c i r c i n a l e and B. f r i g i d u m have not been p r e v i o u s l y r e p o r t e d . Both Hypnum and Brachythecium have a great v a r i e t y of chromosome numbers rep o r t e d , a v a r i a t i o n t h at l i e s not only between sp e c i e s but a l s o w i t h i n the same s p e c i e s . Much of t h i s v a r i a t i o n may be a t t r i b u t a b l e to i n a c c u r a t e observations and u n t i l the counts are c l e a r l y e s t a b l i s h e d d i s c u s s i o n s of t h e i r taxonomic s i g n i f i c a n c e are r e l a t i v e l y menaingless. In c o n c l u s i o n , i t could be s a i d t h a t , w i t h the exception of the d i c t y o t e n e stage, the m e i o t i c sequence i n these mosses conforms c l o s e l y to t h a t found i n most high e r p l a n t s . The d i c t y o t e n e stage, although an unusual f e a t u r e of p l a n t meiosis, may be of common occurrence i n many moss tax a . 25 CHAPTER 2 The Ef f e c t of Heat Stress on Meiosis i n Hypnum c i r c i n a l e Hook. It i s well-known that meiotic stages i n most mosses are stained most e f f e c t i v e l y i f the capsules are immersed i n acetic alcohol f o r a very b r i e f period. With most mosses a 24 hour f i x a t i o n period w i l l reduce the chromosome s t a i n a b i l i t y . t o such an extent that detailed observations are impaired. For t h i s reason, many.authors (Steere et a l . , 1954; Bryan, 1956 et seq.; Anderson and Crum , 1959) have brought the plants d i r e c t l y to the laboratory, and have made the preparations i n the laboratory from l i v i n g material. It has been suggested that plants which are not mature enough fo r meiosis at the time of c o l l e c t i o n can be grown i n the laboratory u n t i l they mature. Usually the plants are kept i n covered p l a s t i c dishes. The above mentioned authors also state that maturing plants can be refrigerated i n order to slow down meiosis. Although there are presumably no abnormalities induced by use of either of these techniques, no controlled experiments have been conducted to substantiate 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 feature of meiosis i n wild populations of mosses: many chromosomal aberr- ations occur and often some bivalents tend to d i s j o i n precoc- iously at metaphase I. To quote Steere et a l . , (1954): "In the present investigation, i t i s observed that c the sporocytes of mosses brought i n from the f i e l d early i n the day were often i n l a t e prophase or tetrad stages. Exposure to l i g h t and warmth, how- ever, induced the young sporocytes to resume meiosis. In view of the many chromosomal aberrations noted at the f i r s t and second meiotic di v i s i o n s , a .careful study of prophase behaviour i s u r g e n t l y needed, e s p e c i a l l y of pachytene stages." I t i s p u z z l i n g that the authors d i d not attempt to connect the two f a c t s and suggest that the a b n o r m a l i t i e s may have a r i s e n as a r e s u l t of the treatment. Bryan (1956b) suggested t h a t clumping of chromatin i n Phascum SMC seemed to be r e l a t e d to the low water content of the c e l l s . The m a t e r i a l she s t u d i e d that p a r t i c u l a r l y showed t h i s phenomenon had been mailed to her and the capsules were f l a c c i d on a r r i v a l . She mentioned that i f the p l a n t s were watered and s t o r e d at reduced ( u n s p e c i f i e d ) temperatures, they had normal meiosis.. She (1956a) observed s i m i l a r clumping and unusual metaphases i n B r u c h i a but no mention was made concerning the treatment given the p l a n t s . Khanna (i 9 6 0 ) , i n a chromosomal study of some Himalayan mosses, has i n d i c a t e d that a l l h i s c y t o l o g i c a l s t u d i e s were c a r r i e d out i n the f i e l d because s t o r e d m a t e r i a l d i d not y i e l d s a t i s f a c t o r y r e s u l t s , but he d i d not say why they were u n s a t i s - f a c t o r y . On the other hand, he i n d i c a t e d t h a t abnormal weather as w e l l as the f i x a t i v e used could cause clumping of m e i o t i c chromosomes. T h i s was e s p e c i a l l y apparent i n Barbula c o n s t r i c t a M i t t , which had chromosomal clumping and other a b n o r m a l i t i e s i n hot dry weather, while i t had normal meiosis i n c o o l wet weather. T h i s appears to be the only o b s e r v a t i o n concerning bryophytes i n which m e i o t i c a b n o r m a l i t i e s were induced by environmental c o n d i t i o n s . In an e a r l i e r study ( D i l l , unpublished d a t a ) , there was a suggestion that b r i n g i n g mosses indoors to r i p e n might cause abnormal m e i o s i s . Mnium venustum, M i t t . , when examined d i r e c t l y from the f i e l d , had 12 c l o s e l y packed b i v a l e n t s at metaphase I . 27 However, m a t e r i a l t h a t had been kept indoors i n a humid c o n t a i n e r f o r 2-3 days had metaphases i n which the chromosomes were l o o s e l y spread throughout the c e l l . In a d d i t i o n , many of the b i v a l e n t s d i s j o i n e d p r e c o c i o u s l y . A c o l l e c t i o n of Hetero- cladium h e t e r o p t e r o i d e s Best was a l s o brought i n t o the l a b o r a t o r y before the capsules had matured. A f t e r 2 weeks the WSMC under- went s e v e r e l y abnormal meiosis (PL. 7, f i g . 3 ) . No good t e t r a d s were observed. Since the capsules were never f l a c c i d i t seemed u n l i k e l y t h a t humidity was r e l a t e d to t h i s anomalous c o n d i t i o n . Studies 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 animals c l e a r l y demonstrate that heat s t r e s s can s e v e r e l y a f f e c t m e i o s i s . The bulk of t h i s work, both c y t o l o g i c a l and g e n e t i c a l , has d e a l t w i t h chiasma frequency at v a r i o u s temperatures (Yanney Wilson, 1959), but some has d e a l t with temperature e f f e c t s on chromosome c o i l i n g , s p i n d l e formation, centromere behaviour, and n u c l e o l i behaviour (Swanson, 1942, 1943; Dorwick, 1957; J a i n , 1957 et sea; and Henderson, 1962). In view of these f a c t s , an experiment seemed j u s t i f i e d to a s c e r t a i n whether or not room temperatures were s u f f i c i e n t l y h i g h to cause such anomalies. Three major questions arose concerning heat and i t s e f f e c t on meiosis i n mosses. F i r s t , i f the room temperature caused m e i o t i c a b n o r m a l i t i e s , how s e n s i t i v e are moss SMC to s l i g h t heat s t r e s s ? Second, i f the s e n s i t i v i t y i s g r e a t , might not n a t u r a l heat s t r e s s i n the environment be a mechanism i n d u c i n g mutations or structuralji'numerical a l t e r a t i o n s of the chromosomes? T h i r d , what i s the mode of response of the SMC to heat s t r e s s ? 28 S e v e r a l experiments were designed to answer some or a l l of these questions.. Although complete answers were not s u p p l i e d f o r a l l the questions, c e r t a i n trends and some d e f i n i t e c o n c l u s i o n s were obtained. 29 METHODS Methods of Treating the Plants: In the following experiments, several different types of heat treatment were applied. In some, special equipment was needed i n order to atta i n a reproducible experimental procedure. The f i r s t experiments involved treating the plants to short term heat stress ( i e . a period of about 4 hours). A longer period did not seem r e a l i s t i c since a longer period of abnormal heat stress would not be expected under natural conditions. Furthermore, p i l o t experiments showed that the shorter period was su f f i c i e n t to cause severe anomalies i n plants subjected to the higher ranges of temperature used (30-37°C). To ensure r e p l i - cation i t was necessary to control the rate of temperature change when heating as well as when cooling the plants, thus a chamber possessing a heating plus a cooling unit, both of which would give approximately similar rates of temperature change, was required. These requirements were p a r t i a l l y met by the use of the Warburg respirometer. The apparatus was used as a water bath and was f i t t e d with an aluminium cooling c o i l which was cooled with tap water. The top of the bath was insulated with blocks of styrifoam. The plants were put into covered 8" - 5" p l a s t i c cake dishes which were then p a r t i a l l y submerged (to within 1/2 " from the upper rim) with weights. A hole was d r i l l e d i n the cover of one of the dishes to insert a thermometer. The humidity of the dishes was not controlled but the plants were well watered and the inside 30 of the containers were kept damp so that moisture was never lacking. Since the humidity outdoors was close to or at 100$ during most of the study, the humidity of the p l a s t i c dishes probably approximated the control humidity quite closely. The heating gradient remained constant (l°C-C> / 5 min.). Cooling was not as eas i l y controlled since the volume of cold water passing through the cooling c o i l s had to be increased as the temperature approached room temperature. Furthermore the system could not cool fast enough at temperatures below that of the room. It was f e l t that t h i s was not c r i t i c a l since the error was introduced at temperatures which were not considered abnormal to the plants. At the beginning of each experiment the water bath was at 12-14 0 . It was f e l t that any temperature under 15°C. would not be deleterious to the plants. Plants i n p l a s t i c containers were placed into the water bath. The temperatures i n the containers and the water bath were allowed to equalize before the heating cycle was begun. In most of the experiments performed using t h i s apparatus, the plants were kept at the desired maximum temperature f o r 30 minutes. The water bath could be held at any desired temperature by using the thermo-regulator. D i f f i c u l t i e s were encountered i n keeping precise control of the temperature within the p l a s t i c containers. The temperature of the p l a s t i c containers tended to l a g behind that of the water bath at a constant rate u n t i l the desired maximum temperature was reached i n the water bath. At t h i s time the thermostat maintained the water bath temperature. The rate of increase i n temperature within the containers dropped considerably at t h i s time, so that t h e i r temperature never reached that of the water bath. For t h i s reason the maximum temperature w i t h i n the p l a s t i c c o n t a i n e r f l u c t u a t e d approximately 1°C. while the water bath remained at a constant temperature-. Thus, maximum temperature f o r a l l experiments are g i v e n with an e r r o r of 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 the 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 c o i l . 