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UBC Theses and Dissertations

Studies on pollen of selected species in Pinaceae Ho, RongHui 1972

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\ 1123 Studies on pollen of selected species i n Pinaceae "by RongHui Ho B.A., National Taiwan University, 1963 M.Sc., National Taiwan University, 1966 M.Sc, University of B r i t i s h Columbia, 1968 A thesis submitted i n p a r t i a l fulfilment of the requirement for the degree of Doctor of Philosophy i n the Faculty of Forestry. We accept this thesis as conforming to the required standard The University of B r i t i s h Columbia April 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed w ithout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT I h e s t u d y o f t h e e x i n e p a t t e r n o f p o l l e n g r a i n s might p e r m i t the s e p a r a t i o n o f v a r i o u s t a x a and - a s s i s t the i n t e r p r e t a t i o n o f t h e i r r e l a t i o n s h i p s . . T f t i r t y - s e v e n specimens f r o m t w e n t y - s i x s p e c i e s i n e i g h t P i n a c e a e g e n e r a were s t u d i e d by means o f l i g h t , s c a n n i n g e l e c t r o n and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . The f i n e p o l l e n s u r f a c e s t r u c t u r e o f twe n t y - t w o s p e c i e s ( o u t o f t w e n t y - s i x ) and t h e v a r i a t i o n s o f p o l l e n m o r p hology i n Tsuga h e t e r o n h v l l a and I . m e r t e n s i a n a were d e m o n s t r a t e d f o r t h e f i r s t t i m e . The p o l l e n g r a i n s o f f o u r P s e u d o t s u g a s p e c i e s were f o u n d t o be e l l i p t i c a l - s h a p e d when t u r g i d , and cup-shaped when d r y . They v a r i e d i n s i z e , but a l l had t h e same s c a b r a t e s c u l p t u r e . The p r o x i m a l f a c e o f g r a i n s b o r e an a n n u l a r t h i c k e n i n g , and a t r i r a d i a t e s t r e a k w h i c h d i v i d e d t h i s f a c e i n t o t h r e e s e c t o r s . Under t h e t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e , s u r f a c e r e p l i c a s r e v e a l e d t h e e x i n e a r e o l a e w h i c h c o n s i s t e d o f i n t e r s e c t i n g r i b s p r o t r u d i n g f r o m t h e p o l l e n s u r f a c e . M i n u t e p r o t r u s i o n s emanated f r o m i n t e r s e c t i o n s o f r i b s . The d i a m e t e r and t h e d e n s i t y o f t h e m i n u t e p r o t r u s i o n s v a r i e d f r o m s p e c i e s t o s p e c i e s . P o l l e n o f t h e s p e c i e s examined h e r e c o u l d be d i s t i n g u i s h e d f r o m s i z e -f r e q u e n c y c u r v e s o f mature g r a i n s , b u t n o t o n t h e b a s i s o f f i n e s t r u c t u r e . I n P s e u d o t s u g a m e n z i e s i i . t h e d i a m e t e r o f t h e m i n u t e p r o t r u s i o n s o f p o l l e n g r a i n s h a r v e s t e d p r i o r t o n a t u r a l I I s h e d d i n g was f o u n d t o "be much g r e a t e r t h a n t h a t o f mature g r a i n s . The r e a s o n m i g h t be t h a t t h e e a r l y h a r v e s t o f p o l l e n c u t s o f f a d d i t i o n a l s u p p l i e s o f t h e n e c e s s a r y p r e c u r s o r s f o r t h e p o l y m e r i z a t i o n and t h e f o r m a t i o n o f t h e e x i n e . The a p p l i -c a t i o n o f a c e t o n e d u r i n g s p e c i m e n p r e p a r a t i o n m i g h t cause e x p a n s i o n o f t h e p r o t r u s i o n s b ecause o f i n c o m p l e t e p o l y m e r i z a -t i o n . Of e i g h t L a r i x s p e c i e s examined, p o l l e n - s i z e d i s t r i b u t i o n c u r v e s o v e r l a p p e d each o t h e r . P o l l e n g r a i n s o f t h e e i g h t s p e c i e s h ad t h e same morphology and e x i n e s c u l p t u r e as d e s c r i b e d f o r P s e u d o t s u g a . w i t h t h e e x c e p t i o n t h a t t h e mature p o l l e n s i z e was s m a l l e r and t h e t r i r a d i a t e s t r e a k was l e s s w e l l d e v e l o p e d . Hence, s p e c i e s were n o t d i s t i n g u i s h a b l e by t h e s e c r i t e r i a . The s i m i l a r i t y o f t h e e x i n e s c u l p t u r e i n s p e c i e s o f P s e u d o t s u g a p o l l e n may be due t o s i m i l a r i t i e s i n chromosomal make-up a f t e r t h e chromosome r e o r g a n i z a t i o n . I t i s a l s o p o s s i b l e t h a t P s e u d o t s u g a and L a r i x have e v o l v e d f r o m common a n c e s t o r s , w i t h t h e p a r a l l e l development o f s i m i l a r c h a r a c t e r s f r o m t h e i r common a n c e s t o r . P o l l e n g r a i n s o f t h e s p e c i e s i n A b i e s , Cedrus» K e t e l e e r i a . P i c e a and Tsuga examined were f o u n d t o be d i s t i n c t f r o m one a n o t h e r , b a s e d on p o l l e n m orphology and f i n e s t r u c t u r e . The s c u l p t u r e p a t t e r n o f t h e e x i n e was m i c r o v e r r u c a t e , e x c e p t i n K e t e l e e r i a w h i c h was r u g u l a t e on t h e p r o x i m a l f a c e . On t h e o t h e r hand, t h e f o u r s p e c i e s o f P i n u s p o l l e n were f o u n d t o be / I l l i n d i s t i n g u i s h a b l e b a s e d on f i n e s t r u c t u r e . The e x i n e p a t t e r n on t h e p r o x i m a l s u r f a c e o f p o l l e n g r a i n s w&s m i c r o v e r r u c a t e . E a c h g r a n u l e o f t h e m i c r o v e r r u c a t e s c u l p t u r e was a l s o ornamented w i t h m i c r o v e r r u c o i d s c u l p t u r e . The s i m i l a r i t y i n f i n e s t r u c t u r e o f P i n u s p o l l e n m i g h t be a t t r i b u t e d t o t h e g e n e t i c s i m i l a r i t y i n t h e s p e c i e s o f t h i s genus. P o l l e n m o r p hology o f Tsuga m e r t e n s i a n a c o m p l e t e l y d i f f e r e d f r o m t h a t o f T. h e t e r o p h v l l a . The d i f f e r e n c e may have been due t o v a r y i n g r a t e s o f e v o l u t i o n a r y change, t o t h e m o d i f i c a t i o n o f r e p r o d u c t i v e s t r u c t u r e f o r t h e i r c o n t i n u e d e x i s t e n c e * o r t o i n t r o -g r e s s i o n . The a u t h o r recommends s p l i t t i n g t h e s e two s p e c i e s i n t o s e p a r a t e g e n e r a . The h i g h e r were t h e r a n k o f t a x a , t h e g r e a t e r were t h e d i f f e r e n c e s i n p o l l e n m o r p h o l o g y . Based on morphology, p o l l e n i n P i n a c e a e was p r o p o s e d t o be g r o u p e d as f o l l o w s : (1) P s e u d o t - s u g a - L a r i x : (2) A b i e s - K e t e l e e r i a - T s u g a ( p o l l e n o f s a c c a t e t y p e e.g., T. m e r t e n s i a n a ) : (3) C e d r u s - P i c e a : (4-) P i n u s : (5) Tsuga ( p o l l e n o f b i c o n v e x - l e n s t y p e e.g., T. h e t e r o p h v l l a ) . I V ACMOWLEDGEMENTS I w o u l d l i k e t o e x p r e s s my s i n c e r e t h a n k s t o my s u p e r v i s o r , D r. 0 . S z i k l a i , P r o f e s s o r , f o r i n t r o d u c i n g me t o t h e f i e l d o f F o r e s t G e n e t i c s , and f o r h i s i n s p i r i n g g u i d a n c e and s u g g e s t i o n s t h r o u g h o u t t h e s t u d y . I t i s my d i s t i n c t p l e a s u r e t o acknowledge t h e i n v a l u a b l e c o n t r i b u t i o n s made by t h e f o l l o w i n g p e r s o n s : D r . K.M. C o l e , P r o f e s s o r , Department o f B o t a n y , D r. E.W. Meyer, F o r e s t P r o d u c t s L a b o r a t o r y ( V a n c o u v e r ) , Dr. G.E. House, P r o f e s s o r , Department o f B o t a n y , and D r . J.W. W o r r a l l , A s s i s t a n t P r o f e s s o r , F a c u l t y o f F o r e s t r y , f o r t h e i r most c o n s t r u c t i v e s u g g e s t i o n s and c r i t i c i s m s . I w i s h a l s o t o t h a n k Dr. G. Erdtman, S t o c k h o l m , Sweden f o r s e r v i n g as t h e E x t e r n a l E x a m i n e r o f t h i s t h e s i s . G r a t e f u l t h a n k s a r e e x t e n d e d t o Dr. S. A r n o , Dr. K.K. C h i n g , Mr. L . I . H s i n , D r. L.C. John s o n , Mr. M.D. Meagher, Dr. A.L. O r r -Ew i n g , D r . S. S a t o o , Mr. J . L . Wang and t h e C u r a t o r , E o y a l B o t a n i c a l Garden, E d i n b u r g h f o r f u r n i s h i n g s amples; Mr. H. M a r s h a l l , f o r r e a d i n g t h e m a n u s c r i p t and Mr. L. Ve t o f o r t e c h n i c a l a d v i c e on e l e c t r o n m i c r o s c o p y . The f i n a n c i a l a s s i s t a n c e o f B r i t i s h C o l u m b i a F o r e s t P r o d u c t s L t d . , ( V a n c o u v e r ) , i n t h e f o r m o f F o r e s t G e n e t i c s S c h o l a r s h i p s , i s g r a t e f u l l y a c k nowledged. V TABLE OF CONTENTS ABSTRACT I ACKNOWLEDGEMENTS I V TABLE OF CONTENTS V L I S T OF TABLES . V I I L I S T OF FIGURES V I I I 1. INTRODUCTION 1 2. LITERATURE REVIEW „. A. L i g h t m i c r o s c o p i c s t u d i e s o f t h e p o l l e n i n P i n a c e a e 4-B. E l e c t r o n m i c r o s c o p i c s t u d i e s o f t h e p o l l e n i n P i n a c e a e , ( 1 ) R e p l i c a t e c h n i q u e s f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e 5 ( 2 ) R e p o r t s o f t h e p o l l e n s u r f a c e s t r u c t u r e by-t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e 13 (3) The a p p l i c a t i o n o f t h e s c a n n i n g e l e c t r o n m i c r o -scope on p o l l e n s t u d i e s 15 3. MATERIAL 18 4. METHODS A. P o l l e n c o l l e c t i o n 19 B. P o l l e n s i z e s t u d i e s 21 C. Specimen p r e p a r a t i o n f o r s c a n n i n g e l e c t r o n m i c r o -scope 21 D. C a r b o n r e p l i c a t e c h n i q u e 25 E. M i c r o p h o t o g r a p h e v a l u a t i o n 28 F. C a l c u l a t i o n o f t h e r a t i o o f a b s o l u t e t o a v e r a g e chromosome l e n g t h 28 5. RESULTS AND DISCUSSION A. The f o r m a t i o n o f t h e D o u g l a s - f i r p o l l e n e x i n e i n t h e l a t e s t a g e o f m i c r o s p o r o g e n e s i s 28 V I B. V a r i a t i o n s o f p o l l e n g r a i n s i n P s e u d o t s u g a macro-c a r p a and P. m e n z i e s i i . ( 1 ) P s e u d o t s u g a m a c r o c a r p a 29 ( 2 ) P s e u d o t s u g a m e n z i e s i i ( a ) P o l l e n s i z e . 41 ( b ) S u r f a c e s t r u c t u r e o f p o l l e n 43 C. I n t e r s p e c i f i c v a r i a t i o n s o f p o l l e n g r a i n s . (1) P s e u d o t s u g a s p e c i e s 45 ( 2 ) L a r i x s p e c i e s 53 (3) A b i e s s p e c i e s 57 ( 4 ) Cedrus s p e c i e s 61 (5) K e t e l e e r i a s p e c i e s «• 63 ( 6 ) P i c e a s p e c i e s 64 ( 7 ) P i n u s s p e c i e s 66 ( 8 ) Tsuga s p e c i e s 70 D. I n t e r g e n e r i c v a r i a t i o n s o f p o l l e n g r a i n s . ( 1 ) P s e u d o t s u g a - L a r i x 78 ( 2 ) A b i e s - K e t e l e e r i a - T s u g a 79 ( 3 ) C e d r u s - P i c e a 80 ( 4 ) P i n u s 81 ( 5 ) Tsuga 81 6. SUMMARY AND CONCLUSION 82 7. LITERATURE CITED 88 8. APPENDIX I . G l o s s a r y o f terms u s e d i n t h e t e x t 158 V I I L I S T OF TABLES 1. R e p o r t s on t h e l i g h t m i c r o s c o p i c s t u d i e s o f t h e p o l l e n i n P i n a c e a e 6 2. R e p o r t s on t h e r e p l i c a t e c h n i q u e s f o r p o l l e n s t u d i e s . 12 3. Da t e s and s o u r c e s o f p o l l e n c o l l e c t i o n i n P i n a c e a e . . 1 9 4. C o l o r c l a s s e s o f m a c r o s p o r a n g i a t e s t r o b i l i , d a t e s o f b r a n c h l e t s c u t , d a t e s o f p o l l e n c o l l e c t e d , and p o l l e n s o u r c e s o f P s e u d o t s u g a m e n z i e s i i ... 22 5. A v e r a g e s i z e o f t h e p o l l e n g r a i n s i n m i c r o n s (i one s t a n d a r d d e v i a t i o n ) and c o e f f i c i e n t o f v a r i a t i o n .... 31 6. The a v e r a g e s i z e and r a n g e o f t h e p o l l e n - s i z e d i s -t r i b u t i o n o f P s e u d o t s u g a and L a r i x s p e c i e s 32 7. The d e n s i t y ( p e r 0.1 mm ) and d i a m e t e r o f m i n u t e p r o t r u s i o n s i n P s e u d o t s u g a and L a r i x s p e c i e s 42 8. Comparisons o f t h e r a t i o o f a b s o l u t e t o av e r a g e chromosome l e n g t h i n P s e u d o t s u g a and L a r i x s p e c i e s . . 4 9 9. The a v e r a g e t o t a l l e n g t h and r a n g e o f t h e p o l l e n -s i z e d i s t r i b u t i o n i n A b i e s , C e d r u s . K e t e l e e r i a . P i c e a , P i n u s and Tsuga s p e c i e s 58 10. The a v e r a g e c o r p u s s i z e and r a n g e o f t h e d i s t r i b u t i o n i n A b i e s , C e d r u s , K e t e l e e r i a , P i c e a , P i n u s and Tsuga s p e c i e s 58 11. The a n g l e o f t h e s a c c a t e a t t a c h m e n t i n A b i e s . Cedrus  K e t e l e e r i a , P i c e a , P i n u s and Tsuga s p e c i e s 59 12. The c o u n t s o f t h e p o l l e n g r a i n s w i t h s a c c i , v e s t i g a l s a c c i , abnormal s a c c u s , and w i t h o u t s a c c u s i n Tsuga m e r t e n s i a n a 72 V I I I L I S T OF FIGURES 1. P o l l e n g r a i n s o f b i s a c c a t e and b i c o n v e x t y p e s 2 3 2 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a m a c r o c a r p a f r o m Oregon 3 3 3 . S i z e - f r e q u e n c y c uves o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a m a c r o c a r p a f r o m C a l i f o r n i a 3 3 4. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a m e n z i e s i i f r o m Vancouver I s l a n d 3 3 5 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a m e n z i e s i i f r o m UBC #2 3 3 6. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a m e n z i e s i i f r o m I n t e r i o r , B.C 3 4 7 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a .japonica 3 4 8. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P s e u d o t s u g a w i l s o n i a n a 3 4 9 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x d e c i d u a 3 4 1 0 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x g m e l i n i i 3 5 1 1 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x l a r i c i n a 3 5 1 2 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x l e p t o l e p i s 3 5 1 3 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x l y a l l i i 3 5 14. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x o c c i d e n t a l i s 3 6 1 5 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x p o t a n i n i i 3 6 16. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f L a r i x s i b i r i c a 3 6 1 7 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f A b i e s g r a n d i s 3 6 I X 18. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f A b i e s n i n s a n o 3 7 1 9 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f Cedrus a t l a n t i c a 3 7 2 0 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f Cedrus d e o d a r a 3 7 2 1 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f Cedrus l i b a n i 3 7 2 2 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f K e t e l e e r i a d a v i d i a n a 3 8 2 3 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f F i c e a o r i e n t a l i s 3 8 24. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P i c e a s i t c h e n s i s 3 8 2 5 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P i n u s b a n k s i a n a 3 8 26. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P i n u s c o n t o r t a 3 9 2 7 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P i n u s p o n d e r o s a 3 9 28. S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f P i n u s s y l v e s t r i s 3 9 2 9 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f Tsuga h e t e r o - p h y l l a 3 9 3 0 . S i z e - f r e q u e n c y c u r v e s o f t h e p o l l e n g r a i n s o f Tsuga m e r t e n s i a n a 40 3 1 . F i n e s t r u c t u r e o f D o u g l a s - f i r p o l l e n showing t h e m i n u t e p r o t r u s i o n s . P o l l e n c o l l e c t e d two weeks b e f o r e a n t h e s i s . S l i t s i n t h e p r o t r u s i o n s a r e a r t e f a c t s 9 5 3 2 . F i n e s t r u c t u r e o f D o u g l a s - f i r p o l l e n showing t h e m i n u t e p r o t r u s i o n s w i t h f a i n t l y marked s l i t s . P o l l e n c o l l e c t e d one week b e f o r e a n t h e s i s 9 6 3 3 . F i n e s t r u c t u r e o f D o u g l a s - f i r p o l l e n showing t h e m i n u t e p r o t r u s i o n s w i t h o u t any s l i t s . P o l l e n c o l l e c t e d a t a n t h e s i s 9 7 3 4 . T u r g i d p o l l e n o f b i g c o n e D o u g l a s - f i r i n e l l i p t i c a l shape 9 8 X 3 5 * T u r g i d p o l l e n o f D o u g l a s - f i r i n e l l i p t i c a l shape 9 8 3 6 . T u r g i d p o l l e n o f Japanese D o u g l a s - f i r i n e l l i p t i c a l shape 9 8 3 7 . T u r g i d p o l l e n o f Eormosan D o u g l a s - f i r i n e l l i p t i c a l shape 9 8 3 8 . B i g c o n e D o u g l a s - f i r f r o m Oregon showing t w o - f u s e d s p h e r e s formed "by a n n u l a r t h i c k e n i n g and t r i r a d i a t e s t r e a k 9 9 3 9 . B i g c o n e D o u g l a s - f i r p o l l e n f r o m C a l i f o r n i a showing t h r e e u n e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s 9 9 40. D o u g l a s - f i r p o l l e n f r o m Vancouver I s l a n d showing two-f u s e d s p h e r e s formed b y a n n u l a r t h i c k e n i n g and t r i r a d i a t e s t r e a k 1 0 0 4 1 . D o u g l a s - f i r p o l l e n o f t r e e No. 1 showing a n n u l a r t h i c k e n i n g and t h r e e e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s . 1 0 0 4 2 . D o u g l a s - f i r p o l l e n o f t r e e No. 1 showing a n n u l a r t h i c k e n i n g and t h r e e u n e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s 101 4 3 . D o u g l a s - f i r p o l l e n o f t r e e No. 2 showing t h a t one o f t r i r a d i a t e s t r e a k s i s i n v i s i b l e 101 4 4 . D o u g l a s - f i r p o l l e n o f t r e e No. 11 showing t h e a n n u l a r t h i c k e n i n g and t h r e e e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s 1 0 2 4 5 . D o u g l a s - f i r p o l l e n o f t r e e No. 12 showing t h e a n n u l a r t h i c k e n i n g , t h r e e e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s and p o r i i n t h e e x i n e 1 0 2 46. D o u g l a s - f i r p o l l e n o f t r e e A showing a n n u l a r t h i c k e n i n g and t h r e e u n e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s 1 0 3 4 7 . D o u g l a s - f i r p o l l e n o f t r e e B showing t h e a n n u l a r t h i c k e n i n g and t h r e e u n e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s 1 0 3 48. D o u g l a s - f i r p o l l e n o f t r e e E showing a n n u l a r t h i c k e n i n g and t h r e e u n e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s 104 4 9 . D o u g l a s - f i r p o l l e n f r o m B.C. I n t e r i o r showing t h a t one o f t h e t r i r a d i a t e s t r e a k s i s i n v i s i b l e 104 5 0 . Japanese D o u g l a s - f i r p o l l e n showing a n n u l a r t h i c k e n i n g and f a i n t l y marked t r i r a d i a t e s t r e a k 1 0 5 5 1 . Formosan D o u g l a s - f i r p o l l e n s howing a n n u l a r t h i c k e n i n g and t h r e e e q u a l l y s p a c e d s t r e a k s 105 X T 5 2 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f b i g c o n e D o u g l a s -f i r f r o m Oregon. A r e o l a e a r e f o rmed by t h e i n t e r s e c t i n g r i b s where t h e m i n u t e p r o t r u s i o n s p r o j e c t f r o m t h e i n t e r s e c t i n g p o i n t s 106 5 3 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f b i g c o n e D o u g l a s - f i r f r o m Oregon 1 0 7 5 4 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f b i g c o n e D o u g l a s - f i r f r o m C a l i f o r n i a 1 0 7 5 5 . P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r f r o m Vancouver I s l a n d 108 5 6 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e No. 1 108 5 7 . P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e No. 2 1 0 9 5 8 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e No. 11 - 1 0 9 5 9 - P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e No. 12 1 1 0 60. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e A 1 1 0 6 1 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e B 111 62. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r t r e e E 111 6 3 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D o u g l a s - f i r f r o m B.C. I n t e r i o r 112 64. S i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f Japanese D o u g l a s - f i r 113 6 5 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f Formosan D o u g l a s - f i r 114 66. P o l l e n o f E u r o p e a n l a r c h showing a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k 115 6 7 . P o l l e n o f D a h u r i a n l a r c h s howing a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k 115 68. T u r g i d p o l l e n o f t a m a r a c k 116 6 9 . P o l l e n o f t a m a r a c k showing a n n u l a r t h i c k e n i n g and i n c o m p l e t e s t r e a k 116 X I I 7 0 . Abnormal p o l l e n o f t a m a r a c k i n p e a r - l i k e shape 116 7 1 . P o l l e n o f Japanese l a r c h showing i n c o n s p i c u o u s a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k 117 7 2 . P o l l e n o f Japanese l a r c h showing w e l l marked a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k 117 7 3 - P o l l e n o f a l p i n e l a r c h s howing i n c o n s p i c u o u s a n n u l a r t h i c k e n i n g 118 7 4 . P o l l e n o f w e s t e r n l a r c h showing a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k 118 7 5 - P o l l e n o f C h i n e s e l a r c h showing a n n u l a r t h i c k e n i n g 119 7 6 . S i b e r i a n l a r c h p o l l e n showing a n n u l a r t h i c k e n i n g and p o r u s , 2 b y 4 m i c r o n s , i n t h e e x i n e 1 1 9 7 7 * H i g h m a g n i f i c a t i o n o f t h e p o r u s i n t h e e x i n e o f S i b e r i a n l a r c h p o l l e n 1 2 0 7 8 . P o l l e n o f S i b e r i a n l a r c h s howing c o a r s e a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k 1 2 0 7 9 - P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f E u r opean l a r c h . A r e o l a e a r e formed by t h e i n t e r s e c t i n g r i b s where t h e m i n u t e p r o t r u s i o n s p r o j e c t f r o m t h e i n t e r s e c t i n g p o i n t s . 121 80. P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f D a h u r i a n l a r c h . 1 2 2 8 1 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f t a m a r a c k 1 2 2 82. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f Japanese l a r c h . 123 8 3 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f a l p i n e l a r c h .... 123 84. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f w e s t e r n l a r c h .. 124 8 5 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f C h i n e s e l a r c h .. 124 8 6 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f S i b e r i a n l a r c h . 1 2 5 8 7 - T u r g i d p o l l e n o f g r a n d f i r 126 88. T u r g i d p o l l e n o f g r a n d f i r a t t h e p r o x i m a l f a c e 126 8 9 . P o l l e n o f g r a n d f i r showing m i c r o v e r r u c a t e s c u l p t u r e and p o r i i n t h e s a c c i 126 9 0 . P o l l e n o f g r a n d f i r showing t h e t r i r a d i a t e s t r e a k on t h e cap 126 X I I I 9 1 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f g r a n d f i r . A r e o l a e a r e f ormed by t h e i n t e r s e c t i n g r i b s where t h e p r o t r u s i o n s p r o j e c t f r o m t h e i n t e r s e c t i n g p o i n t s .... 127 9 2 . T u r g i d p o l l e n o f S p a n i s h f i r showing t h e t h i n e x i n e a t t h e c e n t e r o f t h e cap 128 9 3 - P o l l e n o f S p a n i s h f i r s h o w i n g m i c r o v e r r u c a t e s c u l p -t u r e , g r a n u l a t e s t r u c t u r e a t t h e m a r g i n a l a r e a , and p o r i i n t h e s a c c i 128 94- . P o l l e n o f S p a n i s h f i r showing t h e t r i r a d i a t e s t r e a k on t h e c a p . Two s t r e a k s f o r m a s t r a i g h t l i n e and t h e t h i r d s t r e a k a p p r o a c h e s t h i s l i n e by about 3 0 ° 128 9 5 - F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f S p a n i s h f i r s howing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s and t h e s a c c u s p r o v i d e d w i t h m i n u t e p r o t r u s i o n s 129 9 6 . T u r g i d p o l l e n o f A t l a s c e d a r shows t h a t t h e s e x i n e f o rms a c o n t i n u o u s l i n e f r o m t h e cap t o t h e s a c c i ... 1 3 0 9 7 • P o l l e n o f A t l a s c e d a r showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s 1 3 0 9 8 . P o l l e n o f A t l a s c e d a r showing m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t s 1 3 0 9 9 . P o l l e n o f A t l a s c e d a r showing t h e g r a n u l a r s u r f a c e o f t h e g e r m i n a l f u r r o w 1 3 0 1 0 0 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f A t l a s c e d a r s howing t h e u n d u l a t i n g s u r f a c e w i t h m i n u t e p r o t r u -s i o n s 131 1 0 1 . P o l l e n o f I n d i a n c e d a r showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s 1 3 2 1 0 2 . P o l l e n o f I n d i a n c e d a r showing t h e s t r a i g h t s t r e a k a c r o s s t h e cap i n t h e d i r e c t i o n o f t h e body l e n g t h .. 1 3 2 1 0 3 . P o l l e n o f Lebanon c e d a r showing t h e wavy s u r f a c e o f t h e cap 132 104. P o l l e n o f Lebanon c e d a r showing t h e g e r m i n a l f u r r o w . 