Studies on pollen of selected species in Pinaceae

Ho, RongHui

doi:10.14288/1.0101239

UBC Theses and Dissertations

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UBC Theses and Dissertations

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

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Title	Studies on pollen of selected species in Pinaceae
Creator	Ho, RongHui
Publisher	University of British Columbia
Date Issued	1972
Description	The study of the exine pattern of pollen grains might permit the separation of various taxa and -assist the interpretation of their relationships. Thirty-seven specimens from twenty-six species in eight Pinaceae genera were studied by means of light, scanning electron and transmission electron microscopy. The fine pollen surface structure of twenty-two species (out of twenty-six) and the variations of pollen morphology in Tsuga heterophylla and T. mertensiana were demonstrated for the first time. The pollen grains of four Pseudotsuga species were found to be elliptical-shaped when turgid, and cup-shaped when dry. They varied in size, but all had the same scabrate sculpture. The proximal face of grains bore an annular thickening, and a triradiate streak which divided this face into three sectors. Under the transmission electron microscope, surface replicas revealed the exine areolae which consisted of intersecting ribs protruding from the pollen surface. Minute protrusions emanated from intersections of ribs. The diameter and the density of the minute protrusions varied from species to species. Pollen of the species examined here could be distinguished from size-frequency curves of mature grains, but not on the basis of fine structure. In Pseudotsuga menziesii, the diameter of the minute protrusions of pollen grains harvested prior to natural shedding was found to be much greater than that of mature grains. The reason might be that the early harvest of pollen cuts off additional supplies of the necessary precursors for the polymerization and the formation of the exine. The application of acetone during specimen preparation might cause expansion of the protrusions because of incomplete polymerization. Of eight Larix species examined, pollen-size distribution curves overlapped each other. Pollen grains of the eight species had the same morphology and exine sculpture as described for Pseudotsuga, with the exception that the mature pollen size was smaller and the triradiate streak was less well developed. Hence, species were not distinguishable by these criteria. The similarity of the exine sculpture in species of Pseudotsuga pollen may be due to similarities in chromosomal makeup after the chromosome reorganization. It is also possible that Pseudotsuga and Larix have evolved from common ancestors, with the parallel development of similar characters from their common ancestor. Pollen grains of the species in Abies, Cedrus, Keteleeria, Picea and Tsuga examined were found to be distinct from one another, based on pollen morphology and fine structure. The sculpture pattern of the exine was microverrucate, except in Keteleeria which was rugulate on the proximal face. On the other hand, the four species of Pinus pollen were found to be indistinguishable based on fine structure. The exine pattern on the proximal surface of pollen grains was microverrucate. Each granule of the microverrucate sculpture was also ornamented with microverrucoid sculpture. The similarity in fine structure of Pinus pollen might be attributed to the genetic similarity in the species of this genus. Pollen morphology of Tsuga mertensiana completely differed from that of T. heterophvlla. The difference may have been due to varying rates of evolutionary change, to the modification of reproductive structure for their continued existence, or to introgression. The author recommends splitting these two species into separate genera. The higher were the rank of taxa, the greater were the differences in pollen morphology. Based on morphology, pollen in Pinaceae was proposed to be grouped as follows: (1) Pseudot- suga-Larix: (2) Abies-Keteleeria-Tsuga (pollen of saccate type e.g., T. mertensiana); (3) Cedrus-Picea; (4-) Pinus; (5) Tsuga (pollen of biconvex-lens type e.g., T. heterophylla).
Genre	Thesis/Dissertation
Type	Text
Language	eng
Date Available	2011-03-21
Provider	Vancouver : University of British Columbia Library
Rights	For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
DOI	10.14288/1.0101239
URI	http://hdl.handle.net/2429/32636
Degree	Doctor of Philosophy - PhD
Program	Forestry
Affiliation	Forestry, Faculty of
Degree Grantor	University of British Columbia
Campus	UBCV
Scholarly Level	Graduate
AggregatedSourceRepository	DSpace