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 that the r a t e of temperature change was s i m i l a r f o r c o o l i n g and f o r h e a t i n g . When the bath was cooled to 1 4 ° C , the p l a n t s were removed and immediately taken back to the r o o f where they had been p r e v i o u s l y s t o r e d . Methods of A n a l y z i n g Treated P l a n t s : The methods f o r c y t o l o g i c a l study of these p l a n t s are d e s c r i b e d i n Chapter 1. S a m p l i n g — I n most of the experiments i n v o l v i n g the use of the Warburg apparatus some t r e a t e d sporangia were examined immediately a f t e r t h e i r removal at the end of the heat shock p e r i o d . An attempt was made to o b t a i n r e p r e s e n t a t i v e s of a l l stages of meiosis at each sampling time. T h i s was extremely d i f f i c u l t because only a l i m i t e d number of s l i d e s c ould be prepared at one time. Many of the sporophytes of any s e l e c t e d clump of p l a n t s were e i t h e r too young or too o l d f o r study, so that only about one-half the s l i d e s made at one time were s u i t a b l e f o r a n a l y s i s . I t was e s s e n t i a l to l e a v e enough young capsules f o r f u r t h e r study as w e l l . Because more than one experiment was being conducted at the same time, p r o g r e s s i v e sampling of each treatment was performed approximately every second day. However, day to day 32 sampling was performed when time was a v a i l a b l e . Each s l i d e was examined as soon as i t was made and any me i o t i c i r r e g u l a r i t i e s were noted. I f c e r t a i n stages of meiosis were not represented i n the group of s l i d e s made at one sampling time then f u r t h e r attempts were made to choose capsules which might be at the d e s i r e d stage. A n a l y s i s — L a t e r the 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. An attempt was made to procure some q u a n t i t a t i v e data on the treatments. T h i s was done by t a b u l a t i n g the a b n o r m a l i t i e s found i n c e l l s which had completed or had begun to complete meiosis a f t e r the treatment p e r i o d . T h i s t a b u l a t i o n was made concerning s l i d e s i n which the m a j o r i t y of the SMC were i n l a t e anaphase I I or the t e t r a d stage of d e v e l - opment. F i v e hundred c e l l s from each s a t i s f a c t o r y s l i d e were analysed and t a b u l a t e d as f o l l o w s , u s i n g a l a b o r a t o r y counter: (0) normal t e t r a d s w i t h f o u r c e l l s , a l l o f which have n u c l e i of comparable s i z e and which possess no m i c r o n u c l e i , e x t r a n u c l e a r chromosomes or fragments o f .chromosomes (PL. 3> f i g . 2). (1) t e t r a d s with f o u r c e l l s each with n u c l e i but po s s e s s i n g one e x t r a n u c l e a r chromosome or fragment. The s i z e of the fragment was not p a r t i c u l a r l y considered but some were o b v i o u s l y l a r g e r than any of the b i v a l e n t s of normal metaphase (PL. 5, f i g . 3). (2) same as ( l ) but po s s e s s i n g two e x t r a n u c l e a r chromosomes (PL. 5, f i g . 4). (3) same as ( l ) but pos s e s s i n g 3 or more e x t r a n u c l e a r chromosomes (PL. 5, f i g . 5). 33 (4) degenerate t e t r a d s of SMC showing aborted m e i o s i s . This i n c l u d e d t e t r a d s which had e i t h e r more than, or l e s s than, f o u r c e l l s w i t h n u c l e i . Almost a l l of these had e x t r a n u c l e a r chromosomes, (PL. 5, f i g . 6-8). C o n t r o l p l a n t s were examined and analysed from the same po p u l a t i o n used i n the experiments, and at the same time the experiments were conducted. Observations presented e a r l i e r (Chapter l ) concerning Hypnum c i r c i n a l e were d e r i v e d from the c o n t r o l s . The t e t r a d a n a l y s i s of the c o n t r o l s , however, i s i n c l u d e d with the r e s u l t s of the experiments. M a t e r i a l s : P i l o t experiments were performed i n the s p r i n g of 1963 w i t h p l a n t s of Hypnum subimponens Lesq. from populations c o l l e c t e d i n the 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 #5163). Experiments conducted i n the f a l l of 1963 were performed u s i n g the same popul a t i o n s of H. c i r c i n a l e t h a t were used i n the p r e p a r a t i o n s d e s c r i b e d i n Chapter 1. Since the m a t e r i a l was from w i l d p opulations i t cannot be ' s a i d t h at the sporangia from any one p o p u l a t i o n were g e n e t i c a l l y the same. C e r t a i n l y sporangia from d i f f e r e n t p o p u l a t i o n s would not be g e n e t i c a l l y i d e n t i c a l . However, s i n c e thedense p o p u l a t i o n s of gametophytes of H. c i r c i n a l e probably have been produced, to a l a r g e extent, by asexual means, i t i s suspected that the >;•- gametophyte members are q u i t e uniform g e n e t i c a l l y . In a d d i t i o n , as the sp-ecies i s monoe&ious and the gametophytes probably s e l f - f e r t i l e , the sporophytes of one clump, o r , s m a l l p o p u l a t i o n , a l s o 3 3 would be genetically quite uniform. 34 RESULTS The results of p i l o t experiments on Hypnum subimponens i n the spring of 1 9 6 3 indicated that the species was, during i t s meiotic cycle, extremely sensitive to short term heat stress (4 hour period with maximum temperature range at 3 2 , 3 6 , & 3 7 ° C ) . Many unusual meiotic aberrations were observed, including complete clumping of metaphase I chromosomes, precocious d i s - junction of a l l or some of the bivalents, abnormal tetrad form- ation and complete abortion of meiosis. However, i n s u f f i c i e n t material was available to make a satisfactory analysis. In the f a l l of 1 9 6 3 Hypnum c i r c i n a l e produced abundant sporophytes and was therefore selected as the material with which to continue the work. Unfortunately, due to the low y i e l d of pertinent s l i d e s during sampling even t h i s material was i n s u f f i c i e n t to indicate more than trends ; i n abnormal behaviour. Experiment # 1 : Material—Hypnum c i r c i n a l e , population #1363 . Treatment—The plants were maintained i n the water bath for a period of 4 1 / 2 hours while the temperature was raised to 3 6 . 5 - 0 . 5 ° C and held at that temperature f o r 30 minutes; then returned to 14°Q, Following the heat shock period the plants were stored outside ( 7 - l l ° C ) . Observations—The f i r s t 20 slides were prepared 2 1 / 2 hours after completion of the heat shock period. Each s l i d e contained a l l the SMC of a sporangium. The majority of the sporangia had completed meiosis and only young spores were seen. These were 35 d i s c a r d e d . The f i v e usable s l i d e s f e l l i n t o the f o l l o w i n g c a t e g o r i e s : one had o l d tetrads}, two were mostly t e t r a d s ; one had l e p t o t e n e , zygotene and pachytene; and one had l e p t o t e n e and zygotene. A few metaphase I c e l l s were observed i n the s l i d e s with t e t r a d s and a l l of these were s e v e r e l y clumped ( s i m i l a r to those shown i n PL.7, f i g . 7 ) . Some of the t e t r a d s contained e x t r a n u c l e a r chromatin, i n d i c a t i n g t h a t the l a s t p a r t of meiosis was completed d u r i n g the heat shock p e r i o d . Only two of the s l i d e s with 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 ; these are sporangia 1 and 3 i n Table I . Table I : Tetrad a n a l y s i s of sporangia of experiment #1. Sporan- Time of samp- gium No. l i n g a f t e r the end of the heat shock p e r i o d . 1 2.5 h r s . 88.4 8.4 1.6 0.6 1.0 s l i d e had mostly t e t r a d s 3 2.5 h r s . 91.0 6.0 1.4 0.4 1.2 ti 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 next 12 s l i d e s were made 26 hours a f t e r t e r m i n a t i o n of 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 a t e g o r i e s : f o u r contained young spores!;, two had o l d t e t r a d s with no a c t i v e stages ; two had t e t r a d s with some a c t i v e stages; two had mostly the term " a c t i v e stages" i s used to. denote a l l the stages from d i a k i n e s i s t o telophase I I . fo c e l l s of each a n a l y s i s Comments category of 500 examined c e l l s per sporangium. 0 1 2 3 4 36 Table I I : Tetrad a n a l y s i s of c o n t r o l p l a n t s of p o p u l a t i o n #1363 which were used i n .experiments 1 aand 2. Sporan- $ c e l l s o f each a n a l y s i s category Comments gium No. of 500 examined c e l l s per sporangium. 0 1 2 3 4 1 94.0 4.0 0.0 1.0 1.0 mostly t e t r a d s 5 93.6 5.0 0.0 0.8 0.6 it 7 89.0 5.6 4.6 0.4 0.4 i» 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 + 0.5 + d i c t y o t e n e stages (one of these s l i d e s had some metaphase I c e l l s ) ; and two had l e p t o t e n e and zygotene. The a c t i v e stages " i n a l l of these s l i d e s were abnormal. Some of the metaphase I were clumped, but i n those that were not clumped, over 70$ had b i v a l e n t s that.were not o r i e n t e d on a proper s p i n d l e (PL. 4, f i g . 10). Anaphase I and I I contained many laggards and b r i d g e s . Again prophase I stages could not be d i s t i n g u i s h e d from prophase I stages observed i n the c o n t r o l s . One s l i d e (#6) 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 i n c l u d e d i n Table I (Page 35). Seven s l i d e s were made 48 hours a f t e r the t e r m i n a t i o n of the heat shock p e r i o d . They f e l l i n t o the f o l l o w i n g c a t e g o r i e s : two had young spores; two had predominantly a c t i v e stages; one had p r i m a r i l y m i g r a t i n g pachytene; and two had l e p t o t e n e , zygotene and some pachytene. In one of the s l i d e s c o n t a i n i n g young spores, a l l the spores were s p h e r i c a l and appeared normal. However i n 37 a second s l i d e there were some (approximately 10$) l a r g e c e l l s t h a t were h y a l i n e , i n t h i s r e s p e c t resembling young spores. These c e l l s contained p y c n o t i c chromosomes i n v a r i o u s stages of meiosis; such c e l l s may be termed r e s t i t u t i o n c e l l s . I t i s apparent t h a t these c e l l s were SMC i n which meiosis had aborted. A l l a c t i v e stages were abnormal (PL. 4, f i g . 11, 12; PL. 5, f i g . l ) . Metaphase I stages were sometimes p a r t i a l l y , but never completely clumped. Unclumped metaphaseswwere not organized on a proper s p i n d l e and u n i v a l e n t s and fragments were s c a t t e r e d around the 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, of the c e l l s i t was impossible t o determine what stage of meiosis the chromosomes were i n . Telophase I n u c l e i were clumped i n t o g l o b u l a r masses. Some of these c e l l s , although abnormal, had completed anaphase I without l e a v i n g laggards and fragments ou t s i d e the telophase I n u c l e i , (PL. 5, f i g . 