1 3 2 1 0 5 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f I n d i a n c e d a r s howing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s w i t h m i c r o v e r r u c o i d s c u l p t u r e . The s u r f a c e s o f t h e c o r p u s and s a c c u s a r e c o v e r e d w i t h m i n u t e p r o t r u s i o n s 133 X I V 106. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f Lebanon c e d a r showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s . The s u r f a c e s o f t h e c o r p u s and s a c c u s a r e c o v e r e d w i t h m i n u t e p r o t r u s i o n s 134 1 0 7 . T u r g i d p o l l e n o f c o w t a i l f i r 135 108. P o l l e n o f c o w t a i l f i r s howing r u g u l a t e p a t t e r n o f t h e cap s u r f a c e 1 3 5 1 0 9 . P o l l e n o f c o w t a i l f i r showing p o r i i n t h e s a c c i ..... 1 3 5 1 1 0 . P o l l e n o f c o w t a i l f i r w i t h 3 - s a c c a t e 1 3 6 1 1 1 . P o l l e n o f c o w t a i l f i r w i t h 4 - s a c c a t e 1 3 6 1 1 2 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f c o w t a i l f i r s h owing t h e m a r g i n a l a r e a and s a c c u s . The g r a n u l a t e s u r f a c e o f t h e m a r g i n a l a r e a i s sometimes i n t e r s e c t e d b y t h e r i b s f r o m where some m i n u t e p r o t r u s i o n s a r e e v o l v e d . The m i n u t e p r o t r u s i o n s a r e a l s o p r o j e c t e d f r o m t h e s u r f a c e o f g r a n u l e s and t h e wavy s u r f a c e o f s a c c i 137 1 1 3 . F i n e s t r u c t u r e o f t h e cap s u r f a c e o f c o w t a i l f i r p o l l e n showing c o n v o l u t e d s t r u c t u r e w i t h m i n u t e p r o t r u s i o n s 1 3 8 114. T u r g i d p o l l e n o f o r i e n t a l s p r u c e i n o v o i d - c o n i c a l f o r m 139 1 1 5 . T u r g i d p o l l e n o f o r i e n t a l s p r u c e showing t h e s a c c i r e d u c e d t o r a t h e r s m a l l s i z e s 139 116. P o l l e n o f o r i e n t a l s p r u c e showing smooth m a r g i n a l a r e a and p o r i i n t h e s a c c i 1 3 9 1 1 7 . P o l l e n o f o r i e n t a l s p r u c e showing t h e m i c r o v e r r u e a t e s c u l p t u r e 139 118. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f o r i e n t a l s p r u c e showing t h e g r a n u l a t e s u r f a c e w h i c h i s some-t i m e s d e l i m i t e d by t h e r i b s . The m i n u t e p r o t r u s i o n s a r e e v o l v e d e i t h e r f r o m t h e r i b s o r f r o m t h e g r a n u l e s 140 1 1 9 . T u r g i d p o l l e n o f S i t k a s p r u c e i n o v o i d - c o n i c a l f o r m . 141 120. P o l l e n o f S i t k a s p r u c e showing t h e a r c h e d c o r p u s t o e n c l o s e t h e g e r m i n a l f u r r o w . The m a r g i n a l a r e a s a r e g r a n u l a r and p o r i i n t h e s a c c i 141 121. P o l l e n o f S i t k a s p r u c e showing t h e m i c r o v e r r u c a t e s c u l p t u r e and t h e f a i n t l y marked t r i r a d i a t e s t r e a k .. 141 XV 122. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f S i t k a s p r u c e s howing t h e a r e o l a e w h i c h a r e f o r m e d by t h e i n t e r -s e c t i n g r i b s . The m i n u t e p r o t r u s i o n s a r e e v o l v e d e i t h e r f r o m t h e s u r f a c e o f t h e g r a n u l e s o r f r o m t h e r i b s 142 123. T u r g i d p o l l e n o f j a c k p i n e 143 124. P o l l e n o f j a c k p i n e showing t h e m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t s 143 125. P o l l e n o f j a c k p i n e i n a r c h e d f o r m and w i t h o u t m a r g i n a l c r e s t s 143 126. P i n e s t r u c t u r e o f j a c k p i n e p o l l e n s howing g r a n u l a t e s u r f a c e w h i c h i s sometimes d e l i m i t e d by t h e r i b s . M i n u t e p r o t r u s i o n s a r e e v o l v e d e i t h e r f r o m t h e r i b s o r f r o m t h e g r a n u l e s . M i c r o v e r r u c o i d s t r u c t u r e i s shown on t h e c o r p u s and s a c c a t e s u r f a c e 144 127. T u r g i d p o l l e n o f l o d g e p o l e p i n e 145 128. P o l l e n o f l o d g e p o l e p i n e showing m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t s 145 129. P o l l e n o f l o d g e p o l e p i n e showing s a c c i w i t h p o r i . S a c c i t e n d t o bend i n w a r d t o e n c l o s e t h e g e r m i n a l f u r r o w 145 130. P i n e s t r u c t u r e o f t h e c o r p u s s u r f a c e o f l o d g e p o l e p i n e p o l l e n showing t h e g r a n u l a r s u r f a c e w h i c h i s sometimes d e l i m i t e d by t h e r i b s . M i n u t e p r o t r u s i o n s a r e e v o l v e d f r o m t h e r i b s o r f r o m t h e g r a n u l e s . M i c r o v e r r u c o i d s c u l p t u r e i s shown on t h e g r a n u l e s ... 146 131. T u r g i d p o l l e n o f p o n d e r o s a p i n e showing c o n s p i c u o u s m a r g i n a l c r e s t s 147 132. P o l l e n o f p o n d e r o s a p i n e showing m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t 147 133. P o l l e n o f p o n d e r o s a p i n e i n a r c h e d f o r m 147 134. P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f p o n d e r o s a p i n e showing m i c r o v e r r u c o i d s c u l p t u r e . The g r a n u l a t e s u r f a c e o f t h e c o r p u s i s sometimes d e l i m i t e d by t h e r i b s f r o m where t h e m i n u t e p r o t r u s i o n s a r e e v o l v e d .. 148 135. T u r g i d p o l l e n o f s c o t s p i n e showing m a r g i n a l c r e s t s . 149 136. P o l l e n o f s c o t s p i n e showing no m a r g i n a l c r e s t s and s a c c i w h i c h bend I n w a r d t o e n c l o s e t h e g e r m i n a l f u r r o w 149 X V I 1 3 7 * P o l l e n o f s c o t s p i n e showing m i c r o v e r r u c a t e s c u l p t u r e and f a i n t l y marked t r i r a d i a t e s t r e a k 149 1 3 8 . P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f s c o t s p i n e showing m i c r o v e r r u c o i d s c u l p t u r e . M i n u t e p r o t r u s i o n s a r e e v o l v e d e i t h e r f r o m t h e r i b s o r f r o m g r a n u l e s ... 1 5 0 1 3 9 . T u r g i d p o l l e n o f m o u n t a i n hemlock showing s a c c i w h i c h a r e d i s p l a c e d f u r t h e r t o t h e d i s t a l f a c e 151 140. P o l l e n o f m o u n t a i n hemlock showing s a c c i w h i c h a r e r e d u c e d t o r a t h e r s m a l l s i z e s 151 141. P o l l e n o f m o u n t a i n hemlock w i t h v e s t i g a l s a c c i 151 142. P o l l e n o f m o u n t a i n hemlock w i t h o u t s a c c i ... 151 143. P o l l e n o f m o u n t a i n hemlock w i t h abnormal s a c c u s i n u n f r i l l e d f o r m 151 144. P o l l e n o f m o u n t a i n hemlock w i t h abnormal s a c c u s i n f r i l l f o r m 151 145. P o l l e n o f m o u n t a i n hemlock s h o w i n g s a c c i w h i c h bend i n w a r d t o e n c l o s e t h e g e r m i n a l f u r r o w . No m a r g i n a l c r e s t s a r e p r e s e n t 152 146. P o l l e n o f m o u n t a i n hemlock showing cap i n m i c r o -v e r r u c a t e s c u l p t u r e 1 5 2 147. P o l l e n o f m o u n t a i n hemlock showing m i c r o v e r r u c a t e s c u l p t u r e 1 5 2 148. P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f m o u n t a i n hemlock showing g r a n u l a t e s u r f a c e w h i c h i s d e l i m i t e d b y t h e i n t e r s e c t i n g r i b s . S u r f a c e o f g r a n u l e s a r e wavy. M i n u t e p r o t r u s i o n s a r e e v o l v e d e i t h e r f r o m t h e r i b s o r f r o m t h e g r a n u l e s 153 149. T u r g i d p o l l e n o f w e s t e r n hemlock i n t h e e q u a t o r i a l v i e w showing f r i l l and n a r b e 154 1 5 0 . T u r g i d p o l l e n o f w e s t e r n hemlock a t t h e d i s t a l f a c e s howing f r i l l w i t h s p i n u l e s 1 5 4 1 5 1 . T u r g i d p o l l e n o f w e s t e r n hemlock showing t h i c k p u f f y f r i l l 1 5 4 1 5 2 . T u r g i d p o l l e n o f w e s t e r n hemlock w i t h o u t n a r b e 1 5 4 1 5 3 . P o l l e n o f w e s t e r n hemlock i n s a u c e r shape showing n a r b e a t t h e c e n t e r o f t h e d i s t a l f a c e 1 5 4 X V I I 1 5 4 . P o l l e n o f w e s t e r n hemlock a t t h e d i s t a l f a c e showing g r o o v e a t t h e e q u a t o r i a l a r e a 154 1 5 5 . P o l l e n o f w e s t e r n hemlock showing d e t a i l s o f n a r b e and s p i n u l e s 1 5 5 1 5 6 . P o l l e n o f w e s t e r n hemlock showing groove a t t h e e q u a t o r i a l a r e a 155 1 5 7 . P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f w e s t e r n hem-l o c k a t t h e e q u a t o r i a l a r e a showing s p i n u l e s and g r a n u l a t e s u r f a c e w h i c h i s d e l i m i t e d by t h e i n t e r -s e c t i n g r i b s f r o m where t h e m i n u t e p r o t r u s i o n s a r e e v o l v e d 1 5 6 1 5 8 . P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f w e s t e r n hemlock a t t h e d i s t a l f a c e showing s p i n u l e s , r i b s , and m i n u t e p r o t r u s i o n s 1 5 6 1 5 9• T e t r a d s t a g e i n m i c r o s p o r o g e n e s i s o f D o u g l a s - f i r . The young p o l l e n g r a i n s a r e i n t e t r a h e d r a l arrangement .. 157 1 1. INTRODUCTION P r o g r e s s i n t r e e improvement depends l a r g e l y upon a p p l i c a -t i o n o f t h e g r o w i n g knowledge o f g e n e t i c s and o t h e r s c i e n c e s . E a c h t r e e i s made u n i q u e t h r o u g h p o l l i n a t i o n , f e r t i l i z a t i o n and d i f f e r e n t i a t i o n . C o n t r o l l e d p o l l i n a t i o n i s a n e c e s s a r y p a r t o f s t u d y i n g c r o s s i b i l i t y and h y b r i d v i g o r , and o f s e l e c t i v e b r e e d i n g p r o g r a m s . E f f i c i e n t c o l l e c t i o n , h a n d l i n g and s t o r a g e o f p o l l e n and c o n t r o l l e d p o l l i n a t i o n t e c h n i q u e w o u l d be i m p r o v e d by t h e t h o r o u g h u n d e r s t a n d i n g o f p o l l e n f o r m a t i o n , m o r phology and p h y s i o l o g y . T h i s t h e s i s i n v e s t i g a t e s t h e m o r p h o l o g i c a l a s p e c t s o f p o l l e n . I t h a s be e n r e p o r t e d t h a t t h e e x i n e p a t t e r n o f p o l l e n g r a i n s i s d e t e r m i n e d b y t h e s p o r o p h y t e , a l t h o u g h c o n t r o l by t h e gameto-p h y t e c a n n o t be r u l e d o u t ( H e s l o p - H a r r i s o n , 1 9 6 8 ) . No m a t t e r what c o n t r o l s t h i s p a t t e r n , d i f f e r e n c e s i n g e n e t i c make-up m i g h t p r o d u c e d i f f e r e n t p a t t e r n s o f p o l l e n s c u l p t u r e . T h e r e f o r e , s t u d y i n g t h e t o p o g r a p h y o f p o l l e n s u r f a c e s may p e r m i t t h e s e p a r a t i o n o f v a r i o u s t a x a and a s s i s t t h e i n t e r p r e t a t i o n o f t h e i r r e l a t i o n s h i p s . P o l l e n m o r p h o l o g y h as been a d i a g n o s t i c c h a r a c t e r f o r c l a s s i f i c a t i o n o f f o s s i l and l i v i n g p l a n t s . P o l l e n g r a i n s ( a n d s p o r e s ) a l s o o f f e r a means o f c h e c k i n g t h e e v i d e n c e f r o m m i c r o -f o s s i l s as t o t h e c o u r s e o f e a r l y v a s c u l a r p l a n t e v o l u t i o n . There a r e t e n g e n e r a i n t h e P i n a c e a e f a m i l y , a l l n a t i v e t o t h e n o r t h e r n h e m i s p h e r e ( e x c e p t one s p e c i e s i n P i n u s ) . A b i e s i s w i d e l y d i s t r i b u t e d i n E u r o p e , N o r t h A f r i c a , A s i a , and N o r t h A m e r i c a . Cedrus grows i n t h e S y r i a n M t s . , t h e 2 H i m a l a y a s , t h e A t l a s M t s . , and C y p r u s . Two s p e c i e s o f C a t h a y a were r e c e n t l y d i s c o v e r e d i n s o u t h w e s t e r n and c e n t r a l C h i n a . K e t e l e e r i a i s n a t i v e t o C h i n a , where i t i s w i d e l y d i s t r i b u t e d i n c e n t r a l , w e s t e r n , and s o u t h w e s t e r n p r o v i n c e s , and i n Formosa. Ten s p e c i e s o f L a r i x have been d e s c r i b e d i n N o r t h A m e r i c a , E u r o p e , and A s i a , t h o u g h a few o t h e r s have been named i n n o r t h e r n A s i a . E u r o p e , A s i a M i n o r , t h e Caucasus, S i b e r i a , C h i n a , Formosa, J a p a n , t h e H i m a l a y a s and N o r t h A m e r i c a a r e where P i c e a has been f o u n d . The genus o f P i n u s i s d i s t r i b u t e d t h r o u g h o u t t h e n o r t h e r n h e m i s p h e r e and r e a c h e s i t s s o u t h e r n l i m i t a t about 2P s o u t h l a t i t u d e on Sumatra. P s e u d o l a r i x i s n a t i v e t o e a s t e r n C h i n a . S i x s p e c i e s o f P s e u d o t s u g a have b e e n r e p o r t e d , two b e i n g n a t i v e t o w e s t e r n N o r t h A m e r i c a , t h e r e m a i n d e r o c c u p y i n g a l i m i t e d r a n g e i n s o u t h e a s t e r n and s o u t h w e s t e r n C h i n a , Formosa and J a p a n . Tsuga o c c u r s i n tem-p e r a t e N o r t h A m e r i c a , J a p a n , Formosa, C h i n a and t h e H i m a l a y a s . I n f o r m a t i o n on t h e m i c r o s c o p i c s t r u c t u r e o f p o l l e n g r a i n s i s s c a n t y . A l t h o u g h many s p e c i e s o f c o n i f e r s have b e e n s t u d i e d b y t h e p a l y n o l o g i s t , o n l y few s p e c i e s i n P i n a c e a e have b e e n i n v e s t i g a t e d by t h i n s e c t i o n and c a r b o n r e p l i c a methods. Because b a s i c i n f o r m a t i o n i n t h i s a r e a was l a c k i n g , an i n t r o -d u c t o r y s t u d y o f t h e p o l l e n o f s e l e c t e d s p e c i e s i n P i n a c e a e was u n d e r t a k e n . The o b j e c t i v e was t o p r o v i d e i n f o r m a t i o n on: ( 1 ) a c a r b o n r e p l i c a method f o r t h e s t u d y o f t h e t o p o g r a p h y o f p o l l e n s u r f a c e s ; (2) p o l l e n m o r p hology and p o l l e n s i z e measurements by l i g h t m i c r o s c o p y ; 3 (3) t h e f o r m a t i o n o f D o u g l a s - f i r ( P s e u d o t s u g a m e n z i e s i i ( M i r b . ) F r a n c o ) e x i n e i n t h e l a t e s t a g e o f microspor©genesis; ( 4 ) t h e v a r i a t i o n s i n t o p o g r a p h y o f t h e p o l l e n s u r f a c e i n t h e P i n a c e a e f a m i l y d e m o n s t r a t e d b y t r a n s m i s s i o n and s c a n n i n g e l e c t r o n m i c r o s c o p y . 4 2 . LITERATURE REVIEW A. L i g h t m i c r o s c o p i c s t u d i e s o f t h e p o l l e n i n P i n a c e a e . The s t u d y o f p o l l e n m o r p h o l o g y i s as o l d as most o t h e r b r a n c h e s o f b o t a n i c a l s c i e n c e s . S i n c e t h e d i s c o v e r y o f t h e m i c r o s c o p e t h e s t r u c t u r a l u n i q u e n e s s o f p o l l e n g r a i n s h a s p r o g r e s s i v e l y been r e v e a l e d . The m o r p h o l o g i c a l c h a r a c t e r i s t i c s a r e r e p r e s e n t a t i v e o f t h e v a r i o u s t a x a . However, t h e f i n e s t r u c t u r e o f c o n i f e r o u s p o l l e n i s l a c k i n g . P o l l e n p r e p a r a t i o n s a r e u s u a l l y made by t h e a c e t o l y s i s method i n w h i c h t h e p o l l i n i f e r o u s m a t e r i a l i s t r e a t e d w i t h a m i x t u r e o f n i n e p a r t s o f a c e t i c a n h y d r i d e and one p a r t o f c o n -c e n t r a t e d s u l f u r i c a c i d ( E r d t m a n , 1 9 5 2 ) . The advantage o f t h i s method i s t h a t i t a l l o w s f o r g r e a t e r c l a r i t y o f d e t a i l s o f e x i n e s t r u c t u r e . P o l l e n g r a i n s o f A b i e s , C e d r u s . K e t e l e e r i a , P i c e a , P i n u s , and some s p e c i e s o f Tsuga ( e . g . T. m e r t e n s i a n a ) a r e b i l a t e r a l , a n a l e p t , 2 - s a c c a t e * . P o l l e n g r a i n s o f A b i e s and K e t e l e e r i a have no m a r g i n a l c r e s t s b u t do have a d i s t i n c t d e m a r c a t i o n l i n e between c a p p a and c a p p u l a . A t r i r a d i a t e s t r e a k i s f a i n t l y marked i n A b i e s , b u t n o t i n K e t e l e e r i a ( E r d t m a n , 1 9 6 5 ) . S a c c i o f Cedrus and P i c e a a r e a l w a y s more l a t e r a l l y p l a c e d , l e a v i n g a b r o a d e r and l o n g e r f u r r o w a r e a between them. P o l l e n s i z e o f Cedrus i s s m a l l e r t h a n t h a t o f P i c e a ( E r d t m a n , 1 9 6 5 ; Wodehouse, 1 9 5 9 ) . P i n u s p o l l e n has capp a and c a p p u l a , and i n s e v e r a l s p e c i e s t h e y have more o r l e s s d i s t i n c t m a r g i n a l c r e s t s ( E r d t m a n , 1 9 6 5 ) . The p r o x i m a l s i d e i s c o v e r e d w i t h a t h i c k * G l o s s a r y i s p r o v i d e d f o r t h e te r m s o f p a l y n o l o g y . 5 rugged exine marked "by a heavy, coarse, reticulate granular structure, while the d i s t a l side has an exceedingly thin and f l e x i b l e exine. Larix and Pseudotsuga pollen grains are spheroidal, paraisopolar and analept without furrow, pores or sacci (Erdtman, 1 9 6 5 ; Wodehouse, 1959). The former i s provided with a proximal annular thickening, and the l a t t e r with a tri r a d i a t e streak (Ueno, 1958, 1960). The type of pollen grain found i n other species of Tsuga (e.g., T. heteronhvlla) i s thick-lens-shaped, anatreme, radio-symmetric and more frequently provided with a circular puffy f r i l l (Erdtman, 1 9 6 5 ; Wodehouse, 1 9 5 9 ) . The exine at the proximal face i s thick, reticulate-corrugated; at the d i s t a l face i t i s thinner, less coarse. Studies of the pollen i n Pinaceae reported by these and some other authors are compiled i n Table 1. B. Electron microscopic studies of the pollen i n Pinaceae. (1). Replica techniques for transmission electron microscope. The i n i t i a l suggestion that plastic films could be used for electron microscopic examination of surfaces was f i r s t made by Mahl (1940). Two years later, Pormvar was introduced for making films (Schaefer and Harker, 1942). Heidenreich and Peck (1943) introduced a two-step replica process. A molded polystyrene was u t i l i z e d as the f i r s t r e p l i c a and then s i l i c a was evaporated as the second, thin fi l m r e p l i c a . Table 1 Reports on the light microscopic studies of the pollen i n Pinaceae Species Abies grandis Cedrus atlantica C. deodara .Description Exine of cap, thick and bumpy; no marginal crests; t r i -radiate streak. 2-saccate; marginal crests occasionally present, leptoma smooth; proximal exine thick, undulating; small puncta in saccus. 1-aperturate small sacci, broader and longer furrow; cap circular; marginal crests scarcely developed. Size (ju) e*70x130x100 78x90 Authors Erdtman ( 1 9 6 5 ) Wodehouse ( 1 9 5 9 ) c*45.8x61.5x54,9 Ayfcug (1961) s*37.4x52.7x31.3 c*55x72x60 Erdtman (1965) s*28x72x60 0*43.4x56.2x47.4 Aytug (1961) S*37i1x47.4x27.2 c*54x66x60 Erdtman (1965) s*20x64x47 53x60 70x94x83 c*70x60x68 s*40x50x69 66 65 Ikuse (1956) Nair (1965) Ueno (1958) Campo-Duplan (1950) Wodehouse (1959) c* : corpus s* : saccus Species C. l i b a n i Keteleeria davidiana Larix decidua L. gmelinli L. gmelinii var. .japonic a L. l a r i c i n a L. leptolepis Description Exine thick; leptoma without flecks Exine thick; leptoma with fa i n t l y marked flecks Stray grains with 3 or 4-saccate 1-aperturate Sexine thicker than nexine, no endosexinous rods analept, non-saccate, spheroi dal, exine thin, verrucoid Annular thickening Sexine surface not smooth exine smooth Exine surface undulating, baculum-like rods protrude from nexine Spheroidal Spheroidal, exine thin, intine thick; furrows, pores, and bladders absent Aperturate Exine smooth Monotreme, spheroidal, laesuroid Size (u) c*48.3x58,0x50.9 s*4Q.-4x50.5x34.5 c*54x68x62 s*29x64x50 c*52x70x72 s*29x67x53 c*70x125x85 82x84 c*110x110x98 s*50x64x98 84 80-85 - 90 105 69 65 85-115 71.4x81.6 90 60-90 62.7-80 90 67- 71 83 75 68- 70 Authors Aytug (1961) Erdtman (1965) Erdtman (1965) Erdtman (1965) Ikuse (1956) Ueno (1958) Campo-Duplan (1950) Erdtman (1965) Erdtman, Berglund & Praglowski (1961) Campo-Duplan (1947) Erdtman (1965) Ueno (1958) Campo-Duplan (1950) Erdtman (1965) Chandler and Navrodineanu (1965) Erdtman (1965) Kapp (1969) Wodehouse (1959) Erdtman (1965) Ikuse (1956) Ueno (1958) Campo-Duplan (1950) Yamazaki & Takeoka (1962) Species L. l v a l l i i L. occidentalis L. potaninii L. s i b i r i c a L. s i b i r i c a var baicalensis  Picea sitchensis Pinus banksiana Description Spheroidal, exine thin, intine thick, furrow, pores and bladders absent Sexine thicker than nexine, annular thickening at the proximal face Non-saccate, spherical or e l l i p t i c a l ; exine thin and smooth; annular thickening at the proximal face Sacci small; furrow as a shallow groove; cap circular; no trace of marginal crests; texture of the cap very fine Small 1-aperturate Analept; exine 0.5 Ji at the di s t a l , 2.5-3 u at the proximal pole Exine 0.5 P- at the dis t a l 3 u at the proximal pole; roughness of the surface due to funnel-shaped depressions. Small; cap circular to broad-l y e l l i p t i c a l ; marginal crests best developed. Size (u) 85.5-102 85 70 85 73.3x87.5 8 5 x 9 0 70x78 Author Wodehouse (1959) Erdtman (1965) Erdtman (1965) Ho & House (1970) Erdtman (1965) Wodehouse (1959) Cain (1940) 03 c*31x45x35 s*19x40x32 37-39x43-44 Ikuse (1956) 0*41.0x53.0x48.1 Aytug (1962) s*32.3x41.6x30.0 c*45x55x38 Erdtman, Berglund s*31x37x42 & Praglowski (1961) c*38x42x37 s*31x37x42 44.2x52 Praglowski (1962) Wodehouse (1959) Species Pseudotsuga .japonica P. menziesn P. wilsoniana Tsuga heteronhvlla T. mertensiana Description Exine smooth Proximal-triradiate streak, sexine thicker than nexine Spherical or e l l i p t i c a l without "bladders or furrows, exine smooth and thin Spheroidal, exine psilate Triradiate streak Spheroidal without bladders, pores or furrows, exine smooth Without bladders, aperturate exine verrucate Spherical i n polar, thick-lens-shaped i n side view; narbe at the centre of the d i s t a l face. Bilateral, 2-saccate, without operculum and spinules Size (u) 85 80-85 94.3x128.9 100-115 91.1 -99.2 140 90-100 77-83x70-87 60.5x75-5 65 60 45x78x70 64 69 c*70x58x68 s*20x31x66 Author Ueno ( 1 9 5 8 ) Yamazaki & Takeoka ( 1 9 6 2 ) Ho & S z i k l a i ( 1 9 7 2 ) Kapp (1969) Szi k l a i (1964) Campo-Duplan (1947) Wodehouse ( 1 9 5 9 ) Wang ( 1 9 6 6 ) Ho & S z i k l a i ( 1 9 7 1 ) Ueno ( 1 9 5 7 ) Campo-Duplan ( 1 9 5 0 ) ^ Erdtman ( 1 9 6 5 ) Campo-Duplan ( 1 9 5 0 ) Ueno ( 1 9 5 7 ) Ueno ( 1 9 5 8 ) 1 0 The image c o n t r a s t i n o p t i c a l and e l e c t r o n m i c r o g r a p h s was v e r y p o o r f r o m a l l t h e s e r e p l i c a p r e p a r a t i o n s . I t was n o t i m p r o v e d u n t i l W i l l i a m s and W y c k o f f ( 1 9 4 6 ) d e v e l o p e d a new m e t a l s h a d o w - c a s t i n g t e c h n i q u e whereby t h e c o n t r a s t o f images was g r e a t l y i n c r e a s e d b y d e p o s i t i n g o b l i q u e l y a t h i n f i l m o f m e t a l on t h e m i c r o s c o p i c p r e p a r a t i o n s . A t h i n c a r b o n f i l m f o r t h e r e p l i c a was f i r s t i n t r o d u c e d by K o n i g and H e l w i g (195*0 . The method o f B r a d l e y ( 1 9 5 4 ) d i f f e r e d f r o m t h a t u s e d by them i n t h a t t h e c a r b o n was d e p o s i t e d d i r e c t l y b y vacuum e v a p o r a t i o n . S i t t e ( 1 9 5 3 ) was t h e p i o n e e r i n t h e s t u d y o f p o l l e n w a l l s by s u r f a c e r e p l i c a t e c h n i q u e s . He s t u d i e d t h e s u r f a c e o f p o l l e n and s p o r e e x i n e s by u s i n g o n e - s t e p c o l l o d i o n r e p l i c a t e c h n i q u e s . The e x c e l l e n t s e r i e s o f e l e c t r o n m i c r o g r a p h s o f p o l l e n w a l l s u r f a c e r e p l i c a s r e p o r t e d by M u h l e t h a l e r ( 1 9 5 5 ) were p r e p a r e d by t h e c a r b o n e v a p o r a t i o n method. Carbon i s e s p e c i a l l y s u i t e d f o r t h e p r e p a r a t i o n o f s u p p o r t i n g membranes and r e p l i c a s f o r e l e c t r o n m i c r o s c o p y b e c a u s e o f i t s c o n d u c t i v i t y , e l e c t r o n 11 t r a n s p a r e n c y and l a c k o f s t r u c t u r e ( B r a d l e y , 1 9 5 4 ; M u h l e t h a l e r , 1 9 5 5 ) . V a r i o u s t e c h n i q u e s have been i n t r o d u c e d f o r t h e r e p l i c a -t i o n o f s m a l l specimens by B r a d l e y ( 1 9 5 7 ) . I n 1 9 5 8 , he d e v e l o p e d a d o u b l e - e v a p o r a t i o n c a r b o n r e p l i c a t e c h n i q u e f o r t h e p o l l e n g r a i n . A specimen g r i d was c o a t e d w i t h a Formvar f i l m and t h e s p e c i m e n was mounted on i t . The g r i d was t h e n c o a t e d w i t h 100 A o f c a r b o n f i l m . The Formvar f i l m was washed away, 11 l e a v i n g t h e spe c i m e n embedded i n t h e c a r b o n f i l m w i t h a s m a l l a r e a o f i t s s u r f a c e exposed. The g r i d was i n v e r t e d and a s e c o n d l a y e r o f c a r b o n (150-200 A) d e p o s i t e d on t h e exposed s u r f a c e o f t h e spe c i m e n . The g r i d was t h e n p l a c e d i n a few d r o p s o f s t r o n g s o l u t i o n o f p o t a s s i u m d i c h r o m a t e and permanga-n a t e i n c o n c e n t r a t e d s u l f u r i c a c i d . The spe c i m e n s w e l l e d and n o r m a l l y b r o k e t h e f i r s t c a r b o n f i l m . The g r i d was removed f r o m t h e s o l u t i o n , c o m p l e t e l y immersed i n w a t e r , t r a n s f e r r e d t o s t r o n g h y d r o c h l o r i c a c i d , and f i n a l l y washed t w i c e i n w a t e r . S h a d o w - c a s t i n g was c a r r i e d out t h r o u g h t h e g r i d b a r s . F l y n n and Rowley (1967) a l s o i n t r o d u c e d a s i n g l e - s t a g e r e p l i c a method f o r p o l l e n e x i n e s . F r e s h , d r y o r c h e m i c a l l y t r e a t e d p o l l e n was washed w i t h 5 0 p e r c e n t a c e t o n e . Drops o f t h e s u s p e n s i o n were p l a c e d on f r e s h l y c l e a v e d m i c a s u r f a c e s and 3 5 0 A o f c a r b o n was e v a p o r a t e d onto t h e m i c a . The f i l m was s c o r e d and f l o a t e d on c l e a n w a t e r , t r a n s f e r r e d t o 2 - a m i n o e t h a n o l a t n o t h i g h e r t h a n 5 0°C and t h e n s l o w l y h e a t e d t o 145 - 1 5 5°C f o r 10 m i n s . t o 3 h o u r s d e p e n d i n g on t h e c h e m i c a l r e s i s t a n c e o f t h e e x i n e . The f i l m was t r a n s f e r r e d t o w a t e r a t 90°C f o r 5 m i n u t e s . F i n a l l y i t was f l o a t e d on a c l e a n w a t e r s u r f a c e and p i c k e d up by t h e g r i d . R e p l i c a t e c h n i q u e s r e p o r t e d by some o t h e r a u t h o r s a r e shown i n T a b l e 2. 