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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 . B r a d l e y , D.E. 1 9 5 4 . An e v a p o r a t 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 o r use w i t h t h e e l e c t r o n m i c r o s c o p e and i t s a p p l i c a t i o n t o t h e s t u d y o f p h o t o g r a p h i c g r a i n s . B r i t . J . A p p l . P h y s . 5 : 9 6 - 9 7 . 10 1 9 5 7 - Some 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 e l e c t r o n m i c r o s c o p y , i b i d . 8 : 1 5 0 - 1 5 4 . 11 1 9 5 8 . The s t u d y o f p o l l e n g r a i n s u r f a c e s i n t h e e l e c t r o n m i c r o s c o p e . New P h y t o l . 5 7 : 2 2 6 - 2 2 9 . 11 1 2 . Bringmann, G., and R. Kuhn. 1 9 5 5 - E l e k t r o n e n m i k r o g k o p i s c h e Befunde z u r M o r p h o l o g i e d e r C u t i k u l a v o n B l u t e n g a r t n e r i s c h e r und l a n d w i r t s c h a f t l i c h e r N u t z p l a n z e n . Z. N a t u r f o r s c h . 1 0 6 : 4 7 - 5 9 . 1 3 . B u c h h o l z , T. 1 9 2 0 . Embryo development and polyembryo i n r e l a -t i o n t o t h e p h y l o g e n y o f c o n i f e r . Amer. J . B o t . 7 : 1 2 5-145. 14. C a i n , S. A. 1940. The i d e n t i f i c a t i o n o f s p e c i e s i n f o s s i l p o l l e n o f P i n u s by s i z e - f r e q u e n c y d i s t r i b u t i o n s . Amer. J . B o t . 2 7 : 3 0 1 - 3 0 8 . 1 5 . Campo-Duplan, M. v a n . 1 9 4 7 . Etude de q u e l q u e s g r a i n s de p o l l e n b a l l o n n e t s des A b i e t i n e e s . T r a v . L a b . f o r e s t , de T o u l o u s e , t . 1, v o l . 4 , a r t . 16, 8 pp. 8 9 16 1 9 5 0 - R e c h e r c h e s s u r l a p h y l o g e n i e des A b i e t i n e e s d ' a p r e s l e u r s g r a i n s de p o l l e n , i b i d . t . 2, v o l . 4, a r t . 1, 183 PP. 17 1 9 5 5 * Quelques p o l l e n d ' H y b r i d e s d ' A b i e t i n e e s . Z t c h r . P o r s t g e n e t , und P o r s t p f l a n e n z . 4 : 1 2 3-126. 18 e t H. Gaussen. 1948. 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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 . ( 3 6 , 0 0 0 X ) . ( A ) a r e o l a , (R) r i b , ( P ) p r o t r u s i o n . 107 F i g s . 5 3 - 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 "bigcone D o u g l a s - f i r . ( 3 6 , 0 0 0 X ) . F i g . 5 3 . f r o m Oregon. F i g . 5 4 . f r o m C a l i f o r n i a . 108 F i g s . 5 5 - 5 6 . 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 . ( 3 6 , 0 0 0 X ) . F i g . 5 5 . f r o m Vancouver I s l a n d . F i g . 5 6 t r e e No. 1. 109 F i g s . 57-58. 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 . (56,000X). F i g . 57. t r e e No. 2. F i g . 58. t r e e No. 11. 110 P i g s . 59-60. 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 . ( 3 6 , 0 0 0 X ) . F i g . 59. t r e e No. 12. F i g . 60. t r e e A. 111 F i g s . 61-62. 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 . ( 3 6 , 0 0 0 X ) . F i g . 6 1 . t r e e B. F i g . 62. t r e e E. 112 F i g . 63. 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 . ( 3 6 , 0 0 0 X ) . 113 F i g . 54. 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 D o u g l a s - f i r . ( 3 6 , 0 0 0 X ) . . 114 F i g . 65. 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 . ( 3 6 , 0 0 0 X ) . 115 F i g s . 6 6 - 6 7 . 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 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 ) . ( l O O O X j . P i g . 66. European l a r c h . P i g . 67. D a h u r i a n l a r c h . F i g s . 6 8 - 7 0 . P o l l e n o f t a m a r a c k . F i g . 6 8 . T u r g i d p o l l e n . ( 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 . 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 . (1000X). F i g . 76. 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 by 4 m i c r o n s , i n t h e e x i n e . (1000X). 120 F i g . 77. 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 0 . 0 0 0 X ) . F i g . 78. P o l l e n o f S i b e r i a n l a r c h showing 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 ( a r r o w ) . ( 1 0 0 0 X ) . 121 s e c t i n g p o i n t s . (36,000X) 122 123 F i g s . 82-83. 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 . ( 3 6 , 0 0 0 X ) . F i g . 82. Japanese l a r c h . F i g . 83. A l p i n e l a r c h . 124 F i g s . 84-85. 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 . ( 5 6 , 0 0 0 X ) . F i g . 84. W e s t e r n l a r c h . F i g . 85. C h i n e s e l a r c h . 125 F i g . 86. 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 . ( 3 6 , 0 0 0 X ) . 126 F i g s . 87-90. P o l l e n o f g r a n d f i r . P i g . 87. T u r g i d p o l l e n . (4-50X). F i g . 88. T u r g i d p o l l e n a t t h e p r o x i m a l f a c e . (4-50X). F i g . 89. 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 . ( 1 0 0 0 X ) . F i g . 90. 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 . ( 1 0 0 0 X 7 . 127 F i g . 91. F i n e s t r u c t u r e o f the 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 formed hy 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 . (A) a r e o l a , ( B ) r i b , CP) p r o t r u s i o n . ( 8 , 0 0 0 X ) . 128 9 4 F i g s . 9 2 - 9 4 . P o l l e n o f S p a n i s h f i r . P i g . 9 2 . 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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UBC Theses and Dissertations

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

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

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