2). No 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 f t e r f o u r days f i v e a d d i t i o n a l s l i d e s were made. By t h i s time only a few s u i t a b l e capsules remained i n the t r e a t e d l o t of m a t e r i a l . These s l i d e s f e l l i n t o the f o l l o w i n g c a t e - g o r i e s : two contained mostly r e s t i t u t i o n SMC and some broken t e t r a d s ; one possessed only t e t r a d s ; one had mostly d i c t y o t e n e , but had some d i p l o t e n e , m i g r a t i n g pachytene and metaphase I c e l l s ; and one had a l l d i p l o t e n e and pachytene. About IQfo of the r e s t i t u t i o n SMC were i n d i c t y o t e n e and d i p l o t e n e stages (PL. 5, f i g . 9), the remainder were i n a c t i v e stages that appeared abnormal. 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 i n c l u d e d i n Table I (see page 35). A l l metaphase I stages were d i s r u p t e d and appeared unoriented 38 as the chromosomes were spread a l l over the c e l l . Generally- many -univalents were present. Two observations can be made at t h i s p o i n t . F i r s t , the synchrony of the prophase stages appeared t o be completely d i s r u p t e d ; never were pachytene, d i p l o t e n e , d i c t y o t e n e and metaphase I stages observed i n one pl a n t i n the c o n t r o l m a t e r i a l . Second, the f i r s t d i p l o t e n e phases were seen at the f o u r t h day of sampling. Although t h i s may be due to sampling e r r o r i t suggests that the d u r a t i o n of d i p l o t e n e was p o s s i b l y shortened immediately f o l l o w i n g the heat shock p e r i o d . Experiment #2: Material—Hypnum c i r c i n a l e , p o p u l a t i o n #1363 Treatment—The p l a n t s were maintained i n the water bath f o r a p e r i o d of 3 1/2 hours while the temperature was r a i s e d to 30.0- 0.5° C., h e l d at t h a t temperature f o r 30 minutes and then r e t u r n e d 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 o u t s i d e (7-ll°C.). O b s e r v a t i o n s — T h e f i r s t 8 s l i d e s were prepared immediately a f t e r the end of the heat shock p e r i o d and 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 a c t i v e stages and d i c t y o t e n e stages; two had mostly d i c t y o t e n e with some d i p l o t e n e ; two had m i g r a t i n g pachytene 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 zygotene. Many metaphase I stages were observed i n t h i s m a t e r i a l . About 20$ were clumped (PL. 5, f i g . 10) while the remainder v a r i e d from p a r t i a l l y clumped (PL. 5, f i g . 11,12) to those 39 arranged on an apparently normal s p i n d l e (PL. 6, f i g . 1-3)• Rare metaphase I stages were observed w i t h unoriented and d i v - i d e d b i v a l e n t s (PL. 6£ f i g . 4 ) . The chromosomes of many of the metaphase I c e l l s with o r i e n t e d b i v a l e n t s appeared more h i g h l y condensed and more g l o b u l a r than those of the c o n t r o l s . However, i n many of the c e l l s the chromosomes were i n d i s t i n g u i s h a b l e from those i n the c o n t r o l s . U n f o r t u n a t e l y 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 sporangium 4 i n Table I I I . Table I I I : Tetrad a n a l y s i s of sporangia of experiment #2. Sporan- Time of samp- gium No. l i n g a f t e r the end of the heat shock p e r i o d . 4 0 h r s . 85.6 6.8 0.6 0.6 6.4 mostly t e t r a d s 13 72 h r s . 57.8 22.0 6.6 5.6 8.0 II Of seven s l i d e s made 24 hours a f t e r the t e r m i n a t i o n o f the heat shock p e r i o d , f o u r were d i s c a r d e d because the p l a n t s were much too young f o r m e i o t i c stages. The remainder 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 were i n pachytene, and d i p l o t e n e ; and one had young spores. The prophase I stages and the young spores appeared 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 . Three s l i d e s were made 72 hours a f t e r t e r m i n a t i o n of 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 % c e l l s o f each a n a l y s i s Comments category of 500 examined c e l l s per sporangium. 0 1 2 3 4 40 had old tetrads with about 1$ active stages and two were i n leptotene and pachytene. The s l i d e with tetrads was analysed and i s sporangium 13 i n Table III (page 39). The tetrads contained many abnormalities (PL. 6, f i g . 7-9). The active stages, which were a l l anaphases, contained laggards or exhibited abnormal segregation. Prophase I stages appeared normal. Experiment #3: Material—Hypnum c i r c i n a l e , population #2267 Treatment—The plants were maintained i n the water bath f o r 3 3/4 hours, while the temperature was raised to 31.5 - 0.5° C , l e f t at that temperature f o r 30 minutes, and then lowered to 14° C. Following the heat shock period the plants were stored outside (7-ll°C.) Observations—Five s l i d e s were prepared immediately aft e r the heat shock period. These f e l l into the following classes: one had mostly tetrads with some active stages; one had dictyotene and diplotene; and three had e a r l i e r stages of prophase I. A l l metaphase I stages observed had some degree of clumping, although i t was not as severe as that i n experiment #1. A l l observed anaphases had laggards and prophase I c e l l s appeared normal. One sl i d e was suitable for tetrad analysis and i s sporangium 3 i n T a b l e IV (page 41). Eight slides were made f i v e hours after the end of the heat shock period, and f e l l into the following classes: one had tetrads; one had dictyotene; one had t r a n s i t i o n a l stages between 41 Table IV: Tetrad analysis of sporangia from experiment #3. Sporan- Time of samp- $ c e l l s of each analysis Comments gium No. l i n g after the category of 500 examined end of the heat c e l l s per sporangium. shock period. 0 1 2 3 4 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 hrs. 87.0 3.0 0.0 0.0 10.0 young tetrads many active stages 18 20 hrs. 63-0 7.8 1.2 0.2 27.2 old tetrads 19 20 hrs. 45.6 13.0 3.2 . 1.2 27.0 mostly tetrads 21 5 days 56.6 14.2 8.4 9.0 16.0 tetrads with many active stages 22 5 days 54.8 17.8 9.8 8.4 9.2 mostly tetrads 26 5 days 68.0 12.0 2.2 0.8 17.0 old tetrads 31 7 days 77.0 13.6 1.4 0.8 7.2 active stages with young tetrads 32 7 days 65.2 1.8.-6:: L 5.8 2.4 8.0 .old tetrads diplotene and d±$fgrotene ; four had leptotene to pachytene; and one had diplotene and migrating pachytene. No active 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 within each sporangium was l o s t . In contrast to the findings i n experiment 1 and 2, some diplotene figures were observed. One 4 2 s l i d e w a s 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 i s s p o r a n g i u m 7 i n T a b l e I V ( p a g e 4 1 ) . 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 # 2 2 6 3 w h i c h w e r e u s e d i n e x p e r i m e n t # 3 . S p o r a n - % 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 C o m m e n t s g l u m N o . o f 5 0 0 c e l l s p e r s p o r a n g i u m 0 1 2 3 4 4 9 5 . 6 3 . 6 1 . 0 0 . 0 0 . 4 / o l d t e t r a d s ; s t a i n i n g w o u l d 8 9 9 . 2 0 . 8 J n o t a l l o w ] . a c c u r a t e o b s e r - 1 0 9 4 . 0 4 . 4 0 . 8 0 . 2 0 . 6 v a t i o n s o f V m i c r o n u c l e i S i x s l i d e s w e r e m a d e 2 0 h o u r s a f t e r t h e e n d o f t h e h e a t s h o c k p e r i o d . T h e y f e l l i n t o t h e f o l l o w i n g c l a s s e s : t w o h a d o l d t e t r a d s w i t h s o m e a c t i v e s t a g e s ; o n e h a d a c t i v e s t a g e s , y o u n g t e t r a d s ; o n e h a d m o s t l y d i c t y o t e n e w i t h d i a k i n e s i s a n d r a r e m e t a p h a s e I s t a g e s ; a n d t w o h a d m o s t l y d i c t y o t e n e w i t h e a r l y p r o p h a s e I s t a g e s . M o s t o f t h e a c t i v e s t a g e s i n t h e m o s t a c t i v e s p o r a n g i u m a p p e a r e d n o r m a l ; t w e l v e o f o n e h u n d r e d m e t a p h a s e s c o u n t e d a p p e a r e d t o h a v e s o m e w h a t a b n o r m a l o r i e n t a t i o n o n t h e s p i n d l e . T h e f r e q u e n c y o f l a g g a r d s d u r i n g a n a p h a s e s w a s q u i t e l o w . H o w - e v e r , i n t h e s p o r a n g i a w i t h o l d e r t e t r a d s a l l a n a p h a s e s o b s e r v e d w e r e a b n o r m a l . T h i s d i s c r e p a n c y m a y b e e x p l a i n e d b y t h e f a c t t h a t t h e l a t t e r s p o r a n g i a w e r e a t t h e a c t i v e s t a g e s d u r i n g t h e h e a t s h o c k w h i l e t h e f o r m e r o n e s w e r e i n l a t e p r o p h a s e . T h i s s u p e r - f i c i a l l y s u g g e s t s t h a t t h e h e a t s h o c k a f f e c t e d l e s s s e v e r e l y 43 the l a t e stages of prophase I than i t d i d the metaphase I and l a t e r stages. The prophase stages again showed l a c k o f synchrony but otherwise appeared normal. Three sporangia were s a t i s f a c t o r y f o r t e t r a d a n a l y s i s and are sporangia 17, 18, and 19 i n T a b l e IV (page 41). F i v e days a f t e r the end of the heat shock p e r i o d 9 s l i d e s were prepared. They 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 o l d t e t r a d s and young spores; one had o l d t e t r a d s ; two had t e t r a d s and a c t i v e stages (one of these had only r a r e a c t i v e s t a g e s ) ; one had d i c t y o t e n e and many a c t i v e stages; and f o u r had v a r i o u s unsynchronized prophase I stages. In the sporangia w i t h t e t r a d s and some a c t i v e stages, most of the a c t i v e stages were abnormal. Very few metaphases were observed and most of these were o r i e n t e d on a s p i n d l e , but o f t e n the chromosomes were p a r t i a l l y clumped o r there were d i s j o i n e d b i v a l e n t s . The b i v a l e n t s appeared h i g h l y condensed. Anaphases had many laggards and b r i d g e s , or appeared unoriented (PL. 6, f i g . 