12 T a b l e 2 R e p o r t s on t h e r e p l i c a t e c h n i q u e s f o r p o l l e n s t u d i e s . Y e a r A u t h o r 1 9 5 3 Fukami 1 9 5 4 Fukami 1 9 5 5 Bringmann & Kuhn 1 9 5 6 Hess & S c h a n t z 1 9 5 7 Yamazaki & Takeoka 1 9 5 8 H a r a d a , M i y a z a k i , & Wakashima 1960 Fukami & A d a c h i 1 9 6 3 Takeoka & S t i x 1 9 6 5 Fukami & A d a c h i 1 9 6 6 Rowley & F l y n n D e s c r i p t i o n i n t r o d u c e d e t h y l c e l l u l o s e as b a c k i n g f i l m , t w o - s t a g e r e p l i c a t e c h n i q u e s , u s i n g c o l l o d i o n and e t h y l -c e l l u l o s e f i l m d i r e c t use o f t h e p o l l e n e x i n e gum a r a b i c s i n g l e s t a g e r e p l i c a m e t h y l m e t h a c r y l a t e c a r b o n two-s t a g e r e p l i c a t w o - s t a g e r e p l i c a , u s i n g m e t h y l m e t h a c r y l a t e , c a r b o n o r a l u m i n i u m u s e d epoxy r e s i n by s e l e c t i v e -l y d e p o s i t i n g on t h e uneven a r e a o f t h e r e p l i c a f i l m t o i m p r o v e t h e c o n t r a s t t w o - s t a g e r e p l i c a , u s i n g me-t h y l m e t h a c r y l a t e and c a r b o n u s e d epoxy r e s i n w i t h p h o s -p h o t u n g s t i c a c i d t o i m p r o v e t h e c o n t r a s t s i n g l e - s t a g e r e p l i c a , u s i n g c a r b o n 13 ( 2 ) . R e p o r t s o f p o l l e n s u r f a c e s t r u c t u r e b y t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e . Of a l l t h e s p e c i e s i n A b i e s , o n l y one has been s t u d i e d b y r e p l i c a t e c h n i q u e ( Y a m a z a k i and Takeoka, 1 9 6 2 ) . The s u r f a c e o f t h e c o r p u s o f A. f i r m a S i e b . e t Z u c c . ( J a p a n e s e f i r ) was d e s c r i b e d as b e i n g c o v e r e d w i t h " c a u l i f l o w e r - s h a p e d p r o t r u s i o n s " and d o t t e d w i t h many s m a l l o p e n i n g s and a few m i n u t e s p i n u l e s . The s a c c i were smooth and p r o v i d e d w i t h many s m a l l o p e n i n g s and w a v e - l i k e u n d u l a t i o n s . The c o r p u s and s a c c u s were s m o o t h l y l i n k e d w i t h i n c o n s p i c u o u s p r o t r u s i o n s . The d i a m e t e r o f t h e s m a l l o p e n i n g s was about 3 0 - 2 0 0 mu; t h e l e n g t h o f t h e s p i n u l e s on t h e c a u l i f l o w e r - s h a p e d p r o t r u s i o n s , about 3 0 mu. The p r o -c e s s e s on t h e s u r f a c e o f t h e g e r m i n a l f u r r o w were about 0 . 3 -0 . 8 u l o n g . Y amazaki and Takeoka ( 1 9 5 9 ) showed t h a t t h e p o l l e n s u r f a c e o f K e t e l e e r i a d a v i d i a n a ( B e r t r . ) B e i s s n . ( c o w t a i l f i r ) was c o v e r e d w i t h p r o t r u s i o n s o f t h e " c a u l i f l o w e r t y p e " and some o f t h e p r o t r u s i o n s were s i m i l a r t o "forms o f p i l a " . The s u r f a c e o f t h e p r o t r u s i o n s h a d f i n e s p i n u l e s ( a b o u t 0 . 1 p.) and was d o t t e d w i t h s m a l l o p e n i n g s ( a b o u t 0 . 1 u ) . The f i n e s u r f a c e s t r u c t u r e o f L a r i x l e n t o l e n i s S i e b . and Z u c c . ( J a p a n e s e D o u g l a s - f i r ) h a s been s t u d i e d by Yamazaki and Takeoka ( 1 9 6 2 ) . The s u r f a c e was d e s c r i b e d as b e i n g i r r e g u l a r , w i t h s l i g h t l y v e r r u c o i d p r o c e s s e s i n t e r s p e r s e d w i t h o r b i c u l o i d g r a n u l e s . The o r b i c u l o i d g r a n u l e i t s e l f a l s o had v e r y s m a l l v e r r u c o i d o u t g r o w t h s . 14 P i c e a . i e z o e n s i s ( S . & Z.) C a r r . ( y e z o s p r u c e ) p o l l e n s u r f a c e h a s "been s t u d i e d i n d e t a i l "by Yamazaki and Takeoka ( 1 9 5 9 ) * The p o l l e n s u r f a c e i s o f t h e same c a u l i f l o w e r t y p e as d e s c r i b e d i n J a p anese f i r . The d i a m e t e r o f t h e o p e n i n g s i n t h e e x i n e i s about 3 0 t o 2 0 0 mu and t h e s p i n u l e s about 3 0 mu i n l e n g t h . The s u r f a c e i n t h e g e r m i n a l f u r r o w has g r a n u l o i d p r o c e s s e s . P i n u s s v l v e s t r i s L. ( s c o t s p i n e ) was t h e i r s t s p e c i e s o f c o n i f e r t o be u s e d f o r r e s e a r c h i n g a method t o examine t h e s c u l p t u r e o f p o l l e n s u r f a c e s ( S i t t e , 1 9 5 5 ) * I t was i n d i c a t e d t h a t t h e s u r f a c e had a " r e g u l a r , f i e l d - l i k e s c u l p t u r e " . M u h l e t h a l e r ( 1 9 5 5 ) s t u d i e d t h e p o l l e n g r a i n o f P. n i g r a A r n . ( black" v / . p i n e ) by b o t h t h i n s e c t i o n and r e p l i c a t e c h n i q u e s . The p o l l e n s u r f a c e o f A u s t r i a n p i n e was i r r e g u l a r and had "tumor-like s t r u c t u r e s " w i t h some f i n e g r a n u l e s . P. t h u n b e r g i i F r a n c o ( J a p a n e s e b l a c k p i n e ) p o l l e n has been i n v e s t i g a t e d by r e p l i c a t e c h n i q u e s ( Y a m a z a k i and Takeoka, 1 9 5 7 ) - The s u r f a c e o f t h e c o r p u s c o n s i s t e d o f m a r g i n a l r i d g e s o f complex s t r u c t u r e and was d o t t e d w i t h v e r y s m a l l o p e n i n g s , w h i l e t h a t o f t h e s a c c i showed an even s t r u c t u r e w i t h v e r y s m a l l o p e n i n g s . Yamazaki and Takeoka ( 1 9 5 8 , 1 9 6 2 ) f o u n d t h a t t h e s u r f a c e o f t h e c o r p u s o f P. d e n s i f l o r a S i e b . and Z u c c . ( J a p a n e s e r e d p i n e ) was c o v e r e d w i t h " c a u l i f l o w e r - s h a p e d p r o c e s s e s " w h i c h were p r o v i d e d w i t h some m i n u t e s p i n u l e s , w h i l e t h a t o f s a c c i had s m a l l open-i n g s and m i n u t e s p i n u l e s . 15 By thin section study, Gullvag (1966) reported that the outer layer of the exine of Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) pollen consisted of a three-dimensional network of rounded bars, and the inner was a laminated layer. Pollen of P. .japonica (Shiras.) Beiss. (Japanese Douglas-fir) had a thick granular sexine (Ueno, 1958)* From a re p l i c a study, Yamazaki and Takeoka (1962) reported that the surface of the d i s t a l part of Japanese Douglas-fir was "cauliflower-shaped" with small spinuloid processes, about 20 to 60 mu i n length. The proximal face differed from the d i s t a l face i n only having small, densely spaced spinules and some traces of irregular cracks. Tsuga si e b o l d i i Carr. (Japanese hemlock) i s the only species i n this genus having been studied by replica techniques (Yamazaki and Takeoka, 1958a, 1962). It was stated that the proximal face as well as the d i s t a l face were covered with irregular spaced projections which varied i n length. The exine between the marginal f r i l l and the germinal furrow was covered with minute, irregular processes different from those i n the d i s t a l and proximal faces. ( j ) . The application of the scanning electron microscope to pollen studies. The scanning electron microscope can be used to great advantage for the study of biological specimens which are too small, or have too complicated a surface, to allow the use of r e p l i c a techniques with transmission electron microscope. The technique reported by Thornhill et _al. (1965) for the scanning 16 electron microscope provides great depth of focus and a three-dimensional appearance. Various specimen preparations have "been applied to different materials. The specimen stub i s l i g h t l y coated with a glue, and the sample sprinkled on the surface when the adhesive becomes tacky (Echlin, 1968a). Some specimens with biological polymers are suspended i n l i q u i d . !2he sample may be placed d i r e c t l y onto the specimen stub and the l i q u i d allowed to dry (Echlin, 1968a). For preparing l a b i l e material, freeze drying has proved to be the best method of examination (Echlin, 1968a). In the case of pollen grains, specimens are dusted onto a specimen stub which holds a piece of double-stick Scotch tape (Ridgway and Skvarla, 1969; Hanks and Fairbrothers, 1970). For examining transverse sections of pollen grains to see the con-struction of the various layers, the sectioned specimens are transferred to the specimen stub (Echlin, 1968a). During the examination, the specimens gradually build up a charge which may distort the image. It i s thus necessary to coat the specimens with a fi l m of conducting substance to allow the charge to leak away and to enhance the secondary emission coefficient. It i s also not thick enough to decrease the resolu-tion. Thorahill ejt a l . (1965) applied a fi l m of gold 500 A thick; Echlin (1968a), a 300-500 A thick gold-palladium alloy; Hanks and Fairbrothers (1970), a 200 A thick layer of gold. Chromium was used for coating by Ridgway and Skvarla (1969). A t r i p l e evaporation of carbon on gold on carbon was applied on 17 r o u g h s u r f a c e s by P f e f f e r k o r n ( 1 9 6 9 ) . The u n d e r l y i n g c a r b o n l a y e r was 1 0 0 - 2 0 0 A t h i c k . The g r e a t p e r f o r m a n c e o f t h e s c a n n i n g e l e c t r o n m i c r o s c o p e i s w e l l known, b u t t h e equipment i s n o t w i d e l y a v a i l a b l e . There a r e o n l y two r e p o r t s on t h e m i c r o s c o p i c s t r u c t u r e o f t h e p o l l e n i n P i n a c e a e by t h e a p p l i c a t i o n o f t h e s c a n n i n g e l e c t r o n m i c r o -s c o p e . M a r t i n and Drew ( 1 9 6 9 ) s u g g e s t e d t h a t i t m i g h t be p o s s i b l e t o s e p a r a t e t h e f r e s h p o l l e n g r a i n s o f p i n y o n p i n e s ( P i n u s c e m b r o i d e s Z u c c . and P. e d u l i s Engelm.) f r o m t h o s e o f p o n d e r o s a and l o d g e p o l e p i n e s ( P . p o n d e r o s a Laws, and P. c o n t o r t a D o u g l . ) u s i n g s c a n n i n g e l e c t r o n m i c r o g r a p h s . The f o r m e r e x h i b i t -ed a r u g u l a t e c ap n e a r t h e p o i n t o f s a c c a t e a t t a c h m e n t , w h i l e t h e l a t t e r h a d a m i c r o v e r r u c a t e c a p . They a l s o showed s t r u c t u r a l d e t a i l s o f t h e i n n e r r e t i c u l u m o f t h e p i n e s a c c u s f r o m p a r t l y d e g r a d e d f o s s i l p o l l e n . The i n n e r r e t i c u l u m u n d e r l a i d t h e s a c c a t e w a l l o n l y . I t d i d n o t e x t e n d o v e r t h e body o f t h e g r a i n . The mesh was made up o f l o o s e l y p a c k e d s h o r t r o d s 0 . 3 p-i n d i a m e t e r and e n c l o s i n g l a c u n a e up t o 1 u i n l e n g t h . Engelmann s p r u c e ( P i c e a engelmanni i P a r r y ) p o l l e n was d e s c r i b e d as h a v i n g b a r e l y e v i d e n t m i c r o v e r r u c a t e t e x t u r e on t h e cap ( M a r t i n and Drew, 1 9 6 9 ) . The smooth s a c c a t e - c a p a t t a c h m e n t and h i g h e r r a t i o o f s a c c a t e s i z e t o c o r p u s s i z e d i s t i n g u i s h e d s p r u c e f r o m w h i t e f i r ( A b i e s c o n c o l o r ( G o r d . & G l e n d ) L d l . ) p o l l e n . The cap o f t h e a c e t o l y z e d f i r p o l l e n a p p e a r e d p e r f o r a t e d w i t h e l o n g a t e d g r o o v e s and p i t s , 0 . 9 by 0 . 2 u w i d e . A d i s t i n c t a n n u l a r t h i c k e n i n g 1 . 0 u o r more i n w i d t h was f o u n d i n t h e p o l l e n g r a i n s o f D o u g l a s - f i r ( M a r t i n and Drew, 1 9 7 0 ) . A t h i g h m a g n i f i c a t i o n , t h e s u r f a c e o f t h e e x i n e was n o t 18 smooth, but f a i n t l y pebbled ( m i c r o v e r r u c a t e ) , w i t h i n d i v i d u a l elements about 0.G6 u i n diameter. 3. MATERIAL P o l l e n g r a i n s from two s p e c i e s i n A b i e s , t h r e e i n Cedrus, one i n K e t e l e e r i a . e i g h t i n L a r i x . two i n P i c e a . f o u r i n P i n u s . f o u r i n Pseudotsuga. and two i n Tsuga were used i n t h i s study. The dates of c o l l e c t i o n and the p o l l e n sources are shown i n Table 3- A l l the p o l l e n g r a i n s were s t o r e d i n c l o s e d v i a l s and p l a c e d i n a d e s i c c a t o r w i t h c a l c i u m c h l o r i d e at 0-2°C i n the c o l d room. 4. METHODS A. P o l l e n C o l l e c t i o n M i c r o s p o r a n g i a t e s t r o b i l i of most of the sp e c i e s were c o l l e c t e d from t r e e s on the U n i v e r s i t y of B r i t i s h Columbia (U.B.C.) campus. Twigs w i t h s t r o b i l i were cut from t r e e s at a n t h e s i s . P o l l e n was e x t r a c t e d from the s t r o b i l i at room temperature. B r a n c h l e t s from D o u g l a s - f i r were a l s o c u t , one and two weeks before the p o l l e n was shed; these were p l a c e d i n a j a r w i t h water and the e x t r a c t i o n of p o l l e n g r a i n s from the s t r o b i l i was f o r c e d i n the separate room. I n D o u g l a s - f i r , the c o l o r of macrosporangiate s t r o b i l i of the seven s e l e c t e d t r e e s were c l a s s i f i e d i n t o f i v e c l a s s e s : deep green (GG), p a l e green (GGR), g r e e n i s h r e d (GR), r e d (GRR), and b r i g h t r e d (RR), based on the c l a s s i f i c a t i o n used by G r i f f i t h (1968). D o u g l a s - f i r t r e e numbers ( S z i k l a i , 1964), c o l o r c l a s s e s 19 T a b l e 3 D a t e s and s o u r c e s o f p o l l e n c o l l e c t i o n s i n P i n a c e a e . S p e c i e s A b i e s g r a n d i s L d l . (.grand f i r } A. p i n s a n o B o i s s . rSpanish f i r } Cedrus a t l a n t i c a M a n e t t i ( A t l a s c e d a r } C. d e o d a r a (Roxb.) Loud. ( I n d i a n c e d a r ) C. l i b a n i L o u d . ^Lebanon c e d a r ) K e t e l e e r i a d a v i d i a n a ( B e r t r . } B e i s s n . ( c o w t a i l f i r ) L a r i x d e c i d u a M i l l . ( E u r o p e a n l a r c h ) L. g m e l i n i i ( R u p r . ) ( D a h u r i a n l a r c h ) L. l a r i c i n a ( D u R o i ) K. K o c h , ( t a m a r a c k ) B. l e p t o l e p i s TS. & Z.) G o r d . ( J a p a n e s e l a r c h ) L. l v a l l i i P a r i . T a l p i n e l a r c h ) B. o c c i d e n t a l i s N u t t . ( W e s t e r n l a r c h } L. p n t n n i T i i i B a t a l . ( C h i n e s e l a r c h } L. s i b i r i c a (Munchh.) Ledeb. ( S i b e r i a n l a r c h ) Date o f C o l l e c t i o n S o u r c e A p r . , 17, 1970 A p r . , 13, 1970 S e p t . 15, 1969 O c t . , 1, 1969 O c t . , 1, 1969 Feb., 18, 1970 Mar., 24, 1969 May, 1, 1969 May, 2, 1970 May, 2, 1969 S p r i n g , 1964 Mar., 1968 A p r . , 30, 1970 Mar., 24, 1970 UBC UBC UBC UBC UBC Formosa Vancouver H o k k a i d o , J a p a n UBC Yamanashi,Japan Montana Idaho S c o t l a n d UBC c o n t i n u e d . . . . S p e c i e s P i c e a o r i e n t a l i s ( L . ) C a r r . ( o r i e n t a l sprue e) P». s i t c h e n s i s (Bong.) C a r r . ( S i t k a s p r u c e ) P i n u s b a n k s i a n a Lamb, (jack p i n e ) P. c o n t o r t a D o u g l . Tlodgepole p i n e ) P. p o n d e r o s a Laws. Tponderosa p i n e ) P. s v l v e s t r i s L. Tscots p i n e ) P s e u d o t s u g a .japonica ( S h i r a s . ) B e i s s n . ( J a p a n e s e D o u g l a s - f i r ) P. m a c r o c a r p a T V a s e y ) Mayr ( b i g c o n e D o u g l a s - f i r ) P. m e n z i e s i i ( M i r b . ) P r a n c o ( D o u g l a s - f i r ) P. w i l s o n i a n a H a y a t a (Pormosan D o u g l a s - f i r ' Tsuga h e t e r o p h i l ! a ( E a f . ) S a r g . ( w e s t e r n h e m l o c k ) T. m e r t e n s i a n a (Bong.) C a r r . ( m o u n t a i n h e m l o c k ) 2 0 Date o f C o l l e c t i A p r . , 1 7 , 1 9 7 0 May, 5 , 1 9 7 0 May, 6 , 1 9 6 9 May, 1 3 , 1 9 6 9 May, 6 , 1 9 6 9 May, 1 3 , 1 9 6 9 L a t e Mar. 1 9 7 0 Mar., 2 5 , 1 9 6 9 Mar., 2 7 , 1 9 6 8 May, 1 9 6 7 Feb., 2 0 , 1 9 7 0 May, 2 5 , 1 9 6 8 June, 2 , 1 9 7 0 S o u r c e UBC UBC UBC UBC UBC UBC M i e - k e n , J a p a n ( a ) Oregon ( b ) C a l i f o r n i a ( a ) UBC ( s e e T a b l e 4) ( b ) Van. I s l a n d ( c ) McLeod L a k e , B.C. Formosa UBC Seymour Mt., Vancouver 21 o f m a c r o s p o r a n g i a t e s t r o b i l i , t h e d a t e s o f b r a n c h l e t s c u t , and d a t e s o f p o l l e n c o l l e c t e d a r e c o m p i l e d i n T a b l e 4. B. P o l l e n s i z e s t u d i e s P o l l e n g r a i n s w h i c h were e l l i p t i c a l - s h a p e d were measured a t r i g h t a n g l e s f o r w i d t h and l e n g t h i n m i c r o n s under t h e l i g h t m i c r o s c o p e . Two measurements were a l s o t a k e n on t h e p o l l e n g r a i n s w h i c h were b i c o n v e x - l e n s - s h a p e d ( P i g . 1 ) . I n t h e c a s e o f s a c c a t e p o l l e n , t h e e n t i r e g r a i n and c o r p u s were mea-s u r e d f o r h e i g h t , l e n g t h , w i d t h , and t o t a l l e n g t h ( P i g . 1 ) . O n l y l i v e p o l l e n g r a i n s were measured w i t h t h e e x c e p t i o n o f a l p i n e l a r c h . A hundred r a n d o m l y s e l e c t e d p o l l e n g r a i n s f r o m each s p e c i e s were measured. P i c t u r e s were t a k e n t o measure t h e a n g l e o f t h e s a c c a t e a t t a c h m e n t . The a n g l e i s d e s c r i b e d as ck ( P i g . 1 A ) . C. Specimen p r e p a r a t i o n f o r s c a n n i n g e l e c t r o n m i c r o s c o p e (SEM) D e t a i l s o f t h e specimen p r e p a r a t i o n p r o c e d u r e s a r e as f o l l o w s : ( 1 ) The whole s u r f a c e o f t h e m e t a l s p e c i m e n s t u b was p a i n t e d w i t h an even, t h i n l a y e r o f s i l v e r p a i n t ( A c h e s o n C o l l o i d s , Canada L t d , B r a n t f o r d , O n t a r i o ) . ( 2 ) P o l l e n g r a i n s were s p r e a d e v e n l y on t h e s i l v e r p a i n t , a f t e r i t h a d p a r t i a l l y d r i e d . I f t h e p a i n t was t o o wet, t h e specimens w o u l d s i n k i n t o i t ; i f t o o d r y , t h e specimens w o u l d n o t s t i c k w e l l . ( 3 ) C o a t i n g was c a r r i e d out when t h e s i l v e r p a i n t was d r y . The s p e c i m e n s t u b , p l a c e d on a p l a s t i c s t u b c a r r i e r , was f i x e d on a n e l e c t r i c a l l y d r i v e n r o t a r y s t a g e w h i c h was r o t a t e d a t 10 r.p.m. d u r i n g e v a p o r a t i o n . 2 2 T a b l e 4 C o l o r c l a s s e s o f m a c r o s p o r a n g i a t e s t r o b i l i , d a t e o f b r a n c n l e t s c u t , d a t e o f p o l l e n c o l l e c t e d , and p o l l e n s o u r c e s o f D o u g l a s - f i r . C o l o r C l a s s Date o f Date o f Tree No.* o f f e m a l e s t r o b i l i * * B r a n c n l e t s P o l l e n Cut C o l l e c t e d P o l l e n S o u r c e s 1 - 2 GG Mar 6, 1 9 7 0 Mar 1 0 , 1 9 7 0 UBC 1-1 GG Mar 1 3 , 1 9 7 0 Mar 1 5 , 1 9 7 0 UBC 1 GG Mar 2 0 , 1 9 7 0 UBC 2 GEE A p r 2 , 1 9 6 8 UBC 11 GE Mar 2 5 , 1 9 6 8 UBC 12 EE Mar 2 0 , 1 9 7 0 UBC A GGE Mar 2 5 , 1968 UBC B GGE Mar 2 5 , 1968 UBC E GEE A p r 2 , 1968 UBC * S z i k l a i ( 1 9 6 4 ) ** G r i f f i t h ( 1 9 6 8 ) 23 F i g s . 1. A. P o l l e n of saccate type. B. P o l l e n of b i c o n v e x - l e n s t y p e , (h) h e i g h t , ( l ) l e n g t h , ( t l ) t o t a l l e n g t h , (wj w i d t h , U O angle of saccate attachment. 24 ( 4 ) P a l l a d i u m - g o l d a l l o y (Pd-Au:40:60) i n w i r e f o r m ( d i a m e t e r 0.2 mm) was wound c a r e f u l l y a r o u n d t h e t i p o f a V-shape t u n g s t e n f i l a m e n t . The l e n g t h o f t h e w i r e u s e d was about 8 cm. (5) D i s t a n c e between t h e s o u r c e and t h e t a r g e t was 10-12 cm. The a n g l e o f c o a t i n g was about 45°. ( 6 ) C a r b o n was e v a p o r a t e d o n t o t h e s p e c i m e n a t a t h i c k n e s s o f about 100 A and t h e n Pd-Au a l l o y , o n t o t h e c a r b o n . (7) The s p e c i m e n was s canned a t an a n g l e o f 45° a t 10 KV i n s c a n n i n g e l e c t r o n m i c r o s c o p e ( C a m b r i d g e , Type 96113* Mark 2A, E n g l a n d ) . Specimen p r e p a r a t i o n f o r SEM p r e s e n t e d l i t t l e d i f f i c u l t y , i n c o n t r a s t t o t h e e l a b o r a t e and e x t e n d e d t e c h n i q u e s f o r TEM. Good a d h e s i o n o f t h e s p e c i m e n t o t h e s t u b i s n e c e s s a r y i n o r d e r t o p r o d u c e good e l e c t r i c a l c o n d u c t i v i t y and p r e v e n t t h e b u i l d -up o f an e l e c t r i c a l c h a r g e on t h e s p e c i m e n . Once t h e specimens had been p r e p a r e d and c o a t e d , t h e y were k e p t i n a d u s t f r e e e n v i r o n m e n t t o p r e v e n t c o n t a m i n a t i o n . The method was f o u n d t o be q u i t e s a t i s f a c t o r y f o r a l l t h e s p e c i e s examined. F r e s h l y d r i e d p o l l e n g r a i n s were u s e d . The c o l l a p s e o f p o l l e n g r a i n s was n o t o b s e r v e d when specimens were p l a c e d u n d e r a h i g h vacuum. A t h i g h m a g n i f i c a t i o n s and f o r a p r o l o n g e d s c a n n i n g , g r a i n s d e v e l o p e d c o a t i n g c r a c k s and s w e l l i n g . T h i s s e r i o u s a r t i f a c t was a l s o r e p o r t e d by M a r t i n and Drew ( 1 9 6 9 ) . D e t e r i o r a t i o n o f t h e c o a t i n g i n c r e a s e d w i t h t i m e . The b e s t 2 5 r e s u l t s were o b t a i n e d when f r e s h l y c o a t e d specimens were s t u d i e d . D. C a r b o n E e p l i c a T e c h n i q u e W i t h t h e r e c e n t development o f SEM we have been a b l e t o examine p o l l e n g r a i n s i n t h r e e d i m e n s i o n and i n d e t a i l . D e s p i t e t h e s e a d v a n c e s , i t i s l i k e l y t h a t s u r f a c e r e p l i c a s f o r t r a n s -m i s s i o n e l e c t r o n m i c r o s c o p e (TEM) w i l l be n e c e s s a r y f o r h i g h r e s o l u t i o n o f e x t r e m e l y f i n e s t r u c t u r e s . A m o d i f i e d c a r b o n r e p l i c a t e c h n i q u e f r o m B r a d l e y ( 1 9 5 7 ) was u s e d f o r t h e s p e c i m e n p r e p a r a t i o n f o r TEM. D e t a i l s o f p r o c e d u r e s a r e as f o l l o w s : ( 1 ) An e l e c t r o n m i c r o s c o p e s p e c i m e n s u p p o r t i n g g r i d ( 2 0 0 mesh) was c o v e r e d w i t h Formvar f i l m . A f i l m o f c o r r e c t t h i c k n e s s c o u l d be o b t a i n e d b y d i p p i n g a s l i d e i n t o a 3 p e r c e n t s o l u t i o n o f Formvar i n d i o x a n e and a l l o w i n g i t t o d r a i n s l a n t i n g l y on t o a f i l t e r p a p e r u n t i l t h e s o l v e n t had e v a p o r a t e d . ( 2 ) The g r i d w i t h Formvar f i l m was b e n t s l i g h t l y and p l a c e d on a s l i d e and t h e n p u t i n t h e p e t r i d i s h . The g r i d was wet w i t h a c e t o n e . ( 3 ) P o l l e n g r a i n s were s p r e a d on t h e g r i d e v e n l y b e f o r e t h e a c e t o n e e v a p o r a t e d . ( 4 ) The g r i d , w i t h p o l l e n g r a i n s , was l e f t i n t h e p e t r i d i s h f o r f i v e m i n u t e s . The a c e t o n e j u s t c o v e r e d t h e g r i d on t h e s l i d e . ( 5 ) The a s s e m b l y was l e f t t o d r y un d e r a lamp f o r f i v e m i n u t e s . 26 ( 6 ) The specimens were removed f r o m t h e Pormvar f i l m i n w h i c h t h e r e now r e m a i n e d a p r i m a r y i m p r e s s i o n . T h i s o p e r a t i o n c o u l d be e f f e c t i v e l y p e r f o r m e d u n d e r a b i n o c u l a r d i s s e c t -i n g m i c r o s c o p e . The specimens were removed a f t e r d r y i n g by c a r e f u l l y b r u s h i n g t h e s u r f a c e o f t h e Formvar f i l m w i t h t h e edge o f a l e n s p a p e r . (7 ) The f i n a l r e p l i c a was made by e v a p o r a t i n g c a r b o n on t h e Formvar f i l m . The c a r b o n was d e p o s i t e d onto t h e s u r f a c e o f t h e Formvar w i t h o u t r e m o v i n g t h e g r i d f r o m t h e s l i d e . The t h i c k n e s s o f t h e c a r b o n f i l m was about 2GO A. ( 8 ) L a s t l y , t h e Formvar f i l m was removed by p l a c i n g t h e g r i d on a d i o x a n e - f l o o d e d f i l t e r p a p e r i n a p e t r i d i s h f o r two m i n u t e s . ( 9 ) No more t h a n 3 cm o f Pd-Au a l l o y w i r e was wound c a r e f u l l y a r o u n d t h e t i p o f a V-shaped t u n g s t e n f i l a m e n t , u s i n g a d i s t a n c e between t h e s o u r c e and t a r g e t o f 14 - 1 5 cm. The a n g l e o f shadowing was no more t h a n 3 0 ° . ( 1 0 ) The a l l o y was d e p o s i t e d t h r o u g h t h e g r i d b a r s i n a vacuum e v a p o r a t o r (VE 1 0 , M i k r o s Inc., P o r t l a n d ) , e v a p o r a t e d i t -4 when vacuum was l e s s t h a n 1 0 mm Hg. ( 1 1 ) The r e p l i c a was examined a t 5 0 KV i n a H i t a c h i H S - 7 S e l e c t r o n m i c r o s c o p e . A c o r r e c t t h i c k n e s s o f t h e Pormvar f i l m i s c r i t i c a l . T h i s f i l m s h o u l d be s u f f i c i e n t l y t h i c k n o t t o b r e a k i n t h e p r o c e s s o f r e m o v i n g t h e g r a i n s f r o m t h e f i l m l e a v i n g t h e i m p r e s s i o n s . A one t o f o u r p e r c e n t s o l u t i o n o f Pormvar was f o u n d s a t i s f a c t o r y 2 7 f o r r e p l i c a t i o n ( A g a r and E e v e l l , 1 9 5 6 ; B r a d l e y , 1 9 5 7 ; C h i p p i n d a l e , 1 9 6 0 ) . I n t h i s s t u d y , a 3 p e r c e n t s o l u t i o n o f Formvar i n d i o x a n e was f o u n d t o be t h e s u i t a b l e c o n c e n t r a t i o n f o r a l l t h e p o l l e n g r a i n s examined. The t h i c k n e s s o f t h e c a r b o n f i l m u s e d depends on t h e c o a r s e n e s s o f t h e s u r f a c e s t r u c t u r e u n d e r e x a m i n a t i o n . A f i l m o f 1 0 0 t o 3 5 0 A t h i c k was f o u n d s u i t a b l e f o r p o l l e n g r a i n s ( B r a d l e y , 1 9 5 8 ; Takeoka and S t i x , 1 9 6 3 ; E o w l e y and F l y n n , 1 9 6 6 ; F l y n n and E o w l e y , 1 9 6 7 ) . A t h i c k n e s s o f 2 0 0 A c a r b o n f i l m was u s e d i n t h i s s t u d y . I t was q u i t e s a t i s f a c t o r y even t h o u g h t h e e x i n e s u r f a c e was v e r y r o u g h i n w e s t e r n hemlock. S h a d o w - c a s t i n g n o t o n l y i m p r o v e s t h e c o n t r a s t o f t h e r e p l i c a , b u t a l s o p r o v i d e s a t h r e e - d i m e n s i o n a l e f f e c t . I t i s n e c e s s a r y t o p r o c u r e a p o s i t i v e p r i n t when a n e g a t i v e i m p r e s s i o n i s o b t a i n e d . F o r t h e f i n e r d e t a i l s i t i s c r i t i c a l t o use an amount o f h e a v y m e t a l n e c e s s a r y t o shadow t h e r e p l i c a , b u t n o t enough t o o b s c u r e t h e f i n e s t r u c t u r e . I t i s v e r y i m p o r t a n t t o c o n s i d e r t h e amount o f he a v y m e t a l u s e d , t h e d i s t a n c e f r o m t h e s o u r c e t o t a r g e t , t h e a n g l e o f d e p o s i t i o n and t h e c h o i c e o f shadowing m a t e r i a l s . B r a d l e y ( 1 9 5 7 ) s t a t e d t h a t i f shadowing was t o be u s e d , t h e m e t a l must be d e p o s i t e d t h r o u g h t h e g r i d b a r s . F l y n n and fiowley ( 1 9 6 7 ) r e p o r t e d t h a t shadowing w i t h a heavy m e t a l m i g h t p r e c e d e t h e e v a p o r a t i o n o f c a r b o n . A d e t a i l e d d e s c r i p t i o n o f shadowing has been g i v e n by Kay ( 1 9 6 7 ) . The amount o f m a t e r i a l u s e d h e r e was enough t o impr o v e t h e c o n t r a s t . 28 Rowley and F l y n n (1966) r e p o r t e d t h a t s i n g l e - s t a g e s u r f a c e r e p l i c a s f o r t r e a t e d o r f r e s h p o l l e n g r a i n s c o u l d be made r e a d y f o r t h e e l e c t r o n m i c r o s c o p e i n 1 . 5 h o u r s . The p r e s e n t m o d i f i -c a t i o n i n r e p l i c a t e c h n i q u e o f f r e s h p o l l e n f o r e l e c t r o n m i c r o s c o p y has r e d u c e d p r e p a r a t i o n t i m e t o 1 h o u r o r l e s s . E. M i c r o g r a p h E v a l u a t i o n F o r t h e e v a l u a t i o n o f t h e p r o t r u s i o n s t h e TEM m i c r o g r a p h s were e n l a r g e d . The p r o t r u s i o n s were c o u n t e d i n t e n r a n d o m l y s e l e c t e d a r e a s . The d i a m e t e r o f t h e p r o t r u s i o n s was a l s o measured. F. C a l c u l a t i o n o f t h e r a t i o o f a b s o l u t e t o a v e r a g e chromosome l e n g t h . The measurements o f each chromosome f r o m t h e k a r y o t y p e s and i d i o g r a m s r e p o r t e d by s e v e r a l a u t h o r s ( B a r n e r and C h r i s t i a n s e n , 1960; G u s t a f s s o n and Mergen, 1964; Knaben, 1 9 5 3 ; Sax and Sax, 1 9 3 3 )» were made f r o m ends o f t h e chromosome arms u s i n g t h e d i s s e c t i n g m i c r o s c o p e w i t h 100 d i v i s i o n s i n one cm. The c e n t r o -mere r e g i o n s i n i d i o g r a m s were n o t i n c l u d e d i n t h e measurements. The r a t i o o f a b s o l u t e t o a v e r a g e chromosome l e n g t h was u s e d f o r c o m p a r i n g t h e r e l a t i v e l e n g t h o f each chromosome (Thomas and C h i n g , 1 9 6 8 ) . 5 . RESULTS AND DISCUSSION A. The f o r m a t i o n o f D o u g l a s - f i r p o l l e n e x i n e i n t h e l a t e s t a g e o f m i c r o s p o r o g e n e s i s . Twigs o f D o u g l a s - f i r were c u t and p o l l e n g r a i n s were f o r c e d two weeks b e f o r e a n t h e s i s . A t t h i s p e r i o d , m i c r o s p o r o g e n e s i s o f t h e male s t r o b i l i was a t t h e s t a g e b e f o r e t h e f i r s t v e g e t a t i v e 2 9 d i v i s i o n . When t w i g s were c u t one week b e f o r e a n t h e s i s , t h e y were a t t h e s t a g e o f t h e f i r s t and s e c o n d a r y v e g e t a t i v e d i v i s i o n s . A c c o r d i n g t o H e s l o p - H a r r i s o n ' s r e p o r t ( 1 9 6 2 ) , s p o r o p o l l e n i n o f t h e e x i n e p r e c u r s o r s was t r a n s f e r r e d t o t h e young g r a i n s i n t a p e t a l s e c r e t i o n s , p o l y m e r i z a t i o n o c c u r r i n g on t h e s u r f a c e o f t h e young p o l l e n g r a i n s . The d i a m e t e r o f t h e ecine p r o t r u s i o n s o f p o l l e n g r a i n s h a r v e s t e d p r i o r t o n a t u r a l s h e d d i n g ( F i g s . 3 1 , 3 2 ) was much g r e a t e r t h a n t h o s e o f mature g r a i n s ( F i g . 3 3 ) . The r e a s o n m i g h t be t h a t t h e e a r l y h a r v e s t o f t h e t w i g s suspends t h e f u r t h e r s u p p l y o f t h e p r e c u r s o r s n e c e s s a r y f o r t h e p o l y m e r i z a t i o n and f o r m a t i o n o f t h e e x i n e . The a p p l i c a t i o n o f a c e t o n e d u r i n g t h e s p e c i m e n p r e p a r a t i o n m i g h t c a u s e e x p a n s i o n o f t h e p r o t r u s i o n s b e c a u s e o f i n c o m p l e t e p o l y m e r i z a t i o n . A p p a r e n t s l i t s i n t h e p r o t r u s i o n s a r e a r t e f a c t s . D u r i n g r e p l i c a p r e p a r a t i o n , a more o r l e s s c o n s t a n t amount o f c a r b o n i s a b l e t o f i l l t h e c a v i t i e s o f t h e f i r s t i m p r e s s i o n s f r o m t h e mature g r a i n s , w h i l e t h e same amount o f c a r b o n does n o t c o m p l e t e -l y f i l l t h e l a r g e c a v i t i e s o f e a r l y - h a r v e s t e d p o l l e n g r a i n s , b u t l e a v e s a h o l l o w s t r u c t u r e w h i c h a p p e a r s as a s l i t i n t h e f i n a l r e p l i c a . B. V a r i a t i o n s o f P o l l e n g r a i n s i n P s e u d o t s u g a m a c r o c a r p a and P. m e n z i e s i i . ( 1 ) P s e u d o t s u g a m a c r o c a r p a P o l l e n g r a i n s were e l l i p t i c a l - s h a p e d when wet ( F i g . 3 4 ) . D r y g r a i n s were cup-shaped ( F i g . 3 8 ) and u s u a l l y c o ncave a t t h e d i s t a l f a c e . 3 0 The a v e r a g e s i z e and s i z e - f r e q u e n c y c u r v e s a r e shown i n T a b l e s 5 said 6 and F i g u r e s 2 and 3 , r e s p e c t i v e l y . P o l l e n g r a i n s f r o m P l a c e r v i l l e , C a l i f o r n i a were b i g g e r t h a n t h o s e f r o m C o r v a l l i s , Oregon ( b o t h a r e i n t r o d u c e d t r e e s ) . However, t h e i r c u r v e s o v e r l a p p e d each o t h e r . The c o e f f i c i e n t o f v a r i a t i o n o f s i z e measurements was more o r l e s s t h e same. T h i s s p e c i e s e x h i b i t e d a c o n s i d e r a b l e s i z e v a r i a t i o n ( P i g s . 2 , 3 ) . The v a r i a t i o n s m i g h t be a t t r i b u t a b l e t o t h e d i f f e r e n c e s i n g e n e t i c make-up and e n v i r o n m e n t a l f a c t o r s . C a i n ( 1 9 4 0 ) r e p o r t e d t h a t b a t c h e s o f p o l l e n o f a c e r t a i n s p e c i e s c o l l e c t e d f r o m d i f f e r e n t g e o g r a p h i c r a c e s m i g h t have somewhat d i f f e r e n t s i z e - f r e q u e n c y c u r v e s . The e x i n e s u r f a c e was smooth u n d e r t h e l i g h t m i c r o s c o p e ( P i g . 3 4 ) . However, f r o m t h e SEW m i c r o g r a p h s , i t a p p e a r e d t h a t t h e e x i n e s c u l p t u r e was s c a b r a t e ( P i g s . 3 8 , 3 9 ) • E x i n e on t h e p r o x i m a l f a c e was marked w i t h a t r i r a d i a t e s t r e a k and a n n u l a r t h i c k e n i n g . These two k i n d s o f m a r k i n g s u s u a l l y d i v i d e d t h e p r o x i m a l f a c e i n t o t h r e e s e c t o r s . V a r i a t i o n s o f m a r k i n g s e x i s t e d i n t h i s s p e c i e s . O n l y two s e c t o r s on t h e e x i n e a t t h e p r o x i m a l f a c e c a n be s e e n i n P i g . 3 8 . Three u n e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s a r e shown i n P i g . 3 9 . The v a r i a t i o n s i n t h e m a r k i n g s may be due t o t h e p o l l e n e n v i r o n m e n t whereby t h e f o u r g r a i n s a r e p l a c e d t o g e t h e r i n t h e t e t r a h e d r a l \ arrangement. Under t h e T E M , r e p l i c a s r e v e a l e d t h e e x i n e a r e o l a e w h i c h were f o r m e d b y t h e i n t e r s e c t i n g r i b s . M i n u t e p r o t r u s i o n s emanated f r o m t h e i n t e r s e c t i n g r i b s ( P i g . 5 2 ) . P o l l e n g r a i n s T a b l e 5 A v e r a g e s i z e o f t h e p o l l e n g r a i n s o f P i n a c e a e i n m i c r o n s (- one s t a n d a r d d e v i a t i o n ) S p e c i e s A b i e s g r a n d i s A. p i n s a p o  Cedrus a t l a n t i c a C. d e o d a r a C. l i b a n i  K e t e l e e r i a d a v i d i a n a  L a r i x d e c i d u a L. L. Ii. L. L. L. ii. P i c e a o r i e n t a l i s and c o e f f i c i e n t o f v a r i a t i o n ( c . v . ) . Corpus g m e l i n i i l a r i c i n a l e p t o l e p i s l v a l l i i o c c i d e n t a l i s p o t a n i n i i s i b i r i c a FT s i t c h e n s i s H e i g h t 80.4-2.7 89.3T"4.2 46.1-3.7 48.4-3.9 43.3J3.1 82.5i5.2 P i n u s b a n k s i a n a P. c o n t o r t a P. p o n d e r o s a P. s v l v e s t r i s  P s e u d o t s u g a .japonica P. m a c r o c a r p a a. Oreg. b. C a l i f . P. m e n z i e s i i a. UBC #2 b. Van. I s l . c• I n t . , B•C P. w i l s o n i a n a  Tsuga h e t e r o p h v l l a T. m e r t e n s i a n a 5 9 . 4 4 . 7 71.4-4.2 36.1J2.2 46.0*1.7 50.2|7.7 45.9*3. 2 60.2-3.9 C V . Length. c.v. W i d t h c.v. 3.4 82 .9*1 .7 2.1 84 .3*4 .3 5.1 4.7 82 .3*2 .8 3.4 83 .3*2 .8 3.4 8.0 57 • 7*2 .8 4.9 54 .4*2 .5 4 . 6 8.0 61. .2*2. .5 4.1 55- .5*2. .7 4 .9 7.2 59 .9*2 .5 4 .2 50 .4*2 .6 5.2 6.3 89 .4*4, .3 4 . 8 81 .6*3- .8 4.7 91 .2*6. .1 6.7 80 .4-4. .4 5.5 86 .0*6 .0 7.0 81 .5*4, .6 5 .6 82. .6*4. .5 5.4 77. .1*3. .9 5.1 91. .7±5- .5 6.0 82. .3*3. .6 4 .4 92. .9*4. .6 5 .0 92. .7*4. .0 4 .3 85. .9^5. .2 6.1 78. .1*4, .2 5.4 96. .3|6. .2 6.4 86. .4*4. .9 5.7 86. .7*5. .4 6.2 80, .3*3- .8 4.7 7.9 74. .0*3. .4 4 .6 56. .4*4. .1 7.3 5.9 84, .7*3- .4 4 . 0 72. .0*3. .1 4.3 6.1 43. ,8j2, .4 5.5 40, .1*2. .7 6.7 3.7 52. .2*2. .4 4 .6 49. .1*2. .6 5.3 15.3 60, ,0*4. 0 6.7 54. .3*3. .1 5.7 7.0 52. •9±3. -5 6.6 46. .3*3. .4 7.3 101. ,1*4. 0 4 . 0 85- .9-2. .8 3.3 123. 8*5. 1 4.1 104. .2*3. 7 3.6 132. ,5*6. 0 4.5 107. .7-4, 5 4 .2 118. 1*7. 2 6.1 97. .7*4. 2 4 .3 113. 3±7. 2 6.4 95. 3*4. 4 4 . 6 119. .0*6. 9 5 .8 93. .3*4, 7 5 .0 102. 2*4. 6 4.5 93. .4*3. 4 3 .6 73. 1|3. 3 4-.5 59. 0*2. 4 4.1 6.5 65. 1*0, 7 1.1 61. 9*3. 2 5.2 T o t a l L e n g t h c . v . 105.4* 7.4 7.0 116.8*17.3 14.8 70.6x 5.5 7.5 79.0* 6.2 7.8 74-.3T 4.3 5.8 128.1- 9.7 7.6 85.3T 4.2 4.9 108.2* 6.0 5.5 59-9* 4.1 6.8 69.4* 2.9 4.2 81.7T12.5 15.3 67.6- 3.9 5.8 7 0 . 0 - 2.9 4.1 T a b l e 6 The a v e r a g e s i z e and ran g e o f t h e p o l l e n - s i z e d i s t r i b u t i o n o f P s e u d o t s u g a and L a r i x s p e c i e s S p e c i e s S i z e ( m i c r o n s ) Range o f D i s t r i b u t i o n ( m i c r o n s ) Remarks W i d t h (w) L e n g t h ( l ) P. m a c r o c a r p a 104.2x123.8 107.7x132.5 96.2-114.4 96.2-119.6 111.8-137.8 119.6-145.6 P i g . 2, Oreg. P i g . 3, C a l i f . P. m e n z i e s i i 95.3x113.3 97.7x118.1 93.3x119.0 83.2-106.6 88.4-109.2 83.2-104.0 96.2-130.0 101.4-132.6 101.4-135.2 P i g . 4, Van. I s l . P i g . 5, UBC 2 P i g . 6, I n t . , B.C. P. .ia p o n i c a 85.9x101.1 80.6- 96.2 91.0-114.4 P i g . 7 P. w i l s o n i a n a 93.4x102.2 85.8x101.4 91.0-117.0 P i g . 8 L. d e c i d u a L. g m e l i n i i L. l a r i c i n a L. l e p t o l e p i s 80.4x91.2 81.5x86.0 77.2x82.6 83.3x91.7 67.6- 91.0 72.8- 93.6 67.6-85.5 72.8-91.0 78.0-104.0 75.4-101.4 72.8-93.6 78.0-104.0 P i g . 9 P i g . 10 P i g . 11 P i g . 12 L. l v a l l i i 92.7x92.9 83.2-104 83.2-106.6 P i g . 13 L. o c c i d e n t a l i s 78.1,85.9 67.6-91.0 75.4-98.8 P i g . 14 L. p o t a n i n i i 86.9x96.3 78.0-98.8 85.8-109.2 P i g . 15 L. s i b i r i c a 80.3x86.7 70.2-88.4 72.8-98.8 P i g . 16 3 3 4 0 30 2 0 -£ io in Q. 4 0 -30 -20 -10-0 ' — | r -Pseudotsuga macrocarpa from California /» / * / » Pseudotsuga menziesii from Vancouver Island Pseudotsuga menziesii from U B C - No- 2 104 117 Pollen Size in Microns 130 143 91 Width Length -Figs- 2 - 5 - Size-frequency curves of the pollen groins of Pseudotsuga macrocarpa and P-menziesii-3k 5 0 -40 30 20 - i « 10 r S OMr o a •5 40f- 8 > » o £ 30 3 CT a> £ 20 10 Pseudotsuga japonica oMr Pseudotsuga Wilsoniana 104 117 130 143 Pollen Size in Microns Width Length Figs- 6 - 9 - Size-frequency curves of the pollen grains of Pseudotsuga and Larix species-35 4 0 3 0 2 0 10 13 0U& Width -78 Length-Larix lyallii 91 I04 Pollen Size in Microns II7 I30 Figs- IO -13- Size-frequency curves of the pollen grains of Larix species-3 6 Pollen Size in Microns Height Length Width Total Length Figs- 14-17- Size-frequency curves of the pollen grains of Larix and Abies species-37 Pollen Size in Microns Height Length Width Totol Length Figs- 18-21- Size-frequency curves of the pollen grains of Abies and Cedrus species-3 8 Pollen Size in Microns Height Length Width Total Length — — Figs- 2 2 - 2 5 - Size-frequency curves of the pollen grains of Keteleeria, Picea and Pinus species-3 9 Pollen Size in Microns Height Length Width Total Length Figs- 2 6 - 2 9 - Size-frequency curves of the pollen grains of Pinus and Tsuga species-40 Pol len S i ze in Mic rons Length Width Fig- 30- Size-frequency curves of the pollen groins of Tsuga heterophylla-41 from the two sources had the same microscopic surface structure with the exception that the density of minute protrusions was higher from California and the diameter was smaller than those from the Oregon l o c a l i t y (Pigs. 53» 54) (Table 7 ) . (2). Pseudotsuga menziesii (a) Pollen size Pollen grains from three l o c a l i t i e s were measured. The average pollen size, range of distribution, and size-frequency curves are shown i n Tables 5 and 6, and Figures 4, 5» and 6. The tree from UBC campus showed the least of variation i n width measurements. Campo-Duplan ( 1 9 5 0 ) reported that fresh pollen ranged from 9 0 to 100 u. The diameter of stained pollen was measured 140 u (Wodehouse, 1959). Sziklai (1964) found that the range i n diameter of pollen grains varied among four trees from 91.1 to 99-2 u. Erdtman (1965) indicated that pollen grains were spheroi-dal and. measured 80 u i n the longest axis, but measured 100 u i n a tree from Scania (Sweden). The size reported by Ho and S z i k l a i ( 1 9 7 2 ) was 94.3x128.9 u. Pollen grains are e l l i p t i c a l -shaped when wet (Pig. 35). Therefore, two measurements should be considered necessary. From a l l the reports and present data, i t i s evident that pollen grains of Douglas-fir exhibit considerable size variation. This may be attributed to the effects of different treatments, procedures of mounting, mounting media, and some other factors such as environmental conditions and ecotypic variations. Kessler and Larson (1969) came to similar conclusions. T a b l e 7 The d e n s i t y ( p e r 0.1 mm ) and d i a m e t e r o f p r o t r u s i o n s i n P s e u d o t s u g a and L a r i x s p e c i e s Gen: P s e u d o t s u g a S p e c i e s m a c r o c a r p a m e n z i e s i i j a p o n i c a w i l s o n i a n a S o u r c e s Or e g . C a l i f . I n t . 1 2 11 12 A B E Van. I s l . 16.4 11.5 A v e r a g e Number 13.9 14.3 9.2 10.9 11.0 10.4 10.7 10.5 10.8 10.0 11.5 A v e r a g e 14.1 10.5 D i a m e t e r (mu) 25 23 40 25 25 25 25 25 25 33 25 27 25 A v e r a g e 24 27 Gen: L a r i x S p e c i e s d e c i d u a g m e l i n i i l a r i c i n a l e p t o l e p i s l y a l l i i o c c i d e n t a l i s p o t a n i n i i s i b i r i c a A v e r a g e Number 8.7 8.1 7.7 10.2 10.8 7.3 8.4 7.4 D i a m e t e r (mu) 22 22 25 33 33 44 25 33 43 I t has been r e p o r t e d t h a t t h e s i z e - f r e q u e n c y c u r v e s o f D o u g l a s - f i r p o l l e n f r o m f o u r s e l e c t e d t r e e s v a r i e d f r o m y e a r t o y e a r i n e a c h i n d i v i d u a l t r e e , and f r o m t r e e t o t r e e ( S z i k l a i , 1964). The f r e q u e n c y c u r v e s i n t h i s s t u d y i n d i c a t e t h a t p o l l e n g r a i n l e n g t h i n c r e a s e d f r o m Vancouver I s l a n d t o B.C. I n t e r i o r , w h i l e t h e r e i s no r e g u l a r t r e n d o f w i d t h i n c r e a s e . T h i s i s n o t c o n c l u s i v e , h o w e v e r , p o l l e n g r a i n s f r o m o n l y t h r e e l o c a l i t i e s were measured. The q u e s t i o n o f c l i n a l v a r i a t i o n c a n o n l y be answered b y more s a m p l i n g a t each l o c a t i o n and a f u l l r a n g e o f p o l l e n c o l l e c t i o n f r o m t h e c o a s t t o t h e i n t e r i o r a r e r e q u i r e d , ( b ) S u r f a c e s t r u c t u r e o f p o l l e n E x i n e o f D o u g l a s - f i r p o l l e n g r a i n s have a l w a y s been d e s c r i b e d as smooth w i t h no t r a c e o f s u r f a c e s c u l p t u r e o r a p e r t u r e (Wodehouse, 1959)- The s c u l p t u r e i s d e s c r i b e d as p s i l a t e (Kapp, 1969). From t h e SEM m i c r o g r a p h s i n t h i s s t u d y , i t was e v i d e n t t h a t t h e e x i n e s u r f a c e was n o t smooth, b u t r a t h e r s c a b r a t e . The shape o f d r y p o l l e n g r a i n s and t h e m a r k i n g on t h e i r p r o x i m a l f a c e were as d e s c r i b e d f o r b i g c o n e D o u g l a s - f i r . F i g u r e s 4 1 , 44 and 45 showed t h e a n n u l a r t h i c k e n i n g and t h e t r i -r a d i a t e s t r e a k w h i c h had t h r e e a l m o s t e q u a l l y s p a c e d a n g l e s . When i t d i v i d e d i n t o t h r e e u n e q u a l l y s p a c e d a n g l e s b y t h e t r i r a -d i a t e s t r e a k , t h e t h r e e s e c t o r s were u n e q u a l i n s i z e ( F i g s . 42, 46, 47, 4 8 ) . F i g u r e 40 i n d i c a t e d t h a t p a r t o f t h e a n n u l a r t h i c k e n i n g was i n v i s i b l e . I t was a l s o f o u n d t h a t one o f t h e 44 s t r e a k s c o u l d n o t be s e e n i n F i g u r e s 43 and 49. These m a r k i n g s showed c o n s i d e r a b l e v a r i a t i o n . They d i f f e r e d f r o m t r e e t o t r e e . These v a r i a t i o n s o f m a r k i n g s c o u l d a l s o be f o u n d i n one s i n g l e t r e e . The v a r i a t i o n s i n t h e m a r k i n g s may have been due t o t h e p o l l e n e n v i r o n m e n t . I f f o u r p o l l e n g r a i n s a r e p l a c e d t o g e t h e r i n t h e t e t r a h e d r a l . arrangement ( F i g . 159), i t w i l l be s e e n t h a t e a c h makes c o n t a c t a t one p o i n t w i t h each o f i t s t h r e e n e i g h b o r s , g i v i n g each p o l l e n g r a i n t h r e e e q u a l l y s p a c e d t r i -r a d i a t e s t r e a k s and a n n u l a r t h i c k e n i n g s ( F i g s . 4 1 , 44, 45). Any d e v i a t i o n f r o m t h r e e e q u a l l y s p a c e d c o n t a c t p o i n t s w i l l c a u s e t h e v a r i a t i o n s i n m a r k i n g s . Thus Wodehouse (1959) has s t a t e d t h a t t h e i n t e r n a l e n v i r o n m e n t t e n d s t o c o n t r o l t h e number and a r r a n g e -ment o f t h e i r g e r m i n a l f u r r o w s and p o r e s , and t h e e x t e r n a l e n v i r o n m e n t t e n d s t o m o d i f y t h e i r s c u l p t u r e . P o l l e n g r a i n s f r o m D o u g l a s - f i r t r e e No. 12, w h i c h p r o d u c e d b r i g h t r e d m a c r o s p o r a n g i a t e s t r o b i l i had p o r i i n t h e i r e x i n e ( F i g . 45), T h i s was n o t s e e n i n p o l l e n f r o m o t h e r t r e e s . Whether t h i s c h a r a c t e r i s t i c i s t h e t r a i t o f t r e e s w i t h b r i g h t r e d m a c r o s p o r a n g i a t e s t r o b i l i needs f u r t h e r i n v e s t i g a t i o n . The s u r f a c e s t r u c t u r e o f t h e p o l l e n e x i n e ( F i g s . 55-63) o f n i n e s e l e c t e d t r e e s r e p r e s e n t i n g f i v e c o l o r c l a s s e s o f macro-s p o r a n g i a t e s t r o b i l i and d i f f e r e n t l o c a l i t i e s had t h e same b a s i c s t r u c t u r e where e x i n e a r e o l a e were formed by t h e i n t e r s e c t i n g r i b s f r o m w h i c h m i n u t e p r o t r u s i o n s a r o s e . The e x i n e p a t t e r n o f D o u g l a s - f i r i s s i m i l a r t o t h a t o f b i g c o n e D o u g l a s - f i r . 4 5 P o l l e n g r a i n s o f s e v e n s e l e c t e d t r e e s a t U.B.C. campus had more o r l e s s t h e same d e n s i t y o f m i n u t e p r o t r u s i o n s r a n g i n g f r o m 10 t o 11 ( T a b l e 7 ) . They a l s o h ad t h e same d i a m e t e r o f m i n u t e p r o t r u s i o n s , m e a s u r i n g 2 5 mu, w i t h t h e e x c e p t i o n o f t r e e E w h i c h h a d l a r g e r p r o t r u s i o n s , 3 3 mu. P o l l e n g r a i n s f r o m V a n c o u v e r I s l a n d h ad h i g h e r d e n s i t y o f p r o t r u s i o n s , 1 1 . 5 , "but t h e d i a m e t e r o f t h e p r o t r u s i o n s was t h e same as t h o s e a t t h e U n i v e r s i t y . On t h e c o n t r a r y , p o l l e n f r o m B.C. I n t e r i o r showed t h e h i g h e s t d i a m e t e r o f t h e p r o t r u s i o n s , 40 mu, b u t h a d t h e l o w e s t d e n s i t y o f t h e p r o t r u s i o n s , 9 - 2 . G r e a t v a r i a t i o n e x i s t s i n D o u g l a s - f i r b o t h w i t h i n s m a l l g e o g r a p h i c a r e a s and t h e r a n g e as a w h o l e . M o r p h o l o g i c a l , a n a t o m i c a l , p h y s i o l o g i c a l and g e n e t i c a l d i f f e r e n c e s o f C o a s t a l and I n t e r i o r D o u g l a s - f i r have been r e c o g n i z e d f o r a l o n g t i m e . E l - L a k a n y and S z i k l a i ( 1 9 7 0 ) r e p o r t e d t h a t t h e s e two forms a l s o d i f f e r e d i n n u c l e a r v olumes, DNA c o n t e n t s and r a d i o s e n s i t i v i t y . V a r i a t i o n s i n p o l l e n s i z e , s i z e - f r e q u e n c y c u r v e s , and t h e d e n s i t y and d i a m e t e r o f m i n u t e p r o t r u s i o n s on t h e e x i n e r e p o r t e d h e r e may add t o and s u b s t a n t i a t e t h e s e d i f f e r e n c e s . C. I n t e r s p e c i f i c v a r i a t i o n s o f p o l l e n g r a i n s ( 1 ) . P s e u d o t s u g a s p e c i e s P o l l e n g r a i n s o f b i g c o n e D o u g l a s - f i r and D o u g l a s - f i r have been d e s c r i b e d i n d e t a i l i n t h e p r e v i o u s s e c t i o n , B. These two s p e c i e s have t h e same b a s i c s t r u c t u r e and f i n e d e t a i l o f t h e e x i n e s u r f a c e and c a n o n l y be d i s t i n g u i s h e d by t h e l a r g e r p o l l e n 46 g r a i n s o f b i g c o n e D o u g l a s - f i r , The a v e r a g e s i z e o f b i g c o n e D o u g l a s - f i r p o l l e n i s 105.9x128.2 u, and t h a t o f D o u g l a s - f i r 95.4x116.8 u. P o l l e n g r a i n s o f b i g c o n e D o u g l a s - f i r a l s o have a h i g h e r d e n s i t y o f m i n u t e p r o t r u s i o n s on t h e e x i n e , 14.1, t h a n t h o s e o f D o u g l a s - f i r , 10.5, b u t t h e l a t t e r has l a r g e r p r o t r u s i o n s , 2 7 mu t h a n t h e f o r m e r , 24 mu. The a v e r a g e p o l l e n s i z e and r a n g e o f t h e d i s t r i b u t i o n o f J a p a nese and Pormosan D o u g l a s - f i r ( P s e u d o t s u g a . i a n o n i c a and P. w i l s o n i a n a ) a r e shown i n T a b l e s 5 soaA 6. The c u r v e s of s i z e -f r e q u e n c y o f t h e s e two s p e c i e s a r e diagrammed i n F i g u r e s 7 and 8. The shapes o f t u r g i d and d r y g r a i n s ( P i g s . 36, 37, 50, 51) a r e as d e s c r i b e d f o r b i g c o n e D o u g l a s - f i r . The d i a m e t e r o f Japanese D o u g l a s - f i r p o l l e n r e p o r t e d by Ueno (1958) and Yamazaki and Takeoka (1962) r a n g e d f r o m 80 t o 85 u. Pormosan D o u g l a s - f i r was measured a t 77-85x70-87 u (Wang, 1966). The r a n g e o f t h e p o l l e n s i z e was t a k e n by t h o s e a u t h o r s i n s t e a d o f means, w h i c h were u s e d i n t h i s s t u d y f o r t h e w i d t h and l e n g t h . The d i f f e r e n c e i n s i z e measurements m i g h t be a t t r i -b u t e d t o t h e f a c t o r s as d i s c u s s e d i n D o u g l a s - f i r . The s i z e o f Japanese D o u g l a s - f i r p o l l e n showed t h e l e a s t v a r i a t i o n . Thus t h e r e l a t i v e v a r i a b i l i t y o f t h e p o l l e n s i z e o f t h e f o u r s p e c i e s s t u d i e d d e c r e a s e d i n t h e o r d e r o f D o u g l a s - f i r , b i g c o n e D o u g l a s - f i r , Pormosan D o u g l a s - f i r and Japanese D o u g l a s -f i r . The s i z e - f r e q u e n c y c u r v e s o f Japanese and Pormosan D o u g l a s -f i r p o l l e n o v e r l a p p e d i n l e n g t h , b u t o n l y p a r t i a l l y i n w i d t h ( P i g s . 