10). In the sporangium w i t h predominantly d i c t y o t e n e and a c t i v e stages, t h e r e were many metaphase I stages. These, f o r the most part (71 of 100 c e l l s counted) had normal arrangement on the s p i n d l e . In the metaphase c e l l s w i t h abnormal b i v a l e n t arrangement, the b i v a l e n t s were u s u a l l y h i g h l y condensed. Three sporangia 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 are sporangia 21, 22, and 26 i n T a b l e IV (page 41). Seven days f o l l o w i n g the heat shock 6 more s l i d e s were prepared and 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 spores; one had o l d t e t r a d s ; one had a c t i v e stages and young t e t r a d s ; 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 with r a r e pachytene stages; and one was i n l e p t o t e n e . Again the metaphase I stages ranged from what appeared t o he normal t o those that were abnormal. F i f t y - o n e of the metaphase I stages counted had apparently normal o r i e n t a t i o n on the s p i n d l e . A l s o there were many cases where i t was d i f f i c u l t to e s t a b l i s h whether the o r i e n t a t i o n was normal or abnormal. The chromosomes o f t e n were condensed s l i g h t l y more than those i n the c o n t r o l s (PL. 6, f i g . 11), but there were a l s o those that could not be d i s t i n g u i s h e d from those of the c o n t r o l s (PL. 7, f i g . l ) . Of the stages observed, anaphases were the most abnormal. The chromosomes were not o r i e n t e d and there were many la g g a r d s . A l s o , n o n d i s j u n c t i o n of one or more chromosomes was most commonly observed (PL. 7, f i g . 2). 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 are sporangia 31 and 32 i n Table IV (page 41). Experiment #4: Material—Hypnum c i r c i n a l e . p o p u l a t i o n #2463 Treatment—The p l a n t s were maintained i n the water bath f o r a p e r i o d o f 6 1/2 hours d u r i n g which the temperature was tfaaiseds + o to 25.0 - 0.5 C , kept at th a t temperature f o r 4 hours and lowered t o 14°C. The p l a n t s were placed o u t s i d e f o l l o w i n g the treatment ( 7 - l l ° C ) . O b s e r v a t i o n s — N o s l i d e s were prepared immediately a f t e r the treatment. A f t e r 24 hours 6 s l i d e s were made and 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 a c t i v e stages and young t e t r a d s ; two had d i c t y o t e n e and a c t i v e stages; and three had 45 mostly 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 stages between d i p l o t e n e and d i c t y o t e n e , and a l s o r a r e pachytene. The a c t i v e stages observed d u r i n g t h i s sampling appeared predominantly normal. There was an o c c a s i o n a l metaphase i n which the b i v a l e n t s appeared u n o r i e n t e d and were over-condensed, but i n g e n e r a l behaviour was not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l . Prophase I stages e x h i b i t e d some asynchrony and d i p l o t e n e stages were r a r e . One pl 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 i s sporangium 6 i n Table YI (page 46). T h i r t e e n s l i d e s were prepared 72 days a f t e r the end of the treatment and 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 : f o u r had ol d t e t r a d s w i t h few or no a c t i v e stages; one had mostly t e t r a d s w i t h some a c t i v e stages; two had mostly a c t i v e stages; two had mostly 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 stage; two had d i p l o t e n e and pachytene; and one had l e p t o t e n e , zygotene and r a r e pachytene stages. Again the a c t i v e stages appeared predominantly normal. Only a few (probably l e s s than 5f°) of the metaphases appeared s l i g h t l y u n o r i e n t e d w i t h highly-condensed b i v a l e n t s . The prophase stage e x h i b i t e d some asynchrony s i n c e stages from d i p l o t e n e to metaphase I were observed 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 are sporangia 17, 12, 14 and 19 i n Table VI (page 46). F i v e days a f t e r the end of the heat shock p e r i o d 11 more s l i d e s were prepared and 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 : e i g h t had spores; one had a c t i v e stages and young t e t r a d s ; one 46 Table VI: Tetrad analysis of sporangia of experiment #4, Sporan- Time of samp- fo c e l l s of each analysis Comments glum No. l i n g after 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 active stages and young tetrads 17 72 hrs. 90.2 3.4 0.2 0.4 5.8 old tetrads 12 72 hrs. 73.8 6.6 1.2 1.0 17.4 mostly tetrads 14 72 hrs. 87.4 7.2 1.8 0.2 3.4 " 19 72 hrs. 84.2 5.8 1.8 0.2 8.0 " 21 5 days 80.6 10.4 1.6 0.6 6.8 active stages and young tetrads Table VII: Tetrad analysis of sporangia from population #2463 which were used i n experiments 4-'7. Sporan- $ c e l l s i n each analysis category Comments gium No. of 500 examined c e l l s per sporangium 0 1 2 3 4 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+ 4 7 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 stages; and one had mostly d i c t y o t e n e . The spores observed appeared normal, as d i d the a c t i v e stages, with the exception of a few questionable metaphase I stages. Prophase I stages appeared s l i g h t l y asynchronous. 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 2 1 i n Table VI (page 4 6 ) . Experiment #5: Material—Hypnum c i r c i n a l e , p o p u l a t i o n # 2 4 6 3 T r e a t m e n t — I n an attempt to determine the immediate e f f e c t s of r a i s i n g the temperature to a l e v e l t h a t would induce severe anomalies, the p l a n t s were put i n t o the water bath and the temperature was r a i s e d i n the normal manner to 3 7 - 0 . 2 ° C . The m a t e r i a l was removed at that temperature and s l i d e s were made as soon as p o s s i b l e . O b s e r v a t i o n s — O f the twenty s l i d e s made immediately a f t e r the treatment p e r i o d 9 were d i s c a r d e d because the sporangia were too o l d . The remaining 1 1 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 t e t r a d s ; f o u r had mostly t e t r a d s and some a c t i v e stages; two had mostly d i c t y o t e n e and some metaphase I stages; three had d i p l o t e n e and l a t e pachytene stages; and one had l e p t o t e n e . A l l metaphase I stages observed were s e v e r e l y clumped; the b i v a l e n t s had fused i n t o ' a g l o b u l a r spheres o f chromatin (PL. 7 , f i g . 7 ) . Only a few of the anaphases and telophase stages had laggards, i n d i c a t i n g perhaps t h a t these stages were completed 48 before the temperature was c r i t i c a l l y h i g h. Many of the d i c t y - otene stages had been a f f e c t e d by the heat shock, f o r o f t e n the chromatin appeared reduced i n area. Chromosomes were app a r e n t l y p a r t i a l l y condensed around the chromocentre. The e a r l i e r prophase stages appeared normal (PL. 7, f i g . 5,6). There were o c c a s i o n a l i n s t a n c e s of abnormal p a i r i n g (PL. 7, f i g . 4) but as t h i s anomaly was r a r e i t could not be determined whether i t was caused by the heat shock or whether i t was due to n a t u r a l abnormality. 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 n a l y s i s and are sporangia 4, 10, 2, 7, and 11 i n Table V I I I (page 49). E l e v e n s l i d e s were made 4 hours a f t e r the end of the treatment and 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 mostly o l d t e t r a d s w i t h a few a c t i v e stages; two had many a c t i v e and some t e t r a d s ; one had d i c t y o t e n e and metaphase I stages; and s i x had e a r l y prophase stages i n which d i p l o t e n e was r a r e . Again the metaphase I stages were s e v e r e l y clumped. In c e l l s at telophase I , the chromosomes of the two n u c l e i were clupped i n t o g l o b u l a r spheres. Anaphase stages were not observed, i n d i c a t i n g t h a t a c t i v e movement of chromosomes had been t e m p o r a r i l y a r r e s t e d . The d i c t y o t e n e stages had more heterochromatic areas than those of c o n t r o l s and the chromatin threads appeared t h i c k e r and stuck together i n c e r t a i n areas. In a d d i t i o n , the s i z e o f the chromatin mass at d i c t y o t e n e had been reduced. E a r l y prophase I stages appeared normal. Four 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 are sporangia 12, 17, 20 and 22 i n Table V I I I (page 49). Twelve s l i d e s were made 72 hours f o l l o w i n g the end of the 49 Table V I I I : Tetrad analysis of sporangia of experiment #5 Sporan- Time of samp- $ c e l l s i n each analysis Comments gium No. 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 -. « ~ . 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 .3.8 active sta-ges and young tetrads 7 0 hrs. 73.8 15.0 3.6 2.8 4.8 ti 11 0 hrs. 67.0 14.0 4.6 5.0 9.4 it 12 441 hrg. 90...8 5.6 0.8 0.0 2.8 old tetrads 17 41 ihrs . 84.6 8.4 1.2 1.2 4.6 mostly t e t -rads 20 4 hrs. 65.0 14.0 4.0 4.0 13.0 active stages and tetrads 22 4 hrs. 70.6 14.4 7.0 2.0 6.0 active stages and young tetrads 25 72 hrs. 80.0 6.8 1.6 0.0 11.6 active stages and old tetO- rads 39 5 days 71.8 12.8 3.8 3.0 7.6 old tetrads 42 5 days 46.4 13.8 1.6 1.4 36.8 active stages and tetrads treatment. These f e l l into the following classes: two had young spores; one had active stages and tetrads, one had mostly active stages; two had mostly dictyotene; f i v e had unsynchronized 5 0 e a r l y prophase stages i n c l u d i n g d i c t y o t e n e ; and one had l e p t o t e n e and zygotene. In the s l i d e s with young spores, r e s t i t u t i o n SMC were common, i n d i c a t i n g t h a t meiosis had been completely a r r e s t e d i n some cases. No clumping was observed at metaphase I , but g e n e r a l l y s p i n d l e formation was abnormal. F i f t y - t w o of 200 metaphases, examined appeared to be arranged on a s p i n d l e (PL. 7, f i g . 8; PL. 8, f i g . 12), but i n a l l cases the chromosomes were h i g h l y condensed and d i s j u n c t i o n was.not synchronous. Ana- phases had many laggards but 88 of IQO1 examined telophase I stages e x h i b i t e d no a b n o r m a l i t i e s . There are at l e a s t two p o s s i b l e explanations f o r t h i s d i s c r e p a n c y . One i s t h a t the laggards at anaphase were i n c l u d e d i n the telophase n u c l e i d e s p i t e t h e i r apparent l a c k of movement; the second i s that meiosis had been a r r e s t e d soon a f t e r the treatment and that these c e l l s would e v e n t u a l l y become r e s t i t u t i o n SMC . N e i t h e r e x p l a n a t i o n i s e n t i r e l y s a t i s f a c t o r y . The f i r s t e x p l a n a t i o n i s c o n t r a r y to the f i n d i n g s elsewhere i n these s t u d i e s and the second i s p u z z l i n g because i f meiosis had been a r r e s t e d f o r three days the SMC had not gone i n t o r e s t i t u t i o n , while r e s t i t u t i o n SMC were observed i n p l a n t s with young spores. 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 sporangium 25 i n T a b l e V I I I (page 49). E i g h t s l i d e s were made 5 days a f t e r the treatment and 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 young spores; one had o l d t e t r a d s ; one had t e t r a d s and 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 v a r i o u s e a r l y prophase I stages. The s l i d e s w i t h young spores had many r e s t i t u t i o n SMC and 51 abnormally shaped spores (PL. 8, f i g . 3-5)• Often spores were s p h e r i c a l but were j o i n e d together by threads of c e l l w a l l m a t e r i a l (PL. 8 , f i g . 4). In many cases the spores and r e s t i t u t i o n SMC were clumped i n t o l a r g e aggregated (PL. 8, f i g . 5). The a c t i v e stages were markedly abnormal and probably had been a r r e s t e d . Prophase stages appeared normal. 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 are sporangia 39 and 42 i n Table V I I I (page 49). Experiment #6: Material—Hypnum c i r c i n a l e , p o p u l a t i o n #2463 T r e a t m e n t — I n t h i s experiment the p l a n t s were brought i n t o the l a b o r a t o r y and put i n t o covered p l a s t i c d i s h e s . The l a b o r a t o r y temperature was 22- 2°C. (normal indoor c o n d i t i o n s ) . The purpose of the treatment was to determine the behaviour o f m e i o t i c chromosomes under l a b o r a t o r y c o n d i t i o n s . O b s e r v a t i o n s — T h e f i r s t s l i d e s , made 24 hours a f t e r the p l a n t s had been brought indoors, were markedly abnormal. In 50- 60$ of the metaphase I stages there was no s p i n d l e o r i e n t a t i o n , and i n almost a l l the c e l l s the metaphase I b i v a l e n t s were h i g h l y condensed. Moreover, there were many c e l l s w i t h p a r t i a l l y clum- ped b i v a l e n t s as w e l l as those 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 - v a l e n t s . There were a l s o those c o n t a i n i n g a mixture of the v a r - ious a b n o r m a l i t i e s . Anaphases had many laggards and appeared d i s - organized. Prophase stages were apparently normal. No s a t i s - f a c t o r y d i p l o t e n e was observed; only t r a n s i t i o n a l stages between d i p l o t e n e and d i c t y o t e n e were present. Two s l i d e s were s u i t a b l e 52 for tetrad analysis and are sporangia 1 and 2 i n Table IX, Table IX: Tetrad analysis of sporangia of experiment 6. Sporan- Time of samp- fo c e l l s of each analysis Comments gium No. ^JJnts were category of 500 examined brought indoors, c e l l s per sporangium. 0 1 2 3 4 1 24 nrs. 14.8 5.2 1.2 0.0 78.8 mostly tetrads 2 24 hrs.. 27.2 2.0 0.4 0.2 70.2 " 6 30 hrs. 18.0 10.0 0.0 0.0 72.0 active stages and tetrads 10 j.30 hrs. 39.6 9.2 2.2 0.4 48.6 old tetrads 12* 72 hrs. 1.2 0.0 0.0 0.0 98.8 active stages and 17* 72 hrs. 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 active stages were observed and i n most of the SMC meiosis had aborted before second div- i s i o n began. Thirty hours after plants were brought indoors 6 more slides were prepared. These f e l l into the following classes: two had old tetrads; one had active stages and tetrads; one had dictyotene and active stages; and two had pachytene stages. Again the active 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 with some metaphase orientation had highly condensed bivalents, one or more of which usually displayed precocious disjunction. A l l anaphases had laggards and i n many cases the 53 chromosomes were spread a l l through the c e l l . The pachytene stages appeared normal. 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 are sporangia 6 and 10 i n T a b l e IX (page 52). Seventy-two hours a f t e r the p l a n t s were brought i n t o the l a b o r a t o r y 9 s l i d e s were prepared and 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 young spores and o l d t e t r a d s ; f o u r had a c t i v e stages and t e t r a d s ; one had a c t i v e stages; two had d i c t y o t e n e and a c t i v e stages (there were a l s o some e a r l y prophase I stages i n one slide)'; and one had d i c t y o t e n e . I t was noted t h a t the p l a n t s were maturing extremely r a p i d l y by t h i s time. Many of the capsules t h a t were not f u l l s i z e had begun meiosis prematurely. The sporangium w i t h young spores had about 80$ r e s t i t u t i o n . SMC and many degenerate t e t r a d s . The a c t i v e stages of meiosis were completely aborted (PL. 9, f i g . l ) and i t appeared that i n many cases the complete t e t r a d stage would never be r e v e a l e d . Prophase I stages were asynchronous 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 condensed around the chromocentre ( s i m i l a r to t h a t i n PL. 9, f i g . 4) and many c e l l s were observed i n which the chromosomes were clumped i n t o a t i g h t l y packed b a l l ( s i m i l a r to that i n P l l 9» f i g . 5). T h i s stage c o u l d not be a c c u r a t e l y i n t e r p r e t e d , but was p o s s i b l y an aborted pachytene or d i p l o t e n e . Three 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 are sporangia 12, 17 and 18 i n Table IX (page 52). These t e t r a d stages very seldom had f o u r c e l l s s i n c e meiosis had aborted at or before second d i v i s i o n (PL. 9, f i g . 2, 3) . In a d d i t i o n , i t was o f t e n noted that i n d i v i d u a l chromosomes had formed m i c r o n u c l e i . Observations were made again at 4 and 6 days a f t e r the 54 p l a n t s were brought i n t o the l a b o r a t o r y . M e i o s i s had completely aborted; by 4 days many of the SMC had undergone r e s t i t u t i o n . Those that had not undergone r e s t i t u t i o n possessed e i t h e r aborted a c t i v e stages or prophase stages which were c l o s e l y clumped (PL. 9 , f i g . 5)t as p r e v i o u s l y mentioned. At 6 days the capsules w i t h any s i g n of m e i o t i c a c t i v i t y were extremely s m a l l (about l / 2 f u l l s i z e ) and these had only developed a few a r c h e s p o r i a l c e l l s . M e i o s i s had aborted. Experiment #7: Material—Hypnum c i r c i n a l e , p o p u l a t i o n #2463 Treatment—The p l a n t s were brought i n t o the l a b o r a t o r y and put i n t o open t r a y s . One of the t r a y s was put i n t o a cupboard d u r i n g the evenings and at n i g h t i n order to simulate the normal outdoor photoperiod. The other was l e f t out on a bench. The purpose of t h i s experiment was twofold: (a) to determine whether the humidity i n the p l a s t i c dishes was c o n t r i b u t i n g to or coun t e r b a l a n c i n g the e f f e c t of the indoor temperature, (b) to demonstrate whether a photoperiod response was c o n t r i b u t i n g to 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 from both t r a y s at c e r t a i n times a f t e r the p l a n t s were brought indoors, much as i n experiment 6. The a b n o r m a l i t i e s observed were s i m i l a r to those d e s c r i b e d i n experiment 6, and meiosis had aborted 72 hours a f t e r the p l a n t s had been brought i n d o o r s . Tetrad a n a l y s i s of sporangia from both t r a y s are i n c l u d e d i n Tables X and XI. The analyses i n d i c a t e d t h a t the s e v e r i t y 55 of the anomalies was comparable to those.observed i n experiment 6. Table X: T e t r a d a n a l y s i s of sporangia of experiment 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 . Sporan- Time of samples $ c e l l s of each a n a l y s i s Comments gium No. a f t e r the p l a n t s category of 500 examined were brought c e l l s per sporangium 7 36 h r s . 71.2 5.0 0.0 0.0 23.8 o l d t e t r a d s 12 36 h r s . 68.8 4.2 0.0 0.0 27.0 11 9 36 h r s . 56.0 8.6 1.2 0.0 34.4 mostly t e t r a d s 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 a r r e s t e d a c t i v e stages and t e t r a d s 56 Table XI: Te t r a d a n a l y s i s of sporangia o f experiment 7, p l a n t s under normal photoperiod. Sporan- Time of samp- $ c e l l s i n each a n a l y s i s Comments gium No. l i n g a f t e r ' category of 500 examined c p l a n t s were c e l l s per 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 ve r y o l d t e t r a d s 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 o l d t e t r a d s 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 a c t i v e stages and t e t r a d s 22 72 h r s . 3.0 0.0 0.0 0.0 97.0 mostly t e t r a d s 23 72 h r s . 0.8 0.2 0.0 • 0.0 99.0 a c t i v e stages and t e t r a d s 57 DISCUSSION I t i s r e a l i z e d t h a t the r e s u l t s of these experiments suggest l i t t l e more than trends of abnormal behaviour and that much more meaningful i n f o r m a t i o n could have been d e r i v e d i f the sample s i z e s had been l a r g e r . T h i s was e s p e c i a l l y evident i n those experiments i n which the heat shock was r e l a t i v e l y m i l d and the p o s s i b i l i t y of recovery a f t e r such shock was super- f i c i a l l y suggested from the t e t r a d a n a l y s i s (see experiment 3, page 40). In a d d i t i o n i t i s obvious that the experiments could be r e f i n e d by u s i n g c o n t r o l l e d temperature, humidity, and photo- p e r i o d rooms which would g i v e more accurate and probably more meaningful r e s u l t s . Although the i n t e r p r e t a t i o n of the experiments possess these l i m i t a t i o n s , some i n f o r m a t i v e c o n c l u s i o n s can be drawn. I t was s t a t e d e a r l i e r that the purpose of conducting the heat shock experiments was f o u r f o l d : (1) to determine whether e r r o r s i n i n t e r p r e t a t i o n of chromosomal behaviour would be i n t r o d u c e d i f mosses were brought indoors to mature the sporangia, (2) to determine the s e n s i t i v i t y of moss SMC to s l i g h t heat s t r e s s , (3) to i n d i c a t e the l i k e l i h o o d that n a t u r a l heat s t r e s s i n the environment might be a mechanism of i n d u c i n g mutations or s t r u c t u r a l / n u m e r i c a l a l t e r a t i o n s of the chromosomes, and ( 4 ) to determine the mode of response of the SMC to heat s t r e s s These f o u r c a t e g o r i e s are d i s c u s s e d under separate headings. 