7, 8 ) . T h e r e f o r e t h e s e two s p e c i e s c o u l d be d i s t i n g u i s h e d 47 n o t o n l y f r o m e a c h o t h e r , b u t a l s o f r o m D o u g l a s - f i r and b i g c o n e D o u g l a s - f i r . The m a r k i n g s on t h e p r o x i m a l f a c e o f Japanese and Formosan D o u g l a s - f i r p o l l e n were d e s c r i b e d f o r D o u g l a s - f i r ( P i g s . 50, 5 1 ) . However, t h e t h r e e e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s o c c u r r e d q u i t e u n i f o r m l y i n J a p a n e s e D o u g l a s - f i r a l t h o u g h o c c a s i o n a l l y t h e t r i r a d i a t e s t r e a k was f a i n t l y marked. Ueno (1958) and Yamazaki and Takeoka (1962) r e p o r t e d t h a t t h e t r i -r a d i a t e s t r e a k was o b s e r v e d i n Japanese D o u g l a s - f i r , b u t t h a t t h e a n n u l a r t h i c k e n i n g was a c h a r a c t e r i s t i c o f L a r i x s p e c i e s . T h i s i s n o t s u p p o r t e d i n t h i s s t u d y . The s c u l p t u r e o f t h e p o l l e n e x i n e o f Japanese and Formosan D o u g l a s - f i r ( P i g s . 5 0 , 5 1 , 64, 6 5 ) was t h e same as t h a t o f b i g c o n e D o u g l a s - f i r and D o u g l a s - f i r . However, t h e d e n s i t y o f m i n u t e p r o t r u s i o n s o f Japanese D o u g l a s - f i r was 16.4, h i g h e r t h a n t h e o t h e r t h r e e s p e c i e s ( T a b l e 7 ) . The d i a m e t e r o f t h e p r o -t r u s i o n s was more o r l e s s t h e same among f o u r s p e c i e s . U s i n g t h i n s e c t i o n s , Ueno (1958) r e p o r t e d t h a t t h e s e x i n e o f Japanese D o u g l a s - f i r p o l l e n was g r a n u l a r and t h i c k . Xamazaki and Takeoka (1962) f o u n d t h a t t h e e x i n e o f t h e d i s t a l f a c e was c o v e r e d w i t h " c a u l i f l o w e r - s h a p e d g r a n u l e s " w h i c h were p r o v i d e d w i t h s m a l l s p i n u l o i d p r o c e s s e s about 20-60 mu i n l e n g t h . They a l s o s t a t e d t h a t t h e e x i n e s u r f a c e p a t t e r n o f t h e s i d e s and t h e p r o x i m a l f a c e d i f f e r e d f r o m t h a t o f t h e d i s t a l f a c e . I n t h i s s t u d y e x i n e s u r f a c e p a t t e r n was f o u n d u n i f o r m on a l l f a c e s o f p o l l e n g r a i n s . 48 A c c o r d i n g t o H e s l o p - H a r r i s o n ' s r e p o r t ( 1 9 6 8 ) , a l t h o u g h t h e q u e s t i o n o f s p o r o p h y t i c o r g a m e t o p h y t i c c o n t r o l o f t h e e x i n e p a t t e r n r e m a i n s u n r e s o l v e d , t h e "balance o f g e n e t i c a l e v i d e n c e a p p e a r s t o f a v o r s p o r o p h y t i c c o n t r o l . However, t h e r e i s no e v i d e n c e t h a t f i r m l y e x c l u d e s t h e p a r t i c i p a t i o n o f t h e s p o r e n u c l e u s i t s e l f i n p a t t e r n d e t e r m i n a t i o n . P s e u d o t s u g a s p e c i e s have b e e n r e p o r t e d t o have 12 p a i r s o f chromosomes, 6 p a i r s w i t h m e t a c e n t r i c c e n t r o m e r e s and 6 p a i r s w i t h s u b m e t a c e n t r i c c e n t r o m e r e s ( C h i n g and Doerksen, 1 9 7 0 ; C h r i s t i a n s e n , 1 9 6 3 ; Thomas and C h i n g , 1 9 6 8 ) . The e x c e p t i o n i s D o u g l a s - f i r , w h i c h has 13 p a i r s o f chromosomes, 5 p a i r s w i t h m e t a c e n t r i c c e n t r o m e r e s , 6 p a i r s w i t h s u b m e t a c e n t r i c c e n t r o m e r e s , and 2 p a i r s w i t h t e l o c e n t r i c c e n t r o m e r e s ( B a r n e r and C h r i s t i a n s e n , 1 9 6 2 ; C h i n g and Do e r k s e n , 1 9 7 0 ; Sax and Sax, 1 9 3 3 ; Thomas and C h i n g , 1 9 6 8 ) . A r a t i o o f a b s o l u t e t o a v e r a g e chromosome l e n g t h o f P s e u d o t s u g a s p e c i e s a r e shown i n T a b l e 8. I t i s e v i d e n t t h a t t h e r a t i o o f each chromosome i s n e a r l y c o n s t a n t f o r f o u r s p e c i e s i n P s e u d o t s u g a . P s e u d o t s u g a s p e c i e s have d i f f e r e n t numbers o f chromosome p a i r s . Why do t h e y have t h e same p o l l e n e x i n e p a t t e r n w h i c h i s g e n e t i c a l l y c o n t r o l l e d by t h e s p o r o p h y t e as w e l l as b y t h e gametophyte? B a r n e r and C h r i s t i a n s e n ( 1 9 6 2 ) p r o p o s e d a f r a g m e n t a t i o n h y p o t h e s i s t h a t two t e l o c e n t r i c p a i r s o f chromosomes i n D o u g l a s -f i r h ad e v o l v e d f r o m two chromosomes by s i m p l y b r e a k i n g i n t o two. 4 9 Table 8 Comparisons of the ratio of absolute to average chromosome length i n Pseudotsuga and Larix species as determined by several authors. Chromosome number i n haploid or diploid set Author 1 2 3 4 5 6 7 8 9 1 0 11 12 13 Barner and Douglas-fir* Christiansen ( 1 9 6 2 ) Sax and Sax ( 1 9 3 3 ) Thomas and Ching (1968) Christiansen ( 1 9 6 3 ) Thomas and Ching ( 1 9 6 8 ) Ching and Doerksen ( 1 9 7 0 ) European larch 139 1 3 5 128 1 2 3 122 95 90 89 86 86 76 76 56 148 135 123 121 111 108 96 91 81 81 79 69 54 144 136 128 128 120 99 9 2 87 83 80 77 69 57 128 124 122 Bigcone Douglas-fir* 120 114 110 89 85 85 78 75 74 136 128 124 Formosan Douglas-fir* 121 119 112 88 82 78 74 71 66 133 126 123 Japanese Douglas-fir** 1 2 0 115 1 0 7 88 83 80 76 7 2 65 •Darner ana ^ Christiansen- (i960) 1 133 121 118 114 113 112 94 84 77 76 75 7 2 136 122 118 116 117 112 96 88 79 77 76 73 Gustafsson and 9 Mergen (1964)* 1 3 4 129 1 2 3 1 1 9 115 105 89 87 79 75 73 72 Sax and Sax ( 1 9 3 3 ) 2 1 3 0 121 119 119 1 2 0 100 98 92 74 74 78 74 Japanese 5 larch Sax and Sax ( 1 9 3 3 ) 151 1 2 0 116 112 111 1 0 9 102 94 83 68 67 66 Western larch Enaben ( 1 9 5 3 ) 145 130 117 114 114 114 89 89 76 75 73 7 2 132 116 116 118 112 126 89 87 76 74 7 2 74 Siberian larch Gustafsson ando Mergen (1964)* 143 131 124 116 116 106 88 82 77 73 73 71 * after Thomas and Ching ( 1 9 6 8 ) ; ** after Ching and Doerksen ( 1 9 7 0 ) ; 1 computed from karyotype; 2 computed from idiogram. 50; while Thomas and Ching 6968) suggested that the derivation of the karyotype of Douglas-fir with 26 chromosomes was from a progenitor with 24 chromosomes by translocation and unequal division. If the 13 pairs of chromosomes i n Douglas-fir have evolved from the progenitor with 12 pairs of chromosomes by fragmenta-tion, Douglas-fir thus has the same chromosomal make-up as the progenitor. This progenitor may be one of the extinct or extant Pseudotsuga species. It i s evident that Pseudotsuga species have the same exine pattern and general plant morphology. Gustafsson and Mergen (1964) presented a diagram of karyotype evolution which i l l u s t r a t e d that 2 telocentric chromosomes of 13 haploid set chromosomes i n Pseudotsuga had evolved from 1 metacentric chromosome of 12 haploid set chromosomes i n Larix, a hypothesis not supported i n this study. If the 13 pairs of chromosomes i n Douglas-fir have evolved from a progenitor with 12 pairs of chromosomes by translocation and unequal division rather than simple fragmentation, Douglas-f i r probably does not have the same genetical make-up as the progenitor. The uniformity of the exine pattern i n Pseudotsuga species may be due to duplication of certain genes which do not play a role i n altering the exine pattern, or which alter the exine pattern i n a way that can not be resolved by these techniques. The mechanism of centric fusion by which two telocentric chromosomes are formed into a metacentric chromosome i s another 51 p o s s i b i l i t y f o r e v o l u t i o n o f 12 p a i r s o f chromosomes f r o m 1 3 . H a i r and B e n z e n b e r g ( 1 9 5 8 ) r e p o r t e d t h a t a p r o g r e s s i v e r e d u c t i o n b y f u s i o n i n b a s i c number was a m a j o r f a c t o r i n t h e chromosomal e v o l u t i o n o f P o d o c a r p a c e a e . I f t h i s i s t h e c a s e , t h e p r o g e n i t o r w i t h 13 p a i r s o f chromosomes w i l l have t h e same g e n e t i c a l make-up as t h e o f f s p r i n g w i t h 12 p a i r s o f chromosomes. T h e r e f o r e t h e p r o g e n i t o r and o f f s p r i n g w i l l have t h e same e x i n e p a t t e r n as c i t e d i n P s e u d o t s u g a s p e c i e s . A l t h o u g h P s e u d o t s u g a s p e c i e s d i f f e r i n chromosome numbers 24 and 26, t h e y a l l have t h e same number o f chromosome arms, 48. A l l f o u r s p e c i e s examined i n t h i s genus have t h e same e x i n e p a t t e r n . O v e r a l l morphology o f P s e u d o t s u g a s p e c i e s i s g e n e r a l l y s i m i l a r . T h e r e f o r e t h e i m p o r t a n c e o f g e n e t i c make-up i s n o t t h e number o f chromosomes, b u t t h e t o t a l number o f chromosome arms and t h e i r g e n e s . I t has been r e p o r t e d t h a t i n t h e m a r i n e s n a i l T h a i s l a p i l l u s . chromosome number r a n g e s f r o m 26 t o 3 6 , b u t t h e number o f chromosome arms r e m a i n s c o n s t a n t a t 3 6 (Swanson _et a l . 1 9 6 7 ) . I n P i n a c e a e , t h e number o f chromosome arms i s c o n s t a n t a t 48, a l t h o u g h t h e chromosome number i s d i f f e r e n t , r a n g i n g f r o m 24 t o 44. I t i s p o s s i b l e t h a t t h e s p e c i a t i o n and t h e f o r m a t i o n o f h i g h - r a n k t a x a c o u l d have o r i g i n a t e d by t h e a c c u m u l a t i o n o f g e n e t i c changes and by some mechanism s u c h as chromosome r e -o r g a n i z a t i o n t h r o u g h chromosome b r e a k a g e a t t h e c e n t r o m e r e s f o l l o w i n g t r a n s l o c a t i o n s and i n v e r s i o n s . 5 2 There a r e two s p e c i e s o f P s e u d o t s u g a i n N o r t h A m e r i c a . B i g c o n e D o u g l a s - f i r has r e t r e a t e d t o a n i c h e i n S o u t h e r n and Lower C a l i f o r n i a , w h i l e D o u g l a s - f i r i s w i d e l y d i s t r i b u t e d f r o m n o r t h e r n M e x i c o t o n o r t h e r n B r i t i s h C o l u m b i a . The o t h e r f o u r s p e c i e s a r e a l s o l o c a t e d i n c e r t a i n n i c h e s i n C h i n a , Formosa and J a p a n . Why i s D o u g l a s - f i r w i t h 13 p a i r s o f chromosomes e x t e n d e d o v e r s u c h a wide d i s t r i b u t i o n w h i l e t h e o t h e r f i v e s p e c i e s w i t h 12 p a i r s a r e s i t u a t e d i n s m a l l n i c h e s ? T h i s may be due t o t h e f o l l o w i n g r e a s o n s w h i c h r e s u l t i n a h i g h d e g r e e o f g e n e t i c v a r i a b i l i t y t h e r e b y f a c i l i t a t i n g s u r v i v a l and r e p r o d u c t i o n i n c o n t e m p o r a r y e n v i r o n m e n t s and p r e a d a p t a t i o n t o e n v i r o n m e n t a l change. ( 1 ) The i n c r e a s e i n chromosome number w i t h o r w i t h o u t i n c r e a s i n g t h e g e n e t i c components t h r o u g h chromosome f r a g m e n t a t i o n o r chromosome r e o r g a n i z a t i o n c a u s e s a c e r t a i n change o f gene e n v i r o n m e n t . T h i s w i l l impose t h e change o f gene i n t e r a c t i o n . ( 2 ) D o u g l a s - f i r i s an e x t r e m e l y h e t e r o z y g o t i c s p e c i e s . The a d v a n t a g e s o f gene exchange and r e c o m b i n a t i o n a r e p r o -duced t h r o u g h s e x u a l r e p r o d u c t i o n and c r o s s p o l l i n a t i o n . ( 3 ) A f t e r z y g o t e f o r m a t i o n , t h e p o s s i b i l i t y o f chromosome r e c o m b i n a t i o n o f a s p e c i e s w i t h 13 p a i r s o f chromosomes i s f o u r t i m e s as g r e a t as t h a t o f t h e s p e c i e s w i t h 12 p a i r s i f some o t h e r t h i n g s a r e e q u a l , s u c h as c r o s s i n g -o v e r , i n v e r s i o n and t r a n s l o c a t i o n . 53 ( 2 ) L a r i x s p e c i e s T u r g i d p o l l e n g r a i n s were e l l i p t i c a l - s h a p e d and o c c a s i o n a l l y s p h e r i c a l - s h a p e d ( P i g . 68). D r y g r a i n s were cup-shaped and u s u a l l y c o n c a v e a t t h e d i s t a l f a c e . The a v e r a g e p o l l e n s i z e and r a n g e o f d i s t r i b u t i o n a r e com-p i l e d i n T a b l e s 5 and 6. The p o l l e n - s i z e f r e q u e n c y c u r v e s a r e diagrammed i n F i g u r e s 9 t o 16. SEM and TEM m i c r o p h o t o g r a p h s a r e shown i n F i g u r e s 66 & 67, 69 t o 78 and 79 t o 86. Of t h r e e s p e c i e s i n N o r t h A m e r i c a , t a m a r a c k ( L a r i x l a r i c i n a ) p o l l e n g r a i n s were s m a l l e s t . A l p i n e l a r c h ( L . l y a l l i i ) p o l l e n was c h a r a c t e r i z e d by i t s l a r g e s i z e , e s p e c i a l l y i n w i d t h , and so c o u l d be e a s i l y d i s t i n g u i s h e d f r o m w e s t e r n l a r c h ( L . O c c i d e n t a l i s ) . Among t h e f o u r s p e c i e s o f L a r i x i n A s i a , p o l l e n s i z e o f C h i n e s e l a r c h ( L . p o t a n i n i i ) was c l o s e t o t h a t o f Japanese l a r c h ( L . l e p t o l e p i s ) . D a h u r i a n l a r c h ( L . g m e l i n i i ) t o S i b e r i a n l a r c h s i b i r i c a ) and European l a r c h ( L . d e c i d u a ) t o Japanese l a r c h . A l t h o u g h t h e y were d i f f e r e n t i n s i z e , t h e c u r v e s o f s i z e f r e q u e n c y o v e r l a p p e d . No s i n g l e s p e c i e s c o u l d be i d e n t i f i e d on t h e b a s i s o f s i z e f r e q u e n c y . Of t h o s e s p e c i e s examined, p o l l e n o f D a h u r i a n l a r c h was f o u n d t o be most v a r i a b l e i n s i z e and a l p i n e l a r c h t o be l e a s t . The e x i n e o f l a r c h p o l l e n h a s a l w a y s b e e n d e s c r i b e d as p s i l a t e u n d e r t h e l i g h t m i c r o s c o p e . SEM ( F i g s . 66-78) and TEM m i c r o p h o t o g r a p h s ( F i g s . 78-86), i n d i c a t e d t h a t p o l l e n o f e i g h t s p e c i e s examined had t h e same m a r k i n g s o f a n n u l a r t h i c k e n i n g and t r i r a d i a t e s t r e a k , and s c a b r a t e s c u l p t u r e w i t h some v a r i a t i o n s i n t h e s c u l p t u r a l e l e m e n t s ( T a b l e 7). I n f i n e d e t a i l i t showed t h a t e x i n e a r e o l a e were formed by t h e i n t e r s e c t i n g r i b s where 5 4 t h e p r o t r u s i o n s emanated f r o m t h e i n t e r s e c t i n g p o i n t s ( F i g . , 7 9 ) . T h i s i s t h e same as r e p o r t e d f o r P s e u d o t s u g a s p e c i e s . A n n u l a r t h i c k e n i n g s and t r i r a d i a t e s t r e a k s were f o u n d on t h e p r o x i m a l f a c e s o f p o l l e n g r a i n s . N e v e r t h e l e s s , t h e t r i r a d i a t e s t r e a k was l e s s w e l l d e v e l o p e d i n t h a t o n l y p a r t o f t h e s t r e a k where i t j o i n e d t h e a n n u l a r t h i c k e n i n g was o b s e r v e d ( P i g s . 6 6 , 6 7 , 6 9 , 7 1 , 7 2 , 7 4 , 7 8 ) . T h i s i s d i f f e r e n t f r o m P s e u d o t s u g a s p e c i e s where a c o m p l e t e t r i r a d i a t e s t r e a k c o u l d be s e e n . The a n n u l a r t h i c k e n i n g was e i t h e r f a i n t l y marked ( P i g s . 6 6 , 7 1 , 7 5 , 7 6 ) o r w e l l marked ( P i g s . 7 2 , 7 8 ) . These v a r i a t i o n s m i g h t be due t o t h e p o l l e n e n v i r o n m e n t i n t e t r a h e d r a l a r r a n g e m e n t . Ueno ( 1 9 5 8 , 1 9 6 0 ) r e p o r t e d t h a t a n n u l a r t h i c k e n i n g was f o u n d on t h e p r o x i m a l f a c e o f t h e p o l l e n g r a i n s o f D a h u r i a n and Japanese l a r c h . E u r o p e a n and S i b e r i a n l a r c h have a l s o been r e p o r t e d t o have a n n u l a r t h i c k e n i n g s (Campo-Duplan, 194-7; Ho and House, 1 9 7 0 ) . The l e s s w e l l d e v e l o p e d t r i r a d i a t e s t r e a k i n l a r c h m i g h t be due t o t h e p o l l e n e n v i r o n m e n t . P o u r p o l l e n g r a i n s a r e p l a c e d t o g e t h e r i n t e t r a h e d r a l a r rangement, b u t each does n o t make c o n t a c t a t one p o i n t w i t h each o f i t s t h r e e n e i g h b o u r s , i . e . , t h e r e i s a space i n t h e c e n t e r o f t h e t e t r a h e d r a l a r rangement. T h i s may be t h e r e a s o n t h a t p o l l e n shows an a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k , where o n l y p a r t o f t h e s t r e a k j o i n e d t o t h e a n n u l a r t h i c k e n i n g i s v i s i b l e . Ueno ( i 9 6 0 ) s u g g e s t e d t h a t t h e f o r m a t i o n o f a n n u l a r t h i c k e n i n g was due t o t h e s u c c e s s i v e membrane f o r m a t i o n i n t h e p r o c e s s o f m i c r o s p o r o -5 5 g e n e s i s which, r e s u l t e d i n a b i l a t e r a l arrangement o f f o u r p o l l e n g r a i n s . T h i s i s n o t s u p p o r t e d h e r e because t h e m a r k i n g s o f t h e t r i r a d i a t e s t r e a k and a n n u l a r t h i c k e n i n g c o u l d be s e e n . I n t h e c a s e o f b i l a t e r a l a rrangement, t h e t r i r a d i a t e s t r e a k w i l l n o t be s e e n , b u t o n l y t h e a n n u l a r t h i c k e n i n g . The c o m p l e t e absence o f t h e m a r k i n g s f o u n d i n t a m a r a c k p o l l e n g r a i n s may be because t h e arrangement o f f o u r g r a i n s d e v i a t e s f r o m t h e t e t r a h e d r o n , i . e . , t h e y a r e i n s q u a r e o r r h o m b o i d a l a r r a n g e m e n t s , w i t h o u t any c o n t a c t w i t h one a n o t h e r . C h a n d l e r and M a v r o d i n e a n u ( 1 9 6 5 ) f o u n d t h a t s q u a r e o r l i n e a r a rrangement o f f o u r p o l l e n g r a i n s were o b s e r v e d a t t h e t e t r a d s t a g e o f m i c r o s p o r o g e n e s i s i n t a m a r a c k . P e a r - s h a p e d p o l l e n g r a i n s were f o u n d i n tamarack and Dahu-r i a n l a r c h ( P i g . 7 0 ) . The ca u s e o f t h i s abnormal p o l l e n f o r m a t i o n i s n o t known. I t may be a t t r i b u t e d t o t h e a b e r r a n t m i c r o s p o r o g e n e s i s f r o m t h e p o l l e n mother c e l l s t o t h e t e t r a d s t a g e s as r e p o r t e d b y C h a n d l e r and M a v r o d i n e a n u ( 1 9 6 5 ) . One t o s e v e r a l o v e r - o r u n d e r - s i z e d p o l l e n g r a i n s were formed i n t h e mother c e l l w a l l w h i c h r e s u l t e d i n t h e f o r m a t i o n o f abnormal p o l l e n . A p o r u s was o b s e r v e d i n t h e p o l l e n o f S i b e r i a n l a r c h ( P i g s . 7 6 , 7 7 ) . The ca u s e and f u n c t i o n o f t h i s p o r u s a r e n o t known. A l t h o u g h t h e p o r u s m i g h t be a pas s a g e between gametophyte and t a p e t a l t i s s u e , t h i s was n o t f o u n d i n a l l t h e p o l l e n g r a i n s . 5 6 I n f i n e d e t a i l , i t was d i s c l o s e d t h a t t h e d e n s i t y and d i a -m e t e r o f t h e p r o t r u s i o n s v a r i e d f r o m s p e c i e s t o s p e c i e s ( T a b l e 7 ) . W e s t e r n and a l p i n e l a r c h showed t h e l o w e s t and h i g h e s t d e n s i t y r e s p e c t i v e l y . As t o t h e d i a m e t e r o f t h e p r o t r u s i o n s , E u r o p e a n and D a h u r i a n l a r c h were s m a l l e s t and w e s t e r n l a r c h t h e l a r g e s t . E r dtman e t a l . ( 1 9 6 1 ) r e p o r t e d t h a t t h e e x i n e o f E u r o p e a n l a r c h p o l l e n was v e r r u c o i d . Yamazaki and Takeoka ( 1 9 6 2 ) f o u n d t h a t t h e p o l l e n s u r f a c e was p r o v i d e d w i t h i r r e g u l a r , s l i g h t l y v e r r u c o i d p r o c e s s e s , i n t e r s p e r s e d w i t h o r b i c u l o i d g r a n u l e s . The o r b i c u l o i d g r a n u l e s t h e m s e l v e s h a d v e r y s m a l l v e r r u c o i d o u t g r o w t h s . The o r b i c u l o i d g r a n u l e s were n o t f o u n d i n t h e samples o f l a r c h p o l l e n g r a i n s examined i n t h i s s t u d y . The s i m i l a r i t y o f t h e m a r k i n g s and e x i n e s c u l p t u r e o f l a r c h p o l l e n may be a t t r i b u t e d t o t h e c l o s e n e s s i n g e n e t i c make-up o f L a r i x s p e c i e s . A l l t h e s p e c i e s i n t h i s genus were f o u n d t o have 12 p a i r s o f chromosomes; 6 o f t h e 12 p a i r s a r e d i s t i n c t l y meta-c e n t r i c , and 6 have s u b m e t a c e n t r i c c e n t r o m e r e s ( S a x and Sax, 1 9 3 3 ) . The r a t i o o f t h e a b s o l u t e t o a v e r a g e l e n g t h o f each chromosome i s n e a r l y c o n s t a n t between s p e c i e s , as shown i n T a b l e 8 . S e v e r a l n a t u r a l o r a r t i f i c i a l i n t e r s p e c i f i c h y b r i d s o f L a r i x s p e c i e s have been r e p o r t e d by L a r s e n and W e s t e r g a a r d ( 1 9 3 8 ) , J o h n s o n ( 1 9 3 9 ) , L a r s e n ( 1 9 5 6 ) , C a r l s o n and B l a k e ( 1 9 6 5 ) , A r n o ( 1 9 6 6 ) , and Wang and H s i n ( 1 9 7 1 ) . ]±- x e u r o l e n i s i s a h y b r i d o f Jap a n e s e and European l a r c h . Sax ( 1 9 3 2 ) f o u n d t h a t t h i s h y b r i d 57 showed almost complete chromosome pairing and more than 90 percent of the pollen grains were viable, although the parental species have been isolated for a long period of time. The capability of interbreeding and the f e r t i l i t y of the hybrids substantiate the facts i n the closeness of larch species. (5) Abies species Of two species i n Abies examined, pollen grains of Spanish f i r (A. pinsapo) were larger than those of grand f i r (A. grandis) (Tables 5, 9, 10). However, the corpus size for these two species was more or less the same except for the height (Figs. 17, 18). It appeared that the pollen corpus of this genus was characterized by i t s having greater width than length (Fig. 88). The corpus size of grand f i r pollen, reported by Erdtman (1965), i s 70x130x100 u, while that reported by Wodehouse (1959) i s 78x90 u. The corpus i s elliptical-like-shaped ( e l l i p t i c a l - l i k e « the two short axes are not equal). Therefore three measurements should be considered necessary. Pollen grains of grand f i r had a greater oL angle than Spanish f i r (Table 11). The two sacci of grand f i r pollen were displaced further to the d i s t a l face than those of Spanish f i r (Pig. 87, 92), but both had the same shape of dry grains. The two sacci tended to bend inwards and enclosed the germinal furrow (Figs. 89, 93). Dry grains of white f i r pollen have the same shape (Martin and Drew, 1969). It was also found that grand and Spanish f i r had thick caps but there was a thin area 58 T a b l e 9 The a v e r a g e t o t a l l e n g t h and ran g e o f t h e p o l l e n - s i z e d i s t r i b u -t i o n i n A b i e s . C e d r u s , K e t e l e e r i a , P i c e a , P i n u s and Tsuga s p e c i e s . S p e c i e s T o t a l l e n g t h ( u ) Range o f d i s t r i b u t i o n ( u ) Remarks A b i e s g r a n d i s 105.4 93.6-119.6 P i g . 17 A b i e s p i n s a n o 116.8 IO9.2-I3O.O P i g . 18 Cedrus a t l a n t i c a 70.6 59.8- 80.6 P i g . 19 Cedrus d e o d a r a 79 67.6- 91 P i g . 20 Cedrus l i b a n i 74.3 65 - 83.2 P i g . 21 K e t e l e e r i a d a v i d i a n a 128.1 114.4-143 P i g . 22 P i c e a o r i e n t a l i s 83.5 78-93.6 P i g . 23 P i c e a s i t c h e n s i s 108.2 93.6-122.2 P i g . 24 P i n u s b a n k s i a n a 59.9 52 - 70.2 P i g . 25 P i n u s c o n t o r t a 69.4 62.4- 75.4 P i g . 26 P i n u s p o n d e r o s a 81.7 72.8- 93.6 P i g . 27 P i n u s s v l v e s t r i s 67.7 59.8- 78 P i g . 28 Tsuga m e r t e n s i a n a 70 T a b l e 10 65 - 75.4 P i g . 29 The a v e r a g e c o r p u s s i z e and r a n g e o f t h e d i s t r i b u t i o n C e d r u s , K e t e l e e r i a , P i c e a , P i n u s and Tsuga s p e c i e s . i n A b i e s , Corpus s i z e ( u ) Range o f S p e c i e s h e i g h t x l e n g t h x w i d t h d i s t r i b u t i o n ( u ) Remarks A b i e s g r a n d i s 80.4x82.9x84.3 75.4-93.6 P i g . 17 A b i e s pirns apo 89.3x82.3x83.3 75.4-98.8 P i g . 18 Cedrus a t l a n t i c a 46.1x57.7x54.4 36.4-65 P i g . 19 Cedrus d e o d a r a 48.4x61.2x55.5 39 -67.6 P i g . 20 Cedrus l i b a n i 43.3x59.9x50.4 36.4-65 P i g . 21 K e t e l e e r i a d a v i d i a n a 82.5x89.4x81.6 72.8-98.8 P i g . 22 P i c e a o r i e n t a l i s 59-4x74.0x56.4 46.8-85.8 P i g . 23 P i c e a s i t c h e n s i s 71.4x84.7x72.0 65 -91 P i g . 24 P i n u s b a n k s i a n a 36.1x43.8x40.1 31.2-49.4 P i g . 25 P i n u s c o n t o r t a 46.0x52.2x49.1 44.2-57.2 P i g . 26 P i n u s p o n d e r o s a 50.2x60.0x54.3 44.2-70.2 P i g . 27 P i n u s s v l v e s t r i s 45.9x52.9x46.3 39 -62.4 P i g . 28 Tsuga m e r t e n s i a n a 60.2x65.1x61.9 52 -72.8 P i g . 29 Tsuga h e t e r o p h v l l a 59.0x73.1 57.2-83.2 P i g . 30 59 Table 1 1 The angle of the saccate attachment i n Abies, Cedrus, Keteleeria,  Picea, Pinus and Tsuga species. Angle of saccate attachment Species (degree) Abies grandis 42 Abies pinsapo 38 Cedrus atlantica 32 Cedrus deodara 30 Cedrus l i b a n i 28 Keteleeria davidiana 42 Picea orientalis 3 1 Picea sitchensis 31 Pinus banksiana 3 0 Pinus contorta 3 6 Pinus ponderosa 3 0 Pinus STlvestris 36 Tsuga mertensiana 49 6 0 i n t h e c e n t e r o f t h e cap o f S p a n i s h f i r p o l l e n ( P i g . 