58 ( l ) Chromosomal Behaviour of Mosses under Laboratory C o n d i t i o n s . Experiment 6 (page 51) and 7 (page 54) were p r i m a r i l y undertaken to i n v e s t i g a t e the e f f e c t s om chromosomal behaviour of mosses under l a b o r a t o r y c o n d i t i o n s . • The r e s u l t s c l e a r l y i n d i c a t e t hat severe a b n o r m a l i t i e s may be introduced by b r i n g i n g mosses indoors to r i p e n . •Furthermore, p l a n t s kept i n the very humid c o n t a i n e r s had 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 open and r e l a t i v e l y a r i d t r a y . Although the. sporangia of those i n the open t r a y d i d not become f l a c c i d , they were i n much d r i e r c o n d i t i o n s than those i n the p l a s t i c d i s h e s . In f a c t , a comparison of the a n a l y s i s of experiment 6 and the f i r s t p art of experiment 7 suggests t h a t p o s s i b l y the p l a n t s under the lower r e l a t i v e humidity responded with s e v e r e l y abnormal m e i o t i c behaviour more slow l y than d i d those i n the humid p l a s t i c d i s h e s . T h i s evidence i s not c o n c l u s i v e , however, s i n c e normal v a r i a t i o n i n day-to-day l a b o r a t o r y temperature may account f o r these d i f f e r e n c e s . In any event, a f t e r 72 hours, both of these environments had induced complete a b o r t i o n of m e i o s i s . The second part of experiment 7, i n which the p l a n t s were subjected t o normal photoperiods i n open t r a y s , was not expected to i n d i c a t e any influence' of m e i o t i c response to day l e n g t h . T h i s s u p p o s i t i o n was borne out by observations of meiosis and by the r e s u l t s of the t e t r a d a n a l y s i s (Table XI, page 56).' Although these r e s u l t s are v a l i d only f o r the species t e s t e d (H. c i r c i n a l e ) , i t c e r t a i n l y emphasizes ' the n e c e s s i t y of great care when h a n d l i n g mosses p r i o r to f i x a t i o n . I t i s q u i t e l i k e l y t h a t mosses maturing i n warmer c l i m a t i c a r e a s of the 59 continent or i n l a t e spring or summer i n the cooler climatic regions are not as sensitive to s l i g h t heat stress as are those maturing during cooler temperatures. Steere et a l . (1954) stated that most of the Californian mosses studied matured i n winter and early spring. Many of these species might well be heat sensitive. However, i n t h e i r study they did not indicate how ind i v i d u a l plants were handled so i t i s impossible to evaluate any abnormalities they reported. It i s l o g i c a l , considering the way many of the mosses must have been handled by many authors (Steere et a l . , 1954; Bryan, 1956a et seq.; Anderson and Crum, 1959) that at least some of the unusual features described for meiosis i n mosses ( i e . clumping, multiple association and precocQ ious disjunction of certain bivalents), can be attributed to the ~ influence of heat shock p r i o r to f i x a t i o n . (2) The S e n s i t i v i t y of Moss SMC to.Slight Heat Stress The prresent results indicate that H. c i r c i n a l e i s extremely sensitive to heat stress. However, the temperature l e v e l at which no abnormalities were produced ( i f there i s an absolute le v e l ) was not determined. It i s s i g n i f i c a n t that even the treatment applied i n experiment 4 (heat stress with a maximum temperature of 25°C for 4 hours) was s u f f i c i e n t to cause some reduction i n normal tetrad formation. In experiments 6 and 7, the sustained room temperature caused complete cessation of meiosis after three days i n such an environment.- This suggests that long periods at temperatures between 15-20°C. may produce abnormalities. It i s d i f f i c u l t to compare the s e n s i t i v i t y of H. c i r c i n a l e to other organisms and to higher plants i n pa r t i c u l a r because 60 a wide v a r i e t y of methods have been used i n a p p l y i n g heat s t r e s s . Heat shock experiments such as those performed by Barber (1941, 1942), Emsweller and B r i e r l e y (1943) and Bao and L i (1940) are uncommon i n the l i t e r a t u r e compared to the s u s t a i n e d temperature experiments of E l l i o t (1955), J a i n (1957 et sea..), Yanney Wilson (1959) and Henderson (1962). Even those experiments u s i n g s u s t a i n e d temperatures e x h i b i t c o n f l i c t i n g r e s u l t s because the l e n g t h of time needed to complete the m e i o t i c sequence at a s p e c i f i c temperature was seldom taken i n t o account (Yanney Wilson, 1959). A constant c r i t e r i o n on which to base s e n s i t i v i t y i s a l s o d i f f i c u l t to e s t a b l i s h . Most w r i t e r s d e a l w i t h chiasma frequency at metaphase; only r a r e l y i s mention made of the f i n a l outcome of m e i o s i s . Chiasma frequency i s v e r y d i f f i c u l t t o study i n moss b i v a l e n t s bedause the chromosomes are sma l l and seldom have a s u f f i c i e n t l y d i s t i n c t morphology f o r accurate chiasma counts. J a i n (1957 et seq.) has s t u d i e d heat e f f e c t s on meiosis of a v a r i e t y of Lolium perenne L. Although he was not i n t e r e s t e d i n the f i n a l product of meiosis he observed that p o l l e n mother c e l l s aborted at metaphase, the chromosomes formed r e s t i t u t i o n n u c l e i and no v i a b l e p o l l e n g r a i n s were produced a f t e r treatments o f ( l ) 35- 2°C. f o r 58 hours, and (2) 34 - 3°C. f o r 72 hours, and (3) 33- 2°C. f o r 86 hours. Henderson (1962) showed that i n the desert l o c u s t , S c h i s t o c e r c a gregaria,maintained at 40°C. f o r the d u r a t i o n of meiosis, many of the spermatocytes f a i l e d to undergo cleavage and became p y c n o t i c . Both of these treatments are much more severe and prolonged than those necessary to cause a b o r t i o n of meiosis i n H. c i r c i n a l e . I t i s not s u r p r i s i n g to f i n d t h a t H. c i r c i n a l e 61 and probably many other mosses are p a r t i c u l a r l y s e n s i t i v e t o temperature i n c r e a s e because the optimum m e i o t i c temperature i s much lower than i t i s f o r high e r plants.which f l o w e r i n the summer. I t i s i n t e r e s t i n g to note t h a t Pao and L i (1940) obser- ved a h i g h degree o f univ a l e n c e i n p o l l e n mother c e l l s of V i c i a c r a c c a L. immediately a f t e r a heat shock as m i l d as 25°C. f o r 30 minutes. These p l a n t s were s t u d i e d i n January when the temperature was 7°C. F a l l and winter-maturing mosses are presumably adapted to these lower temperature ranges. In Lolium the optimum m e i o t i c temperature may be between 15-20 °C. The r e l a t i v e d i f f e r e n c e between t h i s temperature and room temp- erature could be considered i n s u f f i c i e n t to produce anomalous me i o s i s . I t seems obvious that comparison of heat s e n s i t i v i t y between v a r i o u s l a t e spring-maturing mosses and f a l l / w i n t e r - maturing mosses would probably a i d i n determining the adaptive s i g n i f i c a n c e of heat s t r e s s . (3) N a t u r a l Occuring Heat S t r e s s as a Mechanism of Inducing Mutation or S t r u c t u r a l / N u m e r i c a l A l t e r a t i o n s of the Chromosomes. Th i s q u e s t i o n i s only a matter f o r s p e c u l a t i o n at t h i s time. The present experiments were n e i t h e r s u f f i c i e n t l y r e f i n e d nor s p e c i f i c a l l y d i r e c t e d to answer t h i s q u e s t i o n . Abno r m a l i t i e s induced by n a t u r a l heat s t r e s s s i m i l a r to those induced e x p e r i m e n t a l l y have been observed i n w i l d pop- u l a t i o n s i n both h i g h e r p l a n t s and mosses. Beamish (1961a, 1961b) has observed severe clumping of metaphase I stages and h i g h l y abnormal anaphases i n p o l l e n mother c e l l s of S a x i f r a g a s p e c i e s under f i e l d c o n d i t i o n s . I t i s suspected (Beamish, 1961b) 62 t h a t these a b n o r m a l i t i e s r e s u l t e d from h i g h temperatures. Khanna (i960) has observed a s i m i l a r e f f e c t i n the moss Barbula c o n s t r i c t a M i t t . One might s p e c u l a t e from these observations that l e s s severe heat s t r e s s may induce l e s s severe and p o s s i b l y i s o l a t e d occurrence of anomalous m e i o s i s . However, the types of anomalies ( i e . mutations, chromosomalaaberrations) that could be produced i s p r e s e n t l y unknown. For i n s t a n c e , i t i s c u r r e n t l y i m p o s s i b l y t o say whether p o i n t mutations are p o s s i b l y produced by t h i s mechanism. On the other hand, i t i s reasonable to c o n s i d e r t h a t chromosomal a b e r r a t i o n s of e i t h e r the s t r u c t u r a l or numerical type might be produced. From the h i g h frequency of u n i v a l e n c y at metaphase observed i n these experiments and 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 aneuploidy, might be the most common anomaly produced by s l i g h t heat s t r e s s . To be s i g n i f i c a n t i n the p o p u l a t i o n , of course, the aneuploid types must of n e c e s s i t y be v i a b l e and a l s o f e r t i l e . N o n d i s j u n c t i o n was observed o c c a s i o n a l y i n the t r e a t e d moss m a t e r i a l used i n the present experiments. However, only i n r a r e anaphases could a l l chromosomes be counted a c c u r a t e l y , so no r e l i a b l e estimate could be made of the frequency of t h i s anomaly. (4) The Mode of Response of the SMC to Heat S t r e s s . The response of the SMC to i n c r e a s e d temperatures was manifest i n s e v e r a l i n t e r r e l a t e d ways. The d i f f e r e n t c a t e g o r i e s can be c l a s s i f i e d g e n e r a l l y as: metaphase I clumping and m u l t i p l e a s s o c i a t i o n of b i v a l e n t s ; extreme condensation or c o i l i n g of the b i v a l e n t s ; precocious d i s j u n c t i o n ; s p i n d l e d e s t r u c t i o n or i n h i b i t - i o n ; and prophase asynchrony and premature m e i o t i c i n d u c t i o n . In no experiment performed here, however, were a l l of these 63 c a t e g o r i e s of response observed. T h i s simply r e f l e c t s the d i v e r s i t y of time and i n t e n s i t y of heat s t r e s s used. Table XII (page 64) i n d i c a t e s i n g e n e r a l the mode of 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 of heat s t r e s s used on the SMC of H. c i r c i n a l e . Clumping and M u l t i p l e A s s o c i a t i o n of the B i v a l e n t s : Severe clumping was observed only under c o n d i t i o n s of very h i g h temperature ( i e . above approximately 33°C.). In experiments 2(page 38) and 3 (page 40) i n which the maximum temperature was between 30° and 3 2°C, some o f the metaphase I stages were clumped immediately a f t e r the treatment, but t h i s clumping was not considered severe. With temperatures above 33°C. the areas of clumped chromatin were g l o b u l a r and shiny. Severe clumping i s a primary response to h i g h temper- atures and does not occur i n c e l l s e n t e r i n g metaphase I a f t e r the p l a n t s have been returned to normal temperatures (see experiments 1 (page 34) and 5 (page 47) . I t i s expected, although not confirmed that s e v e r e l y clumped c e l l s do not continue meiosis to completion. R e l a x a t i o n of the clumped chromatin mass may occur however, but the c e l l s probably undergo r e s t i t u t i o n . M u l t i p l e a s s o c i a t i o n of the b i v a l e n t s was observed c o n s i s t e n - t l y i n a l l experiments except experiment 4 (page 44) and i s r e l a t e d t o the clumping e f f e c t observed at h i g h temperatures. In experiment 2 and 3, mentioned above, a s s o c i a t i o n ranged from clumping to almost no m u l t i p l e b i v a l e n t a s s o c i a t i o n immediately a f t e r treatment. Experiments 6 and 7 i n d i c a t e d t h a t m u l t i p l e a s s o c i a t i o n , occurs at room temperature a f t e r a day i n d o o r s . 64 Table X I I : A g e n e r a l summary i n d i c a t i n g the mode of a c t i o n o f d i f f e r e n t c a t e g o r i e s of heat s t r e s s on the SMC of H. c i r c i n a l e . Heat S t r e s s Primary E f f e c t Secondary E f f e c t 36 ± 1 ° C . Shock -metaphase I clumping - a r r e s t of a c t i v e stages -condensation of d i c t y o t e n e -premature m e i o t i c i n d u c t i o n (not mani- f e s t ) -prophase asynchrony -spindle breakdown, or i n h i b i t i o n -condensation of meta- phase I b i v a l e n t s -asynchrony of ana- phase I , and la g g a r d and bridge formation - r e s t i t u t i o n -prophase asynchrony -severe r e d u c t i o n i n good t e t r a d formation 3 1 -_il°C. Shock -metaphase I p a r t i a l clumping -laggard and bridge format!dsn at ana- phase -normal appearing pro- phase -premature m e i o t i c i n d u c t i o n (not mani- f e s t ) -prophase asynchrony -metaphase I b i v a l e n t a s s o c i a t i o n , precoc- i o u s d i s j u n c t i o n and condensation - p a r t i a l s p i n d l e breakdown or i n h i b i - t i o n -prophase asynchrony - s i g n i f i c a n t r e d u c t i o n i n good t e t r a d pro- d u c t i o n 25 i 1 ° C Shock -not determined, but premature m e i o t i c i n d u c t i o n i s pre- sumed to occur -metaphase I with 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 ana- phases - s l i g h t r e d u c t i o n i n good t e t r a d formation 22 - 2 ° C Maintained -premature m e i o t i c i n d u c t i o n -prophase asynchrony -spindle i n h i b i t i o n and breakdown - d i s o r i e n t a t i o n at metaphase'I -abortion of meiosis 65 M u l t i p l e a s s o c i a t i o n , a primary e f f e c t of moderate temperature s t r e s s , ( i e . heat shock to 31°C.) i s a l s o a secondary e f f e c t of temperature s t r e s s . In experiment 3 , m u l t i p l e a s s o c i a t i o n was observed i n c e l l s at metaphase I 5 days a f t e r the treatment p e r i o d . I t i s expected that these c e l l s were probably i n e a r l y prophase d u r i n g the treatment. Although the prophase stages appeared normal a f t e r the treatment, they must have been a f f e c t e d i n some way. T h i s may be r e l a t e d to the pro- phase asynchrony which i s mentioned l a t e r . Extreme Condensation or C o i l i n g of the B i v a l e n t s : T h i s f e a t u r e was one of the most c o n s i s t e n t anomalies observed i n a l l experiments except #4. Abnormal c o i l i n g phen- omenon a s s o c i a t e d with heat s t r e s s has been noted o f t e n i n other organisms (Pao and L i , 1940; Swanson, 1942, 1942; J a i n , 1957; and Henderson, 1962). Pao and L i (1940) observed severe con- t r a c t i o n of the b i v a l e n t s s i m i l a r to that noted i n H. c i r c i n a l e a f t e r heat shock i n s e v e r a l s p e c i e s . However, i n the sustained temperature experiments performed by the other authors mentioned above, the c o i l i n g was asynchronous and u s u a l l y delayed at metaphase I. The discrepancy may be explained b y . t a k i n g i n t o account the d i f f e r e n t types of treatments u t i l i z e d . In the heat shock experiments there i s i n s u f f i c i e n t time between the t r e a t - ment and the examination f o r the sporangia to e x h i b i t , at meta- phase I, the r e s u l t s of delayed prophase c o i l i n g and asynchrony d u r i n g the treatment p e r i o d . The h i g h condensation of b i v a l e n t s at metaphase I observed i n moderately h e a t - t r e a t e d H. c i r c i n a l e i s p o s s i b l y caused by a f a i l u r e of the metaphase I b i v a l e n t s t o u n c o i l to the extent they do f o l l o w i n g the extreme condensation 6 6 at the clumped d i a k i n e s i s . In the c o n t r o l m a t e r i a l the meta- phase chromosomes do not appear as condensed as those i n d i a - k i n e s i s . T h i s f a i l u r e of the "bivalents to r e l a x at metaphase I may be r e l a t e d to the anomalies i n the s p i n d l e apparatus under s i m i l a r c o n d i t i o n s . Precocious D i s j u n c t i o n : The precocious d i s j u n c t i o n of b i v a l e n t s at metaphase I i s c l o s e l y r e l a t e d to the c o i l i n g anomalies found i n t r e a t e d m a t e r i a l . In a l l treatments except experiment 4, precocious d i s j u n c t i o n of one or more b i v a l e n t s was a s s o c i a t e d w i t h con- densation, m u l t i p l e a s s o c i a t i o n , and abnormal s p i n d l e formation. T h i s d i s j u n c t i o n of b i v a l e n t s i s probably caused by precocious t e r m i n a l i z a t i o n of the chiasmata at d i a k i n e s i s so that the b i v a l e n t members l o s e a l l contact p o i n t s and d r i f t a p a r t . Precocious chiasma t e r m i n a l i z a t i o n may be a d i r e c t e f f e c t of the heat ( i e . speeding up the process) or i t may be caused by a delay i n s p i n d l e formation. Swanson (1942) has i n d i c a t e d that i n h e a t - t r e a t e d m a t e r i a l o f T r a d e s c a n t i a c o i l i n g was asynchronous and delayed. The t e r m i n a l chiasma frequency was much high e r i n the more condensed metaphase I b i v a l e n t s of the c o n t r o l s of T r a d e s c a n t i a than i n the r e l a t i v e l y u n c o i l e d metaphase I b i - v a l e n t s of the heat t r e a t e d m a t e r i a l . I t i s p o s s i b l e that the h i g h c o n t r a c t i o n noted i n heat t r e a t e d H. c i r c i n a l e b i v a l e n t s 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 c o n t r o l s , thus c o n t r i b u t i n g to the e a r l y d i s j u n c t i o n . S p i n d l e D e s t r u c t i o n or I n h i b i t i o n , Prophase Asynchrony and Premature M e i o t i c I n d u c t i o n : 67 S p i n d l e d e s t r u c t i o n or i n h i b i t i o n by heat s t r e s s can only be i n f e r r e d from these data as the a c t u a l s p i n d l e f i b e r s cannot be seen u s i n g present techniques. The degree of b i v a l e n t o r i e n t a t i o n at metaphase I and the e f f e c t i v e n e s s of anaphase movement can be used as c r i t e r i o n denoting the s t a t e of the s p i n d l e . With temperatures above 36°C., i t i s apparent that the s p i n d l e apparatus i s destroyed, probably by a d e n a t u r a t i o n of i t s f u n c t i o n a l s t r u c t u r e . However, at the moderate heat shock (31 - 1°C.) the s p i n d l e was never completely destroyed as u s u a l l y the metaphase I stages v a r i e d from almost normal o r i e n t a t i o n to s e v e r e l y abnormal o r i e n t a t i o n . Complete i n h i b i t i o n of the p r o d u c t i o n 01 an e f f e c t i v e s p i n d l e apparatus was i n f e r r e d from observations of the m a t e r i a l main- t a i n e d at room temperature. As was mentioned e a r l i e r the prophase asynchrony and premature m e i o t i c i n d u c t i o n ( i e . s t i m u l a t i o n of premeiotic SMC to begin meiosis when the capsule i s about 1/2 s i z e ) was evident i n the p l a n t s kept at room temperature. At the same time prophase I was a c c e l e r a t e d so that the process of v i s i b l e chromosomal p a r t i c i p a t i o n was completed i n a p e r i o d of 1-2 days i n s t e a d of 1-2 weeks. I t would seem u n l i k e l y that a l l the s y n t h e t i c mechanisms l e a d i n g to the a c t i v e stages of meiosis could take p l a c e s a t i s f a c t o r i l y i n such a short time; thus i t i s l o g i c a l to suspect that s p i n d l e production i s a c t u a l l y i n h i b i t e d at room temperature. In a d d i t i o n , the s p i n d l e i n h i b i t i o n may not be due d i r e c t l y to the heat s t r e s s but due to a r e d u c t i o n i n time a v a i l a b l e f o r completion of necessary s y n t e h t i c mech- anisms . Asynchrony of development of SMC i n prophase w i t h i n one 68 sporangium f o l l o w i n g v a r i o u s heat treatments i s probably d i r e c t l y r e l a t e d to the mechanism of premature m e i o t i c i n d u c t i o n and again r e f l e c t s an a c c e l e r a t i o n of the m e i o t i c processes. Reducing 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 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 abnormal s p i n d l e e f f e c t i v e n e s s observed s e v e r a l days f o l l o w i n g the treatment. Heat induced s p i n d l e breakdown or i n h i b i t i o n has been noted many times i n previous s t u d i e s ( J a i n , 1957 et seq.; Swanson, 1942; and Henderson, 1962). These were continuous temperature experiments at r e l a t i v e l y h i g h temperatures. F a i l u r e of the s p i n d l e to develop was based on observations of cleavage f a i l u r e . The b i o c h e m i c a l mechanisms of heat induced s p i n d l e f a i l u r e have not been i n v e s t i g a t e d . A study i n v a r i a t i o n s of s y n t h e t i c a c t i v i t y d u r i n g normal and heat shocked prophase may prove to be a u s e f u l t o o l i n a c q u i r i n g a b e t t e r knowledge of the p r o d u c t i o n and f u n c t i o n of the m e i o t i c s p i n d l e . 6 9 SUMMARY 1. An account of meiosis emphasizing the prophase I stages has "been presented f o r two moss s p e c i e s ; Hypnum c i r c i n a l e Hook, and Brachythecium f r i g i d u m (CM.) Besch. In both s p e c i e s , meiosis f o l lowed the g e n e r a l p a t t e r n of events found i n most p l a n t s except f o r a chromosome e l o n g a t i o n stage which followed d i p l o t e n e . Evidence was presented that the stage may be found i n many moss s p e c i e s . 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 animals, has been d e s c r i b e d i n Balsamina h o r t e n s i s (impat- i e n s balsamina). The f u n c t i o n a l s i g n i f i c a n c e of the d i c t y o t e n e stage i n moss meiosis i s p r e s e n t l y unknown. 