9 2 ) . The s c u l p t u r e o f t h e p o l l e n e x i n e o f A b i e s s p e c i e s was m i c r o v e r r u c a t e ( P i g s . 8 9 , 9 0 , 9 3 , 9 4 ) . Grand and S p a n i s h f i r were d i f f e r e n t i n t h e s i z e o f s c u l p t u r a l e l e m e n t s . Those o f g r a n d f i r h a d b i g g e r g r a n u l e s . B o t h s p e c i e s had no m a r g i n a l c r e s t s , b u t t h e g r a n u l a t e s t r u c t u r e was shown a t t h e m a r g i n a l a r e a s o f S p a n i s h f i r p o l l e n ( P i g s . 9 3 , 9 4 ) . P o l l e n g r a i n s o f w h i t e f i r r e p o r t e d by M a r t i n and Brew ( 1 9 6 9 ) r e s e m b l e d t h o s e o f g r a n d f i r . On t h e cap o f g r a n d f i r p o l l e n , i t was f o u n d t h a t t h e t r i -r a d i a t e s t r e a k was e q u a l l y s p a c e d ( P i g . 9 0 ) . However, i n S p a n i s h f i r , a s t r a i g h t s t r e a k i n t h e d i r e c t i o n o f t h e c o r p u s l e n g t h w i t h a n o t h e r s t r e a k a p p r o a c h i n g a t about 3 0 ° was o b s e r v e d on t h e cap ( P i g . 9 4 ) . T h i s d e v i a t i o n f r o m t h r e e e q u a l l y s p a c e d t r i r a d i a t e s t r e a k s m i g h t r e s u l t f r o m t h e d i f f e r e n t arrangement o f t e t r a h e d r a . Under TEM, r e p l i c a s r e v e a l e d t h a t t h e s e two s p e c i e s had d i f f e r e n t s c u l p t u r a l p a t t e r n s on t h e e x i n e s u r f a c e . I n g r a n d f i r , e x i n e a r e o l a e were f o r m e d by t h e i n t e r s e c t i o n o f r i b s where t h e p r o t r u s i o n s a r o s e f r o m t h e i n t e r s e c t i n g p o i n t s ( P i g . 9 1 ) . O c c a s i o n a l l y t h e p r o t r u s i o n s emanated f r o m t h e s u r f a c e o f g r a n u l e s . I n S p a n i s h f i r , t h e r e was no a r e o l a r f o r m a t i o n , b u t t h e u n d u l a -t i n g s u r f a c e was sometimes i n t e r s e c t e d by t h e r i b s . The p r o t r u -s i o n s a r o s e e i t h e r f r o m t h e i n t e r s e c t i n g r i b s o r f r o m t h e n i c h e s . ( P i g . 9 5 ) . 61 There i s no r e p o r t on t h e f i n e s t r u c t u r e o f t h e s e two s p e c i e s , b u t t h e r e i s one on t h a t o f Japanese f i r by Yamazaki and Takeoka ( 1 9 6 2 ) . The c o r p u s s u r f a c e o f Japanese f i r p o l l e n was d e s c r i b e d as b e i n g c o v e r e d w i t h " c a u l i f l o w e r - s h a p e d p r o t r u s i and d o t t e d w i t h many s m a l l o p e n i n g s and a few m i n u t e s p i n u l e s , and t h e s a c c a t e s u r f a c e was smooth, u n d u l a t i n g and p r o v i d e d w i t h many s m a l l o p e n i n g s . As compared w i t h t h e m i c r o g r a p h s i n t h i s s t u d y , t h e f i n e s t r u c t u r e o f S p a n i s h f i r i s more s i m i l a r t o t h a t o f Japanese f i r . ( 4 ) C e d r u s s p e c i e s I n t h i s genus t h e r e a r e f o u r s p e c i e s , t h r e e o f w h i c h were i n v e s t i g a t e d i n t h i s s t u d y . P o l l e n g r a i n s were c h a r a c t e r i z e d b y t h e i r o v o i d - c o n i c a l forms on w h i c h t h e s e x i n e formed a c o n -t i n u o u s l i n e f r o m t h e cap t o t h e s a c c i ( P i g . 9 6 ) . The d r y g r a i n s were a r c h e d i n shape, and t h e s a c c i o r c o r p u s d i d n o t e n c l o s e t h e i r g e r m i n a l f u r r o w s ( P i g s . 97, 99, 101, 1 0 4 ) . The a v e r a g e p o l l e n s i z e and r a n g e o f t h e d i s t r i b u t i o n a r e c o m p i l e d i n T a b l e s 5 , 9 and 10 . The s i z e - f r e q u e n c y c u r v e s were diagrammed i n F i g u r e s 19, 20 and 2 1 . The c u r v e s e i t h e r o v e r -l a p p e d o r s u p e r i m p o s e d on one a n o t h e r . No s i n g l e s p e c i e s c o u l d be d e t e r m i n e d by i t s s i z e - f r e q u e n c y c u r v e . The a n g l e o f t h e s a c c a t e a t t a c h m e n t v a r i e d f r o m s p e c i e s t o s p e c i e s ( T a b l e 1 1 ) . Lebanon c e d a r ( C . l i b a n i ) h ad t h e s m a l l e s t a n g l e . Ueno ( i 9 6 0 ) r e p o r t e d t h a t t h e a n g l e i n Cedrus r a n g e d f r o m 15 t o 20°. The d i f f e r e n c e i n a n g l e measurements m i g h t be due t o t h e d i f f e r e n t t r e a t m e n t s o f p o l l e n g r a i n s . 62 The measurements o f Cedrus p o l l e n c o r p u s have been r e p o r t e d b y A y t u g ( 1 9 6 1 ) , Erdtman ( 1 9 6 5 ) , N a i r ( 1 9 6 5 ) and Ueno ( 1 9 5 8 ) . P o l l e n s i z e o f t h e t h r e e s p e c i e s examined h e r e was c l o s e t o t h o s e r e p o r t e d by A y t u g ( 1 9 6 1 ) , b u t n o t t o t h o s e r e p o r t e d by t h e o t h e r s . The d i f f e r e n c e i n t h e p o l l e n - s i z e measurements may be due t o t h e f a c t o r s as d i s c u s s e d i n P s e u d o t s u g a s p e c i e s . P o l l e n g r a i n s o f t h i s genus h a d u n d u l a t i n g s u r f a c e s where t h e s c u l p t u r e was m i c r o v e r r u e a t e ( P i g s . 97? 9 8 , 1 0 1 , 1 0 3 ) . The m a r g i n a l c r e s t and a s t r a i g h t s t r e a k i n t h e d i r e c t i o n o f t h e c o r p u s l e n g t h were o c c a s i o n a l l y p r e s e n t ( P i g s . 9 8 , 1 0 2 ) . The s u r f a c e o f t h e g e r m i n a l f u r r o w o f A t l a s c e d a r ( C . a t l a n t i c a ) p o l l e n showed g r a n u l a t e s t r u c t u r e ( P i g . 9 9 ) , w h i l e t h e o t h e r two s p e c i e s d i d n o t have t h i s ornament ( P i g . 1 0 4 ) . Erdtman ( 1 9 6 5 ) came t o t h e same c o n c l u s i o n t h a t t h e s u r f a c e o f t h e g e r m i n a l f u r r o w o f I n d i a n ( C . d e o d a r a ) and Lebanon c e d a r p o l l e n was smooth w i t h o u t any f l e c k s . I n f i n e d e t a i l , t h e e x i n e s u r f a c e o f t h e c o r p u s and s a c c i h a d t h e same s t r u c t u r e ( P i g s . 1 0 0 , 1 0 5 , 1 0 6 ) . The s u r f a c e was c o v e r e d w i t h m i n u t e p r o t r u s i o n s w h i c h a r o s e f r o m t h e n i c h e s . The p r o t r u s i o n s were r o d - s h a p e d . P o l l e n o f A t l a s and I n d i a n c e d a r h a d t h e same d e n s i t y o f p r o t r u s i o n s 3 t o 5 ( p e r 0 . 1 mm ) , w h i l e t h a t o f L e b a n o n c e d a r h a d a h i g h e r d e n s i t y o f p r o t r u s i o n s 6 t o 1 0 . The f i n e s t r u c t u r e o f t h e p o l l e n e x i n e a l s o showed t h a t A t l a s c e d a r had a smooth s u r f a c e , b u t I n d i a n and Lebanon c e d a r s were f o u n d t o have a m i c r o v e r r u c o i d s c u l p t u r e on t h e wavy s u r f a c e ( P i g s . 1 0 5 , 1 0 6 ) . 6 3 A l t h o u g h p o l l e n g r a i n s i n t h i s genus c a n not "be d i s t i n -g u i s h e d "by t h e i r s i z e s and f r e q u e n c y c u r v e s , t h e y a r e d i s t i n -g u i s h a b l e f r o m t h e i r f i n e s t r u c t u r e s . ( 5 ) K e t e l e e r i a s p e c i e s T h i s genus h a s t h e l a r g e s t s i z e o f a l l t h e p o l l e n w i t h s a c c i i n P i n a c e a e . The a v e r a g e t o t a l l e n g t h and c o r p u s s i z e o f c o w t a i l f i r ( K . d a v i d i a n a ) p o l l e n a r e shown i n T a b l e s 5 , 9 and 10. A l t h o u g h t h e t o t a l l e n g t h o f t h i s s p e c i e s was much l o n g e r t h a n t h a t o f f i r s , t h e y had more o r l e s s t h e same s i z e o f c o r p u s ( P i g s . 1 7 , 18, 2 2 ) . I t h a s been r e p o r t e d t h a t t h e c o r p u s was 70x125x85 u ( E r d t m a n , 1 9 6 5 ) , and 110x110x98 u (Ueno, 1 9 5 8 ) . I k u s e ( 1 9 5 6 ) and Campo-Duplan ( 1 9 5 0 ) f o u n d t h a t t h e s i z e was 82x84 and 84 u r e s p e c t i v e l y . The d i f f e r e n c e i n s i z e measurements m i g h t be a t t r i b u t e d t o t h e d i f f e r e n t p o l l e n t r e a t m e n t s and s o u r c e s . The a n g l e o f t h e s a c c a t e a t t a c h m e n t i s shown i n T a b l e 11. I t was t h e same as f o u n d i n g r a n d f i r . S a c c i were d i s p l a c e d t o t h e d i s t a l f a c e ( P i g . 1 0 7 ) . The a n g l e r e p o r t e d by Ueno ( i 9 6 0 ) was 3 0 ° . The d i f f e r e n c e i n measurement m i g h t be due t o t h e d i f f e r e n t s p e c i m e n p r e p a r a t i o n . The e x i n e p a t t e r n on t h e cap was r u g u l a t e ( P i g . 1 0 8 ) . T h i s i s a u n i q u e p a t t e r n f o r P i n a c e a e p o l l e n . The e x i n e s u r f a c e o f s a c c i was c o m p a r a t i v e l y smooth compared t o t h a t o f t h e c o r p u s . T h e . s a c c a t e e x i n e was ornamented w i t h some p i t s and p o r i ( P i g s . 108, 1 0 9 ) . 64 The shape o f t h e d r y g r a i n s was as d e s c r i b e d f o r A b i e s s p e c i e s . Two s a c c i were b e n t i n w a r d s and e n c l o s e d t h e g e r m i n a l f u r r o w ( F i g . 1 0 9 ) . The shape m i g h t be a t t r i b u t e d t o t h e s i z e and r a t i o o f c o r p u s and s a c c i as w e l l as t h e a n g l e o f s a c c a t e a t t a c h m e n t . P o l l e n n o r m a l l y had two s a c c i . O c c a s i o n a l l y p o l l e n w i t h 3 - o r 4 - s a c c i , c o u l d be f o u n d ( P i g s . 1 1 0 , 1 1 1 ) . T h i s has a l s o b een r e p o r t e d by Erdtman ( 1 9 6 5 ) , and Yamazaki and Takeoka ( 1 9 5 9 ) . Under TEM, r e p l i c a s r e v e a l e d t h a t t h e cap o f t h e p o l l e n was c o n v o l u t e d where t h e m i n u t e p r o t r u s i o n s a r o s e e i t h e r f r o m t h e r i b s o r f r o m t h e n i c h e s ( F i g . 1 1 3 ) . The m a r g i n a l a r e a had t h e same s t r u c t u r e as t h e cap ( F i g . 1 1 2 ) . The u n d u l a t i n g s u r f a c e o f t h e s a c c i was a l s o c o v e r e d w i t h p r o t r u s i o n s ( F i g . 1 1 2 ) . Yamazaki and Takeoka ( 1 9 5 9 ) d e s c r i b e d t h e s u r f a c e o f t h e c o r p u s as b e i n g c o v e r e d w i t h p r o t r u s i o n s o f " c a u l i f l o w e r t y p e " f r o m w h i c h some f i n e s p i n u l e s p r o j e c t e d and where o p e n i n g s were f o u n d . F i n e s p i n u l e s and o p e n i n g s were a l s o o b s e r v e d on t h e s u r f a c e o f s a c c i . The d e s c r i p t i o n o f " c a u l i f l o w e r - t y p e d " s t r u c t u r e f o r K e t e l e e r i a i s n o t recommended h e r e . The s t r u c t u r e as shown i n F i g u r e s 108 and 113 was c o n v o l u t e d r a t h e r t h a n " c a u l i f l o w e r - t y p e d " . ( 6 ) P i c e a s p e c i e s P o l l e n g r a i n s o f t h i s genus were c h a r a c t e r i z e d by t h e i r e s s e n t i a l l y c o n t i n u o u s l i n e s f r o m t h e cap o f t h e c o r p u s t o t h e s a c c i . The s a c c i were b r o a d a t t h e b a s e , g i v i n g them an o v o i d -c o n i c a l f o r m ( F i g s . 114, 1 1 9 ) . They were a l s o d i s t i n g u i s h e d by t h e i r a r c h e d c o r p o r a w h i c h e n c l o s e d t h e g e r m i n a l f u r r o w , when 65 t h e g r a i n s were d r y ( F i g s . 116, 1 2 0 ) . H a r t i n and Drew (1969) p r e s e n t e d t h e SEM m i c r o p h o t o g r a p h s o f Engelmann s p r u c e p o l l e n w h i c h showed t h e same shape o f d r y g r a i n s . P o l l e n g r a i n s o f S i t k a s p r u c e ( P . s i t c h e n s i s ) were d i s t i n c t i n t h e i r l a r g e s i z e . The c o r p u s l e n g t h o f S i t k a s p r u c e p o l l e n was n e a r l y t h e s i z e o f t h e t o t a l l e n g t h o f o r i e n t a l s p r u c e ( P . o r i e n t a l i s ) p o l l e n ( T a b l e s 5, 9, 1 0 ) . T h e i r s i z e - f r e q u e n c y c u r v e s o v e r l a p p e d e a ch o t h e r s l i g h t l y as shown i n F i g u r e s 23 and 24-, b u t were e s s e n t i a l l y d i f f e r e n t , a l l o w i n g s e p a r a t i o n o f t h e two s p e c i e s . P o l l e n o f o r i e n t a l s p r u c e v a r i e d i n s a c c a t e s i z e . O c c a s i o n -a l l y t h e s a c c i were r e d u c e d as shown i n P i g . 1 1 5 * However, s u c h a v a r i a t i o n d i d n o t e x i s t i n t h e p o l l e n o f S i t k a s p r u c e . P o l l e n g r a i n s o f t h e s e two s p e c i e s had no m a r g i n a l c r e s t s . The m a r g i n a l a r e a s o f o r i e n t a l s p r u c e p o l l e n were smooth ( F i g . 116) b u t t h o s e o f S i t k a s p r u c e p o l l e n g r a n u l a t e ( F i g . 1 2 0 ) . A l s o t h e f o r m e r d i d not have any t r i r a d i a t e s t r e a k ( F i g . 1 1 7 ) , w h i l e t h e l a t t e r was f a i n t l y marked w i t h a s t r e a k ( F i g . 1 2 1 ) . Erdtman ( 1 9 6 5 ) r e p o r t e d t h a t no m a r g i n a l c r e s t s were f o u n d i n s p r u c e . T h i s was a l s o i n d i c a t e d by Wodehouse ( 1 9 5 9 ) i n S i t k a s p r u c e . The p o l l e n s c u l p t u r e o f P i c e a s p e c i e s was m i c r o v e r r u c a t e ( F i g s . 116, 117, 120, 1 2 1 ) . A l t h o u g h t h e s e two s p e c i e s had t h e same s c u l p t u r e , t h e s c u l p t u r a l e l e m e n t s o f o r i e n t a l s p r u c e p o l l e n were s m a l l e r t h a n t h o s e o f S i t k a s p r u c e . The g r a n u l a t e s u r f a c e o f t h e l a t t e r was o c c a s i o n a l l y d e l i m i t e d by t h e i n t e r -66 s e c t i n g r i b s and formed an a r e o l a ( F i g . 1 2 2 ) . T h i s f e a t u r e was n o t f o u n d i n o r i e n t a l s p r u c e p o l l e n ( F i g . 1 1 8 ) . M a r t i n and Drew ( 1 9 6 9 ) f o u n d t h a t a m i c r o v e r r u c a t e t e x t u r e o f t h e cap o c c u r r e d i n t h e pdLen o f Engelmann s p r u c e . The f i n e s t r u c t u r e o f Ja p a n e s e s p r u c e p o l l e n was d e s c r i b e d as " c a u l i f l o w e r t y p e " w h i c h was ornamented w i t h s p i n u l e s ( Y a m a z a k i and Takeoka, 1 9 5 9 ) . ( 7 ) P i n u s s p e c i e s The a v e r a g e p o l l e n s i z e and r a n g e o f t h e d i s t r i b u t i o n a r e c o m p i l e d i n T a b l e s 5 , 9 and 1 0 . The c u r v e s o f s i z e f r e q u e n c y a r e shown i n F i g u r e s 2 5 t o 28. P o l l e n g r a i n s o f P i n u s s p e c i e s were c h a r a c t e r i z e d by t h e i r s m a l l s i z e s . I n t h e f o u r s p e c i e s examined, j a c k p i n e ( P . b a n k s i a n a ) p o l l e n was s m a l l e s t . I t was a l s o i n d i c a t e d by Erdtman t h a t p o l l e n o f t h i s s p e c i e s was one o f t h e s m a l l e s t i n -P i n u s ( 1 9 6 5 ) . The t o t a l l e n g t h o f j a c k p i n e p o l l e n had t h e s i z e o f t h e c o r p u s l e n g t h o f p o n d e r o s a p i n e ( P . p o n d e r o s a ) p o l l e n . The l a t t e r was l a r g e s t and c o u l d be i d e n t i f i e d f r o m t h e o t h e r t h r e e s p e c i e s b a s e d on t h e p o l l e n - s i z e f r e q u e n c y i n t o t a l l e n g t h ( F i g s . 2 5 , 26, 2 7 , 2 8 ) . L o d g e p o l e ( P . c o n t o r t a ) and s c o t s p i n e ( P . s v l v e s t r i s ) have more o r l e s s t h e same p o l l e n s i z e s . These two s p e c i e s were n o t d i s t i n g u i s h a b l e on t h e b a s i s o f t h e i r s i z e - f r e q u e n c y c u r v e s . A l t h o u g h t h e s i z e - f r e q u e n c y c u r v e s o f t h e t o t a l l e n g t h o f t h e s e two s p e c i e s o v e r l a p p e d w i t h t h a t o f j a c k p i n e , t h e c u r v e o f t h e c o r p u s o f t h e l a t t e r c o u l d be i d e n t i f i e d f r o m t h e f o r m e r ( P i g s . 2 5 , 26, 2 8 ) . 67 J a c k and s c o t s p i n e p o l l e n have b e e n w i d e l y i n v e s t i g a t e d . On t h e o t h e r hand t h e r e has been no work r e p o r t e d on p o n d e r o s a and l o d g e p o l e p i n e . C a i n (1940) r e p o r t e d t h a t t h e c o r p u s o f j a c k p i n e p o l l e n was 31x45x35 u, w h i l e I k u s e (1956) f o u n d 37-39x 43-4-5 u. The c o r p u s s i z e o f s c o t s p i n e p o l l e n was f o u n d t o be 41.9x53.0x48.1 u ( A y t u g , 1 9 6 2 ) , 45x55x38 (Erd t m a n , e t a l . , 1 9 6 1 ) , and 38x42x37 u ( P r a g l o w s k i , 1 9 6 2 ) . P o l l e n s i z e r e p o r t e d by Wodehouse ( 1 9 5 9 ) was 44.2x52 u. P o l l e n c o r p u s was e l l i p t i c a l -l i k e - s h a p e d . H e n c e , t h r e e measurements s h o u l d be c o n s i d e r e d n e c e s s a r y . On t h e o t h e r hand, t h e d i f f e r e n c e i n s i z e r e p o r t e d b y t h o s e a u t h o r s and p r e s e n t d a t a m i g h t be a t t r i b u t e d t o t h e f a c t o r s as d i s c u s s e d f o r P s e u d o t s u g a . I n t h e r e l a t i v e v a r i a t i o n o f p o l l e n s i z e , l o d g e p o l e p i n e p o l l e n showed t h e l e a s t c o e f f i c i e n t o f v a r i a t i o n i n t h e f o u r s p e c i e s examined ( T a b l e 5). P o n d e r o s a p i n e p o l l e n were t h e l a r g e s t i n h e i g h t and t o t a l l e n g t h . On t h e cap o f p i n e p o l l e n , e x i n e s c u l p t u r e was m i c r o v e r r u -c a t e ( P i g s . 124, 125, 128, 132, 136, 1 3 7 ) . The s a c c a t e s u r f a c e was c o m p a r a t i v e l y smooth t o t h e c a p . O c c a s i o n a l l y m a r g i n a l c r e s t s were p r e s e n t i n t h e p o l l e n o f j a c k ( P i g . 124) and l o d g e p o l e p i n e ( P i g . 1 2 8 ) . Most o f t h e p o n d e r o s a p i n e p o l l e n h ad c o n s p i c u o u s m a r g i n a l c r e s t s ( P i g s . 131, 132, 1 3 3 ) , b u t s c o t s p i n e p o l l e n s e l dom h a d any ( P i g . 1 3 5 ) . A t r i r a d i a t e s t r e a k was n o t f o u n d on p i n e p o l l e n e x c e p t s c o t s p i n e w h i c h had a f a i n t l y marked one ( P i g . 1 3 7 ) . SPM m i c r o p h o t o g r a p h s o f l o d g e p o l e and p o n d e r o s a 6 8 p i n e p o l l e n p r e s e n t e d by M a r t i n and Drew ( 1 9 6 9 ) had t h e same s t r u c t u r e as r e p o r t e d i n t h i s s t u d y . P o l l e n g r a i n s o f j a c k and p o n d e r o s a p i n e had a ck a n g l e o f 3 0 ° , w h i l e t h o s e o f l o d g e p o l e and s c o t s p i n e had 3 6 ° . I n d r y g r a i n s , t h e f o r m e r was a r c h - s h a p e d and t h e v e n t r a l r o o t s o f t h e s a c c i e n c l o s e d t h e g e r m i n a l f u r r o w ( P i g s . 1 2 5 , 1 3 2 , 1 3 3 ) ; t h e l a t t e r h a d two s a c c i w h i c h t e n d e d t o bend i n w a r d s t o e n c l o s e t h e g e r m i n a l f u r r o w ( P i g s . 1 2 9 , 1 3 5 ) . The d i f f e r e n c e i n t h e shape o f t h e d r y g r a i n s m i g h t be a t t r i b u t e d t o t h e a n g l e o f t h e s a c c a t e a t t a c h m e n t and t o t h e r a t i o o f t h e c o r p u s t o t h e s a c c i . The l a r g e r was t h e a n g l e , t h e f u r t h e r were t h e s a c c i d i s p l a c e d t o t h e d i s t a l f a c e . S i n c e t h e s a c c i were n a r r o w a t t h e b a s e , t h e g e r m i n a l f u r r o w w o u l d be s m a l l . T h i s i s how t h e shape o f d r y g r a i n s l i k e l o d g e p o l e and s c o t s p i n e was fo r m e d . P o l l e n g r a i n s o f f o u r p i n e s p e c i e s examined h a d t h e same f i n e s t r u c t u r e ( P i g s , 126, 1 3 0 , 1 3 4 , 1 3 8 ) . The g r a n u l a t e s u r f a c e o f t h e c o r p u s was o c c a s i o n a l l y d e l i m i t e d by t h e i n t e r s e c t i n g r i b s . M i n u t e p r o t r u s i o n s emanated e i t h e r f r o m t h e i n t e r s e c t i n g r i b s o r f r o m t h e g r a n u l e s . The s u r f a c e o f t h e g r a n u l e s was n o t smooth, b u t c o v e r e d w i t h a m i c r o v e r r u c o i d s c u l p t u r e ( P i g s . 126, 1 3 0 , 1 3 4 , 1 3 8 ) . The s a c c a t e s u r f a c e was a l s o ornamented w i t h m i n u t e p r o t r u s i o n s and m i c r o v e r r u c o i d s c u l p t u r e , b u t s c a r c e l y d e l i m i t e d by t h e r i b s . S i t t e ( 1 9 5 3 ) s t u d i e d t h e s u b m i c r o s c o p i c morphology o f t h e p o l l e n e x i n e and f o u n d t h a t t h e p o l l e n e x i n e o f s c o t s p i n e had 6 9 a r e g u l a r f i e l d - l i k e s c u l p t u r e . He a l s o i n d i c a t e d t h a t t h e s a c c i h a d t h e same s c u l p t u r e as t h e c o r p u s , h u t t h e s c u l p t u r e II was c o a r s e . M u h l e t h a l e r ( 1 9 5 5 ) r e p o r t e d t h a t "black p i n e e x i n e h a d an i r r e g u l a r t u m o r - l i k e s t r u c t u r e w i t h some f i n e g r a n u l e s . Y amazaki and Takeoka ( 1 9 5 8 , 1 9 6 2 ) d e s c r i b e d t h e p o l l e n e x i n e o f Japanese r e d p i n e as b e i n g c o v e r e d w i t h " c a u l i f l o w e r - s h a p e d p r o c e s s e s " w h i c h were p r o v i d e d w i t h some m i n u t e s p i n u l e s . The f i n e s t r u c t u r e o f f o u r p i n e s p e c i e s d e s c r i b e d i n t h i s s t u d y was s i m i l a r t o t h o s e o f s c o t s and b l a c k p i n e r e p o r t e d by S i t t e ( 1 9 5 3 ) and M u h l e t h a l e r ( 1 9 5 5 ) . The p i n e s p e c i e s c o u l d n o t be d i s t i n g u i s h e d f r o m one a n o t h e r by t h e i r f i n e s t r u c t u r e . However, t h e use o f s i z e -f r e q u e n c y c u r v e s and o t h e r c h a r a c t e r i s t i c s s u c h as cL a n g l e , m a r g i n a l c r e s t s and t h e shape o f d r y p o l l e n m i g h t be h e l p f u l i n i d e n t i f y i n g each s p e c i e s , a l t h o u g h t h e y w i l l n o t a l w a y s be s u c c e s s f u l . A y t u g ( 1 9 6 2 ) and C a i n ( 1 9 4 0 ) f o u n d t o o t h a t t h e s i z e f r e q u e n c y c u r v e method was u s e f u l i n d i s t i n g u i s h i n g p i n e s p e c i e s . On t h e o t h e r hand, W h i t e h e a d ( 1 9 6 4 ) s t a t e d t h a t no s i n g l e s p e c i e s c o u l d be i d e n t i f i e d on t h e b a s i s o f s i z e f r e q u e n c y . I n an a t t e m p t t o i d e n t i f y t h e s p e c i e s o f p i n e p o l l e n , t h e a u t h o r w o u l d s u g g e s t t h a t s i z e - f r e q u e n c y c u r v e s s h o u l d n o t be u s e d a l o n e , b u t suppl e m e n t e d by t h e m o r p h o l o g i c a l f e a t u r e s . The s i m i l a r i t y i n t h e f i n e s u r f a c e s t r u c t u r e o f p i n e p o l l e n may be a t t r i b u t a b l e t o t h e g e n e t i c s i m i l a r i t y i n p i n e s p e c i e s . I t has been known t h a t P i n u s s p e c i e s c o m p r i s e 12 p a i r s o f chromosomes, 11 o f w h i c h a r e m e t a c e n t r i c and one s u b m e t a c e n t r i c 7 0 ( S a x and Sax, 1 9 3 3 ; P e d e r i c k , 1 9 7 0 ) . They a r e c y t o l o g i c a l l y v e r y s t a b l e , w i t h v e r y few changes i n chromosome s t r u c t u r e ( S a x , 1960; S a y l o r , 1 9 6 4 ) . D u f f i e l d ( 1 9 5 2 ) i n d i c a t e d t h a t s t r o n g h o m o l o g i e s e x i s t e d between t h e chromosomal make-up o f d i f f e r e n t c o m p a t i b l e s p e c i e s o f t h i s genus. I t was a l s o v e r i f i e d b y P e d e r i c k ( 1 9 6 7 ) t h a t k a r y o t y p e s o f a r e p r e s e n t a t i v e r a n g e o f s p e c i e s showed r e m a r k a b l e u n i f o r m i t y i n t h e l e n g t h o f chromo-somes. I t i s a l s o e v i d e n t t h a t s p e c i e s h y b r i d i n P i n u s o c c a s i o n -a l l y o c c u r s p o n t a n e o u s l y and many a r t i f i c i a l h y b r i d s have been made i n r e c e n t decades ( D u f f i e l d , 1 9 5 2 ) . Most o f t h e h y b r i d s o b t a i n e d a r e v i g o r o u s and r e l a t i v e l y f e r t i l e . A c y t o l o g i c a l s t u d y showed t h a t m e i o s i s was about as r e g u l a r as i t was i n t h e p a r e n t a l s p e c i e s ( S a x , 1 9 6 0 ) . I t ap p e a r s t h a t t h e y a r e s i m i l a r i n chromosome c o n s t i t u t i o n and g e n e t i c c o m p a t i b i l i t y . ( 8 ) Tsuga s p e c i e s T h i s i s a c o n t r o v e r s i a l genus, n o t o n l y t o p a l y n o l o g i s t s b u t a l s o t o f o r e s t e r s and b o t a n i s t s . The p a l y n o l o g i s t s a r e s t r u c k by t h e c o m p l e t e d i f f e r e n c e i n p o l l e n m orphology between m o u n t a i n hemlock and t h e o t h e r h e m l o c k s , w h i l e f o r e s t e r s and b o t a n i s t s b y t h e marked d i f f e r e n c e i n p l a n t m o r p h o l o g y . Engelmann (1880) p r o p o s e d t o d i v i d e genus Tsuga i n t o two s e c t i o n s - E u t s u g a and He s n e r o n e u c e . Lemmon (1890, 1898) s e p a r a -t e d Hesneroneuce f r o m Tsuga as an i n d e p e n d e n t genus w h i c h was s u p p o r t e d b y E y d b e r g ( 1 9 1 2 ) . B e i s s n e r ( 1 9 0 9 ) a d o p t e d Engelmann's p r o p o s a l o f two s e c t i o n s . I n 1 9 2 3 , D a l l i m o r e and J a c k s o n 71 f o l l o w e d t h e i d e a o f two s e c t i o n s , b u t a d o p t e d M i c r o n e u c e i n s t e a d o f E u t s u g a . Ueno ( 1 9 5 7 ) s t u d i e d t h e p o l l e n o f genus Tsuga and s u g g e s t e d two s u b g e n e r a r a t h e r t h a n two s e c t i o n s . Campo-Duplan and Gaussen ( 1 9 4 8 ) p r o p o s e d t h e genus T s u g o - P i c e a f o r m o u n t a i n hemlock. Tsuga m e r t e n s i a n a was f i r s t u s e d f o r w e s t e r n h emlock by C a r r i e r e ( 1 8 6 7 ) . The s c i e n t i f i c name, Tsuga  m e r t e n s i a n a f o r m o u n t a i n h emlock and T, h e t e r o p h v l l a f o r w e s t e r n hemlock was f i r s t e s t a b l i s h e d by S a r g e n t i n 1 8 9 8 . P o l l e n g r a i n s o f m o u n t a i n hemlock were s a c c a t e ( P i g s . 