2. The e f f e c t s of heat s t r e s s of m e i o t i c spore mother c e l l s of H. c i r c i n a l e has been s t u d i e d . Both heat shock and sustained temperature experiments were performed. Severe anomalies such as metaphase I clumping, b i v a l e n t a s s o c i a t i o n and c o n t r a c t i o n , precocious d i s j u n c t i o n , s p i n d l e d e s t r u c t i o n or i n h i b i t i o n , pro- phase asynchrony, premature m e i o t i c i n d u c t i o n , and m e i o t i c a b o r t i o n were observed i n most of the experiments. The r e s u l t s i n d i c a t e t h a t H. c i r c i n a l e i s extremely s e n s i t i v e to heat s t r e s s and that c y t o l o g i s t s working w i t h mosses should be c a r e f u l 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 i n s u r e against heat induced m e i o t i c anomalies. The e f f e c t s 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 , and the mode of response of the SMC to heat 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 Anderson, L.E.. 1961. Chromosome s t u d i e s on some mosses of the southeastern United S t a t e s . 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(to follow page 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 ; showing the a c e n t r i c a l l y placed chromatin at l e p t o t e n e and zygotene; f i g . 1; l e p t o t e n e with chromosome strands extending past the middle of the SMC (strands appear t h i c k because the s l i d e was o l d at the time the photograph was taken and the 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 zygotene, arrow i n d i c a t e s two strands which appear to be i n the process of p a i r i n g (phase c o n t r a s t ) ; f i g . 3; strands w i t h chromomeres, a l l X2400. F i g u r e s 4-9,' H. c i r c i n a l e ; showing v a r i o u s stages of pachy- tene; f i g . 4,5; beginning of m i g r a t i o n of the chromatin to the center of the SMC; f i g . 7,8; mid-pachytene; f i g . 9; l a t e pachytene with chromatin 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 heterochromatic small b i v a l e n t ; f i g . 11; showing one of the l a r g e r b i v a l e n t s with a h e t e r o - chromatic segment (phase c o n t r a s t ) , X2400. P L A T E 1 ^ttr* S N \ _ , 4l Ilk 1 9 * 10 (to f a c e P l a t e 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 squashed SMC showing the d e t a i l e d s t r u c t u r e of the 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 showing a l l s i x b i v a l e n t s , i n c - l u d i n g the heterochromatic s m a l l b i v a l e n t and the n u c l e o l u s (both 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 stage between d i p l o t e n e and the elongation stage (phase c o n t r a s t ) X&400. Fi 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 stage; f i g . 4; elong- a t i o n stage Tarrow i n d i c a t e s a s e c t i o n where the chromo- somes are 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 stage showing the i n t e r p h a s e - l i k e appearance of the c e l l (both 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 ; a SMC wit h d i f f u s e appearance of chromatin. T h i s stage i s probably 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; densely clumped b i v a l e n t s at 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 w i t h 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 sm 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 11, H. c i r c i n a l e ; i nterphase or prophase I I showing a suggestion of w a l l formation between the 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 packed chromosomes and a temporary bridge formation, X2400. P L A T E 2 ' j ' : ••• *• W ~" (to f a c e P l a t e 3) DESCRIPTION OF FIGURES OF PIATE 3 F i g u r e 1, H. c i r c i n a l e ; telophase I I showing the beginning of w a l l formation, X2400. F i g u r e 2, H. c i r c i n a l e : a normal t e t r a d , X2400. Fi g u r e s 3-7, H. c i r c i n a l e ; sporangium #6; d i a k i n e s i s to metaphase I, showing the unusual behaviour of the 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 sep- ar a t e from the chromosomal mass; f i g . 4-6; l a t e d i a k i n e s i s or e a r l y metaphase I, showing a 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 separate from the mass. The other 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 remainder of the chromosomes and i s not d i s t i n g u i s h a b l e ; f i g . 7; a metaphase I i n which the s m a l l e s t b i v a l e n t has not 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 ; showing bridge and l a g g a r d form- a t i o n s at telophase I; f i g . 8; a bridge and two u n i v a l e n t s i n v o l v i n g the l a r g e r chromosomes; f i g . 9; showing the two u n i v a l e n t s of the s m a l l e s t b i v a l e n t l e f t o f f the metaphase p l a t e , both X2400. F i g u r e s 10&11, B. f r i g i d u m ; showing the 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 each c e l l than i s the chromatin and i s of l i g h t e r d e n s i t y than the chromatin, both 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 pachytene showing the beginning of chromosome mi g r a t i o n , X2400.  (to 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 ; mid-pachytene, X2400. F i g u r e 2, B. f r i g i d u m ; d i p l o t e n e . The heterochromatic segment of one of the chromosomes i s shown i n the lower r i g h t hand corner o f the c e l l ; the other dark s e c t i o n i s caused by overlap of chromosomes, X2400. F i g u r e 3&4» B. f r i g i d u m ; SMC i n the e l o n g a t i o n stage. In f i g . 3 the n u c l e a r membrane i s c l e a r l y evident; 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 , both X2400. F i g u r e 5 , B. f r i g i d u m ; showing a clumped d i a k i n e s i s , X2400. F i g u r e 6, B. f r i g i d u m ; prometaphase showing s i x b i v a l e n t s , X2400. F i g u r e s 7&8, B. f r i g i d u m ; metaphase I . In f i g . 7 the l o n g b i v a l e n t i s l o c a t e d at the pe r i p h e r y . T h i s i s the most common s i t u a t i o n , X2400. F i g u r e 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 hours a f t e r treatment; an unoriented metaphase I, X2400. F i g u r e s 10&11, exp. 1, page 34; 48 hours a f t e r treatment; 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 meta- phase I; f i g . 11; d i a k i n e s i s or unoriented metaphase I, both X2400. P L A T E 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 hours a f t e r treatment; f i g . 1; anaphase I or metaphase I with u n i v a l e n t s ; f i g . 2; telophase I showing g l o b u l a r n u c l e i , but no l a g g a r d s , both X2400. F i g u r e s 3-8, exp. 1, page 34; 4 days a f t e r treatment; showing examples of the 4 abnormal a n a l y s i s c a t e g o r i e s ; f i g . 3; category 1; f i g . 4; category 2; f i g . 5; category 3; f i g . 6,7,8; category 4, a l l X2400* F i g . 9, exp. 1, page 34, 4 days a f t e r treatment; 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 at d i p l o t e n e , X2400. F i g u r e 10-12, exp. 2, page 38; immediately a f t e r treatment; f i g . 10; heat induced clumping at 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; normal s p i n d l e formation with mult- i p l e a s s o c i a t i o n of b i v a l e n t s , a l l X2400. P L A T E 5 ^ ^ ^ ^ ^ ^ ^ ^ 4 0 1 7 [71 ••• • (to f ace 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; immediately a f t e r treatment; f i g . 1; metaphase I w i t h normal s p i n d l e formation; f i g . '2; metaphase I w i t h normal s p i n d l e formation but w i t h h i g h l y condensed b i v a l e n t s ; f i g . 3; normal b i v a l e n t s at metaphase I, o r i e n t a t i o n i s s l i g h t l y abnormal, a l l X2400.; F i g u r e s 4&5, exp. 2, page 38; immediately a f t e r treatment; f i g . 4; metaphase I with 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 wi t h l a g g i n g chromosomes,- both X2400. Fi g u r e s 6 , exp. 2, page 38; 72 hours a f t e r treatment; showing an anaphase I d i s p l a y i n g abnormal segregation, X2400. F i g u r e s 7-9, exp. 2, page 38; 72 hours a f t e r treatment; f i g . 7; a f i e l d of abnormal t e t r a d s , X1650; f i g . 8; a t e t r a d of category 2; f i g . 9; a t e t r a d with f i v e n u c l e i , both X2400. F i g u r e 10, exp. 3, page 40J 5 days a f t e r treatment; showing a metaphase I with good s p i n d l e arrangement and s l i g h t l y over-condensed b i v a l e n t s , X2400.  (to face 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 days a f t e r treatment; f i g . 1; a normal appearing 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, Heterocladium h e t e r o p t e r o i d e s ; abnormal a c t i v e stages and t e t r a d s a f t e r 2 weeks indoors, X2500. F i g u r e s 4-7, exp. 5, page 47; immediately a f t e r treatment; f i g . 4; d i p l o t e n e showing what appears to be abnormal 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 of SMC w i t h s e v e r e l y clumped meta- phase I, X1300. F i g u r e 8, exp. 5, page 47; 72 hours a f t e r treatment; showing a metaphase I with some s p i n d l e o r i e n t a t i o n , but with not a l l the b i v a l e n t s o r i e n t e d , X2400.  (to f a c e 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 hours a f t e r treatment; showing SMC w i t h s p i n d l e arrangement, but w i t h highly- condensed b i v a l e n t s e x h i b i t i n g asynchronous d i s j u n c t i o n or f a u l t y o r i e n t a t i o n , both X2400,. F i g u r e s 3-5, exp. 5, page 47; 5 days a f t e r treatment; f i g . 3; a r e s t i t u t i o n SMC which 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 spores and a r e s t i t u t i o n SMC (dotted l i n e s i n d i c a t e the f i n e threads of w a l l m a t e r i a l which j o i n two s p o r e s ) , both X2400; f i g . 5; a low power f i e l d of spores and r e s t i t u t i o n SMC which have c l u s t e r e d i n t o a l a r g e aggregate, X450. F i g u r e s 6-9, exp. 6, page 51; 30 hours a f t e r the p l a n t s were brought ind o o r s ; showing v a r i o u s anomalies ( 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 clumping and precocious d i s s o c i a t i o n ) at metaphase I, a l l X2400.  (to f ace P l a t e 9) DESCRIPTION OF FIGURES OF PLATE 9 F i g u r e s 1-3, exp. 6, page 51; 72 hours a f t e r p l a n t s were brought indoors; f i g . 1; aborted meiosis at f i r s t d i v i s i o n ; f i g . 2; aborted meiosis d u r i n g m i c r o n u c l e i formation, both XL300; f i g . 3; a t e t r a d formed from a SMC a f t e r a b o r t i o n of meiosis, X2400. F i g u r e s 4&5, exp. 6, page 51; 4 days a f t e r p l a n t s were brought indoors; f i g . 4; d i c t y o t e n e with the chromatin p y c n o t i c and clumped around the chromocentre; f i g . 5\ e a r l y prophase i n which the chromatin has clumped i n t o a b a l l , both X2400. 

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