1 3 9 , 140, 145, 14-7), and t h o s e o f w e s t e r n hemlock b i c o n v e x - l e n s - s h a p e d when wet ( P i g s . 149, 1 5 1 , 1 5 2 ) . The av e r a g e p o l l e n s i z e and r a n g e o f t h e d i s t r i b u t i o n o f t h e s e two s p e c i e s a r e shown i n T a b l e s 5 , 9 and 1 0 . I n t h e r e l a t i v e v a r i a b i l i t y , t h e c o r p u s l e n g t h o f m o u n t a i n hemlock p o l l e n was l e a s t . The s i z e - f r e q u e n c y c u r v e s o v e r l a p p e d one a n o t h e r ( P i g s . 2 9 , 3 0 ) , b u t t h e p o l l e n were d i s t i n g u i s h a b l e by t h e i r c o m p l e t e d i f f e r e n c e i n morpho l o g y . I t was f o u n d t h a t t h e r e were some v a r i a t i o n s i n t h e p o l l e n g r a i n s o f m o u n t a i n hemlock ( T a b l e 1 2 ) . Most o f t h e g r a i n s had two s a c c i ( P i g . 1 3 9 ) , b u t some o f t h e s a c c i were r e d u c e d ( P i g . 1 4 0 ) . P o l l e n g r a i n s w i t h u n i s a c c u s ( P i g s . 143, 1 4 4 ) , w i t h v e s t i g a l s a c c i ( P i g . 141) and w i t h o u t any s a c c i ( P i g . 142) were t h e o t h e r t h r e e c l a s s e s f o u n d . Abnormal g r a i n s were t h o s e w i t h u n i s a c c u s w h i c h e n c i r c l e d t h e d i s t a l f a c e and c o v e r e d t h e g e r m i -n a l f u r r o w . There were two t y p e s o f abnormal p o l l e n ; one had 7 2 T a b l e 12 The c o u n t s o f t h e p o l l e n g r a i n s w i t h s a c c i , v e s t i g a l s a c c i , a bnormal s a c c u s , and w i t h o u t s a c c u s i n Tsuga m e r t e n s i a n a . No. o f p o l l e n c o u n t e d P o l l e n w i t h s a c c i 1484 P o l l e n w i t h v e s t i g a l s a c c i  3 0 P o l l e n w i t h abnormal s a c c i  12 P o l l e n w i t h o u t s a c c u s P e r c e n t 9 6 . 9 1 . 9 0.8 0.4 73 t h e r i m o f t h e s a c c u s i n u n f r i l l e d f o r m ( F i g . 14-3), t h e o t h e r i n f r i l l e d f o r m ( P i g . 1 4 4 ) . Campo-Duplan ( 1 9 5 5 ) p r e s e n t e d some p h o t o s w h i c h showed t h e v a r i a t i o n s o f p o l l e n g r a i n s i n Tsugo- K e t e l e e r i a l o n g i b r a c t e a t a ( i . e . Tsuga l o n g i b r a c t e a t a ) and one o f them was p o l l e n w i t h o u t s a c c i . Ueno ( 1 9 5 7 ) a l s o f o u n d t h a t p o l l e n i n m o u n t a i n hemlock had a s a c c u s i n t h e f r i l l f o r m w h i c h e n c i r c l e d t h e e q u a t o r i a l a r e a . I t a p p e a r s t h a t changes i n t h e s i z e o f p o l l e n s a c c i i n m o u n t a i n hemlock have been t o w a r d s r e d u c t i o n . A l t h o u g h o n l y 2 . 3 p e r c e n t o f p o l l e n g r a i n s were f o u n d w i t h v e s t i g a l s a c c i and w i t h o u t any s a c c i , most o f them were s m a l l . However, t h e r e d u c -t i o n o f s a c c i w h i c h marks t h e e v o l u t i o n o f m o u n t a i n hemlock p o l l e n needs a l o n g p e r i o d o f r e c o r d i n g b e f o r e c o n f i r m a t i o n . Wodehouse ( 1 9 5 9 ) r e p o r t e d t h a t t h e e v o l u t i o n o f t h e p o l l e n g r a i n s o f t h e c o n i f e r s was marked t h r o u g h o u t i t s c o u r s e by r e d u c t i o n . I t has l o n g been r e c o r d e d t h a t t h e p o l l e n o f C o r d a i t e s and L e b a c h i a ( f o s s i l g e n e r a i n c o n i f e r s f o u n d i n l a t e P a l e o z o i c and e a r l y M e s o z o i c p e r i o d s ) a r e s u r r o u n d e d by a s i n g l e s a c c u s ( S c a g e l e t a l . , 1 9 6 9 ) . The d i s a c c a t e p o l l e n as s e e n i n P i n a c e a e h a s e v o l v e d f r o m t h e monosaccate p o l l e n o f L e b a c h i a by a s u p p r e -s s i o n o f t h e s u r r o u n d i n g s a c c u s a l o n g t h e p r o x i m a l s u r f a c e o f t h e g r a i n ( S c a g e l e t a l . , 1 9 6 9 ) . I n t h e p o l l e n g r a i n s o f w e s t e r n hemlock, two t y p e s o f g r a i n s - one w i t h o u t n a r b e and t h e o t h e r w i t h n a r b e a t t h e c e n t e r o f t h e d i s t a l f a c e were shown i n F i g u r e s 149, 1 5 1 , 1 5 2 , 153 and 74-1 5 5 - They were a l m o s t e q u a l l y common. The o t h e r v a r i a t i o n was i n t h e p u f f y f r i l l where most o f t h e g r a i n s had f r i l l s as shown i n F i g u r e s 149 and 1 5 2 , and a few had w i d e r f r i l l s as shown i n F i g u r e 1 5 1 . Ueno ( 1 9 5 7 ) s t u d i e d f i v e s p e c i e s o f E u t s u g a and c o n c l u d e d t h a t p o l l e n g r a i n s a l w a y s ha d n a r b e w h i c h was a c h a r a c t e r i s t i c o f t h i s subgenus. Ho and S z i k l a i ( 1 9 7 1 ) r e p o r t e d t h a t a n a r b e was f o u n d i n t h e c e n t e r o f t h e d i s t a l f a c e o f w e s t e r n hemlock p o l l e n . The two s a c c i o f m o u n t a i n hemlock p o l l e n was d i s p l a c e d t o t h e d i s t a l f a c e so t h a t t h e a n g l e o f t h e s a c c a t e a t t a c h m e n t was l a r g e . T h e r e f o r e t h e two s a c c i o f t h e d r y p o l l e n t e n d e d t o bend i n w a r d s and e n c l o s e d t h e g e r m i n a l f u r r o w ( F i g s . 145, 14-7). The d r y p o l l e n o f w e s t e r n hemlock was a l w a y s concave a t t h e d i s t a l f a c e and was s a u c e r - s h a p e d ( F i g . 1 5 3 ) . P o l l e n o f t h e s e two s p e c i e s showed no t r a c e o f a t r i r a d i a t e s t r e a k and no m a r g i n a l c r e s t s ( F i g s . 146, 1 5 4 ) . A g r o o v e was f o u n d a t t h e e q u a t o r i a l a r e a i n t h e p o l l e n o f w e s t e r n hemlock ( F i g s . 1 5 4 , 1 5 6 ) . I t seemed t h a t t h e g r o o v e was s i t u a t e d on t h e a r e a between t h e p u f f y f r i l l and t h e d i s t a l f a c e . The p o l l e n s c u l p t u r e o f t h e two hemlock s p e c i e s examined was m i c r o v e r r u c a t e ( P i g s . 145, 146, 147, 153, 1 5 4 , 155, 1 5 6 ) . The g r a n u l e on t h e p o l l e n s c u l p t u r e o f m o u n t a i n hemlock was p r o -v i d e d w i t h a wavy s u r f a c e and each g r a n u l e was d e l i m i t e d by i n t e r s e c t i n g r i b s ( F i g . 1 4 8 ) . M i n u t e p r o t r u s i o n s a r o s e f r o m t h e i n t e r s e c t i n g r i b s o r f r o m t h e g r a n u l e s . On t h e o t h e r hand, t h e 7 5 g r a n u l e s o n t h e p o l l e n s c u l p t u r e o f w e s t e r n hemlock was ornament-ed w i t h s p i n u l e s and t h e s u r f a c e o f t h e g r a n u l e s was smooth ( F i g s . 1 5 5 , 1 5 6 , 1 5 7 , 1 5 8 ) . On t h e c r e s t o f each g r a n u l e , t h e r e was a l w a y s one s p i n u l e , seldom two. The g r a n u l e s were d e l i m i t e d "by t h e i n t e r s e c t i n g r i b s f r o m w h i c h some m i n u t e p r o t r u s i o n s emanated. Erdtman ( 1 9 5 7 ) a l s o r e p o r t e d t h a t s p i n u l e s were n o t f o u n d on t h e g r a n u l e s o f m o u n t a i n hemlock p o l l e n , b u t were on t h e g r a n u l e s o f w e s t e r n hemlock p o l l e n . From t h e p o l l e n m orphology o f t h e s e two s p e c i e s , i t seems t h a t t h e y may n o t have e v o l v e d f r o m each o t h e r , b u t f r o m a common a n c e s t o r w i t h s u r r o u n d i n g s a c c a t e p o l l e n g r a i n s . - , The — f o l l o w i n g f a c t o r s may have c o n t r i b u t e d t o one t y p e e v o l v i n g i n t o w e s t e r n hemlock p o l l e n by t h e r e d u c t i o n o f two b i l a t e r a l s a c c i , and t h e o t h e r i n t o m o u n t a i n hemlock b y t h e r e d u c t i o n o f t h e f r i l l . ( 1 ) I n e v o l u t i o n , change o f v a r i o u s p a r t s o r p r o c e s s e s i n i n d i v i d u a l s d o m t p r o c e e d a t t h e same r a t e . ( 2 ) R e p r o d u c t i o n i s e s s e n t i a l f o r t h e c o n t i n u e d e x i s t e n c e o f a s p e c i e s . The m o d i f i c a t i o n o f p o l l e n m orphology may have been n e c e s s a r y t o adapt t o t h e new environment f o r s u r v i v a l . ( 3 ) The m o d i f i c a t i o n o f p o l l e n m o r p hology may have been by i n t r o g r e s s i o n . The h y p o t h e s i s o f a p r o p o s e d a n c e s t o r c a n o n l y be s u b s t a n -t i a t e d by f i n d i n g f o s s i l e v i d e n c e . However, t h e p o l l e n o f m o u n t a i n hemlock has been f o u n d w i t h a s a c c u s i n f r i l l f o r m w h i c h e n c i r c l e s t h e e q u a t o r i a l a r e a (Ueno, 1 9 5 7 ) . I n t h i s s t u d y , p o l l e n g r a i n w i t h a s i n g l e s a c c u s was f o u n d i n f r i l l f o r m . A l l 76 t h e s e may he t h e v e s t i g i a l f o rms o f t h e i r a n c e s t o r . S p e c i a t i o n a r i s e s t h r o u g h t h e a c c u m u l a t i o n o f many d i f f e r -e n t g e n e t i c c h a n g e s , a d a p t a t i o n t o d i f f e r e n t h a b i t a t s and s u b s e q u e n t l y g e n e t i c i s o l a t i o n . M o u n t a i n and w e s t e r n hemlock grow i n a d i f f e r e n t h a b i t a t . That t h e g e n e t i c make-up i s d i f f e r e n t and t h e genes i n t e r a c t w i t h d i f f e r e n t e n v i r o n m e n t s may c a u s e v a r y i n g r a t e s o f change i n each s p e c i e s . The d i f f e r -ence i n t h e i r p o l l e n m orphology may be e x p r e s s i o n s o f t h e r e s u l t o f l o n g e v o l u t i o n a r y s t r u c t u r e m o d i f i c a t i o n . D o y l e (1945) has r e p o r t e d t h a t t h e p o l l i n a t i o n mechanisms o f m o u n t a i n and w e s t e r n hemlock a r e d i f f e r e n t . M o u n t a i n hemlock has no e x u d a t e and t h e m i c r o p y l a r edge expands as a s t i g m a t i c f l a r e . A t an e a r l y s t a g e a f t e r p o l l i n a t i o n , t h e n u c e l l u s grows up l i k e a p l u g i n t o t h e m i c r o p y l a r c a n a l and p r o t r u d e s s l i g h t l y . P o l l e n g r a i n s anywhere on t h e s t i g m a t i c a r e a g e r m i n a t e r e a d i l y i n s i t u , t h e t u b e s g r o w i n g d i r e c t l y t o p e n e t r a t e t h e n u c e l l u s . W i t h no e x u d a t e , no f l a r e and no g r o w i n g n u c e l l u s o f w e s t e r n hemlock, p o l l e n g r a i n s may l o d g e anywhere and g e r m i n a t e . C e r -t a i n changes i n p o l l e n m o r phology may have become n e c e s s a r y f o r p o l l i n a t i o n and o r i e n t a t i o n o f p o l l e n g r a i n s on t h e n u c e l l u s . T h i s change, i n t u r n , m i g h t have f a c i l i t a t e d t h e p o l l e n t u b e t o t r a v e r s e t h e n u c e l l u s and t o i n c r e a s e t h e changes o f f e r t i l i z a -t i o n f o r r e p r o d u c t i o n and c o n s e q u e n t l y s u r v i v a l o f t h e s p e c i e s . The p r o c e s s o f i n t r o g r e s s i o n i n e s t a b l i s h m e n t o f a new s p e c i e s m i g h t r e s u l t i n c e r t a i n changes o f t h e g e n e t i c make-up and i n m o d i f i c a t i o n o f p o l l e n m o r p h o l o g y . M o u n t a i n hemlock 7 7 m i g h t have e v o l v e d f r o m t h i s p r o c e s s . Campo-Duplan and Gaussen ( 1 9 4 8 ) have s u g g e s t e d t h a t m o u n t a i n hemlock i s t h e r e s u l t o f an i n t e r g e n e r i c h y b r i d o f w e s t e r n hemlock and S i t k a s p r u c e . A c c o r d i n g t o D u f f i e l d ' s r e p o r t ( 1 9 5 0 ) , t h e h y b r i d i s i n d o u b t . No b o t a n i s t s have f o u n d s u c h h y b r i d swarms i n t h e i r n a t u r a l d i s t r i b u t i o n . C h e m i c a l s i m i l a r i t i e s between t h e s e two g e n e r a have been measured and f o u n d t o be c o n s i d e r a b l e b u t n o t i n d i c a -t i v e o f h y b r i d i z a t i o n ( T a y l o r , 1 9 7 2 ) . The r e l a t e d n e s s among t h e s e t h r e e s p e c i e s c a n be measured by DNA h y b r i d i z a t i o n . On t h e o t h e r hand, chemotaxonomical i n v e s t i g a t i o n s may be u s e f u l i n s y s t e m a t i c and e v o l u t i o n a r y s t u d i e s . T ree b r e e d e r s , t e s t i n g t h e c r o s s a b i l i t y o f t h o s e t h r e e s p e c i e s , may shed more l i g h t on t h e i r r e l a t e d n e s s . Tsuga . i e f f r e v i has been l o n g a c c e p t e d as a h y b r i d between m o u n t a i n and w e s t e r n hemlock ( D u f f i e l d , 1 9 5 0 ) . T a y l o r and Wood ( 1 9 7 1 ) r e p o r t e d t h a t t h i s h y b r i d i z a t i o n c o u l d be s u s p e c t e d . From t h e s e , i t may be s u b s t a n t i a t e d t h a t m o u n t a i n hemlock i s more c l o s e l y r e l a t e d t o w e s t e r n hemlock t h a n t o S i t k a s p r u c e . T a y l o r ( 1 9 7 2 ) s t u d i e d t h e p h y t o c h e m i c a l r e l a t i o n s h i p s i n t h e genus Tsuga by c o m p a r a t i v e p o l y p h e n o l i c pigment a n a l y s e s . He c o n c l u d e d t h a t " c h e m i c a l h y b r i d s " o f m o u n t a i n and w e s t e r n hemlock were r a r e b u t d i d o c c u r . Based on a n a t o m i c a l and m o r p h o l o g i c a l s t u d i e s , Campo-Duplan and Gaussen ( 1 9 4 8 ) f o u n d t h a t m o u n t a i n hemlock r e s e m b l e d w e s t e r n hemlock i n n i n e o u t o f f o r t y c h a r a c t e r i s t i c s i n v e s t i g a t e d . These two s p e c i e s a r e d i f f e r e n t n o t o n l y i n p o l l e n m orphology b u t a l s o i n t h e p o l l i n a t i o n mechanism, i n h a b i t a t , i n p l a n t m o r p hology 7 8 and i n p o l y p h e n o l i c compounds. The a u t h o r recommends s p l i t t i n g m o u n t a i n and w e s t e r n hemlock i n t o s e p a r a t e g e n e r a r a t h e r t h a n two s e c t i o n s o r s u b g e n e r a . T h i s has a l s o been p r o p o s e d by Lemmon ( 1 8 9 0 , 1 8 9 8 ) and R ydberg ( 1 9 1 2 ) . D. I n t e r g e n e r i c v a r i a t i o n o f p o l l e n g r a i n s . ( 1 ) Ps e u d o t s u g a - L a r i x These two g e n e r a d i f f e r . c o m p l e t e l y i n p l a n t m orphology, b u t have common p o l l e n m o r p h o l o g y . The two p o l l e n s t r u c t u r e s a r e n o t d i s t i n g u i s h a b l e u nder t h e l i g h t m i c r o s c o p e . I n f i n e d e t a i l , t h e y a l s o have t h e same p a t t e r n o f e x i n e s c u l p t u r e b u t some v a r i a t i o n s i n t h e d i a m e t e r and d e n s i t y o f m i n u t e p r o t r u s i o n s . They c a n b e s t be s e p a r a t e d by t h e i r d i f f e r e n c e s i n s i z e and m a r k i n g s . The m a r k i n g s o f t h e t r i r a d i a t e s t r e a k i n P s e u d o t s u g a s p e c i e s ( P i g s . 3 8 - 5 0 ) c a n be e a s i l y d i s t i n g u i s h e d f r o m t h e l e s s w e l l d e v e l o p e d ones i n L a r i x s p e c i e s ( P i g s . 6 6 , 6 7 , 6 9 , 7 1 , 7 2 , 7 4 , 7 8 ) . The f u n c t i o n o f t h e m i n u t e p r o t r u s i o n s on t h e p o l l e n e x i n e i s n o t known. I t i s s u g g e s t e d h e r e t h a t i t may s e r v e as a h o l d f a s t on t h e s t i g m a t i c f l a r e d u r i n g p o l l i n a t i o n . The d i f f e r e n c e i n p o l l e n s i z e and m a r k i n g s may be a t t r i b u -t a b l e t o t h e d i f f e r e n c e s i n e n v i r o n m e n t a l f a c t o r s b e s i d e s t h e g e n e t i c make-up. Why do t h e s e two g e n e r a have s i m i l a r p o l l e n m o r p h o l o g y and e x i n e s c u l p t u r a l p a t t e r n ? What i s t h e i r r e l a t e d -n e s s ? Have t h e y e v o l v e d f r o m each o t h e r o r f r o m a common a n c e s t o r and f o l l o w e d p a r a l l e l e v o l u t i o n ? The p o l l i n a t i o n mechanism i n P s e u d o t s u g a p r o b a b l y was 7 9 s l i g h t l y more advanced t h a n i n L a r i x ( D o y l e , 1 9 4 - 5 ) . A l l e n ( 1 9 6 5 ) f o u n d t h a t i n P s e u d o t s u g a t h e p o l l e n r e m a i n e d i n t h e m i c r o p y l a r chamber, h e l d t h e r e i n i t i a l l y by t h e s t i g m a t i c h a i r s ; o n l y t h e p o l l e n t u b e s e v e n t u a l l y r e a c h e d t h e n u c e l l u s . I n c o n -t r a s t , p o l l e n o f L a r i x i s t r a n s f e r r e d b y exudate t o t h e n u c e l l u s a f t e r i t h a s been h e l d t e m p o r a r i l y w i t h i n t h e i n t e g u m e n t t i p ( B a r n e r and C h r i s t i a n s e n , 1 9 6 0 ; D o y l e , 1 9 4 - 5 ) . These two g e n e r a behave s i m i l a r l y i n p o l l e n g e r m i n a t i o n d u r -i n g g a m e t o g e n e s i s . However, t h e body c e l l o f t h e male gameto-p h y t e d i v i d e s i n t o two male c e l l s i n P s e u d o t s u g a (Ho and S z i k l a i , 1 9 7 2 ) , w h i l e t h e body n u c l e u s i n L a r i x p r o d u c e s two male n u c l e i w i t h i n t h e body c e l l (Ho and House, 1 9 7 0 ) . T h i s marks t h e d i f f e r e n c e i n ontogeny o f t h e male gametophyte o f t h e s e two g e n e r a . Prom a l l t h e i n f o r m a t i o n , i t i s c o n s i d e r e d t h a t P s e u d o t s u g a and L a r i x may have been t h e r e s u l t o f p a r a l l e l development o f s i m i l a r c h a r a c t e r s on t h e b a s i s o f t h e c h a r a c t e r s o f t h e i r common a n c e s t o r . K u p r i a n o v a ( 1 9 6 9 ) s t a t e d t h a t s i n c e t h e g e n e r a t i v e c h a r a c t e r s o f p o l l e n and s p o r e s c o r r e s p o n d e d among h i g h - r a n k t a x a , s u c h as g e n e r a , f a m i l i e s and o r d e r s , f e a t u r e s o f p a r a l l e l i s m i n t h e p o l l e n m orphology were f r e q u e n t l y f o u n d i n r e l a t i v e l y remote g r o u p s . ( 2 ) A b i e s - K e t e l e e r i a - T s u g a P o l l e n g r a i n s i n t h i s group have one t h i n g i n common i n t h a t s a c c i a r e d i s p l a c e d t o t h e d i s t a l f a c e s . T h e r e f o r e t h e a n g l e o f t h e s a c c a t e a t t a c h m e n t i s more t h a n 40°. Dry g r a i n s 80 a l s o have t h e same shape and t h e s a c c i t e n d t o bend i n w a r d s t o e n c l o s e t h e g e r m i n a l f u r r o w . P o l l e n o f each genus i n t h i s group c a n be e a s i l y i d e n t i f i e d n o t o n l y by p o l l e n s i z e b u t a l s o by t h e f i n e s t r u c t u r e . K e t e l e e r i a i s c h a r a c t e r i z e d by i t s huge s i z e and r u g u l a t e s c u l p -t u r e o f t h e p o l l e n c a p . A b i e s p o l l e n i s d i s t i n c t f r o m t h e o t h e r s by i t s w e l l marked t r i r a d i a t e s t r e a k , and some s p e c i e s o f Tsuga p o l l e n ( e . g . T. m e r t e n s i a n a ) by i t s s m a l l s i z e and r e d u c e d p o l l e n s a c c i . I n f i n e d e t a i l , e a c h genus has i t s own c h a r a c -t e r i s t i c as shown i n F i g u r e s 9 1 , 9 5 , 1 1 2 , 113 and 148. O v e r a l l p o l l e n m orphology o f K e t e l e e r i a i s more o r l e s s s i m i l a r t o t h a t o f A b i e s . T h i s has a l s o been c i t e d by Erdtman ( 1 9 6 5 ) and Campo-Duplan ( 1 9 5 0 ) . The c h a r a c t e r s o f s a c c a t e p o l l e n o f Tsuga r e s e m b l e t h o s e o f A b i e s . Ueno ( 1 9 5 7 ) came t o t h e same c o n c l u s i o n . The c l o s e -n e s s o f g e n e r a A b i e s and Tsuga was a l s o f o u n d i n p o l l i n a t i o n mechanism ( D o y l e , 194-5) and embryogeny ( B u c h h o l z , 1 9 2 0 ) . ( 3 ) C e d r u s - P i c e a T h i s g r o u p i s c h a r a c t e r i z e d by t h e s e x i n e o f t h e cap and s a c c i m a k i n g an e s s e n t i a l l y c o n t i n u o u s l i n e . The s a c c i a r e b r o a d e s t a t t h e b a s e , g i v i n g them an o v o i d - c o n i c a l f o r m . The a n g l e o f t h e s a c c a t e a t t a c h m e n t i s about 3 0 ° . P o l l e n g r a i n s o f Cedrus s p e c i e s a r e s m a l l . The g e r m i n a l f u r r o w o f t h e d r y g r a i n s a r e n e v e r e n c l o s e d by t h e s a c c i o r c o r p u s . P o l l e n g r a i n s o f P i c e a s p e c i e s however, a r e b i g and t h e 81 g e r m i n a l f u r r o w o f d r y g r a i n s i s e n c l o s e d by t h e a r c h e d c o r p u s . They c a n a l s o be e a s i l y i d e n t i f i e d by t h e i r f i n e s t r u c t u r e . The wavy s u r f a c e o f Cedrus p o l l e n i s ornamented w i t h some m i n u t e p r o t r u s i o n s , w h i l e t h e g r a n u l a t e s u r f a c e o f P i c e a p o l l e n i s de-l i m i t e d by t h e i n t e r s e c t i n g r i b s f r o m w h i c h t h e m i n u t e p r o t r u s i o n s a r i s e . (4-) P i n u s P i n u s i s t h e o n l y genus w h i c h does n o t have some c h a r a c -t e r i s t i c s i n common w i t h o t h e r g e n e r a ( e x c e p t s a c c i ) . P o l l e n g r a i n s a r e r a t h e r s m a l l . A c c o r d i n g t o t h e shape o f d r y g r a i n s , t h i s genus c a n be d i v i d e d i n t o two g r o u p s : O ) t h e a n g l e o f t h e s a c c a t e a t t a c h m e n t i s about 3 0 ° and t h e p o l l e n g r a i n s a r e a r c h e d ( P i g s . 1 2 5 , 1 3 3 ) ; ( 2 ) t h e a n g l e o f t h e s a c c a t e a t t a c h m e n t i s about 3 6 ° and t h e p o l l e n s a c c i t e n d t o bend i n w a r d s and e n c l o s e t h e g e r m i n a l f u r r o w ( P i g s . 1 2 9 , 1 3 6 ) . The shape o f d r y g r a i n s a r e s i m i l a r t o t h e A b i e s - K e t e l e e r i a - T s u g a g r o u p . ( 5 ) Tsuga I n o t h e r s p e c i e s o f Tsuga ( e . g . T. h e t e r o p h y l l a ) , p o l l e n g r a i n s a r e b i c o n v e x - l e n s - s h a p e d when wet and s a u c e r - s h a p e d when d r y . T h i s i s u n i q u e i n t h e P i n a c e a e f a m i l y . The f r i l l s o f p o l l e n g r a i n s i n t h i s group a r e t h o u g h t t o be r u d i m e n t a r y s a c c i (Campo-Duplan, 1 9 5 0 ; Wodehouse, 1 9 5 9 ) . P o l l e n w i t h s a c c i i s more p r i m i t i v e t h a n t h a t w i t h o u t s a c c i (Wodehouse, 1 9 5 9 ) . S c a g e l e t a l . ( 1 9 6 9 ) s t a t e d t h a t P s e u d o t s u g a has e v o l v e d f a i r l y r e c e n t l y . T h e r e f o r e t h i s g r o u p s t a n d s i n t h e e v o l u t i o n a r y s e r i e s between t h o s e g e n e r a w i t h s a c c a t e p o l l e n and P s e u d o t s u g a and L a r i x . 82 6. SUMMARY AND CONCLUSION 1) 37 specimens f r o m 26 s p e c i e s i n 8 P i n a c e a e g e n e r a were s t u d i e d by means o f l i g h t , s c a n n i n g e l e c t r o n and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e s . 2) The d i a m e t e r o f t h e e x i n e p r o t r u s i o n s o f P s e u d o t s u g a  m e n z i e s i i p o l l e n h a r v e s t e d p r i o r t o n a t u r a l s h e d d i n g was much g r e a t e r t h a n t h a t o f mature p o l l e n . The r e a s o n may be t h a t t h e e a r l y h a r v e s t o f t h e t w i g s suspends t h e f u r t h e r s u p p l y o f t h e p r e c u r s o r s n e c e s s a r y t o t h e p o l y m e r i z a t i o n and f o r m a t i o n o f t h e e x i n e . The e x p a n s i o n o f t h e p r o t r u s i o n s may be c a u s e d by t h e a p p l i c a t i o n o f a c e t o n e d u r i n g t h e s p e c i m e n p r e p a r a t i o n b ecause o f i n c o m p l e t e p o l y m e r i z a t i o n . However, a c e t o n e a p p e a r e d t o have no e f f e c t on mature p o l l e n g r a i n s . 3) P s e u d o t s u g a p o l l e n g r a i n s a r e e l l i p t i c a l - s h a p e d when wet and cup-shaped when d r y . P o l l e n c o u l d n o t be d i s t i n g u i s h e d f r o m t r e e t o t r e e and f r o m s p e c i e s t o s p e c i e s , b a s e d on t h e f i n e s t r u c t u r e . These f o u r s p e c i e s c o u l d o n l y be s e p a r a t e d by t h e i r s i z e - f r e q u e n c y c u r v e s . A n n u l a r t h i c k e n i n g s and t r i r a d i a t e s t r e a k s on t h e p r o x i m a l f a c e o f t h e p o l l e n g r a i n s d i v i d e d t h i s f a c e i n t o t h r e e s e c t o r s . T r i r a d i a t e s t r e a k s were e i t h e r e q u a l l y o r u n e q u a l l y s p a c e d . The s c u l p t u r a l p a t t e r n was s c a b r a t e . Under TEM, s u r f a c e r e p l i c a s r e v e a l e d t h a t t h e e x i n e a r e o l a e were formed by t h e i n t e r s e c t i n g r i b s . M i n u t e p r o t r u s i o n s a r o s e f r o m t h e i n t e r s e c t i n g p o i n t s . The d i a m e t e r and d e n s i t y o f t h e s e p r o t r u s i o n s v a r i e d between 83 t r e e s and between s p e c i e s . 4 ) Although. P s e u d o t s u g a s p e c i e s d i f f e r i n chromosome number, t h e y a r e s i m i l a r i n t h e i r e x i n e s c u l p t u r e w h i c h i s c o n t r o l l e d b y s p o r o p h y t e as w e l l as gametophyte. T h i s may be due t o s i m i -l a r i t y i n t h e number o f chromosome arms and chromosomal make-up a f t e r chromosome r e o r g a n i z a t i o n . 5) P o l l e n g r a i n s o f L a r i x s p e c i e s have t h e same p o l l e n morpho-l o g y as t h o s e o f P s e u d o t s u g a s p e c i e s , e x c e p t t h a t t h e L a r i x p o l l e n i s s m a l l e r and t h e t r i r a d i a t e s t r e a k i s l e s s w e l l d e v e l o p e d . I n L a r i x . p a r t o f t h e t r i r a d i a t e s t r e a k was v i s i b l e where t h e s t r e a k j o i n e d t h e a n n u l a r t h i c k e n i n g . T h i s may be due t o a space i n t h e c e n t e r o f t h e t e t r a h e d r a l arrangement a t t h e t e t r a d s t a g e o f p o l l e n development. E i g h t s p e c i e s examined were n o t d i s t i n g u i s h a b l e f r o m t h e i r s i z e - f r e q u e n c y c u r v e s . However, t h e d i a m e t e r and d e n s i t y o f m i n u t e p r o t r u s i o n s i s d i f f e r e n t i n some o f t h e s p e c i e s , b u t cannot be u s e d f o r s p e c i e s i d e n t i f i c a t i o n . The s i m i l a r i t y o f p o l l e n m o r p h o l o g y i n P s e u d o t s u g a and L a r i x may be t h e r e s u l t o f t h e p a r a l l e l development o f s i m i l a r c h a r a c -t e r s f r o m t h e i r common a n c e s t o r . 6) A b i e s . P o l l e n g r a i n s o f g r a n d (A. g r a n d i s ) and S p a n i s h f i r (A. n i n s a n o ) c o u l d be i d e n t i f i e d by t h e i r s i z e - f r e q u e n c y c u r v e s , m a r k i n g s and f i n e s t r u c t u r e , a l t h o u g h t h e y had t h e same m i c r o -v e r r u c a t e s c u l p t u r e . E x i n e a r e o l a e were o n l y f o u n d i n t h e f i n e s t r u c t u r e o f g r a n d f i r p o l l e n . 7) C e d r u s . P o l l e n g r a i n s o f c e d a r s a r e o v o i d - c o n i c a l . I n d r y g r a i n s , t h e s a c c i n e v e r e n c l o s e d t h e g e r m i n a l f u r r o w . P o l l e n 84 s c u l p t u r e was m i c r o v e r r u c a t e . F i n e d e t a i l showed t h a t t h e un-d u l a t e s u r f a c e was c o v e r e d w i t h m i n u t e p r o t r u s i o n s . The s p e c i e s examined c o u l d o n l y he d i s t i n g u i s h e d b y t h e i r f i n e s t r u c t u r e . The g e r m i n a l f u r r o w o f A t l a s c e d a r ( C . a t l a n t i c a ) p o l l e n had a g r a n u l a r s u r f a c e . M i c r o v e r r u c o i d s c u l p t u r e was f o u n d on t h e u n d u l a t e s u r f a c e o f I n d i a n ( C . d e o d a r a ) and Lebanon c e d a r ( C . l i b a n i ) p o l l e n . However, t h e m i n u t e p r o t r u s i o n s were h e a v i l y c o v e r e d on t h e s u r f a c e o f Lebanon c e d a r p o l l e n . 8) K e t e l e e r i a . C o w t a i l f i r ( K . d a v i d i a n a ) has a r u g u l a t e p a t t e r n on t h e cap u n i q u e among t h e p o l l e n i n P i n a c e a e . The f i n e s t r u c t u r e showed t h e cap w i t h a c o n v o l u t e d s c u l p t u r e w h i c h was d e l i m i t e d by t h e r i b s and c o v e r e d w i t h m i n u t e p r o t r u s i o n s . The wavy s u r f a c e o f t h e s a c c i was o n l y p r o v i d e d w i t h p o r i and m i n u t e p r o t r u s i o n s . 9) P i c e a . S p r u c e s a r e c h a r a c t e r i z e d by an o v o i d - c o n i c a l f o r m o f p o l l e n as f o u n d i n c e d a r . The a r c h e d c o r p u s o f d r y p o l l e n e n c l o s e d t h e g e r m i n a l f u r r o w . V a r i a t i o n o f t h e s a c c a t e s i z e was f o u n d i n o r i e n t a l s p r u c e ( P . o r i e n t a l i s ) p o l l e n , b u t n o t i n S i t k a s p r u c e ( P . s i t c h e n s i s ) . B o t h s p e c i e s had t h e same m i c r o v e r r u c a t e s c u l p t u r e , b u t S i t k a s p r u c e p o l l e n was o c c a s i o n a l l y marked w i t h a t r i r a d i a t e s t r e a k . I n f i n e s t r u c t u r e , t h e g r a n u l a r s u r f a c e o f S i t k a s p r u c e p o l l e n was d e l i m i t e d b y t h e r i b s and c o v e r e d w i t h m i n u t e p r o t r u s i o n s , w h i l e t h a t o f o r i e n t a l s p r u c e was o c c a s i o n -a l l y d e l i m i t e d by t h e r i b s . The g r a n u l e s o f t h e l a t t e r were s m a l l e r t h a n t h o s e o f t h e f o r m e r . 10) P i n u s . P o l l e n g r a i n s o f p i n e s a r e s m a l l . P o u r s p e c i e s 85 examined were not d i s t i n g u i s h a b l e on the b a s i s of t h e i r s i z e -frequency curves and f i n e s t r u c t u r e . P o l l e n g r a i n s of j a c k ( P . banksiana) and ponderosa p i n e (P. ponderosa) have about a 30° angle saccate attachment, w h i l e the angle i n lodgepole (P. c o n t o r t a ) and s c o t s p i n e (P. s v l v e s t r i s ) i s about 36°. Dry g r a i n s of the former were arched and those of the l a t t e r had t h e i r s a c c i t e n d i n g t o bend inwards and e n c l o s i n g the germinal furrow. M a r g i n a l c r e s t s were present on most of the p i n e p o l l e n g r a i n s . The g r a n u l a r s u r f a c e of those g r a i n s was i n t e r s e c t e d by the r i b s , ornamented w i t h m i c r o v e r r u c o i d s c u l p t u r e and s c a t t e r e d w i t h minute p r o t r u s i o n s . Pine s p e c i e s had the same: f i n e s c u l p t u r e of the p o l l e n e x i n e . This may be a t t r i b u t a b l e t o t h e i r g e n e t i c s i m i l a r i t y . 11) Tsuga. Mountain hemlock (T. mertensiana) p o l l e n g r a i n s v a r i e d i n saccate s i z e . A c c o r d i n g t o t h e i r saccate s i z e , f o u r types of g r a i n s were found. These were p o l l e n w i t h s a c c i , w i t h v e s t i g i a l s a c c i , w i t h abnormal s a c c i and without saccus. S a c c i of most of the g r a i n s were reduced. One i n two hundred and f i f t y g r a i n s examined was found without saccus. The p o l l e n s c u l p t u r e was m i c r o v e r r u c a t e . The g r a n u l a r s u r f a c e was wavy which was d e l i m i t e d by the r i b s and covered w i t h minute p r o t r u s i o n s . P o l l e n of western hemlock (T. h e t e r o p h v l l a ) i s biconvex-lens-shaped when wet and saucer-shaped when dry. Grains were p r o v i d e d w i t h p u f f y f r i l l s and o c c a s i o n a l l y w i t h narbes at the c e n t e r of the d i s t a l f a c e . The p o l l e n s c u l p t u r e was m i c r o v e r r u -c a t e . Each granule had a s p i n u l e on the c r e s t . I n f i n e 86 s t r u c t u r e , t h e g r a n u l a r s u r f a c e was smooth and i n t e r s e c t e d by t h e r i b s f r o m w h i c h m i n u t e p r o t r u s i o n s a r o s e . 12) From t h e p o l l e n m o r p h o l o g y o f m o u n t a i n and w e s t e r n hemlock, i t a p p e a r s t h a t t h e y may n o t have e v o l v e d f r o m each o t h e r , b u t f r o m a common a n c e s t o r w h i c h may have a morphology w i t h s u r -r o u n d i n g saccus.- The d i f f e r e n c e i n t h e i r morphology may have been due t o v a r y i n g r a t e s o f e v o l u t i o n a r y change, t o t h e m o d i f i -c a t i o n o f r e p r o d u c t i v e s t r u c t u r e s f o r t h e i r c o n t i n u e d e x i s t e n c e , o r t o i n t r o g r e s s i o n . M o u n t a i n and w e s t e r n hemlock a r e d i f f e r e n t n o t o n l y i n p o l l e n m o r p h o l o g y b u t a l s o i n h a b i t a t , p l a n t morphology, p o l l i -n a t i o n mechanism and p o l y p h e n o l i c compounds. I t i s recommended by t h e a u t h o r t o s e p a r a t e them i n t o two g e n e r a . 1 3 ) I t i s h e r e p r o p o s e d t h a t t h e p o l l e n o f P i n a c e a e be g r o u p e d , b a s e d on morphology as f o l l o w s : ( 1 ) P s e u d o t s u g a - L a r i x : ( 2 ) A b i e s - K e t e l e e r i a - T s u g a : ( 3 ) C e d r u s - P i c e a ; ( 4 ) P i n u s ; ( 5 ) Tsuga. P o l l e n o f t h e group ( 1 ) i s w i t h o u t any s a c c u s and has t h e same e x i n e s c u l p t u r e . I t i s s i m i l a r i n group ( 2 ) p o l l e n i n t h a t t h e . i r s a c c i a r e d i s p l a c e d t o t h e d i s t a l f a c e . Group ( 3 ) p o l l e n i s c h a r a c t e r i z e d by i t s o v o i d - c o n i c a l f o r m and i t s s a c c i n e v e r e n c l o s e s t h e g e r m i n a l f u r r o w . P i n e p o l l e n o f group ( 4 ) i s s m a l l and u n i f o r m i n i t s f i n e s t r u c t u r e . The l a s t group o f Tsuga i s d i s t i n g u i s h e d by i t s u n i q u e b i c o n v e x - l e n s - s h a p e . 14) P o l l e n s i z e and s t r u c t u r e c a n be u s e d as a d e f i n i t i v e g e n e r i c c h a r a c t e r i n P i n a c e a e . Low-rank t a x a , s u c h as s p e c i e s , s u b s p e c i e s and r a c e s a r e f r e q u e n t l y f o u n d t o have common c h a r a c -87 t e r s . T h e r e f o r e , p o l l e n m o r phology and f i n e s t r u c t u r e have l i m i t e d v a l u e i n s t u d i e s o f p o l l e n s o u r c e s , taxonomy below t h e g e n e r i c l e v e l and p h y l o g e n y . N e v e r t h e l e s s , t h e y c a n r e i n f o r c e t h e c o n c l u s i o n s b a s e d on o t h e r p l a n t m o r p h o l o g i c a l c r i t e r i a . E x i n e p a t t e r n , c o n t r o l l e d by t h e s p o r o p h y t e , t h e gameto-p h y t e o r b o t h , c a n be a u s e f u l a i d i n i n t e r p r e t i n g g e n e t i c and p h y l o g e n e t i c r e l a t i o n s h i p s . 7 . LITERATURE CITED 1 . A g a r , A. ¥., and R. S. M. R e v e l l . 1 9 5 6 . A s t u d y o f t h e Formvar r e p l i c a p r o c e s s . B r i t . J . A p p l . P h y s . 7 : 1 7 - 2 5 . 2 . A l l e n , G. S. 1 9 6 3 . O r i g i n and development o f t h e o v u l e i n D o u g l a s - f i r . F o r . S c i . 9 : 3 8 6 - 3 9 3 . 3 . A r n o , S. P. 1 9 6 6 . A l p i n e l a r c h ( L a r i x l v a l l i i P a r L ) and i t s n a t u r a l o c c u r r e n c e . S c h o o l o f F o r . , U n i v . o f Montana. 5 2 pp. 4 . A y t u g , B. 1 9 6 1 . Etude des p o l l e n s du genre Cedre ( C e d r u s L i n k ; . P o l l e n e t S p o r e s 3 : 4 7 - 5 4 . 5 1 9 6 2 . Diagnose des p o l l e n s de P i n u s s i l v e s t r i s e t P. u n c i n a t a des P y r e n e e s . P o l l e n e t S p o r e s 4:283-296. 6 . B a r n e r , H., and H. C h r i s t i a n s e n . 1 9 6 0 . The f o r m a t i o n o f p o l l e n , t h e p o l l i n a t i o n mechanism, and t h e d e t e r m i n a t i o n o f t h e most f a v o r a b l e t i m e f o r c o n t r o l l e d p o l l i n a t i o n i n L a r i x . S i l v a e Genet. 9 : 1 - 1 1 . 7 1962. The f o r m a t i o n o f p o l l e n , t h e p o l l i n a t i o n mechanism, and t h e d e t e r m i n a t i o n o f t h e most f a v o r a b l e t i m e f o r c o n t r o l l e d p o l l i n a t i o n i n P s e u d o t s u g a . S i l v a e Genet. 1 1 : 8 9 - 1 0 2 . 8 . B e i s s n e r , L. 1 9 0 9 . Handbuch d e r N a d e l h o l z k u n d e . P a u l P a r e y , B e r l i n . 9 . 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( 4 5 0 2 ) . F i g . 6 9 . Showing a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k ( a r r o w ) . ( 1 0 0 0 X ) . P i g . 7 0 . Abnormal p o l l e n i n p e a r - l i k e shape. ( 1 0 0 0 X ) . 117 P i g s . 7 1 - 7 2 . P o l l e n o f Japanese l a r c h . ( 1 0 0 0 X ) . F i g . 7 1 « Showing i n c o n s p i c u o u s a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i -r a d i a t e s t r e a k . F i g . 7 2 . Showing w e l l marked a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i r a d i a t e s t r e a k . 118 F i g . 73. P o l l e n o f a l p i n e l a r c h showing i n c o n s p i c u o u s a n n u l a r t h i c k e n i n g . ( 1 0 0 0 X ) . F i g . 74. P o l l e n o f w e s t e r n l a r c h showing a n n u l a r t h i c k e n i n g and i n c o m p l e t e t r i -r a d i a t e s t r e a k ( a r r o w ) . ( 1 0 0 0 X ) . 119 F i g . 7 5 . 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T u r g i d p o l l e n showing t h e t h i n e x i n e a t t h e c e n t e r o f t h e c a p . (4-5QX). F i g . 9 3 . Showing m i c r o v e r r u c a t e s c u l p t u r e , g r a n u l a t e s t r u c t u r e a t t h e m a r g i n a l a r e a , and p o r i i n t h e s a c c i . ( 1 0 0 0 X ; . F i g . 9 4 . Showing t h e t r i r a d i a t e s t r e a k on t h e cap where two s t r e a k s f o r m a s t r a i g h t l i n e and t h e t h i r d s t r e a k approaches t h i s l i n e by about 3 0 ° . ( 1 0 0 0 1 ) . 129 F i g . 95- F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f S p a n i s h f i r showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s (C) and t h e s a c c u s ( S ) p r o v i d e d w i t h m i n u t e p r o t r u s i o n s . ( 8 , 0 0 0 X ) . 1 3 0 F i g s . 9 6 - 9 9 . P o l l e n o f A t l a s c e d a r . P i g . 9 6 . T u r g i d p o l l e n shows t h a t t h e s e x i n e forms a c o n t i n u o u s l i n e f r om t h e cap t o t h e s a c c i . ( 4 - 5 0 X ) . F i g . 9 7 . Showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s . ( 1 0 0 0 X ) . F i g . 9 8 . Showing m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t . ( 1 0 0 0 X ) . F i g . 9 9 . Showing t h e g r a n u l a t e s u r f a c e o f t h e g e r m i n a l f u r r o w . ( 2 , 0 0 0 X ) . 131 J i g . 100. P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f A t l a s c e d a r showing t h e u n d u l a t i n g s u r f a c e w i t h m i n u t e p r o t r u s i o n s . ( C ) c o r p u s , ( S ) s a c c u s . ( 8 , 0 0 0 X ) . 132 F i g s . 101-102. P o l l e n o f I n d i a n c e d a r . ( 1 0 0 0 1 ) . F i g . 101. Showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s . F i g . 102. Showing s t r a i g h t s t r e a k a c r o s s t h e cap i n t h e d i r e c t i o n o f t h e body l e n g t h . F i g s . 1 0 3-104. P o l l e n o f Lebanon c e d a r . (1000X) F i g . 1 0 3 . Showing t h e wavy s u r f a c e o f t h e c a p . F i g . 104. Showing t h e g e r m i n a l f u r r o w . 133 F i g . 1 0 5 . P i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f I n d i a n c e d a r showing t h e u n d u l a t i n g s u r f a c e o f t h e c o r p u s (C) w i t h m i c r o v e r r u c o i d s c u l p t u r e (MS). The s u r f a c e s o f t h e c o r p u s and s a c c u s ( S ) a r e c o v e r e d w i t h m i n u t e p r o t r u s i o n s ( P ) . ( 8,000X). 134 F i g . 106. F i n e s t r u c t u r e . o f t h e p o l l e n s u r f a c e o f Lebanon c e d a r showing t h e u n d u l a t i n g s u r f a c o f t h e c o r p u s and s a c c u s w i t h m i c r o v e r r u c o i d s c u l p t u r e tMS). The s u r f a c e s o f t h e c o r p u s (C) and s a c c u s (S) a r e c o v e r e d w i t h m i n u t e p r o t r u -s i o n s ( P ) . ( 8 , 0 0 0 X ) . 1 3 5 F i g s . 1 0 7 - 1 0 9 . P o l l e n o f c o w t a i l f i r . F i g . 1 0 7 . T u r g i d p o l l e n . ( 4 - 5 0 X ) . F i g . 108. Showing t h e r u g u l a t e p a t t e r n o f t h e cap s u r f a c e . OOOOXj. F i g . 109. Showing p o r i i n t h e s a c c i . ( 1 0 0 0 X ) . 136 F i g s . 110-111. P o l l e n o f c o w t a i l f i r . ( 1 0 0 0 X ) . P i g . 110. P o l l e n w i t h 3 - s a c c a t e . P i g . 111. P o l l e n w i t h 4 - s a c c a t e . 137 P i g . 112. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f c o w t a i l f i r showing t h e m a r g i n a l a r e a (C) and s a c c u s (S). The g r a n u l a t e s u r f a c e o f t h e m a r g i n a l a r e a i s sometimes i n t e r s e c t e d hy t h e r i b s (R) f r o m which some minu t e p r o t r u s i o n s ( P ) a r e e v o l v e d . The mi n u t e p r o t r u s i o n s a r e a l s o p r o j e c t e d f r o m t h e s u r f a c e o f g r a n u l e s and t h e wavy s u r f a c e o f s a c c u s . ( 8 , 0 0 0 X ) . 138 F i g . 113. F i n e s t r u c t u r e o f t h e cap s u r f a c e o f c o w t a i l f i r p o l l e n showing c o n v o l u t e d s t r u c t u r e w i t h m i n u t e p r o t r u s i o n s . ( l 4 , 0 0 0 X ) . 139 F i g s . 114-117. P o l l e n o f o r i e n t a l s p r u c e . P i g . 114. T u r g i d p o l l e n i n o v o i d - c o n i c a l f o r m . ( 4 5 0 X ) . P i g . 115 • Showing t h e s a c c i r e d u c e d t o r a t h e r s m a l l s i z e s . ( 4 5 0 X ) . P i g . 116. Showing smooth m a r g i n a l a r e a and p o r i i n t h e s a c c i . ( 1 0 0 0 X ) . P i g . 117. Showing t h e m i c r o v e r r u c a t e s c u l p t u r e . ( 1 0 0 0 X ) . 140 F i g . 118. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f o r i e n t a l s p r u c e showing t h e g r a n u l a t e s u r f a c e w h i c h i s sometimes d e l i m i t e d by t h e r i b s ( E ) . The m i n u t e p r o t r u s i o n s ( P ) a r e e v o l v e d e i t h e r f r om t h e r i b s o r f r o m t h e g r a n u l e s (8,000X) 141 Pigs. 119-121. P o l l e n of S i t k a spruce. P i g . 119. Turgid p o l l e n i n ovoid-conical form. (450X). F i g . 120. Showing the arched corpus to enclose the germinal furrow. The marginal areas are granular and p o r i i n the s a c c i . (1000X). F i g . 121. Showing the microverrucate sculpture and f a i n t l y marked t r i r a d i a t e streak (TS). (1000X). 142 Fig. 122. Fine structure of the pollen surface of Sitka spruce showing the areolae which are formed by the inter-secting r i b s . The minute protrusions are evolved either from the surface of granules or from the r i b s . (A) areola, (R) r i b , (P) protrusion. (8,000X). 143 F i g s . 123-125. P o l l e n of jack pine. F i g . 123. Turgid p o l l e n . (450X;. P i g . 124. Showing the microverrucate sculpture and marginal c r e s t s . (1000X). F i g . 125. In arched form and without marginal c r e s t s . (2,000X). 144 F i g . 126. Fine structure of jack pine p o l l e n showing granulate surface which i s sometimes delimited hy the r i b s ( P ) . Minute protrusions (P) are evolved e i t h e r from the r i b s or from the granules. Microverrucoid structure (MS) i s shown on the corpus (C) and saccate surface ( S ) . (8,000X). F i g s . 127-129. P o l l e n o f l o d g e p o l e p i n e . P i g . 127. T u r g i d p o l l e n . ( 4 5 0 X ) . P i g . 128. Showing m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t s . ( 1 0 0 0 X ) . P i g . 129. Show-i n g s a c c i w i t h p o r i . S a c c i t e n d t o bend i n w a r d t o e n c l o s e t h e g e r m i n a l f u r r o w . ( 2 , 0 0 0 X ) . 146 F i g . 1 3 0 . Fine structure of the corpus surface of lodgepole pine p o l l e n showing the granular surface which i s sometimes delimited by the r i b s (RJ. Minute protrusions (P) are evolved e i t h e r from the r i b s or from the granules. Microverrucoid sculpture (MS) i s shown on the granules. (8,000X). 147 F i g s . 131-133. P o l l e n o f p o n d e r o s a p i n e . F i g . 131. T u r g i d p o l l e n showing c o n s p i c u o u s m a r g i n a l c r e s t s . ( 4 5 0 X ) . F i g . 132. Showing m i c r o v e r r u c a t e s c u l p t u r e and m a r g i n a l c r e s t . ( 1 0 0 0 X ) . F i g . 133. P o l l e n i n a r c h e d f o r m . ( 1 0 0 0 X ) . 148 P i g . 134. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f p o n d e r o s a p i n e showing m i c r o v e r r u c o i d s c u l p t u r e (MS). The g r a n u l a t e s u r f a c e o f t h e c o r p u s i s sometimes d e l i m i t e d by t h e r i b s (E) f r o m w h i c h t h e m i n u t e p r o t r u s i o n s ( P ) a r e e v o l v e d . ( C ) c o r p u s , ( S ) s a c c u s . ( 8 , 0 0 0 X ) . 149 P i g s . 1 3 5 - 1 3 7 . P o l l e n o f s c o t s p i n e . P i g . 1 3 5 . T u r g i d p o l l e n s howing mar-g i n a l c r e s t s ( a r r o w ) . (45OX). P i g . 1 3 6 . Showing no m a r g i n a l c r e s t s and s a c c i w h i c h hend i n w a r d t o e n c l o s e t h e g e r m i n a l f u r r o w . ( 2 . 0 0 0 X ) . F i g . 1 3 7 . Showing m i c r o v e r r u c a t e s c u l p t u r e and f a i n t l y marked t r i r a d i a t e s t r e a k ( T S ) . ( 2 , 0 0 0 X ) . 1 5 0 F i g . 138. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f s c o t s p i n e s howing m i c r o v e r r u c o i d s c u l p t u r e (MS). M i n u t e p r o t r u s i o n s ( P ) ' a r e e v o l v e d e i t h e r f r o m t h e r i b s ( f i ) o r f r o m t h e g r a n u l e s . ( C ) c o r p u s , ( S ) s a c c u s . ( 8 , 0 0 0 X ) . 151 > t P i g s . 139-144. P o l l e n o f m o u n t a i n hemlock. ( 4 5 0 X ) . P i g . 139. T u r g i d p o l l e n showing s a c c i w h i c h a r e d i s p l a c e d f u r t h e r t o t h e d i s t a l f a c e . P i g . 140. Showing s a c c i w h i c h a r e r e d u c e d t o r a t h e r s m a l l s i z e s . P i g . 141. P o l l e n w i t h v e s t i g a l s a c c i ( a r r o w J P i g . 142. P o l l e n w i t h o u t s a c c i . P i g . 143. P o l l e n w i t h abnormal s a c c u s i n u n f r i l l e d f o r m . P i g . 144. P o l l e n w i t h abnormal s a c c u s i n f r i l l f o r m . 1 5 2 P i g s . 145-147. P o l l e n o f m o u n t a i n hemlock. P i g . 145. Showing s a c c i w h i c h bend i n w a r d t o e n c l o s e t h e g e r m i n a l f u r r o w . No m a r g i n a l c r e s t s a r e p r e s e n t . ( 1 0 0 0 X ) . P i g . 146. Showing cap w i t h m i c r o v e r r u c a t e s c u l p t u r e . (1000X;. P i g . 147. Showing m i c r o v e r r u c a t e s c u l p t u r e . ( 2 , 0 0 0 X ) . 153 F i g . 148. F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f m o u n t a i n hemlock showing g r a n u l a t e s u r f a c e w h i c h i s d e l i m i t e d by t h e i n t e r s e c t i n g r i b s ( B ) . S u r f a c e o f g r a n u l e s i s wavy. M i n u t e p r o t r u s i o n s ( P ) a r e e v o l v e d e i t h e r f r o m t h e r i b s o r f r o m t h e g r a n u l e s . ( 8 , 0 0 0 1 ) . 154 F i g s . 149-154. P o l l e n o f w e s t e r n hemlock. F i g . 149. T u r g i d p o l l e n i n t h e e q u a t o r i a l v i e w showing f r i l l CF) and n a r b e ( N ) . ( 4 5 0 X ) . P i g . 1 5 0 . T u r g i d p o l l e n a t t h e d i s t a l f a c e show-i n g f r i l l w i t h s p i n u l e s ( S P ) . ( 4 5 0 X ) . F i g . 1 5 1 . T u r g i d p o l l e n showing t h i c k p u f f y f r i l l ( a r r o w ) . (45OX). P i g . 1 5 2 . T u r g i d p o l l e n w i t h o u t n a r b e . (45OX). F i g . 1 5 3 . P o l l e n i n s a u c e r shape showing n a r b e a t t h e c e n t e r o f t h e d i s t a l f a c e . ( 1 0 0 0 X ) . F i g . 154. P o l l e n a t t h e p r o x i m a l f a c e showing g r o o v e (G) a t t h e e q u a t o r i a l a r e a . (1000X). 155 F i g s . 1 5 5 - 1 5 6 . P o l l e n o f w e s t e r n hemlock. F i g . 1 5 5 . P o l l e n showing d e t a i l s o f n a r b e ( N ) . and s p i n u l e s ( S P ) . ( 2 , 0 0 0 1 ) . F i g . 1 5 6 . P o l l e n showing g r o o v e (G) a t t h e e q u a t o r i a l a r e a . ( 2 , 0 0 0 X ; . 156 F i g s . 1 5 7 - 1 5 8 . F i n e s t r u c t u r e o f t h e p o l l e n s u r f a c e o f w e s t e r n hemlock. ( 8 , 0 0 0 X ) . F i g . 1 5 7 . A t t h e e q u a t o r i a l a r e a showing s p i n u l e s ( S P ) and g r a n u l a t e s u r f a c e w h i c h i s d e l i m i t e d by t h e i n t e r s e c t i n g r i b s ( E ) f r o m w h i c h t h e m i n u t e p r o t r u s i o n s ( P ) a r e e v o l v e d . P i g . 1 5 8 . A t t h e p r o x i m a l f a c e showing s p i n u l e s , r i b s , and m i n u t e p r o t r u s i o n s . 157 F i g . 159. T e t r a d s t a g e i n m i c r o -s p o r o g e n e s i s o f D o u g l a s - f i r . The young p o l l e n g r a i n s a r e i n t e t r a d -h e d r a l a r r angement. ( 4 5 0 X ) . 158 o s s a r y o f terms u s e d i n t h e t e x t . t h i n w a l l a r e a s a t t h e d i s t a l f a c e . a p e r t u r e a t t h e d i s t a l f a c e . a space marked o u t on a s u r f a c e ; s m a l l a r e a s e p a r a t e d by s m a l l g r o o v e s f o r m i n g a nega-t i v e r e t i c u l u m . w i t h two v e r t i c a l p l a n e s o f symmetry; e q u a t o r i a l axes n o t e q u i l o n g . t h e t h i c k e n e d d o r s a l ( p r o x i m a l ) s u r f a c e o f t h e s a c c a t e g r a i n s . c a p . w i t h r e s p e c t t o t h e t h i c k n e s s o f t h e e x i n e o f t h e c o r p u s o f c e r t a i n s a c c a t e p o l l e n t y p e s , a p r o x i m a l a r e a r e f e r r e d as cappa and a d i s t a l as c a p p u l a . t h e c e n t r a l p a r t o f p o l l e n g r a i n s h a v i n g s a c c i . t h a t p a r t o f a p o l l e n s u r f a c e w h i c h i s d i r e c t e d o u t w a r d i n i t s t e t r a d . t h e o u t e r , h i g h l y r e s i s t a n t l a y e r o f t h e p o l l e n w a l l . t h e d i s t a l and p r o x i m a l f a c e s a r e more o r l e s s d i s t i n c t l y d i f f e r e n t as r e g a r d s a p e r t u r e s . i f t h e meshes o f a r e t i c u l u m a r e l a r g e and a r r a n g e d r e g u l a r l y , t h e y may be t ermed l a c u n a e . d e h i s c e n c e f i s s u r e . t h i n w a l l a r e a , f u n c t i o n i n g as an a p e r t u r e . t h e s l i g h t l y p r o j e c t i n g r i m o f t h e c a p . w i t h s c u l p t u r i n g e l e m e n t s as b r o a d a s , o r b r o a d e r t h a n h i g h , none o f t h e d i m e n s i o n s w h i c h exceed one m i c r o n . 1 5 9 o r b i c u l e p a r a i s o p o l a r p r o x i m a l f a c e p s i l a t e r a d i o s y m m e t r i c r u g u l a t e s a c c a t e s c a b r a t e s e x i n e t r i r a d i a t e s t r e a k v e r r u c a t e - s m a l l g r a n u l e s s p r e a d o v e r t b e e x i n e s u r f a c e i n c e r t a i n gymnosperm. - t h e d i s t a l and p r o x i m a l f a c e s a r e s i m i l a r . - t h a t p a r t o f a p o l l e n s u r f a c e w h i c h i s d i r e c t e d i n w a r d i n i t s t e t r a d . - smooth. - w i t h more t h a n two v e r t i c a l p l a n e s o f symmetry. - s c u l p t u r i n g t y p e w i t h i r r e g u l a r e l o n g a t e r i d g e s . - w i t h a i r s a c s . - w i t h s c u l p t u r i n g o f p r o j e c t i o n s , no d i m e n s i o n o f w h i c h e q u a l s o r exceeds one m i c r o n . - t h e o u t e r s c u l p t u r e d p a r t o f t h e e x i n e . - t e t r a d mark o r s c a r . - w i t h s c u l p t u r i n g e l e m e n t s as b r o a d a s , o r b r o a d e r t h a n h i g h , one d i m e n s i o n o f w h i c h exceeds one m i c r o n . 

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